1. No category

List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
Department of Anatomy
Details of PI
Project Title with a brief description
Prof Bay Boon Huat
Peroxiredoxin III as a prognostic marker and therapeutic target in breast
cancer
PI’s email id
[email protected]
Office Address
Department of Anatomy, NUS
Telephone Number
6516 3200
The Peroxiredoxin (Prx) family comprise of six thiol-specific antioxidant
enzymes which catalyze the reduction of hydrogen peroxide (H2O2). Prxs
confer protection against oxidative stress and regulate cell signaling associated
with H2O2 as a secondary messenger, thereby influencing cell differentiation,
proliferation and apoptosis. Prx III (encoded by the PRDX3 gene), is found to
be localized mainly in the mitochondria. Our previous studies have shown that
silencing the Prx III gene enhanced cell proliferation and metastatic potential
in breast cancer in vitro.
The hypothesis in this study is that Prx III is involved in breast carcinogenesis
and could be a potential prognostic biomarker and molecular target for cancer
therapy. Hence, the specific aims of this study are:
• Aim 1:
Perform functional studies of Prx III in breast cancer progression
and chemosensitivity to therapeutic drugs. Silencing and
overexpression of the PRDX3 gene will be performed followed
by:
(a) evaluating the effects on functional processes, viz., cell
proliferation, cell cycle, adhesion, migration, invasion and cancer
cell-endothelial interaction.
(b) establishing mechanistic pathways of Prx III by gene arrays
and proteome analysis to establish which potential signal
transduction pathway(s) are activated following siRNA treatment.
Advanced quantitative mass-spectrometry-based SILAC (Stable
isotope labeling Amino Acids in Culture) will be used for
pathway and network analysis
(c) determining the chemosensitivity of breast cancer cells to a
panel of drugs after silencing the target PRDX3 genes.
• Aim 2: Conduct an in vivo study to verify the effects and deduce putative
effectors of Prx III in tumour metastasis using a metastatic mouse
model for human breast cancer.
• Aim 3:
Determine the utility of Prx III as clinical predictors of tumour
aggressiveness in clinical samples.
This study will lead to new knowledge on the mechanistic pathways involved
in Prx III-mediated breast cancer progression.
A/Prof George Yip
PI’s email id
[email protected]
Office Address
Department of Anatomy, National
University of Singapore, 4 Medical
Drive, Block MD10, Singapore
117594
Expression and Functional Analysis of Glycosaminoglycans and
Proteoglycans in Breast Cancer
Glycosaminoglycans are highly negatively charged molecules made up of
repeating disaccharide subunits consisting of an amino sugar and an uronic
acid. They are covalently linked to core protein backbones to form
proteoglycans. Besides structural roles, glycosaminoglycans and
proteoglycans have important biological functions in regulating cell behaviour
through their interactions with growth factors and signalling molecules. They
are also involved in microRNA- and exosome-mediated regulation of cancer.
In this study, we aim to elucidate the effects of these molecules on cancer cell
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
Telephone Number
6516 3206
activities, to investigate their potential clinical use as biomarkers and
prognostic indicators, and to utilise them for the development of novel
therapeutic targets. A variety of cell and molecular biology techniques will be
employed in these studies, including cell culture, immunohistochemistry,
electron microscopy, ion exchange chromatography, proteoglycan
quantification assays, real-time PCR, in situ hybridisation, gene cloning, and
microarray analysis.
References:
1. Kumar AV et al (2014) HS3ST2 modulates breast cancer cell invasiveness
via MAP kinase- and Tcf4 (Tcf7l2)-dependent regulation of protease and
cadherin expression. Int J Cancer (in press).
2. Ibrahim SA et al (2012) Targeting of syndecan-1 by microRNA miR-10b
promotes breast cancer cell motility and invasiveness via a Rho-GTPaseand E-cadherin-dependent mechanism. Int J Cancer, 131, E884-96.
3. Yip GW (2011) Breast cancer: Novel therapeutic targets. Recent Pat
Anticancer Drug Discov 6:164-165.
4. Koo CY et al (2008) Targeting heparan sulphate proteoglycans in breast
cancer treatment. Recent Pat Anticancer Drug Discov 3:151-158.
5. Yip GW et al (2006) Therapeutic value of glycosaminoglycans in cancer.
Mol Cancer Ther 5:2139-2148.
A/Prof S Thameem Dheen
PI’s email id
[email protected]
Office Address
Department of Anatomy, NUS
Telephone Number
6516 3217
Analysis of miRNAs in mouse embryonic neural stem cells derived from
diabetic pregnancy
Diabetes during pregnancy is of serious concern as it causes spontaneous
abortions and congenital malformations. About 10% of the fetuses from
diabetic pregnancies display congenital malformations in various organ
systems, including neurological systems, leading to perturbations in memory
performance. Recently, several signaling molecules and epigenetic factors
such as microRNAs (miRNAs) have been shown to be critical for brain
development and function.
Neural stem cells (NSCs) are self- renewing multipotent cells, which give rise
to the neuronal and glial cells of the brain. Proper neural tube development
and closure depends on several genes and morphogens, which are involved in
NSC proliferation, migration, adhesion, differentiation and apoptosis.
Disturbances or alterations in gene expression or cellular events that regulate
neural tube closure results in failure of closure of the neural tube (neural tube
defect, NTD).
It has been well established that epigenetic modifications are involved in cell
fate specification of NSCs. In recent years, the role of microRNAs (miRNAs)
in regulating a number of cellular processes including development,
proliferation, differentiation and plasticity has gained attention. miRNAs have
been shown to regulate brain development by altering the expression of
various regulatory genes involved in neurogenesis and gliogenesis during
neurodevelopment. Given that gene expression profiling in cranial neural
tubes of embryos from diabetic pregnancy has revealed altered expression of
several genes in the developing brains exposed to maternal diabetes (Jiang et
al 2008), and that miRNAs fine tune gene expression, it is hypothesized that
maternal diabetes alters expression of miRNAs that regulate genes critical for
neural tube closure and patterning. In order to address this, we performed
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
miRNA expression profiling using mouse embryonic NSCs isolated from
forebrain of embryos from control and diabetic pregnancy.
Preliminary results of miRNA expression profiling in NSCs from embryos of
diabetic pregnancy has revealed differential expression of several miRNAs
that are known to be associated with neural tube development. Thus, in the
proposed study, the student will explore the role of specific miRNAs in
regulation of cell fate specification of NSCs derived from mouse embryos of
normal and diabetic pregnancy. We believe that these findings may explain the
basis for patterning defects observed in the developing brain exposed to
maternal diabetes.
References:
Sukanya et al. (2013) Hyperglycemia modulates the expression of miRNAs
and cell fate specification of mouse embryonic neural stem cells. PLoS One.
11;8(6):e65945.
Jiang et al. (2008) Global gene expression analysis of cranial neural tubes in
embryos of diabetic mice. Journal of neuroscience research 86: 3481-3493.
Fu et al., (2007). Aldose reductase is implicated in high glucose-induced
oxidative stress in mouse embryonic neural stem cells. J Neurochem.
103(4):1654-65.
Department of Biochemistry
Details of PI
Project Title with a brief description
Dr Chen Ee Sin
Anti-cancer drug resistance mechanism through the regulation of
chromatin structure
PI’s email id
[email protected]
Office Address
MD6, Level 14, North
Telephone Number
6516 5616
The genomic DNA of all eukaryotic cells are intricately organized around
structural proteins called histones into a higher order chromatin. The most
fundamental subunits of the chromatin is called nucleosome that contains 8
histone molecules organized around about 146 base pairs of DNA. The
nucleosome is the scaffold on which all DNA-dependent processes including
transcription, DNA repair and recombination occur and the integrity of
nucleosomal organization bears direct impact on important cell cycle
processes such as chromosome segregation and DNA replication. These two
processes are commonly targeted by many anti-cancer drugs to induce
genomic instability in cancer cells. However often, the effects of the drugs
also affect normal cells resulting in profuse side effects associated with
chemotherapeutic treatments. This project is aim at finding mechanisms that
protect chromatin against the damage caused by chemotherapeutic drugs. We
will use fission yeast as a model organism to uncover universal mechanism in
order to facilitate targeted testing in human breast and gastric cancer cells.
A/Prof Caroline Lee
Project 1:
PI’s email id
[email protected]
Colorectal cancer (CRC) is the most common cancer in Singapore with
around 2000 new cases each year. About a third of these cases are
metastatic (stage IV) at diagnosis and around one third of curatively
resected cases (stages I-III) will relapse suggesting that a substantial
proportion of patients may need treatment for metastatic/relapsed CRC.
The two commonest combination drug regimes for this cancer in
Office Address
MD6, Level 14 or Level 6, Lab 5,
National Cancer Centre
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
Telephone Number
6516 3251 or 6436 8353
Singapore are CAPOX (capecitabine and platinum-based oxaliplatin) or
CAPIRI (capecitabine and topoisomerase-I inhibitor) but the response rate
to these regimes is only ~40-45%. Developing a reliable early predictive
biomarker of response to these drugs in metastatic CRC can lead to
appropriate tailoring of treatment for individual patients and help move us
closer to a truly personalized care. It will not only be cost effective but
will also reduce unnecessary side-effects in patients who will not benefit
from the drug treatment and novel treatments can be explored for these
patients. However, there are currently no reliable tests for early prediction
of response to chemotherapy in these patients.
Genetic single-nucleotide-polymorphisms (SNPs) have strongly been
implicated in the determination of differences in drug response and hence can
serve as a useful early predictive biomarker for response. Here, we propose a
novel pathway-based, potentially-functional SNP (pfSNP) approach to
identify SNPs that may serve as predictive biomarker of response to these
drugs in 300 treatment-naïve CRC patients who receive either CAPOX or
CAPIRI. Response that will be assessed include tumor shrinkage according
to standard RECIST criteria, toxicity according to standard NCI-CTC
criteria, progression-free survival and overall survival. A simple, robust,
cost-effective point-of-care genotyping assay will then be developed for
polymorphisms that can predict drug response. We will also automate this
point-of-care test so that it will be capable of translating genetic
information to clinically relevant information about drug response.
Project 2:
Hepatocellular carcinoma (HCC), amongst the top 5 cancers in Singapore
is the third leading global cause of cancer death with five-year survival of
~5%. Although molecular advances have led to an increased understanding
of the genetic changes that occur in HCC, there is still inadequate
knowledge about the full spectrum of molecular mechanisms and epigenetic
events in hepatocarcinogenesis.
Current diagnosis of HCC relies on routine screening of at-risk patients,
including those with cirrhosis due to viral hepatitis, by screening serum
alpha-fetoprotein (sAFP) levels in conjunction with hepatic ultrasonography
but this combination has limitations. Although cost-effective,
ultrasonography has only 60% sensitivity and 97% specificity. sAFP is only
40-60% sensitive as many tumors do not produce AFP or do so at very
advanced stage. Hence, there is an urgent need to identify better, more
reliable non-invasive biomarkers with higher sensitivity and specificity for
early detection of HCC.
Here, we propose to interrogate miRNAs/small RNAs, by themselves or in
combination with current available AFP/Ultrasound biomarkers, as
emerging novel biomarkers for predicting individuals who are likely to
develop cirrhosis or at the early stage of cirrhosis as well as those likely to
develop HCC or at early stages of HCC. Retrospective, as well as
prospectively collected samples with yearly follow-up and sample
collection from 4 groups of individuals, namely, Normal Non-cirrhotic
(NNC), HBV chronic carriers (HBCC), Cirrhotic and HCC will be
examined. Next-Generation Deep Sequencing on prospective samples at
zero time point will be employed to identify novel miRNAs/small RNAs
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
that are that are associated with cirrhosis and/or HCC. Real- time RT-PCR
will be used on the retrospective samples to validate promising miRNAs
while prospective samples collected at 24 month time point will be used to
confirm the robustness of these validated biomarkers. These novel
biomarkers either alone or in combination with current biomarkers may be
clinically useful.
Dr Deng Lih Wen
PI’s email id
[email protected]
Office Address
Department of Biochemistry,
MD6-14-02S
Telephone Number
6516 1239
Targeting MLL5β mediated transcriptional control of the E6/E7
oncogenes as a basis for novel therapeutic strategies for HPV16/18
associated cancers.
Our lab has previously identified a new MLL5 isoform, MLL5β, which
expression was correlated to high-risk HPV16/18 cervical cancers. Our initial
data suggest that MLL5β is involved in the regulation and expression of key
oncogenes E6 and E7 in cervical cancer and targeted silencing of MLL5β
could inhibit HPV16/18 cervical cancer development. We have recently
established that cisplatin, the current gold standard in cervical cancer care
modulates its anti-tumour effects through MLL5β. Our findings have also
suggested that targeted gene silencing of MLL5β has potential applications as
a mode of therapeutic intervention for HPV16/18-related cancers with less
cytotoxic effects compared to cisplatin. Currently we have identified a key
post-translation modification (PTM) on MLL5β, which is essential for the
assembly of the transcriptional activation complex required to initiate E6/E7
transcription. Based on this finding we have moved on to identify potential
small molecular inhibitors of this PTM and are investigating their potential
applications in cervical cancer therapy.
However, much remains to be done to better understand the full role of
MLL5β in E6/E7 expression regulation such as what are other components of
this MLL5β-associated transcriptional complex as well as the exact role of
MLL5β in the complex. A better understanding of these molecular events will
lead to the potential identification of better therapeutic avenues for cervical
cancer therapy. We are looking for an enthusiastic and motivated candidate to
focus on further understanding these molecular events. The candidate will
work on identifying the other components of the MLL5β transcription
activation complex as well as further understand the histone methyltransferase
role of MLL5β in the complex. The outcome of the project will be to harness
this knowledge and translate it to the development of novel molecular targeted
therapeutic strategies for cervical cancer therapy.
Targeting Cancer Metabolism In Inhibiting Tumor Progression
Metabolic switch is an important event during tumor progression. It is
believed that cancer cells reprogram their metabolic processes so as to exhibit
high glycolysis to feed their rapid proliferation, a phenomenon known as
Warburg effect. The adaptation to glucose metabolism in cancer cells produces
high metabolic acid that in turn confers tumor survival and invasion. The
invasion of in situ tumor cells to distant sites, also known as cancer metastasis,
accounts for more than 80% of cancer-associated death. Studies have also
found that cancer cells which acquire high invasiveness are usually highly
resistant to chemotherapy. Conceptually, intertwining cancer metabolism,
particularly the glycolysis process would serve as an effective way to target
cancer progression. However, the inter-link between cancer metabolism and
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
cancer invasiveness is unclear at the moment.
Our preliminary data demonstrated that our compound of interest interfered
with cancer glycolysis and potentially affected cancer metastasis process. In
this project, we are interested in finding the underlying cellular metabolic
changes in cancer cells across different invasive stages and understand how
these metabolic switches affect cancer hostile behavior. We also intend to
screen for pharmacological compounds that exhibit synergistic effects to
achieve anti-cancer survival and anti-metastasis. Findings from this project
will provide further knowledge on cancer progression and shed light in
developing innovative cancer therapy strategy.
Dr Lim Yoon Pin
PI’s email id
[email protected]
Office Address
MD4, level 1. 5 Science Drive 2,
Singapore 119545
Telephone Number
6601 1891
Function and mechanism of a novel WBP2 oncogene in breast cancer and
metastasis
Through cutting-edge phosphoproteomics technology, we have identified a
novel breast cancer associated gene (1). Our lab is the first to demonstrate that
WBP2 transcription co-activator is a new breast oncogene that upon
phosphorylation is able to transform normal mammary cells into cancer cells
and make mild cancer cells aggressive by activating multiple other oncogenic
pathways (2). We have also identified a new interacting partner of WBP2 that
modulates the expression of WBP2 and its Wnt-promoting activities in breast
cancer cells (3). The aim of the new project is to elucidate how WBP2 turns on
other oncogenes and contributes to breast oncogenesis and metastasis using a
wide array of in vitro and animal model assays associated with signal
transduction, transcription and cancer biology. The proposed study is likely to
result in the mapping of signaling cascades and transcriptional networks
associated with the WBP2 thus providing new insights into the molecular
etiology of breast cancer. Our lab also has a strong emphasis on translational
research – for example in the exploitation of WBP2 as a biomarker and drug
target. A patent has been filed for the use of WBP2 in cancer detection and
therapy. Students will have a chance to participate in commercialization of
intellectual properties associated with WBP2.
1.
2.
3.
Dr Long Yun Chau
PI’s email id
[email protected]
Office Address
8 Medical Drive, MD7, #03-13
Chen, Y., et.al and Lim, Y. P. (2007) Differential Expression of
Novel Tyrosine Kinase Substrates during Breast Cancer
Development. Mol Cell Proteomics 6, 2072-2087 (5 year impact
factor: 9.4)
Lim, S. K., et.al. and Lim, Y. P. (2011) Tyrosine phosphorylation of
transcriptional coactivator WW-domain binding protein 2 regulates
estrogen receptor alpha function in breast cancer via the Wnt
pathway. FASEB J 25, 3004-3018 (5 year impact factor: 7.2)
Lim S.K., et.al. and Lim YP (2014) WIP negatively regulates WBP2
expression and WBP2-mediated Wnt activation in breast cancer cells
(manuscript in preparation).
The role of energy sensing network in exercise-induced skeletal muscle
adaptation
The beneficial effects of exercise on health are widely recognized. Exercise
could improve and prevent chronic metabolic disorders such as diabetes and
obesity. However, the cellular mechanism behind these effects is largely
unknown. Optimization of exercise regimen or pharmacological recapitulation
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
Singapore 117597
Telephone Number
6601 2084
of exercise effects requires close examination of intramuscular events that
triggers the exercise adaptations. Our previous and current work has aimed to
define the mechanism that drives exercise-induced metabolic adaptation in
skeletal muscle.
Exercise induces a multitude of cellular disturbances in muscle including
decreased intramuscular energy levels. Exercise-induced energy deficit is
considered an important stimulus for the beneficial metabolic outcomes.
AMP-activated protein kinase (AMPK) is an important signal transducer
which is activated in response to energy stress during exercise. Our previous
work in mouse models provided evidence that activation of this protein kinase
is sufficient to increase skeletal muscle glycogen store, fatty acid utilization
and muscle endurance – benefits that are derived from exercise training.
Conversely, disruption of muscle AMPK signaling accelerated the progression
of muscle fatigue and impaired exercise-mediated metabolic gene expression.
These results provided important evidence that AMPK signaling mediates
some of the exercise-mediated metabolic responses in skeletal muscle. In the
current study, we hypothesize that AMPK plays a role in autophagy and amino
acids metabolism in skeletal muscle, and this effect is critical for metabolic
benefits of exercise and endurance.
Role of insulin-induced glycolysis in the regulation of PGC1α-mediated
myotube lipid metabolism
Our body is subjected to irregular nutrient supply, including the transition
between fasting and feeding. Therefore, the ability of our body to selectively
store and utilize different energy substrates is critical for energy balance.
Given the substantial mass and energy consumption, skeletal muscle plays a
critical role in the regulation of energy balance, and impaired skeletal muscle
metabolism is closely associated with metabolic diseases such as type 2
diabetes and obesity. Thus, investigation of skeletal muscle energy substrate
metabolism is critical for the understanding of pathophysiology of metabolic
diseases. Under postprandial conditions, insulin stimulates skeletal muscle
glucose uptake and utilization via Akt, a protein kinase that activates
downstream effectors of insulin. Conversely, skeletal muscle increases lipid
oxidation under fasting conditions, which coincident with the induction of
PGC1α - a transcription coactivator that has been implicated in the induction
of lipid metabolic gene program. Nonetheless, the interaction between the
disparate insulin and PGC1α pathways which are activated under contrasting
nutritional states remains largely unknown. This research project aims to
establish the role of nutrient (glucose) and hormone (insulin) in the regulation
of PGC1α cultured myotubes, and the impact of this regulation on lipid
metabolic gene program in myotubes.
Role of insulin-like signaling in the regulation of SIRT1 deacetylase
The loss of skeletal muscle mass and metabolic function is a common feature
of muscle pathologies associated with inactivity (atrophy), aging (sarcopenia)
and diseases (cachexia). The loss of muscle function is mainly attributed to
elevated protein degradation and resistance to anabolic effects of growth
hormones such as insulin and insulin-like growth factor 1 (IGF1). These
anabolic hormones are critical signals that stimulate skeletal muscle glycogen
and protein synthesis (anabolic metabolism), predominantly via the Akt kinase
pathway. Conversely, nutrient deprivation activates Silent Information
Regulator T1 (SIRT1) deacetylase and forkhead box protein O1 (FOXO1)
proteins that induce catabolic metabolism in skeletal muscle. The increase of
catabolic over anabolic pathway may be a molecular basis for the loss of
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
muscle mass and function. Nonetheless, the mechanism by which such
delicate balance is regulated remains largely unknown. This proposal aims to
evaluate the novel role of insulin-like signals in the regulation of SIRT1, and
the unknown function of SIRT1 in the regulation of glucose and amino acid
metabolism in cultured myotubes. The specific aims are (1) to determine if
insulin-like signals regulate SIRT1 via nutrient metabolism, (2) to establish the
role Akt in the regulation of SIRT1-induced FOXO1 pathways, and (3) to
examine the impact of AKT-SIRT1 pathways on energy substrate metabolism.
Cultured myotubes will be treated with insulin or IGF1 in the absence of
presence of metabolic inhibitors to determine the function of these hormones
on SIRT1 function. The role of Akt in the regulation of SIRT1-dependent
FOXO1 activity will be evaluated via pharmacological and genetic alteration
of the kinase and deacetylase. The metabolic effects of Akt-SIRT1 pathway
will be determined by direct measurements of glucose and amino acids
metabolism in myotubes to provide direct readout for changes in metabolic
fluxes. The research will provide important insight into the pathophysiology of
skeletal muscle metabolism, via the identification of insulin-like hormones as
regulators of SIRT1. It will also provide critical evidence for SIRT1 and IGF1
as potential drug targets for muscle wasting diseases.
Dr Yeong Foong May
PI’s email id
[email protected]
Office Address
MD4, Level 1
Telephone Number
6516 8866
We are interested in how cells regulate cytokinesis such that it occurs only
after chromosome segregation so as to produce two viable daughter cells. We
plan to study the links between cell cycle regulation and cytokinesis, with
particular focus on how the cell cycle machinery affects endocytosis at the
cytokinesis site. Endocytosis is a key cellular process that leads to the
internalization of proteins and membranes from the plasma membrane. During
cytokinesis, endocytosis occurs at the cytokinesis site and perturbations to
endocytosis can cause a failure in cytokinesis
Our preliminary data suggest that the key cell cycle, the cyclin-dependent
kinase (Cdk1) activity in mitosis has an apparent role in preventing
endocytosis and cytokinesis. Indeed, upon the destruction of the mitotic Cdk1
activity, the both cytokinesis and endocytosis at the neck are triggered. This
was evident from our initial data showing that key endocytic components
accumulate at the neck only during cytokinesis.
We plan to make use of budding yeast as our model for the proposed study,
given that both processes are conserved and that the budding yeast serves as a
good system for complex genetic manipulations, we plan to make use of
budding yeast to understand the regulatory relationship among Cdk1 activity
and endocytosis during cytokinesis. Our lab has the necessary tools and
techniques to execute experiments including genetic and physical screens.
Moreover, we also conduct time-lapsed imaging experiments in addition to
physical interaction assays. We aim to provide insights into how cells
normally prevent cytokinesis until after nuclear division by studying how cells
regulate endocytosis during cytokinesis. The proper execution of cytokinesis is
important, given that premature cytokinesis can cause a loss of cell viability
while failure to execute cytokinesis can lead to tetraploidy in human cells,
which is a precursor to tumourous growths.
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
Department of Medicine
Details of PI
Project Title with a brief description
Dr Chester Lee Drum
A Protein Dynamic Approach to Engineered Translational Therapeutics
PI’s email id
[email protected]
Capitalizing on the prior training of the professor, the lab leverages a structural
biology approach (i.e., Drum et al. Nature 2003) to create clinically relevant
engineered proteins for both diagnostic and therapeutic purposes. “The protein
folding problem” is one of the most interesting in biology --how does a linear
peptide with more conformations than there are atoms in universe, know to
fold into essentially a single functional conformation? Can biochemical
engineering fundamentally effect this process in a way to make it amenable to
oncological or cardiovascular treatments? In this project you will use a novel
form of protein expression invented by our lab to study protein folding
dynamics and novel cellular uptake mechanisms for drug delivery. The project
is a continuation of research begun at Massachusetts Institute of Technology
and will create nanometer scaled uptake vesicles for the delivery of active
protein substrates. Our lab has a full time synthetic chemist, biochemist and
materials engineer in addition to full molecular biology and protein
biosynthesis support which is an excellent setting for a graduate student who
wants an efficient approach to a high impact problem, not bounded by a single
technique. Although Prof. Drum was clinically trained as a cardiologist
(BWH, Harvard Med School) he is also an award winning structural biologist
who is interested in developing talented students into future scientific leaders.
Please enquire if interested.
Office Address
Yong Loo Lin Sch of Medicine, 14
Medical Drive, 08-01, Singapore
117599
Telephone Number
6601 3001
Prof Daniel G Tenen
PI’s email id
[email protected]
Office Address
14 Medical Drive, Centre for
Translational Medicine, MD6,
Level 12, Singapore 117599
Telephone Number
6516 8239
Investigation of mutation on TERT promoter in giloblastoma for
potential therapy target.
Glioblastoma is the most common and most aggressive malignant primary
brain tumor in humans. Recently studies demonstrated that a transcriptional
role of mutations on telomerase reverse transcriptase (TERT) gene promoter in
it. Although evidence showed that these mutations correlated with higher
transcription activation of TERT and a more aggressive phenotype, the deep
mechanism is still unclear. Collaborated with Dr. Vinay in IMCB, here we
found a novel transcriptional factor binding site on the mutation site by using
biostatistical analysis. We propose that this novel binding site will increase the
transcriptional activity of TERT promoter and therefore upregulates TERT
protein level.
To test our hypothesis, we plan to do 1) biochemical analysis to investigate the
deep mechanism to see whether this transcriptional factor indeed binds to
TERT promoter and how it regulates TERT level, 2) in vitro and in vivo assay
to confirm whether with this mutation, this novel transcriptional factor
promotes a more aggressive phenotype, 3) multiple analysis in clinical
samples to confirm whether this transcriptional factor and related pathway are
indeed highly activated and caused more aggressive primary human tumor.
Our study will suggest a novel therapy target for giloblastoma.
Prof Edward H Koo
PI’s email id
The Laboratory for Molecular Neurodegeneration at YLL School of Medicine
was established by Dr. Ling Shuo-Chien and Prof. Edward Koo in late 2013
when we first joined the NUS faculty. Our research focus is on the
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
[email protected]
Office Address
Centre for Translational Medicine,
MD6-10-01Q
Telephone Number
6601 3706
mechanisms of neurodegenerative diseases, a group of brain disorders that is
often age-associated and includes Alzheimer and Parkinson diseases,
amyotrophic lateral sclerosis, frontotemporal dementia, among others. We
take a cell and molecular biology approach to our studies with an emphasis on
animal models and translational research. My laboratory’s philosophy is to
study both the “normal and abnormal biology” of genes and proteins that are
implicated in disease pathogenesis. In many instances, mutations are found in
genes with unknown function and which require thorough investigations of the
basic functions of these genes and proteins before we can appreciate how their
dysfunction contributes to the disease phenotype. My laboratory has a
particular emphasis is on the pathophysiology of Alzheimer disease where we
have concentrated on both basic and translational studies as well as
experimental therapeutics. Ongoing and future projects include:
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Mechanisms of synaptic injury in Alzheimer disease
Role of caspase cleavage of the amyloid precursor protein (APP) in
neurodegeneration
Mechanisms of tau mediated synaptic dysfunction
Investigations into mechanisms of neuronal vulnerability in
Alzheimer disease
Contribution of blood-brain barrier to neurodegeneration
Neurobiological basis of cognitive loss due to chemotherapy
treatment in cancer patients (“chemobrain”)
Prof Lee Kok Onn
Generation of functional insulin-producing cells for diabetes therapy
PI’s email id
[email protected]
Diabetes has become the pandemic metabolic disease affecting over 382
million populations worldwide. Insulin-dependent diabetic patients rely on
multiple injections of insulin daily while still developing various
cardiovascular complications. Transplantation of pancreas or isolated
pancreatic islets to replace the lost insulin-secreting beta-cells is the only
therapy to cure the disease. But the lack of organ donors has severely limited
such promising treatment. To circumvent this obstacle, our research aims to
differentiate stem cells or reprogram somatic cells such as fibroblasts and
adipocytes into functional insulin-producing cells using the knowledge and
technologies of molecular biology, stem cell biology, biochemistry, diabetes
and animal models. These surrogate beta-cells may not only be applied for the
replacement therapy of diabetes but also can be used to define the molecular
mechanism for pathogenesis of diabetes. Our work has been awarded by
American Society for Cell Biology
(http://www.ascb.org/files/2005pressbook.pdf), widely covered by media
(http://www.news.gov.sg/public/sgpc/en/media_releases/agencies/astar/press_
release/P-20081120-1.print.html?AuthKey;
http://www.sciencedaily.com/releases/2008/11/081120130539.htm), featured
in the NUS research gallery (http://www.nus.edu.sg/research/rg155.php), and
supported by competitive national research grants.
Example publications:
Li G.D, Luo R, Zhang J, Yeo KS, Lian Q, Xie F, Tan EKW, Caille D, Kon
O.L, Salto-Tellez M, Meda P, and Lim SK. (2009) Generating mESC-derived
insulin-producing cell lines through an intermediate lineage-restricted
progenitor line. Stem Cell Res, 2:41-55, E-pub
http://dx.doi.org/10.1016/j.scr.2008.07.006
Li G.D, Luo R, Zhang J, Yeo KS, Xie F, Tan EKW, Caille D, Que J, Kon O.L,
Salto-Tellez M, Meda P, and Lim SK. (2009) Derivation of functional insulin-
Office Address
Endocrinology, National
University of Singapore
1E Kent Ridge Road
NUHS Tower Block Level 10
Singapore 119228
Telephone Number
6772 4341
Co supervisor:
A/Prof Li Guodong
[email protected]
Telephone Number
83111022
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
producing cell lines from primary mouse embryo culture. Stem Cell Res, 2:2940, E-pub http://dx.doi.org/10.1016/j.scr.2008.07.004
Shao S, Gao Y, Xie B, Xie F, Lim SK, LI GD. (2011) Correction of
hyperglycemia in type 1 diabetic models by transplantation of encapsulated
insulin-producing cells derived from mouse embryo progenitor. J Endocrinol,
208:245-255, e-pub http://dx.doi.org/10.1530/JOE-10-0378
Prof Nicholas Paton
PI’s email id
[email protected]
Office Address
Department of Medicine, Yong
Loo Lin School of Medicine,
National University of Singapore,
Rm W10009D, NUHS Tower
Block, 1E Kent Ridge Road,
Singapore 119228
Telephone Number
6772 6988
Development of Whole Blood Bactericidal Activity Assay for Tuberculosis
Therapeutics
Tuberculosis (TB) is one of the world's deadliest infectious diseases with an
estimated 9 million new cases and 1.4 million deaths per year. It is caused by
Mycobacterium tuberculosis, which is becoming increasingly resistant to
current drugs, thereby posing a major public health problem in Asia and
worldwide. Our group’s research is focused on investigating new approaches
for delivering improved, more efficient treatments for TB.
The whole blood bactericidal activity assay (WBA) is an ex vivo model for
measuring effects of administered drugs, host factors and strain factors on
mycobacterial sterilization. Mycobacteria added to human whole blood
cultures undergo phagocytosis and remain intracellular in 72-h cultures.
During TB treatment regimens, drug levels in the whole blood cultures mirror
those in the circulation at the time of phlebotomy. If performed in parallel with
pharmacokinetics measurements, the method can be used to evaluate the effect
of drugs throughout the dosing cycle.
To date, the method has not been utilized to investigate the synergy between
the immune system and TB drugs on mycobacterial activity, nor has it been
used to look at the effects of drugs on different TB strains. Both of these are
potentially very useful aspects of this experimental paradigm which deserve to
be explored. The aim of this research project therefore is to establish WBA as
a method in clinical trials investigating new drug regimens against
tuberculosis.
The PhD candidate in charge of this project will develop the method and
extend it to measuring host immune responses and effects on different
mycobacterial strains. He/she will have an opportunity to learn the principles
of multinational clinical trials, will be involved in clinical pharmacokinetics
studies, will acquire specialized in-demand skills to work in BSL-3 laboratory
environment, and will learn to transform data into knowledge for improving
human lives.
Prof Nobuhiro Yuki
PI’s email id
[email protected]
Office Address
#09-01 Neuroscience Research
Centre, Centre for Translational
Medicine, MD6, 14 Medical Drive,
Singapore 117599
Telephone Number
Comparison of IVIG with the different immunoglobulin effects on cell
viability in neuromyelitis optica
Neuromyelitis optica (NMO) is a variant of multiple sclerosis, identified by a
spectrum of autoimmune astrocytopathies, with phenotypic presentation of
optical neuritis and relapsing inflammatory myopathies. Detection of IgG
antibodies against aquaporin 4 (AQP4; water pore proteins present in the cell
membrane), in the sera distinguishes it from other inflammatory demyelinating
neuropathies. It has been recently established that the AQP4-IgG have a
deleterious effect on cell viability and this event is reversible in absence of
human complement.
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
6516 1092
In support to this data, a good response to intravenous immunoglobulin (IVIg)
therapy has been observed among the NMO patients cohort. Therefore, the
main aim of this project is to understand whether this observation is due to the
effect of IVIg therapy on cell viability and how the different components
contribute to it. The effect of different formulations of IVIg and its
components - IgA, IgM and F(ab)’ fragment, in restoring cell viability in
presence of NMO plasma is to be investigated. In addition to this hypothesis,
there is an underlying dispute on the pathomechanism based on the expression
of the two AQP4 variants - M1 and M23 which is to be investigated in this
study.
The human astrocytoma cell line (CRL1718) is the in vitro model of choice
along with HEK293, a human embryonic kidney cell line, both known to
express AQP4. The validation of the cell lines for suitability of AQP4 study,
reactivity of NMO patient sera and expression profile of AQP4 variants are the
prime requisites in this study. Following which the cell viability of these cells
in the presence of IVIg and NMO patients’ sera will be tested via cell-based
assays and flow cytometry.
Identification of true target of antibodies from Lambert-Eaton
Myasthenic Syndrome (LEMS)
LEMS is a rare autoimmune neuromuscular disorder in which patients
manifest both muscle weakness and autonomic nervous system disruption. It is
believed to be due to autoantibodies targeting presynaptic voltage-gated
calcium channels (VGCCs) extracellularly, specifically P/Q- and N- types
VGCCs. However, the distribution of VGCCs and the manifestation of
symptoms suggested that VGCCs may not be the true target.
We hypothesise that there could be an unidentified protein X that is closely
associated with VGCCs which results in the false identification of the true
target. The aim of the study is to prove that VGCCs is not the true target and
identify this protein X as the true target in which antibodies from LEMS
patients bind to. We will use techniques of immunocytochemistry,
immunohistochemistry, immunoprecipitation for the study.
Structure/function analyses of bacterial sialyltransferase Cst-II with site
directed mutation
Sialyltransferase Cst-II exist in either mono-functional (α2,3-sialyltransferase)
or bi-functional (α2,3-/α2,8-sialyltransferase) forms. Three amino acid
residues, Asn51, Leu54, and Ile269, are specific for the bi-functional Cst-II,
but a single mutation (Asn51 → Thr51) is sufficient to induce the monofunctional Cst-II. However, little is known about the correlation between
structural and such functional differences.
The aim of this study is to demonstrate the structure-activity relationship of
mono-functional and bi-functional Cst-II. Molecular biological approach will
be able to create bi-functional mammalian sialyltransferases, no cases are
known in which with bi-function, such as α2,3-/α2,8-sialyltransferase. The
structural differences will be determined by in silico analysis.
The data obtained from in silico will provide helpful information about
structural changes of sialyltransferase following their functional changes by
site direct mutation. Active site mutants of mammalian sialyltransferase will
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
cause a change in enzymatic reaction and structure. Such data will be helpful
to indicate the structure-activity relationship of sialyltransferases.
Identification of the novel antibodies in demyelinating diseases.
Multiple sclerosis (MS) and neuromyelitis optica (NMO) are demyelinating
diseases of the central nervous system. A specific serum autoantibody to the
astrocytic water channel aquaporin-4 plays an important role in the
pathogenesis of NMO. In contrast, antibodies to neurofascin 186 and KIR4.1
have been reported in patients with MS, but these findings have not been
replicated by independent groups.
Guillain–Barré syndrome and chronic inflammatory demyelinating
polyneuropathy (CIDP) are immune-mediated neuropathies of the peripheral
nervous system. Guillain-Barré syndrome is classified into acute inflammatory
demyelinating polyneuropathy (AIDP) and acute motor axonal neuropathy
(AMAN). IgG autoantibodies bind to gangliosides such as GM1 and GD1a at
the nodes of Ranvier in the peripheral motor nerves and subsequent
complement activation cause the development of AMAN. In contrast,
pathogenic autoantibodies remain to be identified in patients with AIDP or
CIDP, although some antigens have been proposed as autoantibody targets.
Autoantibody binding to myelin and subsequent complement activation may
be involved in the pathogenesis of central and peripheral demyelinating
diseases. Our collaborator kindly gives us the purified central and peripheral
nerve myelin proteins. Moreover, we have already prepared sera from patients
with MS, CIDP and AIDP. Our preliminary results have suggested the
probable presence of novel autoantibody reacting with extracted proteins from
mouse tissues (e.g. central nerve and peripheral nerve) in patients sera. We
will identify the targets of autoantibodies in MS, CIDP and AIDP.
Non-radioactive serological diagnosis of myasthenia gravis
Myasthenia gravis (MG) is the most common autoimmune disease of the
neuromuscular junction (NMJ) with a prevalence of 200-300 per million
populations. Most MG patients (~85%) have autoantibodies against the muscle
acetylcholine receptor (AChR), and about 6% of MG patients have
autoantibodies against the muscle specific kinase (MuSK) and low-density
lipoprotein receptor-related protein 4 (Lrp4). Currently, the most sensitive
assays for the detection of the autoantibodies in MG sera have been the
radioimmunoprecipitation assays. However, the use of radioactivity limits
availability of these tests in many diagnostic laboratories. To improve nonradioactive assays for detection of antibodies to the AChR, MuSK and Lrp4,
we plan to perform cell-based ELISA assay using a cell line transfected with
the target auto-antigens. This assay has very high sensitivity and has been
reported to detect auto-antibodies that can not be detected by RIPA. We hope
that this study may possible to develop the assays as a routine practice in
future.
Identification of novel glycolipid X project
Fisher syndrome is characterised by ophthalmoplegia, ataxia, and areflexia.
The anti-GQ1b IgG antibodies often detected in patients with Fisher syndrome
may have a role in the pathophysiology of this ophthalmoplegia. The
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
ganglioside GQ1b is expressed in the paranodal portion of human ocular
motor nerves and is the possible target molecule in this disease.
Previously, Mori et al. reported a patient with acute ataxia and areflexia, but
no ophthalmoplegia, associated with anti-GQ1b IgG antibodies. Clinical
findings, electrophysiological studies, and postural body sway analysis
suggested that this patient’s ataxia was very similar to that seen in patients
with Fisher syndrome. This patient did not present with ophthalmoplegia. The
clinical and laboratory findings suggest that this case is a variant form of
Fisher syndrome.
ELISA showed the presence of IgG antibodies to GQ1b and GT1a, a situation
commonly described in Fisher syndrome, Guillain-Barré syndrome (GBS)
with ophthalmoplegia, GBS without ophthalmoplegia and acuteoropharyngeal
palsy. TLC immunostaining using the patient’s serum reacted with GT1a and
more weakly with GQ1b and unidentified glycolipid.
We hypothesise that this unidentified glycolipid X is novel disease-related
antigen. Identification of glycolipid X using anti-ganglioside antibody assays
and mass spectroscopy is useful for elucidation of the pathophysiology.
Identification of
ophthalmopathy
the
pathogenic
autoantibodies
in
Graves’
Graves’ ophthalmopathy (GO) is a potentially debilitating and disfiguring
orbital inflammatory disease. Its association with autoimmune thyroid
disorders is believed to be caused by a putative cross-reactive antigen
connecting the thyroid gland and the extrathyroidal tissues, the nature of
which remains contentious. A number of orbital auto-antigens have been
suggested: thyroglobulin, extraocular muscle antigens, thyroid stimulating
hormone receptor (TSHR), insulin-like growth factor 1 receptor (IGF-1R).
Although TSHR is considered as one of the major antigens in the orbit, TSHR
antibodies cannot explain the development of ophthalmopathy in patients in
some patients with Graves’ hyperthyroidism, Hashimoto’s thyroiditis or in
newborns with neonatal thyrotoxicosis. Ninety percent of patients with GO
have Graves’ hyperthyroidism; the remaining 10% have hypothyroidism or are
euthyroid.
Thyroid stimulating antibodies recognize the extracellular domain of TSHR.
We hypothesize that patients with GO have autoantibodies against a protein
with similar structure to the TSHR receptor and we aim to identify these
autoantibodies responsible for the development of ophthalmopathy. These
autoantibodies when bound to the TSHR-like receptor expressed by fibroblasts
or adipocytes in eye muscle are able to induce differentiation of adipocytes.
Hence, fibroblasts when exposed to sera from patients with GO would
proliferate and develop GO.
We aim to identify the autoantibodies responsible for the immunopathologic
mechanism in GO. Using bioinformatics analysis of the epitope region of
TSHR, we identified a possible antigen with similar structure to the
extracellular domain of TSHR. Now we aim to target this TSHR mimic
molecule and produce an overexpression plasmid of this molecule. We plan to
transfect with the candidate TSHR mimic molecule overexpression plasmid
and treat with normal serum or GO patient serum. This will be followed by
autoantibody recognition of TSHR and the TSHR mimic molecule evaluated
with immunocytochemistry.
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
Dr Swaine Chen
PI’s email id
[email protected]
Office Address
60 Biopolis Street, Genome #02-01,
Singapore 138672
Telephone Number
6808 8074
Towards curing severe recurrent urinary tract infection by
understanding mechanisms of intracellular infection
Urinary tract infection (UTI), particularly of the bladder (cystitis), is an
extremely common infectious disease, affecting more than half of all women
at some point in their life. Recurrent UTI is especially problematic; patients
often suffer for years with this condition, altering their sexual, dietary, and
social habits. The only treatment currently is long term antibiotic therapy, but
this often does not cure the disease.
UTI research in a mouse model of infection has identified an intracellular
population of bacteria that can persist within the epithelial tissue of the
bladder. These bacteria are protected from antibiotic treatment as well as from
clearance by the host immune response. They can reactivate weeks to months
after the initial infection to cause a subsequent recurrence. A primary focus of
the lab is understanding how these intracellular bacteria persist and cause
recurrent infection; we do this using animal models and are developing tools
to directly study urine samples from human UTI patients.
We have established the mouse model of cystitis in the lab. We have very fine
genetic control of uropathogenic Escherichia coli, which we use to determine
the genetic basis for disease. We have recently also developed single cell
genomics tools that allow us to profile expression of intracellular bacteria from
infected mouse samples. Finally, we have clinical collaborators that can
facilitate collection of samples from human UTI patients.
The project is to apply these tools to gain an understanding of the gene
expression during intracellular expression using these bacterial, animal,
genomics, and clinical tools. The end goal is to identify pathways or genes that
may eventually lead to diagnostic tools and therapeutic targets for improving
our current therapy for recurrent UTI. There is a strong translational aspect to
this project, as all animal studies will be validated in primary infected human
samples. Students will have an opportunity to learn skills in animal models of
disease; bacterial genomics and pathogenesis; next generation sequencing; and
rigorous experimental design and data analysis. A strong background in
bacterial genetics and molecular cloning is preferred. Experience with
computer programming, mathematics, statistics, and/or genomics will also be
important for sequencing data analysis. Highly motivated and creative
graduate students are encouraged to apply.
Department of Microbiology
Details of PI
Project Title with a brief description
Dr Justin Chu Jang Hann
Drug discovery and target validation for medically important mosquitoborne viruses
PI’s email id
[email protected]
The re-emerging infections caused by mosquito-borne viruses such as dengue
and chikungunya are on the raise at an explosively rate in many parts of the
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
Office Address
MD4, Level 5, Dept of
Microbiology, 5 Science Drive 2
Telephone Number
6516 3278
world. In the view of the absence of an effective vaccine and antiviral drug
against these viral pathogens, the developments of effective antiviral strategies
against these viruses are in high demand. The intent of this study is to perform
an established high content cell-based screening platform that could identify
compounds that prevent virus replication by (i) inducing antiviral gene
expression pathways in host cells, (ii) interfering with viral proteins and their
functions (iii) interfering essential host-viral protein interactions. Although the
assay can identify compounds that target distinct steps of the virus replication
cycle, we are interested in those that stimulate cellular responses necessary to
confer initial resistance to virus infection. Bioactive compound libraries will
be screened on the high content cell-based assay in triplicate. All of the
compounds that meet the designated criteria of a “hit” in the primary high
content screening platform will be further confirmed with secondary assays
(infectious plaque assays, protein over-expression and RNA interference
studies). The efficacy of the selected compounds will be further validated in
murine models. In additional, the antiviral targets or mechanisms will be
dissected by a combination of reverse genetics, proteomics and bioimaging
approaches. The research data generated from this study are essential for the
rapid identification of potential prophylactic or therapeutic leads that will be
helpful in better preventive management of individuals from suffering viral
infections. In this proposal, re-emerging flavivirus dengue and chikungunya
viruses will be used as the model for the “proof of concept” development.
Prof Nicholas Gascoigne
Reversing age-related thymic involution
PI’s email id
[email protected]
As we age, the output of new T cells from the thymus is greatly reduced. It has
recently been shown that this process can be reversed by expression of a single
transcription factor, Foxn1, in the epithelial cells of the thymus. The cortical
thymic epithelial cells give signals for positive selection of developing
thymocytes, and mice lacking Foxn1, so-called “nude” mice, lack T cells (as
well as hair). Foxn1 expression decreases with age, and re-expression of this
gene in “middle-aged” mice reversed the involution of the thymus and restored
production of naïve T cells [1]. We aim to develop vectors to allow Foxn1 to
be re-expressed in order to reverse this process, eventually in humans. This
project is to develop expression systems for Foxn1, and to test them in tissue
cultured epithelial cells and then in mice.
Office Address
5 Science Drive 2, Block MD4,
YLLSOM
Telephone Number
6516 3275
1.
Bredenkamp, N., Nowell, C.S., Blackburn, C.C. (2014)
Regeneration of aged thymus by a single transcription factor.
Development 141: 1627
Control of T cell activation by Themis in response to weak antigens
Themis is a protein discovered by this lab that is expressed in the T-cell
lineage [1]. It controls thymocyte development by controlling the strength of
signalling via its interaction with the phosphatase SHP1 (PTPN6) which turns
off the signalling cascade [2]. This project will study how Themis works in
mature T cells, and whether Themis inhibition can be used to enhance
responses to weak antigens such as tumour antigen.
1.
2.
Fu, G. et al., Themis controls thymocyte selection through regulation
of T cell antigen receptor-mediated signaling. Nat Immunol
2009. 10: 848
Fu, G. et al., Themis sets the signal threshold for positive and
negative selection in T-cell development. Nature 2013. 504: 441
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
T-cell control of Type 2 diabetes
Themis is a protein discovered by this lab that is expressed only in the T-cell
lineage, controlling selection of naïve T cells in the thymus [1]. It works
through controlling the selection threshold for T cell receptor signalling at a
particular point in T cell development [2]. We found that Themis-deficient
mice develop obesity and metabolic syndrome, leading to type 2 diabetes
(T2D). This indicates a T-cell mediated role in this disease, which is normally
considered to be inflammatory in nature. The aim of this project is to identify
the type of T-cells that cause the disease, and the mechanism by which they
induce T2D, to use T-cells from Themis-sufficient mice to try to control or
cure the disease, and to investigate a role for Themis in human T2D.
1.
2.
Fu, G. et al., Themis controls thymocyte selection through regulation
of T cell antigen receptor-mediated signaling. Nat Immunol
2009. 10: 848
Fu, G. et al., Themis sets the signal threshold for positive and
negative selection in T-cell development. Nature 2013. 504: 441
Dr Stephan Gasser
I have 2 different projects:
PI’s email id
[email protected]
1) Current clinical biomarkers for cancer are often specific for certain types of
cancer and many biomarkers are not detectable in the serum of patients.
Serum-based markers offer key advantages including taking of samples with
minimal inconvenience for the patients, the possibility to perform longitudinal
studies of cancer patients, and early detection of tumors with a sensitive assay.
We recently discovered the presence of genome-derived DNA with specific
properties, including the presence of distinct retroelements and the potential to
form triplex structures, in the cytosol and exosomes of many mouse and
human tumor cells (Lam et al., Cancer Research 2014). Preliminary
experiments show that the generation of such DNA is linked to replication
stress, which is thought to be prevalent in most cancers. Hence, we plan to
characterize the double-stranded (ds) DNA in serum exosomes of cancer
patients and exosomes secreted by tumor cells. We propose to 1) deepsequence DNA present in serum exosomes of tumor patients and exosomes
secreted by human tumors and tumor cell lines, 2) analyze the DNA sequences
for conserved DNA sequences or properties among the different cancer
samples. 3) develop an assay to detect exosomal dsDNA in the serum of
cancer patients. A diagnostic test for a serum-based general tumor marker will
be of interest to various companies in the diagnostic field.
Office Address
Department of Microbiology, NUS
Telephone Number
6516 7209
2) Anti-viral defense mechanisms partially depend on sensors that recognize
DNA associated with virus infections in the cytosol. The same defense
mechanisms have also been implicated in tumor surveillance in several
settings and the understanding of the molecular basis of tumor recognition is a
topic of intense research interest. We recently demonstrated that B cell
lymphomas and many tumor cell lines of various origins constitutively express
cytosolic DNA as a consequence of oncogene-induced DNA damage and
repair. Our analysis showed that the expression of the cytosolic DNA sensor
ZBP1 is highly upregulated in human B cell chronic lymphocytic leukemia,
one of the most common types of lymphoma in Singapore and the United
States. To investigate the role of Zbp1 in tumorigenesis, we crossed Zbp1-
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
deficient mice to Eμ-Myc mice, a model for non-Hodgkin's B cell lymphoma.
Strikingly, Zbp1-deficient Eμ-Myc mice showed an increased survival and a
lower B cell tumor load. Moreover, around one in five Zbp1-deficient Eμ-Myc
mice showed no signs of lymphoma at death while all Zbp1+/+Eμ-Myc mice
died of lymphoma. Preliminary data indicate that tumor cells in Zbp1-deficient
Eμ-Myc mice accumulate higher levels of DNA damage, upregulate the DNA
damage response and are more prone to apoptosis. Co-immunoprecipitation
experiments suggest that ZBP1 interacts with DNA repair proteins and nuclear
RNA export proteins. Our hypothesis is that ZBP1 prevents the
accumulation of damaged nuclear DNA and contributes to the transport
of DNA to the cytosol for recognition by innate immune sensors. We plan
to 1) study the role of Zbp1 in the tumorigenesis of non-Hodgkin's B cell
lymphoma, 2) characterize the role of Zbp1 in DNA repair and DNA damage
3) identify and characterize proteins and DNA that interact with ZBP1. In
summary, our proposal aims to show that the DNA sensor ZBP1 plays a
critical role in tumorigenesis.
A/Prof Tan Yee Joo
Understanding cell death regulation in hepatitis C virus infection
PI’s email id
[email protected]
Hepatitis C virus (HCV), a positive-stranded RNA virus of the family
Flaviviridae, infects an estimated 3% of people worldwide and is one of the
major causes of liver diseases. The HCV genome encodes a precursor
polyprotein of ~3,000 amino acids that is processed to give rise to three
structural (core, E1 and E2) and seven non-structural (p7, NS2, NS3, NS4A,
NS4B, NS5A and NS5B) viral proteins. Unlike the approximately 24,000
genes in the human genome, the number of HCV proteins is very limited.
Thus, HCV interacts extensively with the cellular machineries in the human
host and uses them to its advantage. Apoptosis and other forms of cell death
have been observed in hepatitis C virus (HCV) infection in vitro and in vivo
but the detailed understanding of this intricate viral-host interplay is still
lacking. Massive apoptosis in the liver leads to acute liver failure, while the
dysregulation of cell death pathways is a major factor in the development of
hepatocellular carcinoma (HCC). Apoptosis of hepatocytes during HCV
infection has been proposed to be a direct cause of liver fibrosis, which can
further lead to cirrhosis and liver failure. Hence, it is important to understand
the cellular mechanisms underlying HCV-mediated apoptosis as well as other
cell death pathways. Our hypothesis is that HCV proteins are engaged in a
network of interaction with a group of interconnected host cell death
regulating proteins to yield a tightly regulated system that ensures optimal
HCV replication and survival in the host cell. In this project, we will use
transcriptomic analysis as well as siRNA library screening to identify host
factors that are regulated by HCV infection and/or interact with the HCV viral
proteins. The functional significance of novel viral-host interactions identified
will be analyzed by using cell culture as well as mouse models of infection.
Office Address
Dept of Microbiology, MD4, Level
3, 5 Science Drive 2, Singapore
117597
Telephone Number
6516 3541
Generating antiviral neutralizing antibodies and characterizing their
mechanism of inhibition
Seasonal influenza A virus causes significant morbidity and mortality yearly
while newly emerged strains continue to pose pandemic threats. Current
strategies against influenza include vaccination and antiviral drug treatment.
However, predicting the major strain that may cause the next pandemic is the
main obstacle in current vaccine development. Moreover, some viruses have
acquired resistance to approved antiviral drugs. Passive immunotherapy is
now increasingly being used to treat human infectious diseases and there is a
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
demand for the development of neutralizing mAbs for passive immunotherapy
in the event of a highly pathogenic flu pandemic as this could be particularly
useful for protecting certain groups of people, such as immuno-compromised
patients or the elderly, who may not respond well to vaccines. As the
hemagglutinin (HA) protein mediates viral entry, it has been the main target
for the preclinical studies on antibody-based immunotherapy and these studies
suggest that it may be a viable option to administer neutralizing HA mAbs as a
form of passive immunotherapy for influenza A infection. However, viral
escape mutants were observed when anti-HA mAbs were used individually.
Combination therapy, where multiple steps in the virus life cycle are inhibited
simultaneously, is highly recommended to minimize the development of
escape viruses. Hence, this study uses a multi-disciplinary approach to
determine if other viral proteins of influenza A virus can stimulate neutralizing
antibodies that prevent viral infection and/or replication. If we can develop
combination therapy by using a mixture of mAbs that bind to different
components of the virus, this will minimize the chance of drug resistant virus
developing and this will help in the establishment of a novel class of antiviral
therapeutic drugs.
Department of Obstetrics & Gynaecology
Prof Ganesan Adaikan
PI’s email id
[email protected]
Office Address
Department of OBGYN, NUHS
Tower Block Level 12, 1E Kent
Ridge Road, Singapore 119228
Telephone Number
6775 9240/9049 8636
Endothelial progenitor cells as a novel therapeutic option in vasculogenic
erectile dysfunction
Erectile dysfunction (ED) is an increasingly common disease, afflicting both
young and old men. The problem is exacerbated by aging population, as well
as changes in diet and lifestyle. Metabolic diseases, a major risk factor in ED,
are also on the rise. Pathophysiological studies demonstrate that ED in these
patients stem from compromised vascular function leading to ischaemic
cavernosa, and resultant loss of erectile function. Consequently, existing
palliative drugs, such as sildenafil, that are unable to restore vascular function
are also ineffective for reversing or curing ischaemic ED found in patients
with metabolic disease.
Based on our interesting pilot study, it was hypothesised that the injection of
endothelial progenitor cells (EPC) are expected to facilitate the formation of
de novo vasculature in the ischaemic penile tissue in animal models of ED and
improve vascular networks and restore erectile function through increased
perfusion, as well as re-innervation.
The project involves validation of penile ischemia and vasculogenic erectile
dysfunction (ED) in rabbit and rodent models of metabolic diseases: (1)
characterization of sub-populations of EPC derived from umbilical cord blood
to stimulate angiogenesis and support nervous networks. (2) evaluation of EPC
therapy in reversing endothelial and erectile dysfunction in animal models of
vasculogenic ED.
Department of Orthopaedic Surgery
Dr Angelo H All
Induced Human Oligodendrocyte Precursor Cell
Improved Functionality after Spinal Cord Injury
Transplants
for
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
PI’s email id
[email protected]
Office Address
NUHS - Dep. of Orthopedic
Surgery, 1E Kent Ridge Road,
NUHS Tower Block, Level 11 and
NUS – SINAPSE Institute:
28 Medical Drive, CeLS Building,
5th floor
Telephone Number
6772 3322 (NUHS)
6601 3198 (NUS)
We propose to directly convert adult human fibroblasts into "induced
human oligodendrocyte precursor” cells and to study the remyelination
capability of these cells both in vitro and in vivo. We will generate patientspecific cells in a shorter time
frame than iPS cells as direct conversion does not require reprogramming to
base cells. These newly generated cells would also have significantly reduced
risk of immune rejection even when compared to properly differentiated OPs
from currently available human iPS cell lines. The goal of this project is to
study whether these cells remyelinate demyelinated axons in the hostile postinjury spinal cord micro-environment in rats. We will also test the cells’
myelin production capabilities in a micro-chamber in vitro.
Hypothermia after Spinal Cord Injury: Early Markers of Recovery
Spinal cord injury (SCI) is a devastating condition that can lead to paralysis of
the limbs below the injury level. Beyond the initial trauma of injury, the
secondary phase of injury comprises inflammation, demyelination and
apoptosis, which are all major pathological factors that exacerbates the
progression of injury. Since mitigating this secondary phase of injury greatly
improves patient outcomes, it has motivated the search for an
early neuroprotective therapy. In animal models, primary focus has been on
the effects of post-SCI hypothermia on motor behavioral and histological
outcomes with only a few studies of the electrophysiological function of
descending spinal cord pathways during cooling. Thus, there is a critical need
for establishing the full benefits of hypothermic neuroprotection after SCI.
Unlike other studies, our unique focus is on the hypothermia-induced
enhancements of the afferent sensory conduction, a vital function of the
sensory-motor system, as assessed by multi-limb somatosensory evoked
potentials. The cooling will be followed by a single recording session to
acquire local field potentials (LFPs) and multi-unit activity (MUA) from the
dorsal pathways in the vicinity of the injury, followed later by somatosensory
evoked potentials (SSEPs), which are cortical response waveforms drawn out
by peripheral stimulation. The acute microelectrode recordings will be used to
develop an early statistical marker to identify animals with maximal long-term
recovery of SSEPs. The standard motor behavioral scoring (BBB) and
histopathology will be used to provide complementary measures of recovery.
The goal of this project is to use the contusive rat model of SCI, followed by
an early, local cooling of the injured region, to optimize a hypothermia
treatment process that can adequately mitigate the pathological factors in the
second phase of injury.
Transplantation of Conjugated Nanoparticles to Limit Spinal Cord
Injury
We propose to synthesize and administer conjugated nanoparticles and study
their capability to limit spinal cord injury (SCI) in a rat model. This treatment
approach will enable us to spare more healthy axons and neuropathways
within the spinal cord parenchyma and slow down progress of injury after
trauma. This is important as SCI is a time-sensitive pathology with a very
short window of treatment. The goal of this proposal is to generate a new
generation of nanoparticles, which will be safe and effective in preventing
progress of trauma in our in vivo model of spinal cord injury.
Crosstalk among Neural Pathways after Incomplete Spinal Cord Injury
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
Spinal Cord Injury (SCI) in the pediatric population is fairly rare but bears
huge socioeconomic consequences. Patients with SCI before their adolescence
have different mechanisms of injury and have a better neurological recovery
potential for incomplete and mild to severe injuries when compared to adults.
Although the SCI is usually diagnosed with the use of MRI, often pediatric
SCI are without radiological anomaly. There is also no evidence for the use
of neuroprotective approaches for the treatment of SCI in children. There is
the need for a better determination of the mechanism of SCI at the epicenter of
injury. An understanding of the mechanisms that help promote the improved
neurological recovery observed in pediatric patients would also enhance future
therapies for all SCI patients. This project deals with incomplete SCI and the
study of post-contusion electrophysiological changes in the vicinity of the
injury for pediatric demographics. The goal of this proposal is to characterize
functional reorganization of neuropathways on the neuronal reconnection
formation at the epicenter of contusion induced SCI.
Behavioral Assessment after Spinal Cord Injury
Spinal Cord Injury (SCI) is a serious condition that severely impairs mobility
and quality of life with no treatment options. After injury, some axons are
anatomically continued but not functional. These neurons are electrically
excitable cells and they are the focus of our treatment in the acute phase. One
approach to treat SCI is through limiting and preventing the destruction of
healthy neurons in the spinal cord parenchyma, thus aiding functional
recovery. A set of motor behavioral assessments, such as BBB open field
locomotion test, thermal sensation, rotorod, tactile allodynia and balance beam
will be proposed to thoroughly investigate the onset of injury as well as longterm progress of injury. We will study phenotypic outcomes and the possible
limitations during the progress of injury. These assessments will be
statistically analyzed and compared with the histological examination as well
as our previous electrophysiological assessments.
Prof Lee Eng Hin
PI’s email id
[email protected]
Office address
NUHS Tower Block, Level 11, 1E
Kent Ridge Road, Singapore
119288
Telephone Number
6516 6576
Effect of substrate patterning on the chondrogenic differentiation of
mesenchymal stem cells.
The main focus of our lab is the optimization of chondrogenesis towards a
normal cartilage phenotype for cartilage tissue engineering. Mesenchymal
stem cell (MSC) differentiation is influenced by its microenvironment.
Manipulation of extracellular biophysical and/or biochemical
microenvironment of stem cell can improve the efficiency of tissue
engineering approaches using MSCs. We have identified specific biochemical
and topographical cues for the directed differentiation of MSCs to specific
cartilage phenotype. However, the mechanisms induced by these
microenvironmental cues have not yet been fully understood. The project will
investigate the effect of incorporating specific biochemical and/or physical
cues in the substrate to achieve the desired lineage and phenotype specification
of the differentiated cells. The study will aim to elucidate the
mechanotransduction mechanisms that affect specific functional outcomes,
including the optimisation of the manipulation conditions, and validation of
the approach in in vivo animal model. Primary stem cell culture techniques
and molecular biology analysis will be employed in these studies, including
immunohistochemistry, confocal microscopy, scanning electron microscopy,
real-time PCR and Western Blot.
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
References:
 Wu Y, Law JBK, He AY, Low HY, Hui JHP, Lim CT, Yang Z, Lee EH.
Substrate topography determines the fate of chondrogenesis from human
mesenchymal stem cells resulting in specific cartilage phenotype formation.
Nanomedicine: NBM 2014 (in press).
 Raghothaman D, Leong MF, Lim TC, Toh JKC, Wan ACA, Yang Z, Lee
EH. Engineering cell matrix interactions in assembled polyelectrolyte fiber
hydrogels for mesenchymal stem cell chondrogenesis. Biomaterials 2014
Mar;35(9):2607-16.
Department of Otolaryngology
Details of PI
Project Title with a brief description
A/Prof Wang De Yun
Quantitative assessment of the virulence of rhinoviruses, influenza viruses and
respiratory syncytial viruses and host defense mechanisms of human nasal
epithelium in vitro
PI’s email id
[email protected]
Office Address
NUHS Tower Block, Level 7,
Department of Otolaryngology
Telephone Number
6772 5373
The viral upper respiratory infections (URIs), including common cold,
influenza and acute rhinosinusitis, are very common disease, affecting millions
of people annually. They can be caused by any of the more than 200 different
strains of viruses, such as rhinovirus, coronavirus, respiratory syncytial viruses
(RSV), influenza, parainfluenza and adenoviruses. With the SARS pandemic
of 2003 and that of H1N1 in 2009, global concerns was raised, stemming from
virulent new strains of viruses such as corona virus (or SARS virus), H1N1,
H5N1, which appear to be more promiscuous and circulate in several species
and are endemic in humans, birds and pigs. In addition to continuous efforts in
surveillance of naturally occurring viruses in humans and domestic animal
hosts, experimental studies elucidating susceptibilities to respiratory viruses
and response to infection by human nasal epithelium, which is the primary
target site for common cold and influenza viruses, are required. In this study,
we aim to develop an in vitro model for screening and quantitative assessment
of the virulence of common cold and influenza viruses, and the host defense
mechanisms of human nasal epithelium using our newly developed in vitro
experimental models of human nasal epithelial stem/progenitor cells
(hNESPCs) and differentiated epithelial cells derived from hNESPCs in an airliquid interface (ALI) culture. In addition, we will investigate the host defense
functions including viral fusion and uncoating, transport of viral
ribonucleoprotein (RNP) complexes from nucleus, replication, transcription
and translation of the viral genome, export of viral RNP complexes from the
nucleus, viral assembly and budding, and potential drug targets, which will
provide us with a useful reference for public health risk assessment,
interventions, disease control and prognosis for viral upper respiratory
infections. This information is particularly useful in preparation for eventual
pandemics or outbreak of viral infection.
Department of Paediatrics
Details of PI
Project Title with a brief description
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
A/Prof Heng Chew Kiat
PI’s email id
[email protected]
Office Address
Department of Paediatrics, NUHS
Tower Block, Level 12, 1E Kent
Ridge Road, Singapore 119228
Telephone Number
6772 3354
Investigation of a newly identified protein, androgen-dependent tissue
factor pathway inhibitor regulating protein (ADTRP), for its role in
coronary artery disease
Recently, a GWAS in the Chinese population has identified rs6903956 within
the C6orf105 gene (now known as androgen-dependent tissue factor pathway
inhibitor “TFPI” regulating protein or ADTRP) on chromosome 6p24.1 as a
novel susceptibility locus for CAD.
ADTRP is a protein encoded by C6orf105 that regulates TFPI expression.
TFPI is the major inhibitor of tissue factor-factor VIIa–dependent FXa
generation. Hence, ADTRP can be considered a protective factor of CAD. As
it is a newly discovered protein, very little is known about its characteristics.
This provides ample scope for investigation.
Co supervisor:
The specific aims of this study are:
Prof Yechiel Friedlander
Email: [email protected]
Please contact A/Prof Heng Chew
Kiat if you are interested in this
project
1.
2.
3.
4.
To determine which of the two known isoforms of ADTRP is the
predominant form through the use of Human Coronary Artery
Endothelial Cell (HCAEC) line.
To determine the changes in ADTRP in response to androgen in
HCAEC.
To select meaningful single nucleotide polymorphisms for
genotyping to determine if any are significantly associated with CAD
in the Singaporean population.
To conduct genetic variant screening in ADTRP gene
The methods employed in this study include cell culture, PCR, qRT-PCR,
genotyping, bioinformatics and statistical analyses.
Identification of rare variants with strong effects on early onset
myocardial infarction
There is an increasing trend of early onset of myocardial infarction (MI) in
patients who are below 40 years. A proportion of these do not have strong
conventional risk factors such as smoking, hypertension and diabetes. In fact
some of the patients with early onset of MI lead physically active lives and do
not appear to have any conventional risk factors except for being male. It is
therefore of interest to investigate the genetic factors underlying this early
onset phenotype. Genome-wide association studies (GWAS) have thus far
identified genetic variants that may predispose individuals to MI. However,
these are mostly variants that are common in the population and are hence
unlikely to account for the small subset of patients who had MI at a young age.
We aim to carry out exome sequencing using next generation sequencing
(NGS) technology for MI patients <40 years old) who have been identified
from the National University Heart Centre in NUH. The number of variants
identified from these patients is expected to be very large. We will employ
filtering strategies to obtain meaningful number of target genes that we can
conduct experiments to verify their functions. We believe be insightful
information could be gleaned from such an approach.
A/Prof Lee Yung Seng
PI’s email id
[email protected]
Prenatal, perinatal, and postnatal determinants of adiposity and
metabolic phenotype in childhood
This research is part of the birth cohort study “Growing Up in Singapore
Towards healthy Outcome” (GUSTO) of the TCR flagship programme
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
Office Address
Pediatrics, NUHS Tower Block,
Level 12, 1E Kent Ridge Road,
S(119228)
Telephone Number
6772 4112
“Developmental Origins: Singapore” (DevOS). Prenatal, perinatal, and
possibly early postnatal factors determine the subsequent growth of the child.
This project aims to uncover the factors which determine the size and body
composition of the newborns, and predictors of the growth of young children
in the first few years of life, including maternal factors (diet and nutrition,
habitus, pregnancy complications), birth events, and the baby’s growth pattern
in the first few months’ of life. Specifically the candidate will determine the
factors which predict catch up growth in the group of children with lower birth
weights, as well as those with growth failure. The student will get
opportunities to study epigenetic changes of candidate genes which predict the
subsequent growth pattern of the children, as well as predictors of failed catch
up growth.
Determining body composition of infants and young children
This research is part of the birth cohort study “Growing Up in Singapore
Towards healthy Outcome” (GUSTO) of the TCR flagship programme
“Developmental Origins: Singapore” (DevOS). The candidate will study the
various modalities of body composition assessment of infants and young
children, including MRI of abdominal fat compartments and whole body fat,
bioelectrical impedance, and air displacement plethysmography. There will
also be opportunity to study correlation of the body composition parameters
with developmental factors and epigenetic biomarkers.
A/Prof Lynette Shek Pei-Chi
PI’s email id
[email protected]
Office Address
Department of Pediatrics
NUHS Tower Block, Level 12, 1E
Kent Ridge Road, Singapore
119228
Telephone Number
6772 4420
Upper airway nasal microbiota and its relationship with persistent
rhinitis and wheezing in early childhood
There is little data on the pattern of nasal microbiome colonization in early
life, and its influence on disease development. A Singapore birth cohort
GUSTO) is followed up for allergic disorders including rhinitis in early
childhood. We have shown that the prevalence of rhinitis for at least 4 weeks
is common (18.8%) at the age of 18 months, and is associated with wheeze
and eczema. We aim to analyze the diversity and abundance of upper
respiratory microbiota from archived nasal swabs taken at regular intervals in
the first 18 months of life and correlate the microbiota data with the clinical
outcomes of early onset rhinitis, and subsequent development of allergic
rhinitis and wheeze/asthma at age of 5 years.
Co supervisor:
Prof Lee Bee Wah
Email: [email protected]
Prof Yap Hui Kim
Role of angiomotin in the pathogenesis of membranous nephropathy
PI’s email id
[email protected]
Idiopathic membranous nephropathy (IMN) is the commonest cause for
nephrotic syndrome in adults, and half of the patients progress to renal failure.
Despite its prevalence, the molecular mechanisms behind IMN are poorly
understood. Current treatment options are therefore empirical and often
ineffective. IMN is a result of immune complex deposition in the glomerular
basement membrane. The triggers for immune complex formation are
unknown.
Office Address
Pediatrics, NUHS Tower Block,
Level 12, 1E Kent Ridge Road,
S(119228)
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
Telephone Number
6772 4411
In our preliminary study, we have identified a local Chinese family with Xlinked recessive membranous nephropathy, Fanconi syndrome and anti-tubular
basement membrane (anti-TBM) antibodies. Using exome sequencing, we
have found angiomotin (AMOT) as a novel candidate gene, with the mutation
p.S50G in the p130-AMOT isoform which is critical in maintaining tight
junctions between cells. In our preliminary work, we have demonstrated that
AMOT is expressed in human podocytes and renal tubular cells, both of which
depend highly on tight junctions for their cellular function. Our hypothesis is
that the mutation p.S50G in the AMOT gene is functional and the anti-TBM
antibodies present in the family are anti-AMOT antibodies.
Our specific aims are to:
1) Study the expression of angiomotin and p130-AMOT isoform in human
podocytes and renal tubular cell cultures, and in animal and human kidney
tissues by immunostaining
2) Study the identity of anti-TBM antibodies present in the family with
p.S50G AMOT mutation.
3) Elucidate the function of the angiomotin mutation p.S50G by its
transfection into HEK293, human podocyte and renal tubular cell cultures,
observing its effect on cell morphology, angiomotin subcellular
distribution and its interactions with known and novel binding partners.
4) Introduce the p.S50G mutation in rats using Transcription activator-like
effector nucleases (TALENs) and studying its phenotypic effects.
Our study will improve the understanding of the mechanisms of IMN, which
can result in more targeted therapy with more efficiency and less side effects.
Role of Soluble Urokinase Plasminogen Activator Receptor (suPAR) in
Pathogenicity of Focal Segmental Glomerulosclerosis (FSGS)
Immune-mediated focal segmental glomerulosclerosis (FSGS) is the most
common cause of acquired end-stage renal disease in children worldwide.
Studies on the pathogenetic mechanism suggested a plausible role for
induction of urokinase plasminogen activator receptor (uPAR) signaling with
increase in circulating soluble uPAR. Several questions, however, remained
unanswered: i) High suPAR levels are also found in non-proteinuric diseases
(eg. sepsis); ii) Is the whole suPAR molecule or one of its subunits, the elusive
proteinuric-inducing circulating factor; iii) Are immune cells the only possible
source of suPAR in FSGS? Our preliminary studies demonstrated increased
suPAR production in monocyte supernatant and injured podocytes. We
therefore hypothesized that:
1. Secreted suPAR from podocytes promotes podocyte migration, contributing
to elevated circulating suPAR levels in FSGS patients;
2. A specific isoform of suPAR may be responsible for podocye foot process
effacement;
3. Filtration of suPAR into urine in the presence of podocyte damage may
explain the discrepant suPAR levels observed in FSGS patients, and the
elevated suPAR levels in the presence of decreased glomerular filtration rate.
Thus, the primary aims are to:
1. Demonstrate suPAR production by injured podocytes and its role in
promoting podocyte migration,
2. Identify the suPAR isoform responsible for foot process effacement,
3. Identify the upstream mediators leading to increased uPAR expression in
podocytes and monocytes/immune cells.
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
The secondary aim is to validate the above findings in both wild type and
Plaur-/- (CD87-/-) mice via introduction of these upstream mediators, to
determine if uPAR is increased on the mouse podocytes resulting in FSGS.
The tertiary aim is to assess the clinical utility of suPAR (plasma+urine) as an
early biomarker of podocyte injury.
Understanding the role of suPAR and its isoforms in podocyte injury will
facilitate the design of novel targeted therapies for this disease, as well as
provide a potential biomarker.
Department of Pharmacology
Details of PI
Project Title with a brief description
Dr Alan Prem Kumar
To Evaluate the Potential Utility of Annexin A1 as a Biomarker of
Response to PPARγ Agonist, Efatutazone, in Breast, Colon and Lung
Carcinoma
PI’s email id
[email protected]
Office Address
MD6, 14 Medical Drive, #11-01M
Telephone Number
6516 5456
NUS-Daiichi Sankyo Material Transfer Agreement L146737 (NUS Ref.
TL2013-077)
Despite the evidence of PPARγ ligands as potential new anti-tumor
therapeutic option, clinical trials with the use of PPARγ ligands for cancer
treatment were not consistent. In addition, the glitazone family of drugs has
shown toxicity in the clinic and has been either withdrawn or no longer
prescribed to patients. CS-7017 (efatutazone), with an in vitro IC50 of 10nM,
is the newest and potent PPARγ investigational drug on clinical trials. In
patients, it’s given orally 0.50mg BID as determined from Phase I studies.
Recently, Daiichi Sankyo, Inc (USA) has completed two Phase II studies on
CS-7017 in advanced colorectal cancer. Our preliminary findings established
identification of a novel biomarker, Annexin A1 (ANXA1) predictive of
response to PPARγ ligands in vitro and in vivo. The objective of this study (a
NUS-Daiichi Sankyo Project) is to further characterize the suitability of
ANXA1 as a predictive and companion marker for CS-7017 therapy in breast,
colon and lung cancers.
Identification and Characterization of Novel Protein Tyrosine Kinase-6
Inhibitors for the Treatment of Breast Cancer
RCA - NUS and GenoMed Inc, USA (NUS Ref. RL2013-007)
Breast cancer is a diverse malignancy; understanding the molecular
mechanisms driving tumor progression will facilitate development of targeted
therapies. For example, tamoxifen, an anti-estrogen agent, used for treating
estrogen receptor (ER)-positive breast tumors, and trastuzumab, a monoclonal
antibody targeting HER2-overexpressing tumors, have benefitted specific
subsets of breast cancer patients. However, the enormous variation in breast
cancer initiation and growth complicate biological approaches to this
malignancy and challenge the effectiveness of current therapies. Elucidation of
novel, targetable signaling pathways in breast cancers would provide means of
addressing current deficiencies in the treatment of the disease. We've found a
SNP in the upstream region of the breast tumor kinase (Brk), also known as
protein tyrosine kinase 6 (PTK6), about 5 kb from the gene's transcription start
site, to be highly associated with ovarian (OR 10.36), breast (OR 5.93), and
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
prostate (OR 5.42) cancers, but not colon, lung, or pancreatic cancers (OR 1).
Although PTK6 is undetectable in the normal mammary gland, it is
overexpressed in more than 60% of human breast tumors and breast cancer
cell lines, with the highest levels in advanced tumors. In multivariate analysis,
the disease-free survival of patients of >240 months was directly associated
with the protein expression level of PTK6 (P=0.001), but was also inversely
associated with nodal status (P=0.001) and tumor size (P=0.01). However, to
date there is no inhibitor available for PTK6. Using molecular docking, we
have identified a series of novel PTK6 inhibitors. The project is to first test the
inhibitory potential of these new drugs in breast cancer models, possibly
extending to ovarian and prostate cancers. The project will then be extended to
decipher the molecular mechanism/targets in vitro and in vivo by which PTK6
inhibition mediates its anticancer property and the underlying events that
“switch on” expression of PTK6 in breast cancer.
A/Prof Bian Jinsong
PI’s email id
[email protected]
Office Address
MD11, Room #05-30A
Telephone Number
6516 5502
Activation of Na+/K+ ATPase (NKA) is a new strategy to treat ischemic
heart diseases
Na+/K+ ATPase (NKA) is responsible for maintaining the electrochemical
gradient, and hence the membrane potential, of the cell membrane. The subunit of NKA is important in maintaining the function of α-subunit. In
oxidative stress, glutathionylation of 1-subunit impairs this interaction and
decreases NKA activity. In chronic heart injury, NKA loss contributes to the
development of CHF. Our preliminary data showed disruption of this
interaction may induce heart dysfunction and injury. In this project, we will
express the soluble extracellular β ectodomains, which lack glutathionylation
site but are still able to interact with and activate α1-NKA, and study the
cardioprotective effects of these ectodomains.
Inhibition of abnormal protein aggregation by hydrogen sulfide: a
potential approach to treat neurodegenerative diseases
Neurodegenerative disorders represent a major cause of disability and death,
with an unmet need for therapies that alter disease progression. Hydrogen
sulfide (H2S) has recently been hypothesized to be an important
neuromodulator in the brain. Abnormal generation and metabolism of H2S
may be actively involved in the pathogenesis of central nervous system (CNS)
diseases. We recently reported that both endogenous and exogenous
application of H2S produces therapeutic effects on Parkinson’s disease and
Alzheimer’s disease. However, the molecular mechanisms are still unclear.
We hypothesize that H2S may have the potentials to retard pathological
process by prevention of abnormal protein aggregation. In this project, we will
examine the effects of H2S on Tau phosphorylation, α-synuclein nitration and
amyloid β aggregation. H2S-induced S-sulfhydration, autophagy preservation,
anti-oxidative stress and mitochondrial protection will be studied.
Dr Pieter Eichhorn
Identification and roles of Dubiquitinating enzymes in the TGFB pathway
PI’s email id
[email protected]
TGF-β is essential for embryogenesis and tissue homoeostasis in multicellular
organisms. Furthermore, in advanced cancers TGF-β can act as an oncogenic
factor and according to growing clinical evidence, TGF-β can be considered a
therapeutic target in cancer. Ubiquitin modification of the TGF-β signaling
pathway is emerging as a key mechanism of TGF-β pathway control.
However, the role of deubiquitinating enzymes (DUBs), which mediate the
Office address
MD6 Level 11 CSI
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
Telephone Number
6516 5475
removal and processing of ubiquitin, is less well understood. I am interested in
studying and elucidating the roles of DUBs in the TGF-β pathway.
Department of Physiology
Details of PI
Project Title with a brief description
A/Prof Celestial TSM Yap
To investigate the roles of cytoskeletal proteins in oncogenic signaling
PI’s email id
[email protected]
Gelsolin (GSN) is an actin-associated cytoskeletal protein involved in binding
and severing actin filaments, thus controlling cytoskeletal turnover in response
to cellular signals (eg. migration, apoptosis). Gelsolin has also emerged as a
new player in pathways regulating signaling and gene transcription. We
previously uncovered a novel role of gelsolin in promoting invasion of
colorectal tumour cells by enhancing extracellular matrix breakdown, through
the upregulation of urokinase plasminogen activator (uPa).
Office Address
Department of Physiology, Block
MD 9 Level 4, 2 Medical Drive,
Singapore 117597
Telephone Number
6516 3294
We aim to ascertain if specific oncogenic signaling pathways interact with
gelsolin to influence cancer cell behaviour. Our preliminary analysis of
microarray data from human breast cancer tissues and in vitro work on cell
lines showed a correlation between gelsolin expression with various oncogenic
signaling pathway modulators, suggesting possible involvement of gelsolin in
regulating oncogenic signaling. The specific signaling pathways identified to
have potential interactions with gelsolin are known to induce aggressive
tumour cell behavior, including invasion and epithelial-mesenchymal
transformation. Gelsolin knockdown by siRNA decreases the expression of
target genes controlled by oncogenic signaling, while gelsolin overexpression
increases signaling activity. In addition, we found that induction of specific
oncogenic signaling in breast cancer cells also increased the expression of
gelsolin, suggesting that the activities of gelsolin may be recruited by certain
oncogenic signaling pathways in a feed-forward loop to enhance their target
effects. Collectively, these data suggest novel regulatory roles of gelsolin in
signaling pathways that promote cancer progression. We will confirm the
interactions between gelsolin and the specific signaling pathways identified by
our laboratory, as well as examine the significance of these interactions on
cancer cell behavior and clinical outcomes.
A/Prof Herbert Schwarz
How does the cytokine receptor CD137 contribute to the pathogenesis of
Hodgkin lymphoma?
PI’s email id
[email protected]
Office Address
CeLS, level 3, Immunology
Programme
Telephone Number
6516 7773
Hodgkin’s lymphoma (HL) is a cancer of the lymphatic system, and one of the
most common cancers among the young adults. No effective therapies exist
for HL. HL is characterized by an extensive tumor stroma which is essential
for the persistence and pathogenesis of HL. This tumor stroma is induced by
the malignant cells in HL, the Hodgkin Reed-Sternberg (HRS) cells, which are
a small minority among the cells of the tumor stroma that consists mainly of
infiltrating leukocytes. It is largely unknown by which mechanisms HRS cells
cause HL.
Recently we found that the tumor necrosis factor receptor family member
CD137 is ectopically expressed by HRS cells, but not by corresponding
healthy cells (Cancer Research. 73(2):652-61, 2013). We have identified
several changes induced by CD137 in HRS cells that may cause or enhance
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
the malignancy of HL. These chances involve the recruitment of leukocytes
that form the tumor stroma, the secretion of growth factors for HRS cells, and
escape from immune surveillance.
The project will characterize the contribution of CD137 to the pathogenesis of
HL, and aim at identifying novel immunotherpeutic approaches for therapy.
Dr Lee Chi Wai
PI’s email id
[email protected]
Office Address
Block MD9, 2 Medical Drive,
Singapore 117597
Telephone Number
65165955
Cellular and molecular mechanisms underlying the neuronal development
and diseases
Synapses are specialized cell membrane domains that facilitate neuronal
communication in the intricate nervous system. These synaptic specializations
develop in response to molecular interactions between pre- and postsynaptic
cells. A major goal of current research in developmental neuroscience is to
elucidate the mechanisms underlying how synapses are assembled. The nervemuscle synapse, neuromuscular junction (NMJ), which controls all muscle
movements, has been considered as the best model for the study of
synaptogenesis due to its large size, simplicity and accessibility. When
neurons and muscle cells are cultured together, functional NMJs are formed
spontaneously. The structure and physiology of mature vertebrate NMJs are
well understood. Currently, our laboratory specifically focuses on the signal
transduction and cytoskeletal mechanisms underlying synapse development,
disease, and regeneration. Three major areas are being pursued in our lab:
(1) postsynaptic receptor trafficking in the pathogenesis of muscular
dystrophy;
(2) cytoskeletal dynamics in neuronal growth cones during axonal
outgrowth and pathfinding;
(3) axonal trafficking of mitochondria in synaptic formation, function,
and elimination.
Using the simple and elegant Xenopus primary culture system, a variety of
techniques, including live-cell time-lapse fluorescence microscopy, superresolution microscopy, molecular biology, immunocytochemistry, and
Western blotting will be applied to these experimental systems to gain
understanding to the cellular and molecular mechanism of synaptic
development. Our goal is to not only gain a mechanistic understanding of the
molecular and cellular aspects of neuronal structure and function, but also
provide insights into the cellular basis for neurological disorders
A/Prof Lina Lim Hsiu Kim
PI’s email id
[email protected]
Office Address
Centre for Life Sciences, 28
Medical Drive, S117456
Telephone Number
6516 5515
Control of PPARγ and NFκB by Annexin-A1 in inflammatory
macrophages and breast cancer
Inflammation occurs through dynamically varying levels of pro- and antiinflammatory cytokines competing for an upper hand either by activation of
signalling cascades or inhibition of downstream signals. NFKB is a master
transcription factor involved in the transcription of a number of proinflammatory as well as anti-inflammatory genes which could control
inflammation and resolution of inflammation. Natural NF-κB inhibitors exist
that would assist to regulate the production of cytokines during inflammation.
One such natural inhibitor of NF-κB is PPAR-γ, which has been shown to be
anti-inflammatory in a number of models of inflammation. In the following
investigation, we will define the regulation of PPAR-γ by another anti-
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
inflammatory molecule we have been working on for more than 10 years,
Annexin-1. This project will focus on the regulation of PPAR-γ by ANXA1 in
the context of inflammatory macrophage function and cancer and will answer
important questions related to PPAR-γ agonist use clinically in the treatment
of inflammatory diseases.
Annexin-A1: a host factor modulating influenza virus replication
The influenza virus infects millions of people each year and can result in
severe or even fatal complications. Understanding host responses to influenza
infection will enable the development of more effective anti-viral therapies.
The host immune system recognizes viral RNA via specific receptors and
intracellular sensors that directly activate anti-viral immune responses.
Previous research has revealed diverse yet important roles for Annexin family
proteins in modulating the course of influenza infection. However, the role of
Annexin-A1 (ANXA1) in influenza virus infection has not been addressed.
ANXA1 is increased in nasal swab samples obtained from influenza infected
patients. In addition, presence of ANXA1 increases virus replication and
results in more weight loss after virus infection. We therefore hypothesize that
ANXA1 may play a critical role in host anti-viral responses. These data will
enhance our understanding of disease pathogenesis and may lead to the
identification of novel targets for anti-viral immunotherapies.
Dr Ling Shuo-Chien
PI’s email id
[email protected]
Office Address
MD6, #10-01R, Centre for
Translational Medicine (CeTM)
Telephone Number
6601 3645
Mechanisms of Neuronal and Synaptic Dysfunctions in Ageing and
Neurodegeneration
Aging poses both a fascinatingly biological question and a growing medical
problem as aging is the leading risk factor for age-associated diseases, many of
which are late adult-onset neurodegenerative diseases, including Alzheimer’s
disease (AD), Parkinson’s disease (PD), Frontotemporal dementia (FTD), and
Amyotrophic Lateral Sclerosis (ALS, also known as motor neuron disease).
The current goal of my lab is to define and understand the processes
underlying normal and pathological brain aging by elucidating the underlying
molecular and cellular mechanisms and how perturbation of the at-risk
systems cause premature failing that leads to neurodegeneration.
The overall strategy is to use disease-related genes as molecular handles to
probe the gene-phenotype relationship systematically in mice that are genetic
mimics of human diseases during aging and neurodegeneration with top-down
and bottom-up approaches. Specifically, we focus on two RNA-binding
proteins, TDP-43 (TAR DNA-binding protein 43 KDa) and FUS/TLS (fused
in sarcoma/translocated in liposarcoma), whose mutations are causal for ALS
and FTD and inclusions of both proteins are the defining pathological
hallmarks in the majority of ALS and FTD patients. On the top-down
approach, we will interrogate molecular and synaptic changes in selective
brain regions and in neuronal populations as well as the contribution of nonneuronal neighboring cells by combining mouse genetics with genome-wide
quantitative methodologies. On the bottom-up approach, we will reconstruct
an in vitro neural network by building a mechanically- and chemically-defined
microfluidic system to analyze at a single cell resolution as well as to
manipulate with temporal and spatial precision. By integrating neuroscience
with leading edge technology, we expect that we will identify quantitative
changes in gene expression that are critical for initiating and/or accelerating
age-dependent neurodegeneration.
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
The long-term goal of our laboratory is to revert the molecular and cellular
alternations to rescue neuronal damages and ultimately to slow down aging
and subsequent neurodegeneration (300 words).
A/Prof Reshma Taneja
Identifying therapeutic targets for skeletal muscle disorders
PI’s email id
[email protected]
De-regulation of epigenetic control is increasingly apparent in many human
pathologies including cancer. Small molecule inhibitors to chromatin
modifying proteins have shown great promise in preclinical trials validating
druggability of epigenetic modulators. In muscle cells, proliferation and
differentiation are mutually exclusive events that are regulated by chromatinassociated proteins. Among these, lysine methyltransferases that mediate
methylation of histone and non-histone proteins play a central role in
maintaining this equilibrium.
Office Address
MD9, Level 4
Telephone Number
6516 3236
We have recently shown that G9a, a lysine methyltransferase inhibits
differentiation of skeletal muscle cells. G9 mediates histone H3 lysine 9 dimethylation (H3K9me2), a post-translational modification associated with
transcriptional repression on myogenic promoters that are expressed during
differentiation. Moreover, G9a also methylates MyoD, a key transcription
factor that is essential for muscle differentiation.
Using muscle cells from control and G9a conditional knockout mice, we will
profile the G9a methylome. These studies allow us to obtain an integrated
view by which G9a-mediated methylation of histone and non-histone proteins
balance growth and differentiation of muscle cells. Our results will help design
therapies for muscle disorders that are characterized by a differentiation
defect.
Prof Shazib Pervaiz
Redox Regulation of protein phosphatase PP2A in carcinogenesis
PI’s email id
[email protected]
Over the years, our work has highlighted the critical role of an altered redox
metabolism on cell survival and death signaling in cancer cells. Using a
variety of model systems such as drug-induced apoptosis, receptor mediated
death signaling, and oncogene-induced cell survival, we demonstrated that
the intracellular ratio between the two main reactive oxygen species (ROS),
superoxide and hydrogen peroxide (O2-:H2O2), determines cancer cell
response to death signals; a tilt in favor of superoxide promotes cell survival
whereas an increase in hydrogen peroxide favors death execution via
activation of the death promoting protein Bax. Of note, we have highlighted
a novel biological activity of Bcl-2 by providing experimental evidence
linking Bcl-2-induced increase in mitochondrial superoxide levels to the
anti-apoptotic activity of Bcl-2. Interestingly, an elevated O2-:H2O2 ratio
induced by either pharmacological inhibition (DDC) or gene knockdown of
Cu/Zn SOD resulted in an increase in phosphorylation of Bcl-2, specifically
at Ser70 (S70), and this site specific phosphorylation of Bcl-2 enhanced the
anti-apoptotic activity of Bcl-2, thereby rendering cancer cells resistant to
chemotherapy-induced apoptosis. To that end, we have uncovered a novel
mechanism in which an increase in intracellular O2- endows cancer cells
with a survival advantage via tyrosine nitration-mediated detachment of
B56δ from PP2A catalytic core, and the eventual accumulation of S70
phosphorylated Bcl-2 with potent anti-apoptotic activity. The mechanism of
redox modulation of PP2A in the context of carcinogenesis is under
Office Address
2 Medical Drive, Building MD9,
Level 4
Telephone Number
6516 6602
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
investigation.
Specific targeting of mutant K-Ras expressing cancers via Aktdependent ROS production
This project involves investigating the molecualr mechanism(s) underlying
the activity of a novel small molecule compound against mutant Kras
expressing human cancers.
Identification of a novel protein TMTC2 in death receptor sensitization of
human cancers
Our preliminary studies indicate that ligation of the death receptors DR4 and
DR5 (TRAIL R1 and TRAIL R2) upregulates the expression of a novel transmembrane protein TMTC2. Upregulation of TMTC2 increases death receptor
sensitivity while its gene knockdown inhibited TRAIL-mediated cell death in
human nasopharyngeal carcinoma cell lines. The sub-cellular localization of
TMTC2 appears to place it at the ER, however this has not been validated in
more than one cell lines. Notably, TMTC2 induction appears to be under the
influence of intracellular reactive nitrogen species (RNS). The mechanism of
induction, protein structure and localization as well as the death sensitizing
activity of TMTC2 is under study.
Identification of surrogate death signaling pathways in cisplatinresistant human cancer cells
Platinum-based compounds are the main line of treatment for a number of
clinical cancers. However, development of drug resistance remains a
therapeutic challenge. We have generated cisplatin-resistant clones from
human lung and ovarian carcinoma cell lines. These cells become resistant
to most chemotherapeutic drugs, thus suggesting a MDR phenotype.
Although, the mechanism of this drug resistance is not clearly understood,
we have made a remarkable observation during the course of these studies,
i.e that cells that become resistant to cisplatin become highly sensitive to
death receptor-mediated apoptosis. The mechanism of activation of this
surrogate death signaling in the context of cisplatin resistance is under
investigation
Mechanism of statin-induced apoptosis in cancer cells
We recently demonstrated that the cholesterol lowering drug, simvastatin,
induces apoptosis in human colorectal and breast carcinoma cells. We
identified a critical role for intracellular ROS and downstream JNK
activation in statin-induced apoptosis. Interestingly, we showed a massive
induction and activation of Rho family of proteins, Rac1, Rho and cdc42 in
statin treated cells, upstream of ROS production. Of note, the Rho family
of proteins is prenylated despite their localization in the cytoplasm as
opposed to the plasma membrane. The mechanism of this non-membrane
dependent prenylation as well as ROS production in this model is under
investigation.
Dr Thai Tran
PI’s email id
[email protected]
Effects of anti-malarial drug, Artesunate, in steroid-resistant model of
asthma
Airway smooth muscle (ASM) cell hyperplasia contributes to airway wall
remodeling (AWR) in asthma. Glucocorticoids, which are used as first-line
List of Potential Supervisors and Projects available for Laboratory Rotation – January 2015 intake
Office Address
MD9, Department of Physiology
Telephone Number
6516 3663
therapy for the treatment of inflammation in asthma, have limited impact on
AWR and protracted usage of high doses of glucocorticoids are associated
with an increased risk of side effects. Moreover, patients with the severe
asthma often show reduced sensitivity to glucocorticoids/steroids.
Artesunate, a semi-synthetic artemisinin derivative used to treat malaria with
minimal toxicity, attenuates allergic airway inflammation in mice but its
impact on AWR in patients with asthma and especially in those that are
resistant to current steroid treatment is not known.
a) Steroid-Resistant Airway Hyperresponsiveness In Vivo Mouse Models
To develop steroid-resistant airway hyperreponsiveness, mice will be given
intratracheal administration of 40 ml IFN-g (1.5 mg) + LPS (50 ng).
Dexamethasone (1 mg/kg) and/or test compound will be given
intraperitoneally for 3 consecutive days, commencing 3 d before IFN-g/LPS
treatment. Airway hyperreponsiveness will be measured 12 h after the
combined cytokine treatment. At the same time point, BAL fluid and lung
tissues will be collected for analysis. For each complete set of experiment,
mice will be divided into 6 treatment groups: (1) PBS control, (2) IFN-g/LPS
treatment (steroid-resistant), (3) IFN-g/LPS + DMSO vehicle control, (4) IFNg/LPS + drug-optimum dose (test compound), (5) IFN-g/LPS +
dexamethasone (1 mg/kg), and (6) IFN-g/LPS + dexamethasone 1 mg/kg plus
test compound (restore steroid sensitivity). Optimum drug dose will be
determined in preliminary studies.
(b) Steroid-Resistant Alveolar Macrophage In Vitro Model
Mouse alveolar macrophages will be isolated and stimulated with PBS, IFN-g
(1.5 mg/ml), LPS (50 ng/ml), or IFN-g (1.5 mg/ml) + LPS (50 ng/ml) for 24 h
and followed by treatment with vehicle, dexamethasone (1 mM), test
compound (10 mM), or dexamethasone (1 mM) + test compound (10 mM for
restoration of steroid sensitivity) for another 24 h. Supernatants and cell
lysates will be collected for analysis. To detect impairment of glucocorticoid
receptor (GR) translocation by IFN-g/LPS, and restoration of GR translocation
by test compound, macrophages will be fixed, permeabilized, probed with
polyclonal antibody to GR and stained with Cy3-conjugated secondary
antibody. The nucleus will be stained with DAPI. GR translocation in
macrophages will be visualized using a fluorescence microscope.