Boston University
OpenBU
http://open.bu.edu
Theses & Dissertations
Boston University Theses & Dissertations
2015
The role of retinoic acid related
orphan receptor alpha in
age-related macular degeneration
Hoang, Hai
http://hdl.handle.net/2144/16259
Boston University
BOSTON UNIVERSITY
SCHOOL OF MEDICINE
Thesis
THE ROLE OF RETINOIC-ACID-RELATED ORPHAN RECEPTOR ALPHA
IN AGE-RELATED MACULAR DEGENERATION
by
HAI HOANG
B.A., Boston University, 2013
Submitted in partial fulfillment of the
requirements for the degree of
Master of Arts
2015
© 2015 by
HAI HOANG
All rights reserved
Approved by
First Reader
Vickery Trinkaus-Randall, Ph.D.
Director, Cell and Molecular Biology Graduate Program
Professor of Biochemistry and Ophthalmology
Second Reader
Neena Haider, Ph.D.
Associate Professor of Ophthalmology
Harvard University, School of Medicine
ACKNOWLEDGMENTS
I would like to acknowledge and thank everyone in the Haider lab group for all of their
support and assistance on this project. I would like to thank Dr. Neena Haider for giving
me an opportunity to perform my thesis work in her lab, as well as, being an invaluable
mentor in guiding me through my project. I want to also thank Dr. Vickery TrinkausRandall for being my first reader and assisting on the writing of my thesis.
iv
THE ROLE OF RETINOIC ACID RELATED ORPHAN RECEPTOR ALPHA
IN AGE-RELATED MACULAR DEGENERATION
HAI HOANG
ABSTRACT
Age-related macular degeneration (AMD) is a prevalent cause of vision loss and
irreversible blindness that affects more than 11 million Americans. AMD is a
multifactorial disease with a number of genetic, demographic, and environmental risk
factors. Currently the etiology of AMD is still unclear and there are no effective cure for
this devastating disease, but recent studies have demonstrated that RORA is a candidate
gene involved in AMD pathophysiology. RORA is a critical regulator of multiple
biological processes and has been implicated in various physiological processes including
circadian rhythm, lipid metabolism, photoreceptor development, autism, and
inflammation. Our current study will explore in depth the role of RORA in AMD. We
will look at the effects of RORA in the retina of mice. Localization studies of retinal
tissues obtained from mice with a conditional knockout of RORA in epithelial cells
showed little effect of RORA on structural cells of the retina. However, there was a
decrease in VEGF and TGF-B proteins in RORA knockout. This is an interesting finding
because VEGF and TGF-B has an important function in angiogenesis and
neovascularization which are pathophysiological effects of AMD. In addition, we will try
to identify gene targets of RORA that have also been linked with AMD. By identifying
the targets of RORA and discovering how RORA regulates these targets, we hope to
better understand the role of RORA in AMD pathophysiology. ChIP-seq and software
v
analysis of the data was performed to identify all genomic targets of RORA linked with
AMD. A number of promising genes were found in both RORA and AMD networks. The
next step of this study is to perform quantitative analysis of these genes and how their
expression is affected by RORA. Also, we will perform additional conditional RORA
knockout models in cone cells and developing retinal cells to further understand the role
of RORA in the retina and AMD pathogenesis.
vi
TABLE OF CONTENTS
TITLE……………………………………………………………………………………...i
COPYRIGHT PAGE……………………………………………………………………...ii
READER APPROVAL PAGE…………………………………………………………..iii
ACKNOWLEDGMENTS ................................................................................................. iv
ABSTRACT ........................................................................................................................ v
TABLE OF CONTENTS .................................................................................................. vii
LIST OF TABLES ............................................................................................................. ix
LIST OF FIGURES ............................................................................................................ x
LIST OF ABBREVIATIONS ........................................................................................... xii
INTRODUCTION .............................................................................................................. 1
Age-Related Macular Degeneration (AMD) ........................................................... 1
Retinoic-Acid-Related Orphan Receptor Alpha(RORA)...................................... 5
SPECIFIC AIMS AND OBJECTIVE ................................................................................ 9
METHODS ....................................................................................................................... 10
RESULTS ......................................................................................................................... 16
DISCUSSION ................................................................................................................... 30
LIST OF JOURNAL ABBREVIATIONS........................................................................ 36
vii
REFERENCES ................................................................................................................. 37
CURRICULUM VITAE ................................................................................................... 43
viii
LIST OF TABLES
Table
1
Title
Risks of AMD include age, smoking, family history,
Page
1
demographics, and diet:
2
RORA targeted genes that may have associations with
AMD
ix
25
LIST OF FIGURES
Figure
1
Title
Dry AMD contains the presence of drusen while Wet
Page
4
AMD contains the presence of neovascularization in the
retina:
2
RORA is a transcriptional factor that has many
6
physiological functions.
3
Genotype analysis shows that all mice used contained
17
mutations of both RORA- floxed and Cdh5-cre:
4
IHC of structural proteins in retina shows no change
19
between RORA knockout and WT
5
IHC of Growth Factors in retina shows decrease
20
expression in RORA knockout:
6
IHC of Photoreceptors in retina shows no difference
22
between RORA mutant and WT
7
IHC of Synaptophysin and Chx10 shows no change
23
between RORA knockout and WT:
8
Association between AMD genes and fatty acid metabolic
26
genes that are targets of RORA
9
Association between AMD genes and GPCR genes that
x
27
are targets of RORA.
10
Association between AMD genes and cell signaling genes
28
that are targets of RORA.
11
Association between AMD genes and transcriptional
regulator genes that are targets of RORA.
xi
29
LIST OF ABBREVIATIONS
AMD ............................................................................ Age-Related Macular Degeneration
CHIP ..................................................................................Chromatin Immunoprecipitation
CNV ........................................................................................Choroidal neovascularization
GCL.......................................................................................................Ganglion Cell Layer
GFAP .................................................................................... Glial Fibrillary Acidic Protein
GPCR ....................................................................................... G-protein-coupled receptors
IHC ................................................................................................... Immunohistochemistry
INL ........................................................................................................ Inner Nuclear Layer
IPL...................................................................................................... Inner Plexiform Layer
NFL .......................................................................................................Neurofilament Light
ONL ..................................................................................................... Outer Nuclear Layer
OPL ................................................................................................... Outer Plexiform Layer
RORA ........................................................ Retinoic-Acid-Related Orphan Receptor Alpha
RPE ........................................................................................... Retinal Pigment Epithelium
TGF-β .............................................................................. Transforming Growth Factor Beta
VEGF ............................................................................. Vascular Epithelial Growth Factor
xii
INTRODUCTION
Age-Related Macular Degeneration (AMD)
Age-related macular degeneration (AMD) is a prevalent cause of vision loss and
irreversible blindness that affects more than 11 million Americans according to the
BrightFocus Foundation (2013). AMD is a multifactorial disease with a number of
genetic, demographic, and environmental risk factors. The strongest risk factors include
age, cigarette smoking, diet, and race (Coleman et al., 2008).
Risk factors
beyond your
control
Facts
Age
Family History
Gender
Coloring
Adults over age 50
AMD runs in families (up to three times the risk)
Women are more vulnerable (up to two times the risk)
Those with light colored irises and skin may be more susceptible
Risk factors
within your
control
Facts
Diet
Low intake of antioxidant vitamins and minerals puts you at higher risk.
Canadians 50+ often do not consume Canada Food Guide’s recommendations
of 5 to 10 servings of fruits and vegetables daily
Smokers are more susceptible (up to six times the risk)
Extended sunlight exposure associated with 10 year incidence of early AMD
Smoking
Excessive
exposure
to sunlight
Excessive
weight /obesity
Excessive weight increases risk, and a high body mass index increases risk of
progression
Table 1: Risks of AMD include age, smoking, family history, demographics, and
diet: AMD is a multifactorial disease with a number of risk factors. This chart discusses
the most common risks of AMD. Obtained from Family Health Magazine, 2012
1
AMD causes vision impairment through deterioration of the macula, the center of
the retina responsible for central vision. This is a slow progressing disease that develops
over a number of years, often asymptomatically in the early stages. (Coleman HR et al.,
2008). Early and intermediate stages of AMD are characterized by drusen formation and
pigmentary abnormalities, and have a high chance of progression to advanced AMD
(Age-Related Eye Disease Study Research Group (AREDSRG), 2005). Drusens are
extracellular debris and lipid deposits that localize in between the retinal pigment
epithelium (RPE) and Bruch’s membrane. They range in size, color, distribution, and
shape. They also, tend to increase with age, and are linked to RPE atrophy and
dysfunction (Abdelsalam et al., 1999; Bhutto and Lutty, 2012). RPE cells are essential for
vision by being responsible for many complex functions that involve supporting retinal
cells (Strauss, 2005). They provide support to retinal cells by providing nutrients,
removing waste products, healing wounds, secreting growth factors, and sustaining the
choroid and photoreceptors cells (Marshall, 1987; Streilein et al., 2002; Nita et al., 2014).
There are two forms of advanced AMD: dry and wet AMD. Dry AMD is the more
common form of AMD, consisting of around 85-90% of total cases. Dry AMD, also
known as non-exudative or geographic atrophy, is characterized by atrophy of the cells in
the macula responsible for central vision. In dry AMD, drusen accumulates between the
retina and choroid which leads to breakdown of retinal pigment epithelial (RPE) cells.
RPE atrophy (Geographic atrophy) results in degradation of the photoreceptors leading to
decrease in central visual function. Dry AMD progresses slowly, but can manifest into
wet AMD in 10% of patients (Sunness et al., 1999).
2
Wet AMD, also known as exudative or neovascular AMD, occurs in 10% of all
AMD cases. Choroidal neovascularization (CNV) is a signature clinical hallmark of wet
AMD. CNV is caused by decrease in oxygen and growth factors due to atrophy and
dysfunction of the RPE (Bird, 2003). Formation of new blood vessels in the choroid can
invade into the retina through Bruch’s membrane and the RPE (Bhutto and Lutty, 2012;
Das and McGuire 2003). These vessels are more susceptible to leaking blood and fluids,
causing damage and scars, and leading to acute, severe central vision loss (Ding et al.,
2008).
Currently there are treatments for some cases of wet AMD, which includes laser
treatments and intravitreal injections of vascular epithelial growth factor inhibitors to
combat progression of the disease (Keane et al., 2015). In addition, studies have shown
taking antioxidant multivitamins and zinc have helped to slow the progress of dry AMD
(AREDSRG, 2001). Even with the recent advancement in treating patients with AMD,
the etiology and pathogenesis of AMD is poorly understood. Currently, there is no known
cure of AMD or effective prevention of the disease. Therefore it is important to discover
new methods that can help detect early manifestation of AMD. Due to the progressive
nature of the disease, it is beneficial to understand the development and progression of
the disease to provide better treatments.
3
Figure 1: Dry AMD contains the presence of drusen while wet AMD contains the
presence of neovascularization in the retina: This figure illustrates the
pathophysiological findings of advanced AMD. Dry AMD is characterized by drusen
formation causing RPE dysfunction which leads to retinal cells degeneration. Wet AMD
is characterized by choroidal neovascularization, which can cause leaking blood and fluid
into the retina. Figure obtained from BrightFocus foundation, 2013.
4
Retinoic-Acid-Related Orphan Receptor Alpha(RORA)
Recent studies on the interaction between genetic and environmental factors in the
disease have implicated a number of genes in the pathogenesis of AMD (Scholl et al.,
2007). A promising study, performed previously in this research lab, have linked retinoicacid-related orphan receptor alpha (RORA or NR1F1) to the pathogenesis of AMD
(Silveira et al., 2009). RORA is a member of the nuclear receptor superfamily. Nuclear
receptors are a type of transcription factor because they can directly bind to DNA to
regulate gene expression. Many nuclear receptors have ligands that cause conformational
change once bound. This conformational change activates the nuclear receptor enabling
to interact with specific response elements, and subsequently alter gene expression
(Olefsky, 2001). Nuclear receptors are a diverse family, with a total of 48 known human
nuclear receptors (Zhang et al., 2004). They are involved in the etiology of many diseases
and functions in almost all physiological processes including development and
homeostasis. Currently, it is a promising area of research, which has major implications
in physiology, therapeutic treatments, and drug development.
Orphan receptors are a group of nuclear receptors that currently do not have any
known ligands. RORA is an orphan receptor since its endogenous ligand is not fully
agreed upon. Many recent studies have identified potential ligands that can modulate this
NR to identify/understand its physiological role, but none has been fully agreed upon
(Solt et al., 2012). RORA is a critical regulator of multiple biological processes and has
been implicated in various physiological processes including circadian rhythm, lipid
metabolism, photoreceptor development, autism, bone morphogenesis, and inflammation
5
(Jetten et al., 2009). Its expression is distributed widely thorough several organs and
tissues including brain, liver, testis, skin, and bone (Giguere et al., 1994).
Figure 1: RORA is a transcriptional factor that has many physiological functions.
This diagram illustrates how RORA binds to its response element (AGGTCA preceded
by 6 A/T) to affect the transcription of many target genes. RORA has a wide variety of
gene targets in almost all physiological functions. It is also been implicated in a number
of diseases, which makes it an ideal target for novel therapeutic treatments. Obtained
from National Institute of Environmental Health Sciences, 2011
RORA has a direct role in the retina, specifically in photoreceptor development.
RORA expression is found in GCL, INL, and cone photoreceptors in mouse adult retinas.
6
It is also highly expressed throughout the retina during embryonic development where it
is involved in retinal cone photoreceptor cells development (Fujieda et al., 2009).
RORA has been found to have a role in circadian rhythms. Circadian rhythms
have an essential role in regulation of almost all normal physiology processes and
behavior. Many cells contain its own circadian oscillator that operates in an independent
manner. However, all of the cells are coordinated by the master circadian clock located in
the brain (Ko and Takahashi, 2006). The master clock is in turn regulated in part by the
light-dark input by the visual photoreceptor systems in the retina. A number of genes
(BMAL1, CLOCK, PER, CRY) that are essential in the molecular circuitry of circadian
clock are regulated by RORA through positive and negative feedback loop pathways
(Isojima 2003). Circadian rhythms have a role in metabolism thus implicating RORA to
have an intermediate role to couple circadian clock with metabolism. Fluctuations in
daily levels of glucose, lipids, insulin, and other important molecules in metabolism
suggest that the circadian cycle plays an important role in regulation of metabolic
pathways. Disruption of this cycle can result in manifestation of metabolic disease such
as obesity, diabetes, and cardiovascular defects which all have AMD risk associations
(Duez and Staels 2008).
In addition, RORA has been directly implicated in cellular metabolism especially
lipid and cholesterol metabolism. Reduced expression of cholesterol transporters
(ABCA1, ABCA8, APOA1) is a proposed mechanism in which RORA affects
cholesterol and lipoproteins levels (Lau et al., 2008). Also, cholesterol has been reported
to be a potential natural endogenous ligand of RORA (Kallen el al., 2002). This is a
7
possible pathway in which RORA can affect AMD, since lipid metabolism plays a role in
formation of drusen and angiogenesis.
Angiogenesis is one of the clinical manifestations of advanced neovascular AMD.
Low oxygen levels play an important role in inducing angiogenesis, vasculogenesis, and
tumorigenesis (Besnard et al., 2001). Studies have shown that RORA is upregulated in
not only hypoxic conditions, but also under oxidative stress. Oxidative stress has also
been implicated in AMD due to its pro-inflammatory responses that leads to retinal
damage (Beatty et al., 2000). AMD development has been strongly linked to the immune
system and inflammatory pathway (Klein et al., 2005). This is another pathway where
RORA and AMD are linked.
8
SPECIFIC AIMS AND OBJECTIVE
Age-related macular degeneration is a devastating disease that affects millions of
people worldwide, yet the etiology and pathogenesis of AMD is not fully understood. A
recent study by Jun et al. (2011) has demonstrated RORA as a candidate gene involved in
AMD pathophysiology. RORA has numerous physiological functions which are common
in AMD pathology. Our current study will further explore the role of RORA in AMD.
We will look at the effects of RORA in the retina of mice by immunohistochemistry.
Antibodies against retinal cell-specific markers and proteins of interest will be used to
detect their expression in the retina. In addition, we will try to identify gene targets of
RORA that have also been linked with AMD. By identifying the targets of RORA and
discovering how RORA regulates these targets, we hope to better understand the role of
RORA in AMD pathophysiology. This study is an important step to understanding this
complex disease and is necessary to form novel therapeutic treatments in the future.
9
METHODS
MICE
All mice were bred and maintained under standard conditions at Schepens Eye
Research Institute. Rora(flox/flox) were crossed with Cdh5-Cre to produce offsprings
with both mutations (Rora(flox/flox) x Cdh5-Cre). Tissues were harvested from control,
wild type C57BL6/J (WT) mice, Rora(flox/flox) mice, and Cdh5-cre.
GENOTYPING
Mice were genotyped by PCR to ensure each mutation were present. Tail samples
were collected and DNA was extracted for genotyping using the isopropanol method.
Tail samples were submerged in a solution of 250µl tail buffer and 7µl proteinase K, and
incubated for 2 hours at 65℃, vortexing every 10 minutes. 200µl of 5M ammonium
acetate was added to the solution, and then incubated on ice for 15 minutes. Solution was
centrifuged for 20 minutes at 13,000 RPM. Afterwards, the supernatant was transferred to
a new tube containing 750µl isopropanol, and then centrifuged for 10 minutes at 16,000
RPM. The supernatant was removed, and 1 ml of 70% ethanol was added. Solution was
centrifuged for 5 minutes at 16,000 rpm. Supernatant was discarded, and pellet was dried
for 5 minutes on heat block at 55 ℃. 30µl of DEPC treated water was added, and tube
was incubated for 10 min at 55℃. The concentration of DNA extracted was measured
using Thermo Scientific NanoDrop 2000 Spectrophotometer.
PCR master mix component (per sample): 10x PCR buffer (1µl) (Roche Life
Sciences), 40mM dNTP (0.20 µl) (Roche Life Sciences), 10µM forward primer (0.25 µl),
10
10µM reverse primer (0.25µl), Taq Polymerase 5U/µl (0.1 µl) (Roche Life Sciences),
DNA sample 50 ng/µl (0.7µl). Final reaction = 10 µl. The following primers were used:
RORA-flox (Forward, 5’- TCT GAA TCC ACC ATA CTT CC -3’, Reverse, 5’- AGG
TCT GCC ACG TTA TCT G -3’) (Eurofins MWG Operon), Cdh-Cre (Forward, 5’- GTG
AAA CAG CAT TGC TGT CAC TT -3’, Reverse, 5’- GCG GTC TGG CAG TAA AAA
CTA TC -3’) (Eurofins MWG Operon).
All reactions were performed in 200µl PCR tubes, and were run in a Bio-Rad
C1000 Touch Thermal Cycler. Cycle parameters for Rora-floxed: 1) 95℃ for 3 minutes,
2) 95℃ for 15 sec, 3) 65℃ for 30 seconds, decrease 1℃/cycle, 4) 72℃ for 40 seconds, 5)
Go to step 2 for 10 cycles, 6) 95℃ for 15 seconds, 7) 55℃ for 30 seconds, 8) 72℃ for 40
seconds, 9) Go to step 6 for 30 cycles, 10) 4℃ hold until refrigerate product. Cycle
parameters for Cdh-cre: 1) 95℃ for 3 minutes, 2) 95℃ for 30 seconds, 3) 51.7℃ for 1
minute, 4) 72℃ for 1 minute, 5) Repeat steps 2-4 for 34 cycles, 6) 72℃ for 2 minutes, 8)
12℃ hold. Products were analyzed on 2% agarose gel with ethidium bromide staining.
Product size: 323 bp WT RORA, 394 bp mutant RORA-flox, 100 bp Cre product.
IMMUNOHISTOCHEMISTRY
Eyes from wild type B6 mice, Rora-floxed, Cdh-Cre, and Rora/Cdh mice were
collected for paraffin embedding. Eyes were stored in Methanol: Acetic Acid (3:1)
fixative or 4% Paraformaldehyde (PFA) fixative overnight at 4℃. Eyes were then placed
in embedding cartridges with the following solution: 70% ethanol for 2 hours, 95%
11
ethanol for 2 hours, 100% ethanol for 2 hours, xylene for 1 hour, paraffin overnight.
Tissue blocks were sectioned at 5µm and collected on coated slides.
Deparaffinization of tissue sections were performed using xylene twice for 5
minutes each. Afterwards, tissues were dehydrated in decreasing ethanol concentration
(100%, 95%, 70%: twice for 5 minutes each). PFA sections were heat treated with citric
acid before incubating with primary antibodies. Citric acid treatment of slides involved
microwaving slides for 60 seconds submerged in 10mM sodium citrate buffer (pH 6.0).
Sections were then blocked with 2% horse serum diluted in PBS for 45 minutes at room
temperature to prevent non-specific binding of primary antibodies. Tissue sections were
incubated with primary antibodies at 4℃ overnight. Secondary antibodies were incubated
for 45 minutes at room temperature. Cell nuclei were counter stained with 4',6diamidino-2-phenylindole (DAPI, diluted 1:200). Finally, sections were mounted with
glass cover slips using Vectashield (Vector Lab, H-1200). Fluorescence images were
examined by confocal laser scanning microscope (Leica TCS SP8).
The following primary antibodies were used: mouse polyclonal antibodies to
Vascular Epithelial Growth Factor (VEGF, Abcam, Ab1316), rabbit polyclonal
antibodies to Transforming Growth Factor Beta (TGF-β, Abcam Ab66043), rabbit
polyclonal antibodies to Glial Fibrillary Acidic Protein (GFAP, EMD Millipore,
Ab5804), sheep polyclonal antibodies to Chx10 (CHEMICO International, Ab9014),
mouse polyclonal antibodies to Calbindin D-28K (Swant, Cb300), rabbit polyclonal
antibodies to Parvalbumin (Abcam, Ab11427), rabbit polyclonal antibodies to Collagen
IV (Abcam, ab6586), rabbit polyclonal antibodies to Beta-Tubulin III (Sigma-Aldrich,
12
T2200), rabbit polyclonal antibodies to Neurofilament Light (NFL, EMD Millipore,
Ab9568), rabbit polyclonal antibodies to Synaptophysin (Abcam, ab52636), rabbit
polyclonal antibodies to Protein Kinase C alpha (PKC-α, Abcam, Ab31), goat polyclonal
antibodies to OPN1SW (Santa Cruz Biotechnology, SC-14363), mouse polyclonal
antibodies to Rhodopsin (EMD Millipore, MAB5316), and rabbit polyclonal antibodies
to Red/Green Opsin (EMD Millipore, ab5405). Dilutions of primary antibodies were as
followed: VEGF 1:100, TGF-β 1:100, GFAP 1:200, Chx10 1:200, Calbindin 1:200,
Parvalbumin 1:200, Collagen IV 1:400, β-Tubulin III 1:400, NFL 1:200, Synaptophysin
1:200, PKC-α 1:200, OPN1SW 1:200, Rhodopsin 1:200, Red/Green Opsin 1:200. The
following secondary antibodies were used: Alexa Fleur 488 goat anti-rabbit IgG (Life
Technologies, A11008), Alexa Fleur 488 goat anti-mouse IgG (Life Technologies,
A11001), Alexa Fleur 555 donkey anti-sheep IgG (Life Technologies, A21436). All
secondary antibodies were diluted 1:400. Both primary and secondary antibodies were
diluted using 2% horse serum.
CHROMOTIN IMMUNOPRECIPITATION
Chromatic immunoprecipitation (ChiP) was perfomed as previously described
(Haider et al., 2009). 8 retinas from B6 mice were obtained. The retina was disrupted
using a pestle and mortar. 37% formaldehyde was added to the tissue for 60 minutes at
room temperature in order to crosslink proteins with DNA. The samples were then
sonicated to shear the DNA. Samples were sonicated with 10 pulses for 1 second. This
was done 20 times with 10-second pause between pulses. RORA antibody (1μg) was
13
added to bind to cross-linked protein/DNA. Samples were incubated overnight at 4°C on
a rotating platform. Solution went through a number of washes in order to obtain
fragments of interest. The remaining fragments were reverse cross-linked by incubating
with 200mM NaCl and 10mg of Protinase K (to remove proteins) for 5 hours at 65°C.
Qiagen purification kit was used to purify the DNA samples and concentrations were
measured Thermo Scientific NanoDrop 2000 Spectrophotometer. Samples were sent to
for sequencing.
PATHWAY ANALYSIS
Data from the ChIP assay was used to find statistically significant genes of interest
regulated or interacted with RORA. Data were analyzed using Ingenuity Pathway
Analysis (IPA, Ingenuity Systems, www.ingenuity.com) as described in a previous study
(Jelcick el al., 2011). Networks were generated based on their connectivity. Gene
identifiers and statistically significant expression values were uploaded into Ingenuity.
Default cutoffs were set to identify genes whose expression was significantly
differentially regulated and overlaid onto a global molecular network developed from
information contained in the Ingenuity Pathways Knowledge Base. Networks were
algorithmically generated based on their connectivity. Genes or gene products in the
networks are represented as nodes, and the biological relationship between two nodes is
represented as an edge (line). Dashed edges represent a weaker relationship than solid
edges. All edges are supported by at least 1 reference from the literature, from a textbook,
14
or from canonical information stored in the Ingenuity Pathways Knowledge Base. Nodes
are displayed using various shapes that represent the functional class of the gene product.
15
RESULTS
MICE GENOTYPING
A mouse model was produced with a conditional knockout of RORA in
endothelial cells by using Cre-lox recombination technology. Rora (Floxed/floxed) was
crossed with Cdh5-cre to create a new mouse strain with both mutation: RORA
(floxed/floxed) x Cdh5-cre. This strain is a conditional knockout of RORA in only cells
that expresses VE-Cadherein which are endothelial cells.
Genotyping was performed to ensure that the mice have both mutations (Figure
3). Positive control, negative control, and B6 control samples were used to ensure the
correct mutations were analyzed. RORA-floxed product size was 394bp and RORA WT
product size was 323bp. Cdh5-Cre product size was 100bp. The following offspring of
crossed RORA-floxed and Cdh-cre were shown to have both mutations: 79, 80, 81, 82,
83, 84, 85, 103, 104, 105 112, 113, 114, 115. The following did not have Rora-flox
mutations but contained only Cdh-cre: 100, 101, 102, 106, 107, 116.
16
Figure 2: Genotype analysis shows that all mice used contained mutations of both
RORA- floxed and Cdh5-cre: Mice were genotyped to determine if mutations were
present in genome. RORA mutant band is 394 bp and WT RORA is 323 bp. Cdh5-cre
product is 100bp. The following animals are homozygous for RORA mutant as well as
Cdh5-cre: 79-85, 103-107, 112-115.
IMMUNOHISTOCHEMISTRY
Structural proteins (GFAP, NFL, Collagen IV, β-tubulin III):
Glial fibrillary acidic protein is a type III intermediate filament and functions to
maintain cell shape and structure. It is a major constituent in astrocytes. It serves as a cell
specific marker to distinguish between astrocytes from other glial cells. Astrocytes are
found in the nerve fiber layer (NFL) and ganglion cell layer (GCL) where they provide
17
support for the ganglion cells. GFAP immunoreactivity was confined to the GCL in
normal B6 mice as well as in the RORA KO (Figure 4).
Neurofilament light (NFL) are intermediate filament that are major components of
the neuronal cytoskeleton. It is found primarily in the axons of neurons where its function
is to provide structural support for axon and regulate axon diameter and shape.
Immunoreactivity of NFL is found mainly in ganglion cells and horizontal cells in the
GCL and OPL (Figure 4).
Collagen IV is a type of collagen that is a major constituent in the basement
membrane of tissues. Immunoreactivity of collagen IV is seen in the GCL, IPL, OPL, and
RPE (Figure 4).
Beta Tubulin III (β-tubulin III) is a major component of microtubules that is
found only in neurons. It is a marker to distinguish between neurons and glial cells in
samples of neural tissues. The functions of β-tubulin III include axon guidance and
maintenance. Immunoreactivity of β-tubulin III is seen in the IPL and OPL (Figure 4).
18
Figure 4: IHC of structural proteins in retina shows no change between RORA
knockout and WT: GFAP, Collagen IV, NFL all has similar staining across all four mice.
GFAP had staining in GCL and OPL. Collagen IV had staining at GCL, IPL, OPL, and
RPE. NFL had staining at GCL, IPL, and OPL. Scale bar: 100 µm. Nuclei of cells are
stained blue and proteins of interest are stained green (GFAP, Collagen IV, NFL, BTubulin III)
Growth factors (VEGF, TGF-β):
Vascular epithelial growth factor (VEGF) is responsible for stimulating
angiogenesis, epithelial cell growth, and vasculogenesis. VEGF plays a role in wet AMD
19
and diabetic retinopathy where there is new blood vessels formation in the retina causing
loss in visual acuity. VEGF is expressed in the retina by Muller cells, endothelial cells,
ganglion cells, RPE cells, and astrocytes. Immunoreactivity of VEGF is seen in the GCL
and OPL. There seems to be an absence of VEGF staining in the knockout strain, and
interestingly a decrease in the Cdh5-cre mice (Figure 5).
Transforming growth factor beta (TGF-β) is a cytokine whose many functions
include control of cell growth, cell proliferation, cell differentiation, and apoptosis. It is
also linked with vascular barrier function, endothelial permeability, and angiogenesis
which suggests it has a role in neovascularization (Walsh et al., 2009). It is expressed in
ganglion cells, endothelial cells, and photoreceptors. Immunoreactivity of TGF-β is seen
in the GCL and OPL in normal WT mice. There seems to be a decrease of staining in the
knockout mice (Figure 5).
Figure 5: IHC of Growth Factors in retina shows decrease expression in RORA
knockout: VEGF and TGF-B is involved in angiogenesis and cell growth.
20
Immunoreactivity of both VEGF and TGF-B is low in Rora-floxed x Cdh5-Cre (D,F)
compared to WT (A-C, E-G). Scale bar: 100 µm. Nuclei of cells are stained blue and
proteins of interest are stained green (VEGF, TGF-B)
Retinal Cell Specific Markers (Calbindin, Parvalbumin):
Calbindin D-28K is a calcium binding protein. It is a retinal cell specific marker
for horizontal cells in the retina. Parvalbumin is a calcium binding protein. It is a retinal
cell specific marker for amacrine cells in the INL. IHC results show similar
immunoreactivity in all mouse tissues for calbindin and parvalbumin.
Photoreceptors (Blue opsin, Green opsin, Rhodopsin):
Blue opsin, green opsin, and rhodopsin are markers for photoreceptor cells in the
retina. Blue opsin are found in S-cone photoreceptor, green opsin are found in M-cone
photoreceptor, and rhodopsin are found in rod photoreceptors. They are all expressed in
the photoreceptor layer staining the different photoreceptors. All of the immunostaining
were similar in the four different strains of mice (Figure 6).
21
Figure 6: IHC of Photoreceptors in retina shows no difference between RORA
mutant and WT: S-cone, M-cone, and rod cells were stained by blue opsin, green opsin,
and rhodopsin Ab respectively. All immunoreactivity of each protein were similar in both
WT and mutant (A-L). Scale bar: 100 µm. Nuclei of cells are stained blue and proteins of
interest are stained green (Blue opsin, Green opsin, Rhodopsin)
Bipolar cells (Chx10, Synaptophysin, PKCα):
Synaptophysin is a protein found in neurons involved in synaptic transmission. Its
function is currently unknown but it is a part of the synaptic vesicle complex. It is found
in both the inner plexiform layer (IPL) and outer plexiform layer (OPL) since this is
where synaptic transmission occurs in the retina. Synapses of photoreceptors with bipolar
22
cells and horizontal cells occur in the OPL and Synapses of bipolar cells with ganglion
cells and amacrine cells in the IPL. Immunoreactivity of synaptophysin is seen in the IPL
and OPL as expected in WT, Rora-floxed, Cdh5-cre, and Rora-floxed x Cdh5-cre
(Figure 7).
Protein Kinase C alpha (PKCα) is a protein kinase that has many physiological
roles and targets. It has been suggested that PKCα regulates bipolar cells signal
transduction (Ruether et al., 2009). In the retina, it is mainly expressed in bipolar cells
which are found in the inner nuclear layer (INL), OPL, and IPL.
Chx10 is a protein that is involved in retina development. It is necessary in the
developing retina where it helps retinal progenitor cells differentiate into mature cells.
Chx10 expression is high in the developing retina, but in mature retina, it is confined to
bipolar cells expressed at low levels. Immunoreactivity of Chx10 is seen in the INL as
expected in WT, Rora-floxed, Cdh5-cre, and Rora-floxed x Cdh5-cre (Figure 7).
23
Figure 7: IHC of Synaptophysin and Chx10 shows no change between RORA
knockout and WT: Chx10 and Synpatophysin immunoreactivity were similar between
WT and Rora-floxed x VE-Cdh-Cre tissues. Scale bar: 100 µm. Nuclei of cells are
stained blue and proteins of interest are stained red (Synaptophysin, Chx10).
CHROMATIN IMMUNOPRECIPTIATION – SEQUENCING (CHIP-SEQ)
Chromatin immunoprecepitation with massively parallel sequencing is very useful
in mapping DNA-protein interactions on a genomic-wide level. ChiP assay is an effective
assay for transcription factors because binding of TF are very sequence specific, which
leads to very localized ChiP-seq signals in the genome. ChIP data reveals numerous gene
targets of RORA. 1150 genes were found to be statistical significant binding targets of
RORA.
INGENUITY: INTEGRATED PATHWAY ANALYSIS
AMD genes and RORA pathway genes were inputted into Ingunity IPA to
analyze any known associations between these genes. Multiple pathways were found and
linked to AMD (Figures 8-11). These genes were then compared to the list of RORA
targets from the ChIP data. A list of genes appearing in both lists are our genes of
interests which are RORA targeted genes that are linked with AMD (Table 2).
24
AMD Genes
CFH
ELOVL4
FBLN5
FLT1
MPDZ
PLEKHA1
TLR4
Proteins that genes code for
Complement factor H
Elongation of very long chain fatty acids protein 4
Fibulin-5
Vascular epithelial growth factor receptor 1
Multiple PDZ protein
Pleckstrin homology domain-containing family A member 1
Toll-like receptor 4
GPCR genes
DRD3
RAMP2
Proteins that genes code for
D3 dopamine receptor
Receptor activity monitoring protein 2
Transcriptional
Regulator genes
CLDN3
CNPY2
HIST1H2AD
MYCBP
RBM22
RNF113A
Proteins that genes code for
Cell signaling
genes
ARHGAP19
EXOSC4
PSD
RAB40C
Proteins that genes code for
Fatty Acid
Metabolism genes
IGFBP1
N-cor
VLDLR
ZBTB9
Proteins that genes code for
Claudin 3
Canopy FGF Signaling Regulator 2
Histone H2A type 1-D
C-Myc binding protein
RNA binding motif protein 22
Ring finger protein 113A
Rho GTPase-activating protein 19
Exosome component 4
Pleckstrin and Sec7 Domain Containing
Ras-related protein Rab-40c
Insulin-like growth factor binding protein 1
Nuclear receptor co-repressor 1
Very low density lipoprotein receptor
Zinc finger and BTB domain containing 3
Table 2: RORA targeted genes that may have associations with AMD: Genes from
ChIP data was compared with Ingenuity pathway analysis to determine genes that are
common. These genes are targets of RORA and genes that are also linked with AMD.
25
Figure 8: Association between AMD genes and fatty acid metabolic genes that are
targets of RORA.: AMD genes are purple while fatty acid metabolism genes are orange.
Lines and arrows show associations between the genes. Figure was produced by
Ingenuity IPA. Genes of interest include: N-cor, PPARG, Smad, VLDLR, LDLR,
IGFBP1, RARRES2, APOE3.
26
Figure 9: Association between AMD genes and GPCR genes that are targets of
RORA. AMD genes are purple while GPCR genes are green. Lines and arrows show
associations between the genes. Figure was produced by Ingenuity IPA. Genes of
interests include: Beta arrestin, GPCR, RAMP2, DRD3
27
Figure 10: Association between AMD genes and cell signaling genes that are targets
of RORA. AMD genes are purple while cell signaling genes are light blue. Lines and
arrows show associations between the genes. Figure was produced by Ingenuity IPA.
Genes of interst include ERK1/2, RAB40C, CLDN, PRKAC
28
Figure 11: Association between AMD genes and transcriptional regulator genes that
are targets of RORA. AMD genes are purple while transcriptional regulator genes are
red. Lines and arrows show associations between the genes. Figure was produced by
Ingenuity IPA. Genes of interest include HIST1H2AD, PINKX1, 60S ribosomal subunit,
E1F1B, PKA, MYCBP.
29
DISCUSSION
A conditional knockout model was used to explore the effects of RORA in the
retina. In the knockout model, RORA was conditionally knocked out in endothelial cells.
The retinal pigment epithelium is a layer in the retina that supports, nourishes, and
protects the retinal cells so that they can maintain normal visual function. Since the
dysfunction of this layer of cells is critical in the development of AMD, we will study the
effect of RORA knockout in these types of cells.
Immunohistochemistry was performed to detect the levels of expression of
various proteins and cell markers in the retina. Immunostaining of GFAP, NFL, Collagen
IV, and β-Tubulin III were checked to see if RORA had any effect on structural proteins
and structural changes in the retina. In addition, cell specific markers were also checked
to see any changes in the different cells in the retina. Blue opsin, green opsin, rhodopsin,
parvalbumin, and calbindin stains S-cone cells, M-cone cells, rod cells, amacrine cells,
and horizontal cells respectively. IHC results shows that there is not much difference in
levels of expression in either WT or RORA knockout (Figures 4, 6). In addition, we
checked synaptophysin because it is important in normal neuron functions through its
involvement in synaptic transmission. Synaptophysin expression was normal (Figure 7).
Since RORA and Chx10 both are important in retinal cell differentiation and
development, levels of Chx10 expression were also investigated which resulted in similar
expression in WT and knockout (Figure 7).
VEGF and TGF- β are growth factors whose functions include angiogenesis,
vascular permeability, cell growth, cell differentiation, and vasculagenesis. IHC of VEGF
30
and TGF- β shows decrease levels of expression in RORA knockout compared to WT
(Figure 5). This is an interesting finding due to the fact that neovascularization in the
retina often leads to severe acute visual loss in wet AMD. CNV is a significant clinical
manifestation of neovascular AMD, and recent studies have suggested the upregulation of
angiogenic factors (VEGF, TGFB, angiostatin) to be involved in the formation of CNV
(Zhang, 2007). In addition, a previous study suggested the presence of TGF-β in AMD
pathophysiology (Silveira et al., 2010). Also, current treatment for wet AMD involves
injections VEGF inhibitors to delay the progression of the disease (Kovach et al., 2012).
Additional conformational experiments will be done to confirm that these changes are
noted.
Another important part of this study is to investigate the vast RORA pathway and
how it is linked to AMD pathogenesis. Since RORA is a nuclear receptor that has been
implicated in a variety of physiological functions (Solt et al., 2012), it will be invaluable
to identify the gene targets of RORA. Using the ChIP-seq technique, we were able to
determine the sequences of DNA that RORA binds to in vivo. Then, with software
analysis, a list of statistically significant RORA gene targets was determined. Comparing
this lists with a list of commonly known AMD genes, we determined a number of genes
to further investigate (Table 2, Figures 8-11). Our results shows that AMD may have
some associations with genes in a number of pathways including fatty acid metabolism,
G-protein-coupled receptors, transcriptional regulation, and cell signaling.
Fatty acid metabolism has been shown to be involved in AMD pathology and
development. Cholesterol and lipids are major components of drusens, whose presence is
31
a clinical hallmark of AMD. In addition, recent studies have linked AMD risks to ATP
binding cassette transporters, which are a major regulator of cholesterol and
phospholipids (Allikmets, 2000). Our results have shown that IGFBP1, VLDLR, NCOR,
and ZBTB9 are linked with AMD and are potential gene targets of RORA (Table 2,
Figure 8). IGFBP1 is a binding protein that binds to IGF to extend its half-life to regulate
its cellular availability (Juul, 2003). IGF has been shown to regulate metabolic processes
and cell growth and development (Thissen et al., 1994), and recent studies have
suggested that IGF1 and IGFBP1 have a role in AMD pathogenesis through the
inflammatory pathway (Chiu et al., 2011). VLDLR is a transmembrane protein receptor
that is involved in cholesterol uptake and fatty acid metabolism. Studies of VLDLR have
shown in the absence of VLDLR, angiogenesis increases in the retina, a major
complication in wet AMD (Hu et al., 2008; Jiang et al.2009).
G-protein coupled receptors (GPCR) are the largest class of receptors and have
roles in many physiological processes, including fatty metabolism, cell signaling, and
transcriptional regulation. One example of a GPCR in the retina is rhodopsin, which is
found in photoreceptors and is responsible for light transduction (Hamm, 2000). GPCR
has been linked to the production of oxidation in the retina (Chen et al., 2012). Oxidative
stress contributes to photoreceptor degeneration and damage to the retina, especially the
RPE, which has implications in the development of AMD (Beatty et al., 2000). In
addition, one current treatment for dry AMD is taking antioxidant multivitamins, which
are shown to slow progression of the disease (AREDSRG, 2001).
32
Our data suggests that Rab40c and ARHGAP19 is targeted by RORA and is
associated with the AMD pathway (Table 2, Figure 10). Rab40c and ARHGAP19 are
small GTPase, which also links it to the GPCR pathway. Recent studies have shown that
Rab40c is linked with formation and regulation of lipid droplets, which stores lipids and
cholesterol (Tan et al., 2013). Lipid droplet dysfunction has a role in many metabolic
diseases such as obesity and diabetes, which share risk factors for AMD (Greenberg et
al., 2011). By linking AMD and RORA we can better understand the pathway in which
AMD progresses and develops.
Cell signaling is an important system that controls cellular activities and
coordinates cellular actions. One example is that TGF-β downstream pathway includes
many extracellular signal-regulated kinases (ERK/MAP) targets in order to induce its
effects on the cells (IKushima and Miyazono, 2010). In addition, an important signaling
pathway of AMD involves the immune system. Immune-mediated responses and
inflammatory processes has a role in drusen formation and promoting CNV in the retina
(Hageman et al., 2001). Chemokines (IL-6, CCL2, CCR2, CX3CR1, TNF), cell signaling
molecules in immune processes, have been linked to AMD since they have shown
damaging effects on Bruch membrane, RPE, and the retina (Ambati, Atkinson, and
Gelfand, 2013). Also, AMD risk has been strongly linked with the complement cascade
system; drusens contain almost all the complement proteins. Genetic variations in
complement genes (CFH, C3) have shown an increase risk for the disease (Klein et al.,
2005).
33
Transcriptional regulation affects gene and protein expression. RORA is a
transcriptional factor that regulates many different gene targets (Jetten et al., 2009), and it
would be expected that RORA also have some targets that has a function in transcription.
This will be important in elucidating a pathway on how RORA has an effect in AMD
pathogenesis.
The next step of this study is to further explore the targeted RORA genes that
have been linked to AMD. Quantitative real-time PCR will be performed to investigate
how these gene expression levels are affected in the conditional RORA knockout mice.
Genes that show altered expression levels will be promising targets to further analyze. In
addition, we will further investigate the role of RORA in the retina and in the etiology of
AMD. Future studies will explore the effect of RORA in the retina of conditional
knockout of RORA in cone cells and Chx10. By exploring these knockout models, we
will gain a better understanding of the role of RORA in the photoreceptors and the
developing retina.
AMD is the one of the most prevalent causes of decrease in visual acuity and
irreversible blindness in the world. AMD is a complex genetic disease that has a number
of genes and environmental factors that is implicated in its etiology. There is currently no
cure for AMD, but because of its negative effect on quality of life, high economic costs,
and public health implications, there is an increasing urgency for research in this area
(Day et al., 2011; Soubrane et al., 2007). RORA has been linked with AMD
pathogenesis, and it is a promising area to explore to because of its effects in the retina
and numerous physiological pathways. This study is one small step towards elucidating
34
both the etiology of AMD and the role of RORA in AMD. This will be very beneficial to
developing novel treatments and therapeutic drugs targets to cure this disease in the
future.
35
LIST OF JOURNAL ABBREVIATIONS
Arch Ophthalmol
Archives of Ophthalmology
Circ Res
Circulation Research
Diab Vasc Dis Res
Diabetes & Vascular Disease Research
Endocr Rev
Endocrine Reviews
Genes Dev
Genes and Development
Growth Horm IGF Res
Growth Hormone & IGF Research
Hum Mol Genet
Human Molecular Genetics
Invest Ophthlmol Vis Sci
Investigative Ophthalmology & Visual Science
J Biol Chem
Journal of Biological Chemistry
J Neurochem
Journal of Neurochemistry
Nat Struct Mol Biol
Nature Structural & Molecular Biology
Physiol Rev
Physiological Reviews
Prog Retin Eye Res
Progress in Retinal and Eye Research
Surv Ophthalmol
Survey of Ophthalmology
Vision Res
Vision Research
36
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42
CURRICULUM VITAE
Hai Hoang
Hhoang6959@gmail.com
YOB: 1991
203-522-0440
Permanent Address:
1842 Main Street
Stratford, CT 06615
Education:
Boston University School of Medicine–Boston, MA
M.A., Medical Science, Expected May 2015
Boston University –Boston, MA
B.A., Biology, conc. in Molecular, Cell Biology and Genetics, May 2013
B.A., Economics, May 2013
Research
Schepens Eye Research Institute – Boston, MA
Experience: Research Intern – September 2014 - present
Worked on an independent project investigating the role of RORA in
macular degeneration disease. Utilized a variety of lab techniques such as
immunochemesitry, qPCR, genetic pathway analysis, and genotyping.
Completed my master thesis in this lab.
Harvard School of Public Health, GCD – Boston, MA
Research Intern – January 2012 – May 2013
Assisted lab members with their projects and learning advanced laboratory
techniques such as flow cytometry, ELISA, and tissue culture. Worked
alongside a post-doc doing supplementary projects to assist with his thesis.
Assisted lab manager with lab duties such as making buffers, ordering
supplies, stocking benches and hood. Personal project involved examining
the pathway of inflammation regulation and more specifically how
mitochondria are involved in this pathway.
St. Vincent Medical Center, Emergency Department– Bridgeport, CT
Research Associate – May 2011 – August 2011
Surveyed and enrolled patients in a tobacco cessation study. Interacted
with patients and hospital staff in a friendly and proficient manner.
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Yale School of Medicine, Dept. of Infectious Disease – New Haven, CT
Research Assistant – May 2010 - August 2010
Assisted research with a post-doctorate at Yale who was conducting
research on HIV treatment through the use of siRNA. Utilized lab
techniques such as gel analysis, protein purification, amplification, and
extraction.
Additional Burlington Eye Associates - Burlington, MA
Experiences: Medical Scribe – July 2014 – present
I am responsible for assisting the physician with documenting in the EMR
and performing diagnostic eye procedures. There was a lot to learn
including medical terminologies and common medications involved in
ophthalmology. In addition, I had to learn how to use the EMR software,
and be able to navigate the program in order to document patients’
medical record. I found it is crucial to have an open mind and a positive
attitude in order to learn new things quickly.
BostonCares Volunteer – Boston, MA
Community Service volunteer – February 2014 – present
I volunteered at many different opportunities including tutoring adults to
achieve their GED, serving meals to the homeless and veterans, building
computers for schools in third world countries, packing food packages for
the American Red Cross, and building beds for the homeless.
X-Cel Adult Education Tutor- Boston, MA
Math Tutor – February 2014- present
At X-Cel, I volunteered weekly to tutor adults who are working towards
their GED. I find it very inspiring to see those who value their education
and put in an effort into learning. Over time, I learned that the key is to
have patience with the students, and be flexible by explaining concepts in
different ways. Witnessing as well as having a part in their successes has
been a very rewarding experience.
Brigham’s And Women’s Hospital – Boston, MA
Office Assistant – February 2014 – September 2014
I volunteered at the front office checking patients in, answering calls, and
scheduling appointments. I was also responsible for filing charts, and
preparing paperwork for patients. I communicated frequently with nurses,
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physicians, and staff regarding patients’ charts and status. Working in the
office, I was able to observe the various members of the healthcare team
interact and work together to provide the best care for the patients.
Hospital Ambassador – October 2012 – May 2013
Assigned various task within the hospital. Delivered specimens and blood
samples to different labs and departments. Admitted patients into their
room, discharged patients, and transported patients to various places
within the hospital. Frequently interacted directly with patients in a
friendly manner. Worked closely with nurses and hospital staff in a
proficient manner.
Activities Information Maven – September 2009-May 2013
Student Activities Office, Boston University – Boston, MA
Worked in an office as an information desk and selling tickets. Provide
campus information to students and visitors. At nighttime, also had the
role of the Escort Security Service. Maintained the student union by
opening/locking doors that student groups needed to access. Also
promoted various events on campus by social media and through selling
tickets. Learned how to professionally interact with and serve
customers/students. Worked extensively with social media to promote
event and the office to get more reach and influence.
Publication: Jiujiu Yu, Hajime Nagasu, Tomohiko Murakami, Hai Hoang, Lori
Broderick, Hal M. Hoffman, and Tiffany Horng. Inflammasome activation
leads to Caspase-1–dependent mitochondrial damage and block of
mitophagy. PNAS 2014 111: 15514-15519.
Misc.:
BU Finance and Investment Club: September 2010 – May 2012
BU Ultimate Frisbee Club: September 2010- May 2011
Computer literacy: Microsoft (Word, Excel, PowerPoint, Access), HTML
coding
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