Project Title: Mechanobiology of Neural Stem Cells
Project Description: Millions of patients currently suffer from neurodegenerative diseases like Alzheimer's and Parkinson's disease, and with increasing life-expectancies in many countries that number is predicted to increase dramatically. The discovery that cells in our brains can and do generate new neural cells even in adulthood (neurogenesis) presents exciting avenues of research towards possible treatments or preventative therapies for these diseases. Neurogenesis is attributed to small populations of neural stem cells, and thus understanding the signals received by these cells and how they are processed into certain behaviors, like proliferation or differentiation into a specific cell type, is key to developing medical therapies.
The level and specificity of the proliferation and differentiation of neural stem cells has been correlated with the environment the organism is in as well as the presence of various soluble factors, including several compounds studied in our group. However, there has been recent work showing that not only biochemical signals can affect stem cell differentiation, but that mechanical signals, like the stiffness of the surrounding extracellular matrix, or the intracellular contractility of the stem cells can affect their behavior. My research project focuses on exploring how adult neural stem cells convert mechanical input signals into behavioral outputs. Our approach to tackle this question is to use "outside-in" methods by synthesizing new stress-tunable substrates for the cells to grow on as well as genetic and pharmacological studies which directly probe intracellular mechanical signaling pathways.

Project Title: Engineering Biomaterials for Neural Tissue Regeneration
Project Description: Neurogenesis, or the creation of new neurons, is an essential process in the formation of healthy, functioning nervous systems. Not until recently has it been widely accepted that this vital process occurs in the adult mammalian brain, while only continually occurring within two specific regions. This functional disparity begs the question as to what makes these regions neurogenic. Both contain neural stem cells (NSCs), which have the capacity for continuous self-renewal and multipotent differentiation into various neural cell types. These cells, along with several other cell types, contribute to a local microenvironment within neurogenic regions of the brain that is conducive to their own proliferation and eventual differentiation. Understanding the mechanisms behind the directed differentiation of NSCs into neurons is essential in understanding how and why neurogenesis occurs. Biomaterials can serve as easily tunable mediums to study the effects of various chemical and mechanical signals on NSC differentiation. They also allow one to create multivalent peptide constructs to maximize the potency of a cell or tissue scaffold. My research focuses on utilizing biomaterials to elucidate the various mechanisms behind NSC differentiation. Specifically, my goal is to create a functionalized biomaterial that can mimic the neurogenic niche of the brain to direct NSC differentiation into neurons. This engineered tissue scaffold will be tested in vivo for its ability to regenerate neurons and thus may have clinical applications for patients with various neurodegenerative diseases, such as Alzheimer’s or Parkinson’s.

Project Description: With the successful culturing of mouse embryonic stem cells in 1981 and human embryonic stem cells in 1998, scientists have used embryonic stem cells as a platform not only for medical therapies but also for understanding developmental processes. In early development, cells are pluripotent with the ability to turn into many cell types. As development progresses, cells become more strictly defined and may only give rise to a few cell types. In 2006, Shinya Yamanaka reversed this differentiation process by virally inducing four transcription factors (c-myc, Klf4, Oct4, Sox2) into mouse embryonic fibroblasts and selecting for pluripotent cells by both culture conditions and drugs. After culturing for approximately three weeks, this process created pluripotent, stem-cell like colonies or induced pluripotent stem (iPS) cells. While reprogramming had been achieved before through cloning, this approach has already been successfully replicated in many labs and holds enormous therapeutic potential as cells from one patient can be reprogrammed and differentiated into any cell type. My project is to understand the core regulation of iPS cells as well as find methods to increase the reprogramming efficiency.

Project Title: Neurogenesis and Aging

Project Description:
Adult neurogenesis has only recently been established as a mechanism of neural adaptation in the adult nervous system. The adult neural progenitor cells (ANPCs) that are responsible for this continual generation of new neurons in adult humans are tightly regulated by environmental factors, and the rates of adult neurogenesis have intriguing and suggestive correlations with conditions such as learning, memory, anxiety, depression, and stress. Therefore, gaining better control over these stem cell niches has implications for the development of new therapeutic treatments for conditions such as spinal injuries, stroke, Alzheimer's, depression, etc. Furthermore, ANPCs could possibly be harnessed to improve cognitive abilities, or counteract the effects of anxiety, stress, and aging. However, a great deal of research is required to understand how ANPCs determine their fate and how we can manipulate them to achieve a desired therapeutic effect.
One of the greatest challenges to achieving that goal is elucidating the molecular mechanisms of progenitor proliferation and differentiation. What are the intracellular pathways by which a signal is relayed from the cell's surface to its genetic code? And which of those pathways is most critical for signal transduction? By quantitatively studying these pathways, we can understand the primary mechanisms by which proliferation and differentiation signals are relayed through the cell, thereby allowing for the initial design of in vivo strategies directing ANPC proliferation and differentiation for the treatment of neurological disease and injury.
Several extracellular molecules influencing ANPC fate have been discovered. I am most interested in basic fibroblast growth factor (FGF-2) and the mechanisms by which the FGF-2 signal directs ANPC proliferation, producing more ANPCs, rather than differentiating into neural tissues. My approach for studying FGF-2 signaling will be two-fold: 1) quantifying canonical FGF-2 signaling mechanisms, and 2) studying novel mechanisms of FGF-2 signaling as revealed through functional genomics. Once we have a better understanding of these alternate signaling pathways, we can begin to quantify their relative importance in FGF-2 signaling, allowing the development of strategies to control ANPC fate in the hopes of treating neurological injury and disease.

Project Title: Molecular Dynamics of HIV Latency
Project Description: Following successful integration into the host genome, HIV-1 \replication is primarily controlled at the level of transcription. Once integrated, the 5’ long terminal repeat (LTR) functions as the viral promoter, regulating viral gene expression and replication. In both experimental and clinical settings, HIV-1 can establish a quiescent, latent state within resting CD4+ T cells. Current therapeutic strategies are only effective in the context of active viral replication; therefore the establishment of this latent state presents a significant barrier to the complete eradication of the HIV from the host.
HIV-1 encodes a unique transcriptional feedback loop involving the LTR and the Tat viral protein, By binding to a structured RNA element (TAR) in the leader of nascent HIV transcripts, Tat dramatically upregulates LTR mediated transcription, potentially functioning both as a transcriptional initiation and elongation or processivity factor. Tat interacts with numerous cellular factors, which serve to remodel the local chromatin environment and activate transcriptional elongation by RNA Polymerase. Notably, Tat interacts with the histone acetyltransferase, p300, members of the SWI/SNF chromatin remodeling complex, and positive, Transcriptional Elongation Factor b (p-TEFb). In a model HIV transcriptional system containing the LTR driving the expression of Tat with GFP as reporter of transcriptional activity (LGIT) it has been shown1 that clonal, single copy LGIT integrations have the potential to exhibit phenotypic bifurcation in Jurkat T cells, whereby some cells within a population are ON while others are OFF. The OFF state is analogous to latent state as LTR mediated transcription is effectively repressed in OFF cells. We seek to understand the molecular features and dynamics of PheB that lead to heterogeneity within a clonal population. Specifically, we seek to understand how the chromatin environment and the interaction of the LTR and Tat with cellular transcription factors at the proviral integration site may contribute to the latent state. The recent development of a system to directly quantitative transcriptional activity2 (mRNA FISH) in single cells will permit a detailed of transcriptional noise and activity in the context of the HIV transcriptional feedback circuit. We will employ an integrated systems level approach using targeted genetic approaches to understand the molecular logic of HIV transcriptional regulation and latency.

Project Description:
Adeno-associated virus (AAV) has received much attention recently as a gene therapy vector; many attributes of the virus AAV can stably express the transgene episomally for long periods of time or repair genomic mutations quite efficiently through homologous recombination both in vitro and in vivo. AAV vectors currently represent the vector in many of the current gene therapy clinical trials, proving especially effective for gene therapy in immuno-priviliged organs such as the retina and brain.
Despite AAV’s potential as a gene delivery vector, many factors hinder more widespread use. A large majority of the human population carry neutralizing antibodies against AAV2, the serotype most commonly used in clinical trials obstructing its use as a gene therapy vehicle. While the transgene product elicits no cellular immune response, infected cells can cross-present viral capsid proteins, eliciting an immune response even when AAV serotypes for which the host is seronegative are utilized. The use of receptors expressed globally through an organism prevents viral tissue specificity. Also despite the wide tissue tropism, many tissues are non-permissive to AAV infection.
An approach that can potentially circumvent these limitations is through a reversible bioorthogonal reaction. The aldehyde can be used to selectively append targeting groups to produce AAV with altered cellular tropism, as well as polymers that can prevent an immune response to the viral vector. Re-targeting of the viral capsid through a bioorthognal linkage allows targeting not available by protein expression, such as through small molecules and peptoids. The use of a reversible hydrazone linkage could allow for more efficient transduction by allowing the release of the targeting group upon endosomal acification, decreasing steric interference of the targeting group during subsequent steps in infection. Use of a library with the peptide insertion would allow the site-specific conjugation to polymer chains to decrease antibody neutralization. Previous work in our lab and other labs has shown the conjugation of polyethylene glycol (PEG) or HMPA to the surface of AAV can produce moderate decreases in antibody neutralization, as well as ablation of native tropism. The incorporation of a formylglycine allows a method by which to control the location and homogeneity of AAV-PEG conjugates that could be more efficient at immune evasion.

Project Title: Fuctionalizing Biomaterials for Stem Cells
Project Description:
The goal of tissue engineering is to help repair damaged tissue by introducing biomaterials that encourage new cell growth. Hydrogels made of polymers such as polyacrylamide and polyethylene glycol are promising new materials for tissue engineering because of their biocompatibility. These hydrogels can be injected or implanted at the site of injury to aid in tissue regeneration. The properties of the polymer matrix such as its mechanical stiffness and the biochemical signals it presents to cells can be tuned to obtain a hydrogel that mimics the extracellular matrix of a specific tissue. Stem cells can also be incorporated into the gel. These cells can then help regenerate the damaged tissue as the hydrogel degrades.
The focus of my project is on the peptides grafted onto the polymer network. The signals presented to cells via these peptides can encourage cells to attach to the surface of the hydrogel. Since polyethylene glycol is known to have little protein adsorption, cell attachment to the matrix can be controlled by grafting peptides that encourage cell attachment onto the polymer network surface. This completely synthetic matrix could then be used to culture stem cells without the need for a mouse feeder cell layer.

Project Title: Engineering light-regulatable protein systems

Project Title: Regulation of neural stem cell maturation and lineage specification by investigating the mechanical properties of the aNSC microenvironment.
Project Description: Adult neural stem cells (aNSC) have the capacity to differentiate into multiple cell types of the adult nervous system. My research involves investiginag the mechanobiology role of aNSCs to direct their differentiation towards specific neurotransmitter and motor neuron subtypes, such as dopaminergic, GABAergic, glutamarigic, serotonergic, and cholinergic neurons.
Adult rat hippocampous derived NSCs are cultured in microenvironments engineered with specific material and biochemical properties, to emulate the characteristics of the natural niche. I am focused on engineering the biophysical and dynamic properties with well defined knowledge on soluble, cellular, and extracellular matrix factors in the microenviroment in vitro to direct aNSC differentiation into desired subtypes by using a synthetic polyacrylamide gel culture system.
Input on these studies may play important future role in drug discovery and application of regenerative medicine from the devastating effects of neurological disorders, such as Parkinson’s Disease, Alzheimer’s Disease, Schizophrenia, which requires specific functional neuronal subtypes.

Project Title: Gene Targeting to Human Embryonic Stem Cells using Adeno-Associated Virus
Project Description: Human embryonic stem cells (hESCs) represent an exciting area of research because they have the ability to grow and divide in an undifferentiated state or to differentiate into any type of cell in the human body. The ability to control the differentiation of hESCs into a desired cell type through gene targeting has many applications, including enabling the investigation of disease mechanisms and developmental processes, allowing high throughput drug discovery and drug toxicity studies, and developing therapeutic gene correction to cure diseases. However, the current methods of gene targeting have a very low overall efficiency. My research involves using directed evolution to engineer adeno-associated virus vectors that can mediate efficient gene delivery and gene targeting to hESCs.

Project Title: Profiling Glycans on hESCs and Adult Rat Hippocampal Progenitors
Project Description:
The enthusiasm surrounding human embryonic stem cell research comes from the exceptional ability of these cells to differentiate into any cell type in the adult body. While these stem cells hold a tremendous amount of potential for therapeutic use, very little is known about which cellular and molecular factors are involved in their differentiation and self-renewal.
Our approach in identifying these factors is to profile and understand the functions of carbohydrates that exist on their cell surface.
Carbohydrates decorate the surfaces of all cell types. They can be attached to polypeptides or lipids to form glycoconjugates such as glycoproteins and glycolipids. Most classes of carbohydrates are found on cell surfaces and have important roles in development, growth and the function of organisms by mediating cell-cell interactions such as adhesion and signaling. These carbohydrates are also in the position to be cellular markers, which distinguishes one cell type from another based on their cell surface expression. Thus far, only a few pluripotent cell surface markers have been identified, two of which are glycolipids SSEA-3 and SSEA-4.
In our model system, we differentiate human embryonic stem cells and adult hippocampal progenitors into neurons and then identify and track changes in carbohydrates. The primary aim of our research is to characterize known and novel carbohydrates in order to investigate their function and correlate their expression with cellular phenotype. I am a graduate student in Carolyn Bertozzi's lab collaborating with David Schaffer.

Project Title: Viral Purification
Project Description:
Viral vectors have had billions of years to evolve very efficient systems for gene delivery. Consequently, viral vectors show great promise in delivering therapeutic genes. However, one of the setbacks the field is facing is obtaining pure virus for both research and medical use.
Current methods for purification do not provide very clean viral stocks. This means higher doses are needed, and these doses might be accompanied by immunogenic responses to non-viral contaminants in the viral stock.
I plan to develop a method for viral purification that has several advantages to purification options that are currently available. The hope is to make the purification easy to scale up, faster, and modular, meaning it can be used with any enveloped virus.

Project Title: HIV Evolution
Project Description:
Without a HIV vaccine in sight and 40 million HIV-positive people worldwide, HIV treatment must be improved. One of the major problems in HIV therapy is the ability for HIV to evolve around the therapies faster than new therapies can be formulated. This is primarily due to HIV being a retrovirus. The ability to curb HIV's mutation rate would not only make current therapies more efficacious, but also give us the power to force HIV into an unfit quasispecies from which it cannot escape.
I plan on studying the biology and dynamics of HIV retrotranscriptase evolution and its effect on viral fitness. Hopefully, this research will give us some design guidelines for better antiviral therapies.

Project Title: Molecular Engineering of Novel AAV Variants for the Transduction of Müller Cells
Project Description:
Numerous retinal diseases, such as retinitis pigmentosa, glaucoma, and age-related macular degeneration (AMD), afflict millions of individuals worldwide, leading to progressive and irreversible blindness. Neuronal cell death, either in retinal ganglion cells or photoreceptors, ultimately underlies the pathophysiology of each of these conditions. Biomedical strategies that focus on the delivery of neuroprotective factors to afflicted areas using gene therapeutic techniques present a promising approach for the treatment of these disorders.
For retinal gene therapy, retinal-spanning Müller glial cells would be an ideal target for the sustained production and secretion of neuroprotective factors, such as glial-derived neurotrophic factor and fibroblast growth factor-2, which have previously been shown to slow down neuronal cell death in the retina. However, current viral vectors are ineffective in transducing this cell type.
My research focuses on engineering adeno-associated viral vectors for the modification of viral tropism to enhance specific transduction of Müller cells.

Project Title: HIV-1 Infection Dynamics
Project Description:
The goal of using gene therapy to treat genetic disorders is very exciting and promising. However, issues ranging from cell specific targeting and the efficient delivery of genetic materials to the controlled expression of genetic information remain obstacles to this hope. Active research is currently underway to surmount these challenges. Within the past few years, questions concerning the control of gene expression have begun to become an area of particular interest. Already, there are a few systems developed that control gene expression using small molecules that either turn on or off gene expression. Though, there remains a significant amount of work to be done in this area.
My project focuses on modeling and developing systems that examine the interplay between different regulatory elements that govern gene expression. It is our hope to further the fundamental and quantitative understanding of the regulation of gene expression, with the goal of developing the knowledge for application to the logical design, implementation, and control of gene expression.