Performed testing of identified metabolites activity by checking for cellular responses in vitro with transfection of a Sentinel reporter system.

FACS, ELISA, PCR, RT-PCR, q-PCR, and proprietary HTS methods used to quantify cellular response.

Targeting initiation and development of drug discovery for validated disease pathways, identification of new indications for existing drug candidates and known drugs, identifying novel genes, and deciphering gene pathways and their relationships.

With my work in the Zamvil Lab, I turned my focus from developmental neurobiology to neuroimmunology research. The Zamvil lab historically focused on the EAE (experimental autoimmune encephalomyelitis) model of Multiple Sclerosis (MS), but I was brought onto the team to work on a project concerning what used to be once thought of as a MS-variant, called Neuromyelitis Optica (NMO). NMO patients present with optic neuritis and acute myelitis, and it was not until the discovery of a particularly unique antibody, called NMO-IgG, in the blood of these patients that they were recognized as having a condition unique from MS. In 2004, NMO-IgG was then discovered to be an antibody against a water channel called AQP4, and renamed Anti-AQP IgG. It was my work to try and characterize what parts of this water channel, normally expressed in the kidneys and astrocytes, were triggering such an auto-immune response.

In astrocytes, AQP4 is highly expressed in perivascular endfeet (blood-brain barrier function), perisynaptic astrocyte processes (clearance of neurotransmitters), and processes that are in contact with the nodes of Ravier and nonmyelinated axons (implicated in K+ clearance). Astrocytes’ main functions are to optimize the extra-cellular space for synaptic transmission; to do this job, they have highly efficient systems for the clearance of extracellular K+. When these systems are disrupted, the extracellular K+ concentrations lead to severely compromised CNS function. There is much information further linking this water channel, AQP4, to NMO: loss of AQP4 expression on astrocytes in lesions, NMO-IgG titers at disease exacerbation being proportional to the length of the spinal cord lesions, serum titers correlating to clinical disease activity, and the observation that in areas where there is normally high AQP4 expression that there is IgG deposition in such NMO lesions.

I began this work by performing peptide scanning of water channel AQP4 for discovery of T cell epitopes that may be relevant to Neuromyelitis Optica (NMO) pathogenesis.  Working initially alongside Dr. P Nelson, I used techniques involving in vivo immunizations as well as in vitro assays. We did 20-mer scans along the whole length of the AQP4 protein, and eventually did find several targets that elicited T cell responses. Knowing that T-cell epitope prediction software has been on the rise, I compared our manual scanning results to several databases’ predictions of relevant sites, and concluded that we could not rely solely upon epitope prediction yet, but that our manual scanning work could help such predictive algorithms improve. After publication of our paper, I did additional characterization — investigation of cytokine production, immunogenicity for replicating disease, generating clones for development of transgenic mice, and spearheading T cell polarization for adoptive transfer studies.

My comfort with graphic design, data analysis, independently learning/optimizing/teaching protocols, scientific writing, and my ability to use predictive modeling algorithms ultimately led to a swift publication when given the chance.

Continuing my previous work at the Grabel Lab in developmental neurology, I joined the Pleasure Lab at UCSF to further advance both my techniques and depth of research. Like the Hedgehog (Hh) signaling pathway, the Wnt signaling pathway has been identified as critical in proper development of the embryo, controlling such processes as body axis patterning, cell fate specification, cell proliferation, and cell migration. The discovery of Wnt signaling, originally done in the scope of cancer research, and its characterization led to the Nobel Prize in Physiology/Medicine, and as the Wnt proteins are highly conserved across species, I found this area of research quite moving. The laboratory used a broad array of embryologic and molecular genetic techniques to understand these processes in the normal developing state as well as in animals with developmental anomalies.

Development of the neocortex and hippocampus is a highly ordered process with a number of important steps. Cells must be directed to the proper fate in the ventricular zone, must migrate to the appropriate laminar location once born and must establish connectivity with their targets. Our research focused on several distinct aspects of the regulation of neuronal cell fate, migration and axon guidance using the developing hippocampus as the model system.

Additionally, my work with Dr. Grant Li, used these same techniques and principles targeted at understanding the formation of the hippocampus in working to define the mechanisms of formation of the corpus callosum (the tract of neurons that connects the two hemispheres of the brain). The corpus callosum, the largest fibre tract in the brain, helps to integrate both motor and sensory information of the two sides of the body, as well as working to affect executive function, social interaction, and language. Agenesis of the corpus callosum is a common brain malformation, and our experiments using both timed-pregnant mice with electroporated constructs, and gene knock-out mice were designed to study and unravel the formation of this structure from several angles.

For a variety of experiments, I cloned, amplified, and purified constructs that were then used for electroporation in vitro into cultures, as well as into particular neural targets in the embryos of timed pregnant mice. What impressed me the most about the work in this particular lab was that I was able to have the materials at hand to work from plasmid cloning all the way through animal studies and sectioning/histology. I also cloned, amplified, and purified other constructs for transfection into retroviral constructs, and then self-taught how to use these constructs to make a final concentrated retrovirus, with a fluorescent tag, to be able to study our targets of interest in another fashion. (Additional support I provided included lab management, maintenance of animal colonies for our experiments, genotyping, lab ordering, lab functional organization, training of new members/volunteers/students, protocol optimization and keeping adherence to standards of practice (SOP).)

In a slight shift from working in immunology and studying cell signaling involved in tumorigenesis, I began to focus on developmental neurobiology and stem cell research. Majoring in neuroscience & behavior, studying neural stem cells appealed not only for the potential treatment possibilities for neurodegenerative conditions or cases of nerve damage, but also for understanding the role of how signaling goes awry in certain cancers. Intersecting developmental biology with neuroscience, the Grabel Lab is where I learned to work with embryonic stem cell (ESC) cultures and neural stem cell cultures (N-ESC).

The primary lab focus was to differentiate embryonic stem cells into neural progenitors, looking at the Hedgehog signaling pathway and Hedgehog’s role as a mitogen or survival factor. Sonic Hedgehog (Shh) has been implicated to be a vital factor necessary for neural development, especially in adult hippocampal neural stem cells. On the oncological side, medulloblastomas and basal cell carcinomas have been associated with inappropriate activation of Hh signaling, further making the understanding of Hh signaling a relevant priority. Much has been discovered on the Hedgehog signaling pathway, yet the role of Hedgehog signaling in neurogenesis and maintenance of neuronal progenitors was still not clear.

Working under the guidance of Dr. Jeffrey Thorne, I began investigating the effects of Sonic Hedgehog agonists on the differentiation of cultures into progenitor populations using embryoid body intermediates, with a particular cell line that would help us visualize the both the timing and localization of cell differentiation. Following this work, I continued to differentiate ESCs into neural stem cells (NSCs) with Dr. Chunyu (Hunter) Cai; however, this time we used a monolayer culture for differentiation, with a Sox1-GFP ESC line to visualize NSC so that we could better understand the timing of cell fate.

The outer visceral endoderm layer of the embryoid body is a source of hedgehog. Upon receipt of Hh signal, the core responds by upregulating downstream genes such as Ptc1. Ptc1 Expressing cells should be found in the core of the embryoid body & can be visualized by using a cell line of Ptc +/- that expresses LacZ under the control of the Ptc1 promotor. Patched inhibits Shh signaling in the absence of ligand, therefore, mutations in the Patched gene activate the pathway constitutively. Growing Ptc+/- cells according to a monolayer protocol, I sought to observe the timecourse of differentiation, the effects of Hedgehog antagonists, and characterize the involvement of Hedgehog signaling through observing LacZ expression.

Working with Dr. H Cai, I studied agonists and antagonists of Shh, and used immunostaining, labeled cell lines, and RT-PCR of mRNA for characterization of possible interactions with signaling pathways. We also tested multiple alternative protocols, such as the previously mentioned embryoid body (EB) protocol, for differentiation of ES cells into neural progenitors. In the interests of following Hedgehog and the expression of stage-specific and cell fate markers through neuronal differentiation, I antagonized Sox1 GFP cells – a Green fluorescent protein (GFP) knock-in reporter ES cell line used to examine the process by which ES cells acquire neural identity. Sox1 is the earliest known specific marker of neuroectoderm in the mouse embryo, first expressed in the neural plate and subsequently maintained in neuroepithelial cells through the entire neuraxis, and then downregulated during neuronal and glial differentiation.

Lastly, further broadening the scope of the work we were undertaking at the Grabel Lab, our project’s aims fell within a larger-scale research grant that would use our differentiated neural stem cell populations in transplantation studies to potentially alleviate ischemia in animal models of stroke. The work I did in the Grabel Lab helped lead to three publications after my graduation. 

The Kavathas lab focused on studying the interaction of cellular membrane proteins involved in oncogene regulation and consequent cell regulatory behavior. It was under the supervision of Dr. Kavathas that I first learned to set up a PCR and to clone into a plasmid (for the hopeful identification of new candidate genes coding for cellular receptors). My training had a strong emphasis on developing basic skills in cell culture and microbiology techniques such as immunoprecipitation, cell culture maintenance, histochemistry, DNA purification (miniprep/maxiprep), sequence analysis, UV-Vis, in-situ hybridization, electrophoresis (SDS/PAGE and agarose gels), cloning/plasmids, and also gave me the opportunity to self-teach techniques such as Western blotting when there were no experienced researchers to teach them to me. I provided support for a number of projects, but my efforts there primarily revolved around the CD8 receptor, using western blot analysis to characterize glycosylation and phosphorylation of the CD8 and CD4 membrane proteins.

It was also at this time that I began to learn to provide support in the grant process, working through the writing and submittal process. Developing these skills at Yale made me a sought out candidate for science writing and grant support, such that I was asked to be a graduate grant-writing tutor in university (while still being an undergraduate). Furthermore, I was hired as a grant-coordinating contractor at a large pharmaceutical company, Affymetrix, to coordinate and bring all their grants and contracts up to date prior to a NIH (National Institutes of Health) audit – all before I even graduated college.