2017 SRF Summer Scholar Profile: Alefia Kothambawala

Alefia Kothambawala

SRF Summer Scholar, Class of 2017

Sanford Consortium for Regenerative Medicine

My name is Alefia Kothambawala, and I am a rising junior at the University of California, Davis, studying Biomedical Engineering. When I came across the SENS Research Foundation, I was immediately drawn to the projects being researched. I noticed the unique way SENS scientists think about problems – preventing disease before they manifest – and wanted to contribute to this mentality.

Growing up, I hardly thought of aging as a disease as opposed to a natural result of life. However, as I focused on muscle atrophy during a summer internship in Dr. Michael Conboy’s lab, I soon saw the larger scope of the problem. I researched the role of atypical signaling from individual fibroblast growth factors (FGFs), namely FGF-2 and FGF-19, on the activation of dormant skeletal muscle cells isolated from mice in hopes of enhancing their proliferative ability. First, I used an assay to determine which cells were multiplying by observing whether they take up bromodeoxyuridine (BrdU), a thymine analog, in newly synthesized DNA. To accomplish this, I prepared a cell plate consisting of adult mice satellite cells that have not yet entered the cell cycle. Two wells of the cell plate were dedicated to each of the following: a negative control, a positive control, cells with FGF-6, and cells with FGF-19. The positive control consisted of satellite cells treated with a known activating medium, and the negative control consisted of satellite cells in pure growth media without any growth factors. I also stained the cells for the myogenic (muscle cell) marker desmin to confirm whether the cells were myogenic in origin. The positive control had the greatest number of satellite cells that were actively proliferating (61%), while the negative control had the least (32%). The FGF-2 and FGF-19 conditions respectively had 53% and 45% of proliferating cells. Based on this statistically significant evidence, I concluded that that FGF-2 and FGF-19 are potential activators of the muscle regeneration machinery.

After this experience, I sought to better understand tissue growth, wanting to branch out beyond atrophy. At UC Davis, I supplemented my courses with research in Dr. Kent Leach’s tissue engineering lab, primarily working to enhance osteogenesis, or the formation of bone. First, I worked to construct composite scaffolds to induce the formation and regeneration of bone tissues when seeded with mesenchymal stem cells (MSCs). MSCs are bone marrow-derived stem cells that can differentiate into mesenchymal tissues, namely bone, cartilage, ligament, and tendon. In addition, I sought to determine the minimum number of days scaffolds needed to be placed in bioreactors to generate bone in order to use this information for in vivo implantation. The scaffolds were sectioned after 7, 14, and 21 days of culture, and bone formation at each timepoint was estimated by assaying the levels of collagen and calcium using immunohistochemical stains. The greatest increase of both these materials occurred between 7 and 14 days of bioreactor culture, with minimal increase occurring between 14 and 21 days. Thus, we concluded that 14 days of culture was sufficient for bone tissue growth in scaffolds in vitro. To determine the viability of these scaffolds in vivo, seeded scaffolds were implanted subcutaneously in mice at four locations and extracted 14 days later. We then quantified the presence of proteins from the osteoconductive genes A2P, collagen-1, osteopontin, and osteocalcin in scaffolds harvested 14 days after implantation. We detected significant increases in osteoconductive proteins, indicating MSCs can be successfully used to culture bone tissue. However, further testing of pore size, mechanical properties, and vascularization is necessary to confirm the viability of these scaffolds.

Investigating the Role of Clozapine on CRMP-2 Pathways in Alzheimer’s Disease

Both schizophrenia and Alzheimer’s Disease (AD) are neurodegenerative illnesses prevalent in society. Schizophrenia is a psychotic illness, mainly characterized by hallucinations, illogical thinking, delusions, and thought disorder; however, psychosis is not unique to this disorder. Though AD is a dementia characterized by deficits in memory, spatial skills, and language, over half of AD patients also display psychotic symptoms. The shared psychiatric symptoms between schizophrenia and AD suggest common molecular pathophysiology. Furthermore, previous research has shown that the two diseases bear similarities in neural pathology and biochemical dysfunction. Thus, it would be of interest to study the novel use of antipsychotics, particularly clozapine, to investigate AD psychosis. Clozapine has been documented as particularly effective compared to other anti-psychotic agents to mitigate cognitive impairment, improve attention, and refine verbal fluency.

As a SRF Summer Scholar, I will be working in Dr. Evan Snyder’s lab to explore the relationship between AD, clozapine, and CRMP2. Based on prior research, clozapine causes significant changes in the expression of cytoskeletal, synaptic, and regulatory proteins, with alterations in the collapsin response mediator protein CRMP2 being the most spectacular [1]. CRMP2 is a protein involved in cell differentiation, nervous system development, synaptic plasticity, calcium homeostasis, neurotransmitter release, and the regulation of axon extension and signal transduction. Due to its significance in these biological pathways, the downstream expression of CRMP2 may elucidate the pathology of neural disease. Previously, using a 2D-DIGE (Fig1), the lab isolated a group of proteins up-or-down-regulated by CRMP2 [2]. Using bioinformatic software, we then compared these findings to all proteins associated with schizophrenia. Thus, we have information on which protein associations are shared by CRMP2 and lysates isolated from schizophrenia patients.

Figure 1. Summary of 2D DIGE analysis.

Two-dimensional difference gel electrophoresis (2D DIGE) is a modified form of 2D electrophoresis (2DE) that allows one to compare two or three protein samples simultaneously on the same gel. The proteins in each sample are covalently tagged with different color fluorescent dyes that are designed to have no effect on the relative migration of proteins during electrophoresis. Proteins that are common to the samples appear as ‘spots’ with a fixed ratio of fluorescent signals, whereas proteins that differ between the samples have different fluorescence ratios [3].

With this information, my goal is to find a pathway connection between clozapine, AD, and CRMP2. I aim to do so in two-stages: map protein expression downstream of CRMP2 and determine the effect of clozapine on this pathway. First, we will obtain protein lysates from three cell lines: a knock-in (KI) where CRMP2 is constitutively active, a knock-out (KO) where no CRMP2 expression is present, and a wild-type (WT) where CRMP2 function is unchanged. Then, to immunostain (an antibody-based method to detect a specific protein in a sample) for proteins impacted by CRMP2, we will use a series of Western Blots in hopes of elucidating the downstream proteins affected by CRMP2. Second, we believe that clozapine impacts CRMP2 expression in a manner that mitigates psychotic symptoms manifested in AD. In order to determine the impact of clozapine on the CRMP2 pathway, we will use clozapine-treated lysates and a similar procedure to stain for downstream CRMP2 activity. From previous literature, particular proteins of interest that are downstream of CRMP2 and are impacted by clozapine include Cav2.2, WAV1, GFAP, and calmodulin. We will blot for each protein independently, hoping to find a pathway linking each to each other as well as to CRMP2.

Future Plans:

Science and engineering are constantly amalgamating: science is needed to determine the root cause of a problem, and engineering is then required to find the solution. That is why I enjoy being at the intersection of both, using biomedical engineering to reconcile two realms. Post-graduation, I seek to obtain my PhD and continue researching in the field of regenerative medicine using the power of induced pluripotent stem cells (iPSCs). Some of the most serious medical conditions are due to abnormal cell expression, and a more complete understanding of the genetic and molecular controls of these processes may suggest new strategies for therapy. I know I won’t be able to make an impact overnight; I must first cultivate myself not only as a student but also as a scientist. I would like to thank the SENS Research Foundation for giving me an opportunity to do so this summer.


[1] Oncoproteomic Approaches to Cancer Marker Discovery: The Case of Colorectal Cancer – Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/280238750_ fig1 _Fig-2-Basic-workflow-of-gel-based-proteomic-approaches-The-2D-PAGE-separates-proteins [accessed 3 Jul, 2017].

[2] Kedracka-Krok, S., Swiderska, B., Jankowska, U., Skupien-Rabian, B., Solich, J., Buczak, K. and Dziedzicka-Wasylewska, M. (2015). Clozapine influences cytoskeleton structure and calcium homeostasis in rat cerebral cortex and has a different proteomic profile than risperidone. Journal of Neurochemistry, 132(6), pp.657-676.

[3] Tobe, B., Crain, A., Winquist, A., Calabrese, B., Makihara, H., Zhao, W., Lalonde, J., Nakamura, H., Konopaske, G., Sidor, M., Pernia, C., Yamashita, N., Wada, M., Inoue, Y., Nakamura, F., Sheridan, S., Logan, R., Brandel, M., Wu, D., Hunsberger, J., Dorsett, L., Duerr, C., Basa, R., McCarthy, M., Udeshi, N., Mertins, P., Carr, S., Rouleau, G., Mastrangelo, L., Li, J., Gutierrez, G., Brill, L., Venizelos, N., Chen, G., Nye, J., Manji, H., Price, J., McClung, C., Akiskal, H., Alda, M., Chuang, D., Coyle, J., Liu, Y., Teng, Y., Ohshima, T., Mikoshiba, K., Sidman, R., Halpain, S., Haggarty, S., Goshima, Y. and Snyder, E. (2017). Probing the lithium-response pathway in hiPSCs implicates the phosphoregulatory set-point for a cytoskeletal modulator in bipolar pathogenesis. Proceedings of the National Academy of Sciences, 114(22), pp.E4462-E4471.

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