Bea Carbone

= Clubs =


 * Signer for the Blacksmiths Guild from spring 2011 until fall 2012
 * Signer for Comics Collective from spring 2012 until spring 2013
 * Signer for FiCom from fall 2011 until spring 2013

= Research =

Lab Experience:


 * Biology:
 * For the last two summers, 2013 and 2012, I have been employed in the lab of Dr. Wendell Yarbrough MD, MMHC, FACS. Dr. Yarbrough is an oral surgeon, though he also works with ENT oncology. His lab’s work with oral cancers and HPV, along with a number of other things, has had a major impact. Initially, I was at Vanderbilt University before his lab moved at the end of the summer of 2012 to its current location at Yale University. While at Vanderbilt in 2012, I had the opportunity to work on several ongoing projects. My main project was working with Dr. Natalia Isaeva, PhD, on the transfection of three cell lines with an anti-hygromycin B plasmid vector that expressed HPV associated oncoproteins E6 or E7. The goal was to create a cell line that stably expressed one of these two oncoproteins This was done to try and isolate the individual expressions of E6 or E7 and to uncover how best to work with the oncoproteins to manipulate them to the benefit of medical science. HPV related cancers are not only growing more common, but they are easier to treat than traditional squamous cell cancers. Perhaps working with these types of cancers can give hints to the nature of non-HPV related squamous cell carcinomas. I had to return to school before the success of the cell line could be verified with sequencing, but there were many colonies that survived the hygromycin antagonist
 * I also assisted the lab manager, Brandee Brown, by running pre-isolated DNA samples through PCR to test for HPV status by expression of E6 and E7. I would run anywhere from 15 to 40 samples at a time. This was in the hopes of identifying positive cell lines to compare to the normal tumor cells and experiment with. Another project I worked on was the amplification and sequencing of a region of DNA in cancerous tissue-derived cell lines possibly containing a mutation PIK3CA. In the lab, I was also asked to extract DNA from blood and cell cultures using kits and quantify the results for further dilution and use. In addition to this, I took the initiative to assist the lab in other means and organized and compiled the data from uncatalogued mouse cage cards and paraffin blocks. I also catalogued the freezer space and liquid nitrogen tank to help the lab prepare for its move to Yale University. Working in a university-based laboratory has helped me develop my ability to focus on multiple projects through time management skills. These skills have been useful in the rest of my research career to learn how to manage complex projects more efficiently.
 * During the summer of 2013, I maintained my position as a research intern in the lab of Dr. Wendell Yarborough, MD at the lab’s new location at Yale University. My duties and research share some similarities with those from last summer. I verified the identity of several cell lines using microsatellite testing through PCR and sequencing, along with working on an ENT resident’s project identifying E6 and E7 oncoproteins in various paraffin embedded tissue samples from the Republic of Mozambique. I also worked to identify the interaction between LZAP/C53 and the ubiquitin fold modifier protein Ufm1. As this is very preliminary work and has not been done in the lab before, my work will assist in building a basis of knowledge for further work. We performed some exploratory Ufmylation assays and upregulated Ufm1 production in a squamous cell carcinoma cell line, before exposing the results of each to a western blot.. The highlight of my summer, however, is shadowing Dr. Yarbrough through his clinical care work, while interacting with patients and assisting with tasks as needed.
 * Last year, I worked with Div III student Nicole Dhruv, gathering DNA and cortisol samples for her thesis. I also ran PCR and nested PCR to identify the genotype of the 5-HTTLPR region of the serotonin transporter, discussed below.
 * Along with my independent research, in the spring of 2013 I was a lab-based TA for the Methods in Molecular Biology course with the same professors as the advanced course. It is a more introductory course that accepts students with all levels of skill. My main purpose was to instruct the students on the basic techniques of molecular biology. I was one of five teaching assistants and worked with a small group of five people. I instructed them on how to perform a BCA assay and analyze the results from the spectrophotometer; made bacterial gel and plates before transforming competent E-Coli with an empty GFP vector and growing up the colonies overnight; extracted DNA from the E-Coli colonies; made our own DNA ladder with the other groups in the class by taking the extracted DNA and cutting it using PCR; ran restriction digests using various enzymes; showed students how to split and plate cells, maintain cell culture, transfect cells using Fugene lipofection, and extract mRNA using Trizol. Once they had been introduced to these basic methods the students chose an independent project to work on. I worked with a group on my research with P19’s, wherein they continued my research and I assisted them in developing their own sub projects within my research. During the course of the class, I was in lab frequently during the week. I was often the main reference for students in the lab who had questions about basic techniques, even if those students were not in my group.


 * Behavioral Science
 * During the fall of 2010, I worked for Dr. Sarah Partan, collecting and analyzing literature for incorporation into her research done with grey squirrels and Anolis Sagrei


 * Chemistry:
 * In organic chemistry, I assisted Professor Rayane Moriera during the year of 2012 at Hampshire College with the creation of a Diels-Alder reaction catalyst that allows the typically solvent-heavy reaction to proceed at manageable levels in water. Also with Professor Moriera, I have worked with nuclear magnetic resonance (NMR) in the identification of simple lipids.
 * In inorganic chemistry, I worked with Professor Dulasiri Amarasiriwardena  in the spring of 2011 on analyzing acid mine drainage along with general lab work involving spectroscopy and IR.

P19 Differentiation (Project began in October 2012-present)
This last year I have been working with Visiting Assistant Professor John Castorino and a few classmates on the development of a protocol for the differentiation of these p19 cells. Typically, the method for differentiation used, discovered in 1982 (Jones-Villeneuve et al. 1982), is a complex and involved aggregation method. The cells are removed from the plate with trypsin and plated on bacterial dishes so they will not settle on the plate and will aggregate. These aggregates are known as embryoid bodies. The embryoid bodies are then differentiated using varying concentrations of all-trans retinoid acid, which is a metabolite of vitamin A, and is well known for its differentiating properties in mammalian cells (Duester 2008).

The cell differentiation protocol itself will be completed by the end of the semester, as well as initial work towards deducing the type of cell that is created. The differentiated cells have already been identified as neurons by immunofluorescence staining of alpha tubulin and then comparing the visible structures to those seen in Jones-Villeneuve et al. (1982). The cells grown using my method were entirely comparable to the initial photos. This tubulin stain also showed a strong similarity between some of the non-neuron cells fibroblasts, and as there is literary backing in the development of fibroblasts in the P19 strain using RA to differentiate, I believe they are fibroblasts. The next step I plan to take is to use anti-glial fibrillary acidic protein (GFAP) to identify glial cells in the system. This will be done with several iterations of differentiation to determine which variant of the differentiation protocol produces what amount of glial cells.

Using the data from the glia stain, I can identify whether or not these cells are from the peripheral or central nervous system via identification of the presence of Schwann cells on the matured axons. If they are found to be peripheral neurons, there will be fewer types of neuron that they could be. If this distinction is not seen, then in accordance with the research previously done to identify p19 neurons (Jones-Villeneuve et al. 1982; McBurney et al. 1988), I will be testing for levels of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) and the receptors within using various methods to be determined by resources. If neither of those neurotransmitters are found to be expressed, then the course of action will be decided upon.

Due to several sightings of cells encapsuling axons, like Schwann cells are known to do, I believe that these cells are peripheral nervous system cells. Previously, when using the aggregate method of differentiation, strong evidence has been found supporting the identification of these cells as central nervous system neurons using tetanus toxin (Jones-Villeneuve et al. 1983), presence and expression of nestin mRNA (Jin et al. 2009), and the presence of the Sox family of proteins (Hamada-Kanazawa et al. 2004). These markers indicate that the cells differentiated using very different conditions and concentrations of various media additives like fetal bovine serum (FBS) and calf serum, and RA are most likely central nervous system cells. However, as seen in Hu et al. (2013), simple variation on the concentration of FBS resulted in drastic differences in expression of retinal progenitor cell multipotentiality. In 2002, Wichterle et al. differentiated a mouse embryonic stem cell into a motor neuron that had previously been differentiated into a central nervous system cell, leading me to believe that this hypothesis is not entirely unfounded.

References:


 * 1) Duester, G. (2008). Retinoic acid synthesis and signaling during early organogenesis. Cell, 134(6), 921–31. doi:10.1016/j.cell.2008.09.002
 * Hu, Y., Ji, J., Xia, J., Zhao, P., Fan, X., Wang, Z., Zhou, X., et al. (2013). An in vitro comparison study: The effects of fetal bovine serum concentration on retinal progenitor cell multipotentiality. Neuroscience letters, 534, 90–5. doi:10.1016/j.neulet.2012.11.006
 * 1) Jin, Z., Liu, L., Bian, W., Chen, Y., Xu, G., Cheng, L., & Jing, N. (2009). Different transcription factors regulate nestin gene expression during P19 cell neural differentiation and central nervous system development. The Journal of biological chemistry, 284(12), 8160–73. doi:10.1074/jbc.M805632200
 * 2) Jones-Villeneuve, E. M., Rudnicki, M. a, Harris, J. F., & McBurney, M. W. (1982). Retinoic acid-induced neural differentiation of embryonal carcinoma cells. Molecular and cellular biology, 3(12), 2271–9
 * 3) Jones-Villeneuve, E. M., Rudnicki, M. a, Harris, J. F., & McBurney, M. W. (1983). Retinoic acid-induced neural differentiation of embryonal carcinoma cells. Molecular and cellular biology, 3(12), 2271–9. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/18987612
 * 4) Kodama, T., Guerrero, S., Shin, M., Moghadam, S., Faulstich, M., & Du Lac, S. (2012). Neuronal classification and marker gene identification via single-cell expression profiling of brainstem vestibular neurons subserving cerebellar learning. The Journal of neuroscience : the official journal of the Society for Neuroscience, 32(23), 7819–31. doi:10.1523/JNEUROSCI.0543-12.2012
 * 5) McBurney, M. W., Reuhl, K. R., Ally, A. I., Soma, N., Bell, J. C., & Craig, J. (1988). Differentiation and Maturation Neurons in Cell Culture of Embryonal Carcinoma-Derived Neurons in cell culture. Journal of Neuroscience, 8(March).
 * 6) Wichterle, H., Lieberam, I., Jeffery, A., & Jessell, T. M. (2002). ES Cell-Derived Motor Neurons Directed Differentiation of Embryonic Stem Cells into Motor Neurons ES Cell-Derived Motor Neurons. doi:10.1016/S0092867402008322

Serotonin transporter
Understanding is the root of all scientific processes. The desire to understand how, why, and where things work along with what comes from that working. This desire has been around since the beginning on our history on this planet, from the desire to understand the wheel to the desire to understand the nuances of human behavior.

One of the biggest reasons for why I do what I want to do is that mental illness, especially that of major depression and bipolar spectrum disorders, is very poorly understood overall. Though it seems to have been around since humans began looking a bit like humans, we reached the moon before we found an effective medication for some mental illness and even longer before we tweaked it to make it non-lethal. Since then, technology has been reaching the point at which it is able to more clearly do the research required to understand and improve the lives of those millions with mental disorders.

My research is closely tied to that of Nicole Druhv’s. We are both studying the interactions of serotonin (5-hydroxytryptamine or 5-HT) and its interaction with the pre-synaptic serotonin transporter (5-HTT). The serotonin transporter is important in research regarding depression, bipolar spectrum disorders, and a number of other mental illnesses as it is the site of action for many commonly used antidepressants. One of the most common types of these is the selective serotonin re-uptake inhibitor (SSRI), though there are many types, ranging from benzodiazepines, to monoamine oxidase inhibitors (MAOI’s), to tricyclic antidepressants (TCA).

This specific transporter is mainly affected by SSRI’s (selective serotonin reuptake inhibitors) that have a similar shape to serotonin in that they share the five and six membered rings with a carbon-based tail and potentially have a hydroxy group. These inhibitors will prevent serotonin that isn’t binding well enough to the receptors across the synapse in the post-synaptic cell from being transported through the pre-synaptic membrane back into the cell that released it in the first place to be digested by monoamine oxidase.

Another reason for the importance of the SERT (5-HTT) is a specific polymorphism found in humans. This polymorphism is found in the promoter region of the SLC6A4 gene that codes for the transporter (5-HTT). In this case, a polymorphism indicates the presence of more than one genotype within the same species. There are two possible alleles, a short and long. The long (l) allele is 16 repeats of a 23 base pair (bp) long segment of cytosine and guanine rich DNA, while the short (s) allele is only 14 repeats. The short allele has been found to be less efficient when it comes to coding for the messenger RNA used in the creation of the SERT protein itself.

This polymorphism region, called the serotonin transporter linked polymorphic region (5-HTTLPR) has been linked to many of the major mental illnesses like major depression, bipolar spectrum disorders, some eating disorders, increased aggression, etc… Specifically, the short homozygous genotype has been found to be fairly highly correlated with the aforementioned illnesses and has been found to be present in a large number of patients that commit violent suicide.

The overall goal of this research is to grow neurons in vitro and transfect them with varying wild type cDNA in an attempt to observe the varying expression of SERT and serotonin re-uptake.


 * 1) Canli, T., & Lesch, K.-P. (2007). Long story short: the serotonin transporter in emotion regulation and social cognition. Nature neuroscience, 10(9), 1103–9. doi:10.1038/nn1964