Programmed cell death (PCD) is a biological process that shapes human development. Yet, cancer cells are insusceptible to this process leading to the proliferation of tumors. Research on PCD can produce cancer therapies which increase tumor susceptibility to PCD for tumor eradication.

The exact mechanisms of PCD are currently unknown. My research aims to uncover the role of the gene outsiders in the scheme of PCD in Drosophila melanogaster (fruit fly) embryos. During embryogenesis, Drosophila germ cells travel across the embryo to the gonads for proper development. Mutants with the outsiders gene respond less to PCD resulting in the correct number of germ cells in the gonads, but an excess outlying the peripherals.

To decipher the mechanisms involved in PCD, outsiders will be excised from the genome using the CRISPR-Cas9 genetic engineering technique. This knock-out phenotype will provide insight on the network of PCD for human health applications.

Zebrafish (Danio rerio) serve as a very useful model organism because they have a fast generation time, clear embryos and a well mapped genome. In this research project, the students in the Developmental Biology lab course and the students in the Freshmen Research Initiative have used these characteristics to conduct a screening of the zebrafish genome in order to identify genes that are required for development. The CRISPR/Cas9 system (a protein that creates double strand breaks at specific sites in the genome that are then repaired by the cellular machinery) was recently specialized for the use in zebrafish. However, there are usually mistakes made when repairing the break. By using this system we can create mutations at specific sites in the genome and even delete entire sections. We can then observe if the mutation has created any notable phenotypes in the developing embryo. That information can give us insight into what the genetic requirements are for development or how those mechanisms can go wrong in diseases such as cancer.

As a developing country with a population surpassing 1 billion, India faces a myriad of challenges in securing the access and provision of quality healthcare services. To develop my understanding of the multi-dimensional health system of India, I traveled to rural Maharashtra for a two month program. I shadowed doctors in privately-owned clinics, government-run centers and non-profit organizations, visited a consultation and treatment center for those living with HIV/AIDS, and traveled to remote villages with a free mobile clinic. I developed a portfolio documenting my daily log of hours, weekly activities and personal reflections. My time in the general consultation clinics contributed greatly to my knowledge of national vaccination strategies, diagnostics, treatment, regional prevalence of infectious diseases, and challenges faced in securing effective patient interactions at each of these stages. The conditions I most commonly observed were tuberculosis, malnutrition, and respiratory, gastrointestinal and dermal infections. By stepping beyond textbooks and personally facing the conditions of vulnerable communities in rural India, I witnessed the crowded populations, hazardous housing, poor sanitation, and insufficient infrastructure that perpetuate the spread of infection and disease. My research cemented the reality and complexity of healthcare problems in developing countries, concluding that future solutions cannot be successful without addressing the intertwined epidemics of poverty and education.

Long non-coding RNAs (lncRNAs) are important players in epigenetic regulation of gene expression during development and disease (Niland et al, 2012). A number of mechanisms have been proposed for lncRNA action, however, few functional studies of lncRNAs have been described. We are using Transcription Activator-Like Effector Nucleases (TALENs), engineered site-specific nucleases, to create targeted mutations in a novel zebrafish lncRNA. We previously mapped a highly penetrant retinal tumor model to transgene disruption of the zebrafish lncRNAis18 gene. The objective of this project is to isolate a second zebrafish lncRNAis18 allele that contains a deletion of part of the lncRNAis18 gene. Two TALEN pairs were designed to simultaneously target double-strand breaks to exons 2 and 5 of lncRNAis18. Injection of 25-40pg of the TALENs targeting individual exons into zebrafish embryos resulted in efficient mutagenesis of the target sites. To isolate the lncRNAis18 deletion allele we co-injected embryos with the TALEN pairs targeting both exons 2 and exon 5. We predicted co-injection of TALEN pairs targeting exons 2 and 5 of lncRNAis18 would create a 147kb deletion after loss of the intervening sequence and repair by the non-homologous enjoining pathway. PCR products spanning the fusion of exons 2 to 5 were amplified from somatic tissue in 9 out of 14 co-injected embryos. We verified the deletion allele by sequencing PCR products from 3 embryos. We have identified one founder that transmits the deletion allele to the F1 generation. F1 embryos are being raised to establish a new lncRNAis18del line. The lncRNAis18 deletion allele will provide a new genetic tool to study the function of lncRNAis18 in zebrafish development and cancer.

Successful genome editing is associated with the ability to generate double strand breaks (DSB) efficiently at specific chromosomal locations. Recently, enzymes called TALENs (transcription activator like effector nucleases) have been tested for this purpose. TALENs consist of a DNA binding domain and a DNA cleaving or nuclease domain. They function as dimers; two TALEN proteins interact together to produce a DSB in the DNA. The efficiency of TALENs is influenced by the ability to access and bind the target site and of the two TALENs to dimerize. We have assembled a transient assay system in maize to test which TALENs are most effective at generating DSBs. We generated a modified fluorescent protein reporter gene that contains the DNA binding site for the TALENs. When this gene is expressed following DNA bombardment into maize embryos, it is non-functional and no fluorescence is observed. Genes encoding TALENs are then co-bombarded with the reporter gene plasmid. When DSBs occur, DNA repair machinery of the maize cells will repair the reporter gene generating a functional fluorescent protein. The number of fluorescent cells recorded is a measure of TALEN activity. Results from several TALENs and other DSB enzymes, such as RNA-guided cas9 will be discussed.

The enteric nervous system (ENS) consists of neurons and glia that control motility, secretions, and blood flow within the gastrointestinal tract. Using the zebrafish, Danio rerio, as a model we aim to understand ENS development and how this process might go array in disorders such as Hirschsprung’s, in which children are born lacking neurons in portions of their gastrointestinal tract. The number of markers for enteric glia is relatively small and many of the current immunohistochemical approaches are confounded by the uncertainty of cross-reactivity patterns between species. In preliminary experiments using established glia markers, we obtained unexpected results identifying glia in mutants lacking the ENS, suggesting a lack of glia marker specificity, or the presence of an uncharacterized subpopulation of glia in our mutants. To distinguish between these possibilities, we have cloned other markers to examine glia populations in normal larvae and larvae with defects in ENS development.

Transposable Elements (TEs) are segments of DNA that can move throughout the genome. TEs are present in most species and can cause insertions and deletions in the genetic sequence. TEs encode a transposase enzyme which excises the TE and inserts it elsewhere. This is considered standard transposition. Alternative Transposition occurs when transposase acts on the termini of two different TEs. This action can result in major chromosomal rearrangements or chromosomal breakage. Alternative Transposition has been observed previously with maize Ac/Ds elements; here, we are asking whether it also occurs in Petunia hybrida. Petunias have dTph1 TEs which are small, non-autonomous transposons. The S857 allele contains two copies of dTph1 located approximately 30 bp apart and facing in opposite orientations. PCR (Polymerase Chain Reaction) will be used to identify petunias with both elements, and PCR experiments with primers facing in the same direction will be used to test for Alternative Transposition. A product should be generated only if Alternative Transposition has occurred and the primers are re-oriented to face one another. The results may provide evidence that Alternative Transposition can occur in multiple species, suggesting that it may have had a significant impact on the evolution of plant genomes.

Colorectal cancer kills over 50,000 people per year in the United States. While individual drugs can be somewhat effective, the median survival remains only 25-28 months. New therapies are needed, and dual targeted inhibitors are a promising area. A total of 7 cell lines, 4 of which are presented, were treated in varying concentrations of TAK228, an mTORC1/2 inhibitor, and trametinib, a MEK1/2 inhibitor Proliferation, apoptosis, and viability assays as well as immunoblotting were performed to determine the mechanism and efficacy. Immunoblotting determined that the target of TAK228 is mTORC1/2 and that Survivin may be a mechanism for the anti-proliferative effects. The study indicates that TAK228 and trametinib are viable combination partners for the possible future treatment of PI3K mutated cancers, especially within the RAS-mutant area.

The pollen grains of Arabidopsis thaliana produce a fast-growing pollen tube. The cell wall of this pollen tube can model lignocellulosic biomass. This project utilized a fucose analog to label the distribution of fucose in pollen tube cell wall during biosynthesis. Pollen grains were germinated on medium containing this alkyne-modified fucose analog, called FucAl. This allowed pollen grains to incorporate FucAl, rather than fucose, into cell walls. Following tube growth, cell walls containing FucAl were exposed to Alexa 594 and Alexa 647 Azides in a copper-catalyzed “click” reaction. This reaction between the azide and alkyne yields fluorescent product at the FucAl molecule’s location. This allowed us to localize where fucose belongs in the cell wall. These results confirm the success of the click reaction and suggest that fucose may be taken up by the pollen grain during tube elongation. Further studies are needed to shed light on the critical steps of this process. Once the methodology is optimized, it will be expanded to include biomass degradation. This simple biomass model was suitable for effective mapping of polysaccharide distribution during growth, and this project utilized correlative optical and fluorescence microscopy to study fucose delivery and utilization during cell wall biosynthesis.

Amyotrophic lateral sclerosis (or Lou Gehrig's disease) is a neurodegenerative disease caused by the death of motor neurons in the spinal cord and the brain. Once these nerve cells die, the patient's muscle cells degenerate, resulting in paralysis and eventually death. Most cases of ALS are sporadic, meaning that a clear molecular and genetic understanding of the mechanisms by which the motor neurons die is lacking. Ubiquitin C-terminal hydrolase-L1 (UCHL1) is one gene that has been implicated in ALS, as well as several other neurodegenerative diseases. To gain a better understanding into the function of UCHL1 in non-diseased neurons, we are using TAL-effector nucleases (TALEN) to create UCHL1 mutant zebrafish. Specifically, I have generated these TALENs and begun to inject them to generate the mutant fish. Once created, these fish will provide insights into the normal function of UCHL1 and, hopefully, allow us to create a new zebrafish model of nerve cell degeneration.