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.

Stem cells are a promising option for human medical therapy. While pluripotent embryonic stem cells have an almost unlimited differentiation potential to become any type of cell, multipotent adult stem cells have a much more limited ability to differentiate. In order to better understand how adult stem cells choose their fate, two main factors were investigated: intrinsic versus extrinsic influences. This was accomplished by implanting rat adult hippocampal progenitor/stem cells (AHPCs) - which normally differentiate into neurons, astrocytes, and oligodendrocytes - into zebrafish embryos during the blastula stage, an environment with almost unlimited potential for stem cells. These embryos were then allowed to grow to three or five days post-fertilization (dpf), at which point they possess a developed nervous system and are free-swimming. The fish were then imaged using fluorescence microscopy to monitor the fate and localization of the AHPCs in relation to the embryo. The results showed a majority of the AHPCs migrating to the outer eye region (38.5%), central nervous system (26.7%), and superficial skin layer (20.7%). Immunolabeling procedures to identify differentiated cell-types revealed the highest percentage of transplanted AHPCs were TuJ1-labeled (73.4%), which distinguishes immature neurons. No significant difference in AHPC survival between 3 dpf and 5 dpf were found. This preliminary analysis reveals that these brain stem cells are likely influenced by intrinsic cell machinery - regulation of gene expression - than their environment when choosing their fate. Through this knowledge, adult stem cells and their cell regulation behaviors can be further investigated to potentially find similar drug options that may mimic the regulation of their expression.

My research project is for Dr. Michael Kimber regarding schistosomes and the effect of Praziquantel (a drug) on the immune system. This is part of an overall larger project working with his ongoing research of schistosomes. Schistosomiasis affects between 230-440 million people worldwide. Praziquantel is currently the only main drug to treat this disease and it’s largely unknown how it works. Not knowing why it helps may be problematic in the future if the disease develops a resistance to Praziquantel. My project was an independent effort broken into three parts. Initially I was responsible for tissue cultures of frozen J774 macrophages. My responsibility was to pass several generations of these cells in the sterile lab for later testing. Later in the project, I switched to using Bone Marrow Derived Macrophages (BMDM) for the testing of Praziquantel. The second portion of my project was testing for the 5HT2B receptor in these cells. This receptor must be present for my data collection project to be carried over as useful information to humans being treated with Praziquantel. In the final phase, after proving this receptor is present in the BMDM cells, I treated these cells with Praziquantel and tested for any changes in the immune response for these cells. My results showed that overall there does not appear to be a significant change in the immune response of BMDM cells treated with either S-PZQ or R-PZQ, but another replicate of my project would need to be performed to determine any significance of data. This information could possibly help drug developers in their efforts to design an additional drug to treat Schistosomiasis.

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.

Recent studies estimate that there are at least 30 different types of retinal ganglion cell in the mouse eye. These cells are responsible for the connection between the eye and the brain and, therefore, have important functions in image formation. In addition, these are the cells that die in glaucoma, the second leading cause of blindness. Even though the 30 types have been characterized by their physiology, the genetics of each type is unknown. This includes those genes that distinguish one type from another. The goal of our project is to begin to characterize the differences between these cells on a genetic level. In the lab, there is a mouse that has been engineered to express a red fluorescent protein in 8 different types of ganglion cells. Previously, students isolated red cells from the mouse retinas and identified sets of mRNAs that were expressed in each cell. Through these mRNAs, predictions have been made as to which genes define which ganglion cells. However, since the number of cells analyzed by this method was small, larger scale validations need to be performed. Our objective was to take genes that were identified in this initial screen and analyze their expression in retinal ganglion cells in much more detail.

Endothelial tubulogenesis is the formation of functional blood vessels. The purpose of this research is to understand how actin drives this process. To observe the function of actin, certain transgenic gene lines will be characterized. These transgenic gene lines are LifeAct, Moesin, Moesin-Actin-Binding Domain, and Alpha-Catenin. These genes will allow us to see how actin contributes to vascular formation. This formation will be visualized using fluorescent proteins RFP and GFP. These proteins will act as markers on each transgenic gene line to monitor the process of endothelial tubulogenesis in zebrafish using confocal microscopy. The zebrafish will express fluorescents in these gene lines using the GAL4-UAS system. This system is expressed in the vascular system, and binds to the UAS sequence to activate fluorescents for the gene of interest. Based on our research, actin acts as structural units to form vasculature. Actin is a functional protein that forms microfilaments that can be used in this process. This is based on where actin was expressed in zebrafish during endothelial tubulogenesis. Actin was expressed from major vessels, and expressed more in areas with continual vasculature formation. This shows that actin is an important factor in endothelial tubulogenesis.

During development of the maize ear, mutations may occur during cell division. These mutations can form twin sectors, or a sector of the maize ear that has a different coloration or pattern in the pericarp, seed coat, of the maize ear.

We studied the effects of Activator (Ac) transposon (a chromosomal segment that can undergo transposition) in the development of twin sectors on maize ears containing the allele P1-ovov454. This allele conditions orange-variegated pericarp and cob due to the presence of the Ac transposon in the p1 gene which controls kernel pericarp color. We screened for ears with multi-kernel dark red/orange sectors twinned with sectors of colorless pericarp. From this visual screen, we identified 6 different twin sectors on two maize ears. A series of screening PCR tests were developed to classify the type of structural changes present in each case. Then the junctions of the Ac transposon and flaking DNA were amplified by PCR and sequenced. Based on the results, we determined the structure of each new allele and proposed a mechanism of origin for most cases. Some were formed by simple Ac transposition, while others were generated by more complex recombination events.

These findings showed that twin sectors develop in parallel with adjacent sectors, in addition to demonstrating how gene structure and gene expression can be altered by the Ac transposable element during ear development.