SOURCE 2019

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Enrique Reyes (Graduate Student) - Is fecal matter an appropriate proxy for the intestinal tract in studies of the gut microbiome?
As many health phenomena seem to be affected directly and indirectly by the microbiome, gut microbiome research has increased in the last decade. Issues like allergies, cancer, obesity, and other health phenomena have been researched and found to be influenced by the microbiome. Most of gut microbiome research is done by collecting and sequencing the DNA of the microbiome of the fecal matter from model organisms or human subjects. Studies that use this method of sample collection and analysis assume that fecal matter microbiomes are identical to intestinal microbiomes. No published studies exist which compare stool microbial composition and intestinal microbial composition. Fecal matter bacterial composition and intestinal bacterial composition was assessed by extracting and sequencing DNA from stool and intestine (ileum cecum, and colon) of 4 C57BL/6 wild-type male mice. Microbial community comparison showed no significant difference between the species evenness and microbial diversity of all three sections of the gut and stool. Species composition does vary between sample sites. Although more data is required, the preliminary results support the assumption that stool microbial composition is a proxy for intestinal microbial composition, at least in terms of species evenness and diversity.

Silvia Guiterrez and Jennifer Magana - Effects of Prenatal Testosterone Exposure on Female Reproduction: Alterations in Ovarian Function
Polycystic ovarian syndrome (PCOS) is an endocrine disorder affecting 7-10% of women. PCOS causes reproductive and metabolic abnormalities such as multicystic ovaries, hyperandrogenism (excess testosterone), irregular menstrual cycles, obesity, and infertility. A PCOS mouse model can be created by injecting dihydrotestosterone (DHT) into mice to produce high levels of androgens that mimic symptoms seen in women with PCOS. Mice treated postnatally with DHT for 90 days have previously shown increased Claudin (Cldn) 3 and 11 gene expression in the ovary. In this study, mice were treated prenatally with DHT to examine the effects of excess testosterone on ovarian function and compare to a previously established postnatal model. Mice were also treated with flutamide, an androgen receptor inhibitor, to determine the mechanism through which testosterone affects ovarian function. We examined age of vaginal opening, anal genital distance, estrous cycle, and body weight as measurements of ovarian and metabolic phenotypes. At 8 weeks, mouse ovaries were used for quantitative Reverse Transcriptase PCR to examine the expression of Cldn 3 and 11 genes. Ovarian morphology was also observed. Prenatal DHT treatment did not affect Cldn 3 and 11 gene expression. Prenatal DHT treatment did not affect body weight consistently, however it did demonstrate reproductive abnormalities in vaginal opening, anal genital distance, and estrous cycle. The effects of DHT on ovarian function varied in the prenatal model compared to the postnatal model, which can provide a different model for determining the etiology of PCOS.

Naomi Bryant (Graduate Student) - Endogenous Gene Tagging of pfr2 and pfr5 in Trypanosoma cruzi using CRISPR/Cas9
Trypanosoma cruzi, the causative agent of Chagas disease, is a single-celled parasite that contains a unique structure called the paraflagellar rod (PFR). The PFR is a lattice-like structure of cytoskeletal filaments that extends along the length of the flagellum. The exact functions and protein composition of the PFR has yet to be determined, although several major and minor proteins have been identified. The PFR is not only a complex structure shown to be crucial for cell motility, but it is also part of a group of proteins that have shown to protect mice from a lethal dose of T. cruzi following immunization. PFR5 is a proposed component of the PFR, containing a region of homology (PFR domain) which has been found within several well-characterized PFR proteins. Currently, it is unknown if PFR5 is associated with the PFR, with one of the focuses of this study being on its localization. PFR5 localization is being determined using a new gene tagging technique that utilizes CRISPR/Cas9 technology, with PFR2 localization serving as a proof of principle since its localization is well known. This technique allows for the precise insertion of a small hemagglutinin tag at the end of a gene of interest, with subsequent protein product also containing the tag. Localization of the tagged proteins can then be visualized using fluorescent antibodies. Successful utilization of this technique, as well as localization of the PFR5 protein, will help to further research and understanding of this unique structure.

Katherine Boyd (Graduate Student) and Trevor May - Locating a new gene involved in behavioral adaptation to serotonin in the roundworm C. elegans
Using the roundworm Caenorhabditis elegans, a genetic screen was performed to identify genes responsible for behavioral adaptation to chronic exposure to serotonin. When exposed to serotonin, C. elegans will slow their movement, eat more, and lay more eggs, but when they are exposed chronically, they adapt to those behaviors and regain speed, eat less, and lay fewer eggs in a process known as behavioral adaptation. One of the mutants, def-1, was found to not adapt to serotonin and has an additional behavioral phenotype we are studying. This social feeding behavior that has been previously uncharacterized is called “depressed foraging”. The specific purpose of this study is to determine the genetic location of a gene altered in def-1 worms. We are in process of using restriction fragment length polymorphism (RFLP) mapping to localize the mutation. We have localized it to Chromosome I, and are continuing to interval map to a more specific region on the chromosome. Once narrowed to several possible genes, we will sequence these genes to find the one with the mutation. Alternatively, we will use RNA interference techniques to inactivate these genes in wild-type worms in order to see if they mimic the phenotype of def-1 mutants. Another mutant from that original genetic screen, def-2 was found to have identical phenotypes and we are using complementation tests to determine if they have mutations in the same genes. Identifying genes regulating how worms develop adaptation will provide insight to this behavior is regulated at the cellular level.

Darby Wedekind and Michael Riley - Development of Procedures for Modifying Variables in the Brewing Process Using Sensory Analysis
In the beer industry scientific methods and best practices for recipe and process development are rarely shared due to the investment individual companies have in their product. As craft brewing has gained more scientific attention, the industry has the opportunity to experiment more thoroughly on the direct effects of recipe modification. Our experiments serve the purpose of broadening the public understanding of these modifications, and examining the effect on the final product using sensory analysis. Impacts on beer flavor and aroma may be able to be traced back to these specific modifications, therefore through our project we aim to find their direct causes. After preliminary research concerning yeast strain modification, our experiments begin with investigating the effects of variation in fermentation temperatures. For these purposes a single beer is brewed and separated into fermentation vessels to be pitched with yeast of the same strain and amount, while held at different temperatures during fermentation. Upon completion the three variations are evaluated using traditional sensory analysis methods by a panel of students for significant differences in the finished product. Our results will n