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- Creators: School of Life Sciences
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Description
Many factors are at play within the genome of an organism, contributing to much of the diversity and variation across the tree of life. While the genome is generally encoded by four nucleotides, A, C, T, and G, this code can be expanded. One particular mechanism that we examine in this thesis is modification of bases—more specifically, methylation of Adenine (m6A) within the GATC motif of Escherichia coli. These methylated adenines are especially important in a process called methyl-directed mismatch repair (MMR), a pathway responsible for repairing errors in the DNA sequence produced by replication. In this pathway, methylated adenines identify the parent strand and direct the repair proteins to correct the erroneous base in the daughter strand. While the primary role of methylated adenines at GATC sites is to direct the MMR pathway, this methylation has also been found to affect other processes, such as gene expression, the activity of transposable elements, and the timing of DNA replication. However, in the absence of MMR, the ability of these other processes to maintain adenine methylation and its targets is unknown.
To determine if the disruption of the MMR pathway results in the reduced conservation of methylated adenines as well as an increased tolerance for mutations that result in the loss or gain of new GATC sites, we surveyed individual clones isolated from experimentally evolving wild-type and MMR-deficient (mutL- ;conferring an 150x increase in mutation rate) populations of E. coli with whole-genome sequencing. Initial analysis revealed a lack of mutations affecting methylation sites (GATC tetranucleotides) in wild-type clones. However, the inherent low mutation rates conferred by the wild-type background render this result inconclusive, due to a lack of statistical power, and reveal a need for a more direct measure of changes in methylation status. Thus as a first step to comparative methylomics, we benchmarked four different methylation-calling pipelines on three biological replicates of the wildtype progenitor strain for our evolved populations.
While it is understood that these methylated sites play a role in the MMR pathway, it is not fully understood the full extent of their effect on the genome. Thus the goal of this thesis was to better understand the forces which maintain the genome, specifically concerning m6A within the GATC motif.
To determine if the disruption of the MMR pathway results in the reduced conservation of methylated adenines as well as an increased tolerance for mutations that result in the loss or gain of new GATC sites, we surveyed individual clones isolated from experimentally evolving wild-type and MMR-deficient (mutL- ;conferring an 150x increase in mutation rate) populations of E. coli with whole-genome sequencing. Initial analysis revealed a lack of mutations affecting methylation sites (GATC tetranucleotides) in wild-type clones. However, the inherent low mutation rates conferred by the wild-type background render this result inconclusive, due to a lack of statistical power, and reveal a need for a more direct measure of changes in methylation status. Thus as a first step to comparative methylomics, we benchmarked four different methylation-calling pipelines on three biological replicates of the wildtype progenitor strain for our evolved populations.
While it is understood that these methylated sites play a role in the MMR pathway, it is not fully understood the full extent of their effect on the genome. Thus the goal of this thesis was to better understand the forces which maintain the genome, specifically concerning m6A within the GATC motif.
ContributorsBoyer, Gwyneth (Author) / Lynch, Michael (Thesis director) / Behringer, Megan (Committee member) / Geiler-Samerotte, Kerry (Committee member) / School of Life Sciences (Contributor) / Department of Psychology (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Many bacteria actively import environmental DNA and incorporate it into their genomes. This behavior, referred to as transformation, has been described in many species from diverse taxonomic backgrounds. Transformation is expected to carry some selective advantages similar to those postulated for meiotic sex in eukaryotes. However, the accumulation of loss-of-function alleles at transformation loci and an increased mutational load from recombining with DNA from dead cells create additional costs to transformation. These costs have been shown to outweigh many of the benefits of recombination under a variety of likely parameters. We investigate an additional proposed benefit of sexual recombination, the Red Queen hypothesis, as it relates to bacterial transformation. Here we describe a computational model showing that host-pathogen coevolution may provide a large selective benefit to transformation and allow transforming cells to invade an environment dominated by otherwise equal non-transformers. Furthermore, we observe that host-pathogen dynamics cause the selection pressure on transformation to vary extensively in time, explaining the tight regulation and wide variety of rates observed in naturally competent bacteria. Host-pathogen dynamics may explain the evolution and maintenance of natural competence despite its associated costs.
ContributorsPalmer, Nathan David (Author) / Cartwright, Reed (Thesis director) / Wang, Xuan (Committee member) / Sievert, Chris (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Introduction: Human papillomavirus (HPV) infection is seen in up to 90% of cases of cervical cancer, the third leading cancer cause of death in women. Current HPV screening focuses on only two HPV types and covers roughly 75% of HPV-associated cervical cancers. A protein based assay to test for antibody biomarkers against 98 HPV antigens from both high and low risk types could provide an inexpensive and reliable method to screen for patients at risk of developing invasive cervical cancer. Methods: 98 codon optimized, commercially produced HPV genes were cloned into the pANT7_cGST vector, amplified in a bacterial host, and purified for mammalian expression using in vitro transcription/translation (IVTT) in a luminescence-based RAPID ELISA (RELISA) assay. Monoclonal antibodies were used to determine immune cross-reactivity between phylogenetically similar antigens. Lastly, several protein characteristics were examined to determine if they correlated with protein expression. Results: All genes were successfully moved into the destination vector and 86 of the 98 genes (88%) expressed protein at an adequate level. A difference was noted in expression by gene across HPV types but no correlation was found between protein size, pI, or aliphatic index and expression. Discussion: Further testing is needed to express the remaining 12 HPV genes. Once all genes have been successfully expressed and purified at high concentrations, DNA will be printed on microscope slides to create a protein microarray. This microarray will be used to screen HPV-positive patient sera for antibody biomarkers that may be indicative of cervical cancer and precancerous cervical neoplasias.
ContributorsMeshay, Ian Matthew (Author) / Anderson, Karen (Thesis director) / Magee, Mitch (Committee member) / Katchman, Benjamin (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
An introduction to neuroscientific thought aimed at an audience that is not educated in biology. Meant to be readable and easily understood by anyone with a high school education. The first section is completed in its entirety, with outlines for the proposed final sections to be completed over the next few years.
ContributorsNelson, Nicholas Alan (Author) / Olive, M. Foster (Thesis director) / Brewer, Gene (Committee member) / Barrett, The Honors College (Contributor) / Department of Psychology (Contributor) / School of Life Sciences (Contributor) / School of Historical, Philosophical and Religious Studies (Contributor)
Created2014-05
Description
ABSTRACT
Environmental and genetic factors influence schizophrenia risk. Individuals who have direct family members with schizophrenia have a much higher incidence. Also, acute stress or life crisis may precede the onset of the disease. This study aims to understand the effects of environment on genes related to schizophrenia risk. It investigates the impact of sleep deprivation as an acute environmental stressor on the expression of Htr2a in mice, a gene that codes for the serotonin 2A receptor (5-HT2AR). HTR2A is associated with schizophrenia risk through genetic association studies and expression is decreased in post-mortem studies of patients with the disease. Furthermore, sleep deprivation as a stressor in human trials has been shown to increase the binding capacity of 5-HT2AR. We hypothesize that sleep deprivation will increase the number of cells expressing Htr2a in the mouse anterior prefrontal cortex when compared to controls. Sleep deprived that mice express EGFP under control of the Htr2a promoter displayed anteroposterior gradients of expression across sagittal sections, with concentrations seen most densely within the prefrontal cortex as well as the anterior pretectal nucleus, thalamic nucleus, as well as the cingulate gyrus. Htr2a-EGFP expression was most densely visualized in cortical layer V and VI pyramidal neurons within the lateral prefrontal cortex of coronal sections. Furthermore, the medial prefrontal cortex contained significantly cells expressing Htr2a¬-EGFP than the lateral prefrontal cortex. Ultimately, the hypothesis was not supported and sleep deprivation did not result in more ¬Htr2a-EGFP expressing cells compared to basal levels. However, expressing cells appeared visibly brighter in sleep-deprived animals when compared to controls, indicating that the amount of intracellular Htr2a-GFP expression may be higher. This study provides strong visual representations of expression gradients following sleep deprivation as an acute stressor and paves the way for future studies regarding 5H-T2AR’s role in schizophrenia.
Environmental and genetic factors influence schizophrenia risk. Individuals who have direct family members with schizophrenia have a much higher incidence. Also, acute stress or life crisis may precede the onset of the disease. This study aims to understand the effects of environment on genes related to schizophrenia risk. It investigates the impact of sleep deprivation as an acute environmental stressor on the expression of Htr2a in mice, a gene that codes for the serotonin 2A receptor (5-HT2AR). HTR2A is associated with schizophrenia risk through genetic association studies and expression is decreased in post-mortem studies of patients with the disease. Furthermore, sleep deprivation as a stressor in human trials has been shown to increase the binding capacity of 5-HT2AR. We hypothesize that sleep deprivation will increase the number of cells expressing Htr2a in the mouse anterior prefrontal cortex when compared to controls. Sleep deprived that mice express EGFP under control of the Htr2a promoter displayed anteroposterior gradients of expression across sagittal sections, with concentrations seen most densely within the prefrontal cortex as well as the anterior pretectal nucleus, thalamic nucleus, as well as the cingulate gyrus. Htr2a-EGFP expression was most densely visualized in cortical layer V and VI pyramidal neurons within the lateral prefrontal cortex of coronal sections. Furthermore, the medial prefrontal cortex contained significantly cells expressing Htr2a¬-EGFP than the lateral prefrontal cortex. Ultimately, the hypothesis was not supported and sleep deprivation did not result in more ¬Htr2a-EGFP expressing cells compared to basal levels. However, expressing cells appeared visibly brighter in sleep-deprived animals when compared to controls, indicating that the amount of intracellular Htr2a-GFP expression may be higher. This study provides strong visual representations of expression gradients following sleep deprivation as an acute stressor and paves the way for future studies regarding 5H-T2AR’s role in schizophrenia.
ContributorsSchmitz, Kirk Andrew (Author) / Gallitano, Amelia (Thesis director) / Stout, Valerie (Committee member) / Maple, Amanda (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Efforts to quantify the diversity of the T cell repertoire have generally been unsuccessful because not all factors accounting for diversity have been considered. In order to get an accurate representation of the T cell repertoire, one must incorporate analysis of germline gene diversity, diversity from somatic recombination, joining diversity from N- and P- nucleotides, and TCR chain pairing diversity. Because of advances in high-throughput sequencing techniques, estimates have been able to account for diversity from TCR genes. However the ability to account for chain pairing diversity has been more difficult. In order to do so, single cell sorting techniques must be employed. These techniques, though effective, are time consuming and expensive. For this reason, no large-scale analyses have been done on the immune repertoires using these techniques. In this study, we propose a novel method for linking the two TCR chain sequences from an individual cell. DNA origami nanostructure technology is employed to capture and bind the TCRγ and TCRδ chain mRNA inside individual cells using probe strands complementary to the C-region of those sequences. We then use a dual-primer RT and ligation molecular strategy to link the two sequences together. The result is a single amplicon containing the CDR3 region of the TCRγ and TCRδ. This amplicon can then be easily PCR amplified using sequence specific primers, and sequenced. DNA origami nanostructures offer a rapid, cost-effective method alternative to conventional single cell sorting techniques, as both TCR mRNA can be captured on one origami molecule inside a single cell. At present, this study outlines a proof-of-principle analysis of the method to determine its functionality. Using known TCRγ and TCRδ sequences, the DNA origami and RT/PCR method was tested and resulting sequence data proved the effectiveness of the method. The original TCRγ and TCRδ sequences were linked together as a single amplicon containing both CDR3 regions of the genes. Thus, this method can be employed in further research to elucidate the γδ T cell repertoire. This technology is also easily adapted to any gene target or cell type and therefore presents a large opportunity to be used in other immune repertoire analysis and other immunological studies (such as the rapid identification and subsequent production of antibodies).
ContributorsPoindexter, Morgan Elizabeth (Author) / Blattman, Joseph (Thesis director) / Yan, Hao (Committee member) / Schoettle, Louis (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
We examined the evolutionary morphological responses of Drosophila melanogaster that had evolved at constant cold (16°), constant hot (25°C), and fluctuating (16° and 25°C). Flies that were exposed to the constant low mean temperature developed larger thorax, wing, and cell sizes than those exposed to constant high mean temperatures. Males and females both responded similarly to thermal treatments in average wing and cell size. The resulting cell area for a given wing size in thermal fluctuating populations remains unclear and remains a subject for future research.
ContributorsAdrian, Gregory John (Author) / Angilletta, Michael (Thesis director) / Harrison, Jon (Committee member) / Rusch, Travis (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
The transition from high school to college is, for many, a drastic change in lifestyle, social networks, and dietary choices. The prevalence of obesity in college students has been steadily increasing. Freshmen weight gains have been associated with a decrease in fruits and vegetables and an increase in unhealthy items such as desserts, alcohol, and late night snacking after dinner. A survey of college students was constructed to gauge students' perceptions of nutrition how these perceptions influenced dietary practices and behaviors. Survey results indicated that awareness of nutrition and health does not translate to dietary practices, aligning with results from previous studies. Several sex differences were noted in regards to dietary choices and perceptions, knowledge seeking behavior, and sources of information. While there were some similarities, it is clear from the results obtained that men and women have different approaches and thoughts with regard to nutrition. The results showed that college students who actively seek our nutritional information are more likely to do so in the form of social media or Internet sources. This study could be useful for those planning on conducting college-based nutritional programs in that the results indicate patterns and trends that should be taken into consideration in order for a successful nutrition intervention
ContributorsKeahon, Gabriela Estrada (Author) / Jehn, Megan (Thesis director) / Williams, Deborah (Committee member) / Barrett, The Honors College (Contributor) / School of Human Evolution and Social Change (Contributor) / School of Life Sciences (Contributor) / School for the Science of Health Care Delivery (Contributor)
Created2015-05
Description
Cancer poses a significant burden on the global health system and represents a leading cause of death worldwide. For late-stage cancers, the traditional treatments of chemotherapy, radiation, and surgery are not always viable, and they can pose unnecessary health risks to the patients. New immunotherapies, such as adoptive cell transfer, are being developed and refined to treat such cancers. T cell immunotherapies in particular, where a patient’s T cell lymphocytes are isolated and amplified to be re-infused into the patient or where human cell lines are engineered to express T cell receptors for the recognition of common cancer antigens, are being expanded on because for some cancers, they could be the only option. Constructing an optimal pipeline for cloning and expression of antigen-specific TCRs has significant bearing on the efficacy of engineered cell lines for ACT. Adoptive T cell transfer, while making great strides, has to overcome a diverse T cell repertoire – cloning and expressing antigen-specific TCRs can mediate this understanding. Having identified the high frequency FluM1-specific TCR sequences in stimulated donor PBMCs, it was hypothesized that the antigen-specific TCR could be reconstructed via Gateway cloning methods and tested for expression and functionality. Establishing this pipeline would confirm an ability to properly pair and express the heterodimeric chains. In the context of downstream applications, neoantigens would be used to stimulate T cells, the α and β chains would be paired via single-cell or bulk methods, and instead of Gateway cloning, the CDR3 hypervariable regions α and β chains alone would be co-expressed using Golden Gate assembly methods.
ContributorsHirneise, Gabrielle Rachel (Author) / Anderson, Karen (Thesis director) / Mason, Hugh (Committee member) / Hariadi, Hugh (Committee member) / School of Life Sciences (Contributor, Contributor) / School of Sustainability (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
The world of podcasting has exploded in popularity in recent years. This medium is being used in education as well as in the public sector to share ideas, news, and stories. This paper reviews the research behind podcast success as a news form and in the educational sector and the implications of these findings for the future. Podcast listeners tend to listen to podcasts for entertainment and, notably, to diversify their time while completing other tasks. New ways to directly stream media from portable devices and advances in the internet have helped bolster the popularity of this media form. Podcasting proved to be successful in higher education as students tended to perform better when given access to podcasts. However, they were only successful when using podcasts as classroom adjuncts. This implies that educational podcasts must be produced differently than ones intended for the public. By reviewing the neuroscience behind language, emotion and memory, it was found that narrative formats that also evoked emotions had a positive ability in enhancing the listeners learning and memory. Keeping this in mind, the developed podcast aimed to bridge educational material to the general public by utilizing narrative as a vessel in which to deliver complex information about medicine, science and neuroscience. The accessibility and virtually non-existent barriers to the podcasting world offer a breadth of knowledge and opinions that have numerous factors of social influence. The impact of podcasting on the modern world deserves more research in sociology and psychology as it continues to grow in popularity.
ContributorsCharbel, Milad (Author) / Sirven, Joseph (Thesis director) / Reddy, Swapna (Committee member) / School of Life Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2019-05