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- Creators: School of Life Sciences
Description
Immediate early genes (IEGs) are rapidly activated in response to an environmental stimulus, and most code for transcription factors that mediate processes of synaptic plasticity, learning, and memory. EGR3, an immediate early gene transcription factor, is a mediator of biological processes that are disrupted in patients with schizophrenia (SCZ). A microarray experiment conducted by our lab revealed that Egr3 also regulates genes involved in DNA damage response. A recent study revealed that physiological neuronal activity results in the formation of DNA double-stranded breaks (DSBs) in the promoters of IEGs. Additionally, they showed that these DSBs are essential for inducing the expression of IEGs, and failure to repair these DSBs results in the persistent expression of IEGs. We hypothesize that Egr3 plays a role in repairing activity- induced DNA DSBs, and mice lacking Egr3 should have an abnormal accumulation of these DSBs. Before proceeding with that experiment, we conducted a preliminary investigation to determine if electroconvulsive stimulation (ECS) is a reliable method of inducing activity- dependent DNA damage, and to measure this DNA damage in three subregions of the hippocampus: CA1, CA3, and dentate gyrus (DG). We asked the question, are levels of DNA DSBs different between these hippocampal subregions in animals at baseline and following electroconvulsive stimulation (ECS)? To answer this question, we quantified γ-H2AX, a biomarker of DNA DSBs, in the hippocampal subregions of wildtype mice. Due to technical errors and small sample size, we were unable to substantiate our preliminary findings. Despite these shortcomings, our experimental design can be modified in future studies that investigate the role of Egr3 in activity-induced DNA damage repair.
ContributorsKhoshaba, Rami Samuel (Author) / Newbern, Jason (Thesis director) / Gallitano, Amelia (Committee member) / Marballi, Ketan (Committee member) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Damage to DNA can affect the genes it encodes; if this damage is not repaired, abnormal proteins may be produced and cellular functions may be disturbed. DNA damage has been implicated in the initiation and progression of a variety of diseases. Conversely, DNA damage has also been discovered to contribute to beneficial biological processes. Madabhushi and colleagues (2015) determined that activity-dependent DNA double strand breaks (DSBs) in the promoter region of immediate early genes (IEGs) induced their expression. EGR3 is an IEG transcription factor which regulates the expression of growth factors and synaptic plasticity-associated genes. In a previously conducted microarray experiment, it was revealed that EGR3 regulates the expression of genes associated with DNA repair such as Cenpa and Nr4a2. These findings inspired us to investigate if EGR3 affects DNA repair in vivo. Before conducting this experiment, we sought to standardize and optimize a method of inducing DNA damage in the hippocampus. Electroconvulsive stimulation (ECS) is utilized to induce neuronal activity. Since neuronal activity leads to the formation of DNA DSBs, we theorized that ECS could be used to induce DNA DSBs in the hippocampus. We predicted that mice that receive ECS would have more DNA DSBs than those that receive the sham treatment. Gamma H2AX, a biomarker for DNA damage, was utilized to quantify DNA DSBs. Gamma H2AX expression in the dentate gyrus, CA1 and CA3 regions of the hippocampus was compared between mice that received the sham treatment and mice that received ECS. Mice that received ECS were sacrificed either 1 or 2 hours post-administration, constituting treatment conditions of 1 hr post-ECS and 2 hrs post-ECS. Our results suggest that ECS has a statistically significant effect exclusively in the CA1 region of the hippocampus. However, our analyses may have been limited due to sample size. A power analysis was conducted, and the results suggest that a sample size of n=4 mice will be sufficient to detect significant differences across treatments in all three regions of the hippocampus. Ultimately, future studies with an increased sample size will need to be conducted to conclusively assess the use of ECS to induce DNA damage within the hippocampus.
ContributorsAden, Aisha Abubakar (Author) / Newbern, Jason (Thesis director) / Gallitano, Amelia (Thesis director) / Marballi, Ketan (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
In vitro gametogenesis (IVG) research has been growing in countries like Japan, US, and China after the development of stem cell research and other scientific advancements as well as because of the perception of infertility as a domestic and international problem. IVG research’s progress has been deliberated internationally, with discussion of questions, challenges, and possibilities that have arisen and may arise in the future as the technology is adopted by different countries. The first section introduces the meaning of IVG, explains the importance of review by scientists and citizens for IVG, and describes a rise in infertility reported in multiple developed countries that could be addressed by IVG. The second section discusses IVG’s applications and implications using 5 ethical categories articulated by Obama’s Presidential Commission for the Study of Bioethical Issues: Public Beneficence, Responsible Stewardship, Intellectual Freedom and Responsibility, Democratic Deliberation, and Justice and Fairness. These five ethical principles were intended for analysis of emerging technologies, and IVG is an emerging technology with possible integration into clinical settings. Among the principles, it seemed that a major weak point of inquiry concerns LGBT+ and disability inclusion, especially of gender dysphoric and transgender people who may experience higher rates of infertility and have a harder time conceiving due to a mix of discrimination, gender dysphoria, and infertility due to hormone replacement therapy (HRT) treatment or gender/sex reassignment surgeries (GRSs/SRSs) that may impair or remove reproductive body parts. A number of other ethical considerations arise about this technology.
ContributorsVillarreal, Lance Edward (Author) / Maienschein, Jane (Thesis director) / Ellison, Karin (Committee member) / Wilson-Rawls, Jeanne (Committee member) / School of Life Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Small cell carcinoma of the ovary (SCCOHT) is a rare ovarian cancer affecting young women and characterized by mutation in SMARCA4 and silencing of SMARCA2, two tumor suppressors that function as ATPases in the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex. SCCOHT patients face a 5-year survival rate of only 26%, but recently we have identified sensitivity of SCCOHT models to a natural product, triptolide. This study aims to ascertain the mechanism of action of triptolide. Previous SCCOHT epigenetic drug research has shown that some drugs reverse SMARCA2 epigenetic silencing to inhibit tumor growth, therefore it is hypothesized that triptolide acts the same and restores SWI/SNF function. Cells treated with triptolide have no change in SMARCA2 expression, suggesting that re-expression of epigenetically silenced tumor suppressor gene does not underlie its mechanism of action. Growth rates following triptolide treatment were observed in the presence and absence of SMARCA4, but no difference in sensitivity was observed. Thus, it is not likely that triptolide acts by restoring SWI/SNF. Others have observed that triptolide acts on xeroderma pigmentosa type B protein (XPB), a component of super-enhancers, which are DNA regions with high levels of transcription that regulate genes responsible for cell identity and oncogenes driving tumorigenesis. Both SCCOHT-1 and BIN67 cell lines treated with triptolide displayed lower expression of the super-enhancer associated MYC oncogene compared to untreated cells, supporting the theory that triptolide could be inhibiting super-enhancers regulating oncogenes.. A western blot confirmed reduced protein levels of RNA polymerase II and bromodomain 4 (BRD4), two essential components found at high levels at super-enhancers, in BIN67 cells treated with triptolide. ChIP-sequencing of Histone H3 Lysine-27 Acetylation (H3K27ac) marks in BIN67 and SCCOHT-1 cell lines identified super-enhancers in SCCOHT using tools CREAM and ROSE, which were mapped to neighboring genes associated genes and compared with the COSMIC database to identify oncogenes, of which the top 11 were examined by qRT-PCR to ascertain whether triptolide reduces their expression. It has been found that 6 out of 11 of the oncogenes examined (SALL4, MYC, SGK1, HIST1H3B, HMGA2, and CALR) decreased in expression when treated with triptolide. Thus, there is reason to believe that triptolide’s mechanism of action is via inhibition of super-enhancers that regulate oncogene expression.
ContributorsViloria, Nicolle Angela (Author) / Lake, Douglas (Thesis director) / Hendricks, William (Committee member) / Lang, Jessica (Committee member) / School of Life Sciences (Contributor) / School of Human Evolution & Social Change (Contributor) / School of International Letters and Cultures (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Lactase persistence is the ability of adults to digest lactose in milk (Segurel & Bon, 2017). Mammals are generally distinguished by their mammary glands which gives females the ability to produce milk and feed their newborn children. The new born therefore requires the ability to breakdown the lactose in the milk to ensure its proper digestion (Segurel & Bon, 2017). Generally, humans lose the expression of lactase after weaning, which prevents them being able to breakdown lactose from dairy (Flatz, 1987).
My research is focused on the people of Turkana, a human pastoral population inhabiting Northwest Kenya. The people of Turkana are Nilotic people that are native to the Turkana district. There are currently no conclusive studies done on evidence for genetic lactase persistence in Turkana. Therefore, my research will be on the evolution of lactase persistence in the people of Turkana. The goal of this project is to investigate the evolutionary history of two genes with known involvement in lactase persistence, LCT and MCM6, in the Turkana. Variants in these genes have previously been identified to result in the ability to digest lactose post-weaning age. Furthermore, an additional study found that a closely related population to the Turkana, the Massai, showed stronger signals of recent selection for lactase persistence than Europeans in these genes. My goal is to characterize known variants associated with lactase persistence by calculating their allele frequencies in the Turkana and conduct selection scans to determine if LCT/MCM6 show signatures of positive selection. In doing this, we conducted a pilot study consisting of 10 female Turkana individuals and 10 females from four different populations from the 1000 genomes project namely: the Yoruba in Ibadan, Nigeria (YRI); Luhya in Webuye, Kenya; Utah Residents with Northern and Western European Ancestry (CEU); and the Southern Han Chinese. The allele frequency calculation suggested that the CEU (Utah Residents with Northern and Western European Ancestry) population had a higher lactase persistence associated allele frequency than all the other populations analyzed here, including the Turkana population. Our Tajima’s D calculations and analysis suggested that both the Turkana population and the four haplotype map populations shows signatures of positive selection in the same region. The iHS selection scans we conducted to detect signatures of positive selection on all five populations showed that the Southern Han Chinese (CHS), the LWK (Luhya in Webuye, Kenya) and the YRI (Yoruba in Ibadan, Nigeria) populations had stronger signatures of positive selection than the Turkana population. The LWK (Luhya in Webuye, Kenya) and the YRI (Yoruba in Ibadan, Nigeria) populations showed the strongest signatures of positive selection in this region. This project serves as a first step in the investigation of lactase persistence in the Turkana population and its evolution over time.
My research is focused on the people of Turkana, a human pastoral population inhabiting Northwest Kenya. The people of Turkana are Nilotic people that are native to the Turkana district. There are currently no conclusive studies done on evidence for genetic lactase persistence in Turkana. Therefore, my research will be on the evolution of lactase persistence in the people of Turkana. The goal of this project is to investigate the evolutionary history of two genes with known involvement in lactase persistence, LCT and MCM6, in the Turkana. Variants in these genes have previously been identified to result in the ability to digest lactose post-weaning age. Furthermore, an additional study found that a closely related population to the Turkana, the Massai, showed stronger signals of recent selection for lactase persistence than Europeans in these genes. My goal is to characterize known variants associated with lactase persistence by calculating their allele frequencies in the Turkana and conduct selection scans to determine if LCT/MCM6 show signatures of positive selection. In doing this, we conducted a pilot study consisting of 10 female Turkana individuals and 10 females from four different populations from the 1000 genomes project namely: the Yoruba in Ibadan, Nigeria (YRI); Luhya in Webuye, Kenya; Utah Residents with Northern and Western European Ancestry (CEU); and the Southern Han Chinese. The allele frequency calculation suggested that the CEU (Utah Residents with Northern and Western European Ancestry) population had a higher lactase persistence associated allele frequency than all the other populations analyzed here, including the Turkana population. Our Tajima’s D calculations and analysis suggested that both the Turkana population and the four haplotype map populations shows signatures of positive selection in the same region. The iHS selection scans we conducted to detect signatures of positive selection on all five populations showed that the Southern Han Chinese (CHS), the LWK (Luhya in Webuye, Kenya) and the YRI (Yoruba in Ibadan, Nigeria) populations had stronger signatures of positive selection than the Turkana population. The LWK (Luhya in Webuye, Kenya) and the YRI (Yoruba in Ibadan, Nigeria) populations showed the strongest signatures of positive selection in this region. This project serves as a first step in the investigation of lactase persistence in the Turkana population and its evolution over time.
ContributorsJobe, Ndey Bassin (Author) / Wilson Sayres, Melissa (Thesis director) / Paaijmans, Krijn (Committee member) / Taravella, Angela (Committee member) / School of Earth and Space Exploration (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Rhesus (Macaca mulatta) and cynomolgus (M. fascicularis) macaques are the most commonly used nonhuman primate models in biomedical research. It is therefore critical to correctly infer each study animal's ABO blood group phenotype to prevent fatal transfusion- and transplantation-induced immune responses. While most macaques can be efficiently and accurately phenotyped using a DNA-based assay, we have identified some animals that are unable to be classified as type A, B, or AB and therefore exhibit an indeterminate phenotype. The purpose of this study was to develop a protocol for resolving indeterminate blood group phenotypes and consequently determine if these animals do indeed belong to an O blood phenotype. We attempted both direct and cloning-based sequencing of 21 animals phenotyped as A, B, AB, or indeterminate in order to assess variation at the functional mutation site in exon 7 of the macaque ABO gene. Although direct-from-PCR Sanger sequencing was unable to generate reliable sequence results, our cloned plasmid protocol yielded high quality sequences consistent with known blood group-specific alleles and as such can be used to identify informative polymorphisms at this locus.
ContributorsVizor, Choice Popsira (Author) / Kanthaswamy, Sreetharan (Thesis director) / Oldt, Robert (Committee member) / Department of Information Systems (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Egr3 is an immediate early gene transcription factor that shows genetic association with schizophrenia, and is found in decreased levels in the brains of schizophrenia patients. Schizophrenia patients also exhibit cognitive and memory deficits, both of which Egr3 has been shown to play a crucial role in. Additionally, high levels of DNA damage are found in the brains of schizophrenia patients. A recent study has shown that DNA damage occurs as a result of normal physiological activity in neurons and is required for induction of gene expression of a subset of early response genes. Also, failure to repair this damage can lead to gene expression in a constitutive switched on state. Egr3 knockout (Egr3-/-) mice show deficits in hippocampal synaptic plasticity and memory. We were interested in characterizing downstream targets of EGR3 in the hippocampus. To determine these targets, electroconvulsive seizure (ECS) was carried out in Egr3 -/- versus wild type (WT) mice, and a microarray study was first done in our lab. ECS maximally stimulates Egr3 expression and we hypothesized that there would be gene targets that are differentially expressed between Egr3 -/- and WT mice that had been subjected to ECS. Two separate analyses of the microarray yielded 65 common genes that were determined as being differentially expressed between WT and Egr3 -/- mice after ECS. Further Ingenuity Pathway Analysis of these 65 genes indicated the Gadd45 signaling pathway to be the top canonical pathway, with the top four pathways all being associated with DNA damage or DNA repair. A literature survey was conducted for these 65 genes and their associated pathways, and 12 of the 65 genes were found to be involved in DNA damage response and/or DNA repair. Validation of differential expression was then conducted for each of the 12 genes, in both the original male cohort used for microarray studies and an additional female cohort of mice. 7 of these genes validated through quantitative real time PCR (qRT-PCR) in the original male cohort used for the microarray study, and 4 validated in both the original male cohort and an independent female cohort. Bioinformatics analysis yielded predicted EGR3 binding sites in promoters of these 12 genes, validating their role as potential transcription targets of EGR3. These data reveal EGR3 to be a novel regulator of DNA repair. Further studies will be needed to characterize the role of Egr3 in repairing DNA damage.
ContributorsBarkatullah, Arhem Fatima (Author) / Newbern, Jason (Thesis director) / Gallitano, Amelia (Committee member) / Marballi, Ketan (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
As a biology major, many of my classes have included studying the fundamentals of genetics or investigating the way genetics influence heritability of certain diseases. When I began taking upper-division psychology courses, the genetic factors of psychological disorders became an important part of the material. I was exposed to a new idea: that genes were equally important in studying somatic diseases as they were to psychological disorders. As important as genetics are to psychology, they are not part of the required courses for the major; I found many of my peers in psychology courses did not have a grasp on genetic fundamentals in the same way biology majors did. This was a disconnect that I also found in my own life outside the classroom. Growing up, my mother consistently reminded me to limit my carbs and watch my sugars. Diabetes was very prevalent in my family and I was also at risk. I was repeatedly reminded of my own genes and the risk I faced in having this biological disorder. However, my friend whose father was an alcoholic did not warn her in the same way. While she did know of her father's history, she was not warned of the potential for her to become an alcoholic. While my behavior was altered due to my mother's warning and my own knowledge of the genetic risk of diabetes, I wondered if other people at genetic risk of psychological disorders also altered their behavior. Through my thesis, I hope to answer if students have the same perceived genetic knowledge of psychological diseases as they do for biological ones. In my experience, it is not as well known that psychological disorders have genetic factors. For example, alcohol is commonly used by college students. Alcohol use disorder is present in 16.2% of college aged students and "40-60% of the variance of risk explained by genetic influences." (DSM V, 2013) Compare this to diabetes that has "several common genetic variants that account for about 10% of the total genetic effects," but is much more openly discussed even though it is less genetically linked. (McVay, 2015)This stems from the stigma/taboo surrounding many psychological disorders. If students do know that psychological disorder are genetically influenced, I expect their knowledge to be skewed or inaccurate. As part of a survey, I hope to see how strong they believe the genetic risk of certain diseases are as well as where they gained this knowledge. I hypothesize that only students with a background in psychology will be able to correctly assign the genetic risk of the four presented diseases. Completing this thesis will require in-depth study of the genetic factors, an understanding of the way each disease is perceived and understood by the general population, and a statistical analysis of the survey responses. If the survey data turns out as I expect where students do not have a strong grasp of diseases that could potentially influence their own health, I hope to find a way to educate students on biological and psychological diseases, their genetic risk, and how to speak openly about them.
ContributorsParasher, Nisha (Author) / Amdam, Gro (Thesis director) / Toft, Carolyn Cavaugh (Committee member) / Ostwald, Madeleine (Committee member) / Department of Psychology (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
This study focused on the connection between the EnvZ/OmpR two-component regulatory system and the iron homeostasis system in Escherichia coli, specifically how a mutant form of EnvZ11/OmpR is able to reduce the expression of fepA::lacZ, a reporter gene fusion in E. coli. FepA is one of several outer membrane siderophore receptors that allow extracellular siderophores bound to iron to enter the cells to power various biological processes. Previous studies have shown that in E. coli cells that expressed a mutant allele of envZ, called envZ11, which led to altered expression of various iron genes including down regulation of fepA::lacZ. The wild type EnvZ/OmpR system is not considered to regulate iron genes, but because these envz11 strains had downregulated fepA::lacZ, this study was undertaken to understand the connection and mechanisms of this downregulation. A large number of Lac+ revertants were obtained from the B32-2483 strain (envz11 and fepA::lacZ) and 7 Lac+ revertants that had reversion mutations not directly correcting the envZ11 allele were further characterized. With P1 phage transduction genetic mapping that involved moving a kanamycin resistance marker linked to fepA::lacZ, two Lac+ revertants were found to have their reversion mutations in the fepA promoter region, while the other five revertants had their mutations mapping outside the fepA region. These two revertants underwent DNA sequencing and found to carry two different single base pair mutations in two different locations of the fepA promoter region. Each one is in the Fur repressor binding region, but one also may have affected the Shine-Dalgarno region involved in translation initiation. All 7 reveratants underwent beta-galactosidase assays to measure fepA::lacZ expression. The two revertants that had mutations in the fepA promoter region had significantly increased fepA activity, with the revertant with the Shine-Dalgarno mutation having the most elevated fepA expression. The other 5 revertants that did not map in the fepA region had fepA expression elevated to the same level as that found in the wild type EnvZ/OmpR background. The data suggest that the negative effect of envZ11 can be overcome by multiple mechanisms, including directly correcting the envZ11 allele or changing the fepA promoter region.
ContributorsKalinkin, Victor Arkady (Co-author) / Misra, Rajeev (Co-author, Thesis director) / Mason, Hugh (Committee member) / Foy, Joseph (Committee member) / Biomedical Informatics Program (Contributor) / School of Life Sciences (Contributor) / W. P. Carey School of Business (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Mammary gland development in humans during puberty involves the enlargement of breast tissue, but this is not true in non-human primates. To identify potential causes of this difference, I examined variation in substitution rates across genes related to mammary development. Genes undergoing purifying selection show slower-than-average substitution rates, while genes undergoing positive selection show faster rates. These may be related to the difference between humans and other primates. Three genes were found to be accelerated were FOXF1, IGFBP5, and ATP2B2, but only the latter one was found in humans and it seems unlikely that it would be related to the differences between mammary gland development at puberty between humans and non-human primates.
ContributorsArroyo, Diana (Author) / Cartwright, Reed (Thesis director) / Wilson Sayres, Melissa (Committee member) / Schwartz, Rachel (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05