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Description
Transient Receptor Potential (TRP) ion channels are a diverse family of nonselective, polymodal sensors in uni- and multicellular eukaryotes that are implicated in an assortment of biological contexts and human disease. The cold-activated TRP Melastatin-8 (TRPM8) channel, also recognized as the human body's primary cold sensor, is among the few TRP channels responsible for thermosensing. Despite sustained interest in the channel, the mechanisms underlying TRPM8 activation, modulation, and gating have proved challenging to study and remain poorly understood. In this thesis, I offer data collected on various expression, extraction, and purification conditions tested in E. Coli expression systems with the aim to optimize the generation of a structurally stable and functional human TRPM8 pore domain (S5 and S6) construct for application in structural biology studies. These studies, including the biophysical technique nuclear magnetic spectroscopy (NMR), among others, will be essential for elucidating the role of the TRPM8 pore domain in in regulating ligand binding, channel gating, ion selectively, and thermal sensitivity. Moreover, in the second half of this thesis, I discuss the ligation-independent megaprimer PCR of whole-plasmids (MEGAWHOP PCR) cloning technique, and how it was used to generate chimeras between TRPM8 and its nearest analog TRPM2. I review steps taken to optimize the efficiency of MEGAWHOP PCR and the implications and unique applications of this novel methodology for advancing recombinant DNA technology. I lastly present preliminary electrophysiological data on the chimeras, employed to isolate and study the functional contributions of each individual transmembrane helix (S1-S6) to TRPM8 menthol activation. These studies show the utility of the TRPM8\u2014TRPM2 chimeras for dissecting function of TRP channels. The average current traces analyzed thus far indicate that the S2 and S3 helices appear to play an important role in TRPM8 menthol modulation because the TRPM8[M2S2] and TRPM8[M2S3] chimeras significantly reduce channel conductance in the presence of menthol. The TRPM8[M2S4] chimera, oppositely, increases channel conductance, implying that the S4 helix in native TRPM8 may suppress menthol modulation. Overall, these findings show that there is promise in the techniques chosen to identify specific regions of TRPM8 crucial to menthol activation, though the methods chosen to study the TRPM8 pore independent from the whole channel may need to be reevaluated. Further experiments will be necessary to refine TRPM8 pore solubilization and purification before structural studies can proceed, and the electrophysiology traces observed for the chimeras will need to be further verified and evaluated for consistency and physiological significance.
ContributorsWaris, Maryam Siddika (Author) / Van Horn, Wade (Thesis director) / Redding, Kevin (Committee member) / School of Molecular Sciences (Contributor) / Department of English (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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
Description
Alternative polyadenylation (APA) is the biological mechanism in which the same gene can have multiple 3'untranslated region (3'UTR) isoforms due to the presence of multiple polyadenylation signal (PAS) elements within the pre mRNAs. Because APA produces mRNA transcripts that have different 3'UTR isoforms, certain transcripts may be subject to post-transcriptional regulation by regulatory non-coding RNAs, such as microRNAs or RNA binding proteins defects of which have been implicated in diseases such as cancer. Despite the increasing level of information, functional understanding of the molecular mechanisms involved in transcription is still poorly understood, nor is it clear why APA is necessary at a cell or tissue-specific level. To address these questions I wanted to develop a set of sensor strain plasmids capable of detecting cleavage and polyadenylation in vivo, inject the complete sensor strain plasmid into C. elegans and prepare stable transgenic lines, and perform proof-of-principle RNAi feeding experiments targeting genes associated with the cleavage and polyadenylation complex machinery. I demonstrated that it was possible to create a plasmid capable of detecting cleavage and polyadenylation in C. elegans; however, issues arose during the RNAi assays indicating the sensor strain plasmid was not sensitive enough to the RNAi to effectively detect in the worms. Once the problems involved with sensitivity and variability in the RNAi effects are resolved, the plasmid would be able to better address questions regarding the functional understanding of molecular mechanisms involved in transcription termination.
ContributorsWilky, Henry Patrick (Author) / Mangone, Marco (Thesis director) / Newbern, Jason (Committee member) / Blazie, Stephen (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Understanding glycosaminoglycans’ (GAG) interaction with proteins is of growing interest for therapeutic applications. For instance, heparin is a GAG exploited for its ability to inhibit proteases, therefore inducing anticoagulation. For this reason, heparin is extracted in mass quantities from porcine intestine in the pharmaceutical field. Following a contamination in 2008, alternative sources for heparin are desired. In response, much research has been invested in the extraction of the naturally occurring polysaccharide, heparosan, from Escherichia coli K5 strain. As heparosan contains the same structural backbone as heparin, modifications can be made to produce heparin or heparin-like molecules from this source. Furthermore, isotopically labeled batches of heparosan can be produced to aid in protein-GAG interaction studies. In this study, a comparative look between extraction and purification methods of heparosan was taken. Fed-batch fermentation of this E. coli strain followed by subsequent purification yielded a final 13C/15N labeled batch of 90mg/L of heparosan which was then N-sulfated. Furthermore, a labeled sulfated disaccharide from this batch was utilized in a protein interaction study with CCL5. With NMR analysis, it was found that this heparin-like molecule interacted with CCL5 when its glucosamine residue was in a β-conformation. This represents an interaction reliant on a specific anomericity of this GAG molecule.
ContributorsHoffman, Kristin Michelle (Author) / Wang, Xu (Thesis director) / Cabirac, Gary (Committee member) / Morgan, Ashli (Committee member) / Barrett, The Honors College (Contributor) / School of International Letters and Cultures (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
The Dorrance Center for Rare Childhood Disorders is a unique research division at TGen (The Translational Genomics Research Institute) that provides personalized care to children and young adults facing rare, undiagnosed diseases. TGen scientists believe that the answers to these enigmatic disorders can often be found in a person's genetic code. They aim to solve these genetic mysteries using whole exome sequencing, a method that prioritizes the protein-coding portion of the genome in the search for disease-causing variants. Unfortunately, a communication gap sometimes exists between the TGen scientists and the patients they serve. I have seen, first hand, the kind of confusion that this study elicits in the families of its participants. Therefore, for my thesis, I decided to create a booklet that is meant to provide some clarity as to what exactly The Dorrance Center for Rare Childhood Disorders does to help diagnose children with rare disorders. The purpose of the booklet is to dispel any confusion regarding the study by providing a general review of genetics and an application of these lessons to the relevant sequencing technology as well as a discussion of the causes and effects of genetic mutations that often times are linked to rare childhood disorders.
ContributorsCambron, Julia Claire (Author) / LaBelle, Jeffrey (Thesis director) / Huentelman, Matt (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Schizophrenia affects 1.1% of the population worldwide. Schizophrenia is a complex, multifactorial disorder. Stress can trigger psychotic episodes and exacerbate schizophrenic symptoms. For humans, one gene implicated in stress and schizophrenia in humans is the early growth response 3 (EGR3). Patients with genomic variations in EGR3 have reduced levels of EGR3 in the prefrontal brain region compared with healthy patients. Schizophrenic patients also have less serotonin 2A receptor (5HT2AR), which is coded by the gene Htr2a, in their prefrontal cortex. Mice that are Egr3-deficient also have decreased levels of 5HT2AR, suggesting that Egr3 may be involved in the regulation of 5HT2AR. The purpose of the experiment is to determine if EGR3 binds to the Htr2a gene promoter region by using a Chromatin immunoprecipitation (ChIP) assay. We will use ECS to increase EGR3 expression. Previously we have identified two upstream sites of interest where EGR3 potentially binds to the Htr2a gene, one which is distal and one proximal to the transcription start site. After ECS, increased binding is seen in the Htr2a distal region with EGR3 via the ChIP assay. Increased binding was not observed at either of the promoter sites; however, the t-test comparing the distal site of the ECS and the No ECS groups to have a p-value of 0.056, suggesting that increasing the number of animals (n=7) could possibly give a more accurate representation to test our hypothesis. However, the experiment still suggests increased expression and that EGR3 may bind to the distal site of Htr2a. Keywords: stress, environment, genetics, schizophrenia, EGR3, chromatin immunoprecipitation
ContributorsMishra, Abhinav (Author) / Buetow, Kenneth (Thesis director) / Gallitano, Amelia (Committee member) / Zhao, Xiuli (Committee member) / Barrett, The Honors College (Contributor) / School of Politics and Global Studies (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
The primary objective of this project is to further the knowledge about SCL26 family of anion transporters. The goals of the experiment were to find the lowest sulfate concentration where the yeast without Sulp1 and Sulp2 is able to grow, but it grows very slowly, and to find a higher sulfate concentration where the yeast grows quickly, with or without the sulfate transporters. The lowest sulfate concentration where the yeast without the sulfate transporters is able to grow was determined to be 2-4 mM, however, this range can likely be refined by more quantitative analytical methods. At a sulfate concentration of 20 mM sulfate or higher, the yeast is able to grow quickly without high-affinity sulfate transporters. The next step in the project is to re-introduce the Sulp1 and Sulp2 genes into the yeast, so that growth in low and high sulfate conditions can be compared with and without the Sulp1 and Sulp2 proteins. The long-term goals of the project are to bring experience with yeast to Dr. Nannenga’s structural discovery lab, to determine if yeast sulfate transporters respond in the same way to drug candidates as human sulfate transporters, and to determine the structure of the proteins using cryo-electron microscopy.
ContributorsCall, Nicolas I (Author) / Nannenga, Brent (Thesis director) / Wang, Xuan (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
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
The NCAA recently declared sickle cell trait (SCT) to be a risk factor for sudden illness and death among student athletes. Fetal hemoglobin (HbF) concentration in adults is negatively correlated with disease severity in sickle cell anemia, although its effect on SCT is not fully understood and the concentration is found to have high variability across populations. Two single nucleotide polymorphisms (SNPs) at the human beta globin gene cluster, rs7482144 and rs10128556, contribute to the heritable variation in HbF levels and are associated with increased HbF concentrations in adults. A sample population of NCAA football student athletes was genotyped for these two polymorphisms, and their allele frequencies were compared to those of other populations. The minor allele of both polymorphisms had allele frequencies of 0.091 in the sample population, which compared closely with other populations of recent African heritage but was significantly different from European populations. The results of this study will be included in a larger study to predict whether these among other polymorphisms can be used as markers to predict susceptibility to heat-related emergencies in NCAA student athletes with SCT, although the small sample size will delay this process until participation in the study increases. Since both rs7482144 and rs10128556 exhibit high levels of linkage disequilibrium, and as their contributions to the heritable variability of HbF concentrations tend to differ greatly between populations of different ancestry, further investigations should be aimed at distinguishing between the effects of each SNP in African American, European, and other populations represented in NCAA football before conclusions can be drawn as to their practical use as genetic markers of heat susceptibility in student athletes with SCT.
ContributorsGrieger, Ryan Wayne (Author) / Stone, Anne C. (Thesis director) / Rosenberg, Michael (Committee member) / Madrigal, Lorena (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2014-05