Matching Items (18)
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- All Subjects: Genetics
- Creators: Lemery-Chalfant, Kathryn
- Creators: School of Molecular Sciences
Description
The goal of this project was to design and create a genetic construct that would allow for <br/>tumor growth to be induced in the center of the wing imaginal disc of Drosophila larvae, the <br/>R85E08 domain, using a heat shock. The resulting transgene would be combined with other <br/>transgenes in a single fly that would allow for simultaneous expression of the oncogene and, in <br/>the surrounding cells, other genes of interest. This system would help establish Drosophila as a <br/>more versatile and reliable model organism for cancer research. Furthermore, pilot studies were <br/>performed, using elements of the final proposed system, to determine if tumor growth is possible <br/>in the center of the disc, which oncogene produces the best results, and if oncogene expression <br/>induced later in development causes tumor growth. Three different candidate genes were <br/>investigated: RasV12, PvrACT, and Avli.
ContributorsSt Peter, John Daniel (Author) / Harris, Rob (Thesis director) / Varsani, Arvind (Committee member) / School of Molecular Sciences (Contributor) / Department of Psychology (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
Externalizing behaviors are pervasive, widespread, and disruptive across a multitude of settings and developmental contexts. While the conventional diathesis-stress model typically measures the disordered end of the spectrum, studies that span the range of behavior, from externalizing to competence behaviors, are necessary to see the full picture. To that end, this study examined the additive and nonadditive relations of a dimension of parenting (ranging from warm to rejecting), and variants in dopamine, vasopressin, and neuropeptide-y receptor genes on externalizing/competence in a large sample of predominantly Caucasian twin children in toddlerhood, middle childhood, and early adolescence. Variants within each gene were hypothesized to increase biological susceptibility to both negative and positive environments. Consistent with prediction, warmth related to lower externalizing/higher competence at all ages. Earlier levels of externalizing/competence washed out the effect of parental warmth on future externalizing/competence with the exception of father warmth in toddlerhood marginally predicting change in externalizing/competence from toddlerhood to middle childhood. Warmth was a significant moderator of the heritability of behavior in middle childhood and early adolescence such that behavior was less heritable (mother report) and more heritable (father report) in low warmth environments. Interactions with warmth and the dopamine and vasopressin genes in middle childhood and early adolescence emphasize the moderational role gene variants play in relations between the rearing environment and child behavior. For dopamine, the long variant related to increased sensitivity to parent warmth such that the children displayed more externalizing behaviors when exposed to rejection but they also displayed more competence behaviors when exposed to high warmth. Vasopressin moderation was only present under conditions of parental warmth, not rejection. Interactions with neuropeptide-y and warmth were not significant. The picture that emerges is one of gene-environment interplay, wherein the influence of both parenting and child genotype each depend on the level of the other. As genetic research moves forward, gene variants previously implicated as conferring risk for disorder should be reexamined in conjunction with salient aspects of the environment on the full range of the behavioral outcome of interest.
ContributorsO'Brien, T. Caitlin (Author) / Lemery-Chalfant, Kathryn (Thesis advisor) / Eisenberg, Nancy (Committee member) / Enders, Craig (Committee member) / Nagoshi, Craig (Committee member) / Arizona State University (Publisher)
Created2011
Description
Fibromyalgia (FM) is a chronic musculoskeletal disorder characterized by widespread pain, fatigue, and a variety of other comorbid physiological and psychological characteristics, including a deficit of positive affect. Recently, the focus of research on the pathophysiology of FM has considered the role of a number of genomic variants. In the current manuscript, case-control analyses did not support the hypothesis that FM patients would differ from other chronic pain groups in catechol-O-methyltransferase (COMT) and mu-opioid receptor (OPRM1) genotype. However, evidence is provided in support of the hypothesis that functional single nucleotide polymorphisms on the COMT and OPRM1 genes would be associated with risk and resilience, respectively, in a dual processing model of pain-related positive affective regulation in FM. Forty-six female patients with a physician-confirmed diagnosis of FM completed an electronic diary that included once-daily assessments of positive affect and soft tissue pain. Multilevel modeling yielded a significant gene X environment interaction, such that individuals with met/met genotype on COMT experienced a greater decline in positive affect as daily pain increased than did either val/met or val/val individuals. A gene X environment interaction for OPRM1 also emerged, indicating that individuals with at least one asp allele were more resilient to elevations in daily pain than those homozygous for the asn allele. In sum, the findings offer researchers ample reason to further investigate the contribution of the catecholamine and opioid systems, and their associated genomic variants, to the still poorly understood experience of FM.
ContributorsFinan, Patrick Hamilton (Author) / Zautra, Alex (Thesis advisor) / Davis, Mary (Committee member) / Lemery-Chalfant, Kathryn (Committee member) / Presson, Clark (Committee member) / Arizona State University (Publisher)
Created2011
Description
The present study tested the factor structure of the externalizing disorders (e.g. attention-deficit hyperactivity disorder (ADHD), conduct disorder (SE), and substance experimentation (SE) ) in adolescence. In addition, this study tested the influence of the GABRA2 gene on the factors of the externalizing spectrum. Confirmatory factor analyses were used to test the factor structure of the externalizing spectrum. Specifically, three competing alternate confirmatory factor analytic models were tested: a one-factor model where all disorders loaded onto a single externalizing factor, a two-factor model where CD and SE loaded onto one factor and ADHD loaded onto another, and a three-factor model, where all three disorders loaded onto separate factors. Structural equation modeling was used to test the effect of a GABRA2 SNP, rs279858, on the factors of the externalizing spectrum. Analyses revealed that a three-factor model of externalizing disorders with correlated factors fit the data best. Additionally, GABRA2 had a significant effect on the SE factor in adolescence, but not on the CD or ADHD factors. These findings demonstrate that the externalizing disorders in adolescence share commonalities but also have separate sources of systematic variance. Furthermore, biological mechanisms may act as a unique etiological factor in the development of adolescent substance experimentation.
ContributorsWang, Frances L (Author) / Chassin, Laurie (Thesis advisor) / Lemery-Chalfant, Kathryn (Committee member) / Geiser, Christian (Committee member) / Arizona State University (Publisher)
Created2012
Description
In most bird species, females disperse prior to their first breeding attempt, while males remain close to the place they were hatched for their entire lives (Greenwood and Harvey (1982)). Explanations for such female bias in natal dispersal have focused on the potential benefits that males derive from knowing the local environment to establish territories, while females search for suitable mates (Greenwood (1980)). However, the variables shaping dispersal decisions appear more complex (Mabry et al. (2013), Végvári et al. (2018)). There are a number of different variables that could act as a driving force behind dispersal including the social mating system, food competition, inbreeding avoidance, predation, and others. Here, we investigate whether females are the dispersing sex in great-tailed grackles, which have a mating system where the males hold territories and the females choose which territory to place their nest in (Johnson et al. (2000)). We used genetic approaches to identify sex biases in the propensity to disperse. In the experiment, we found that the male grackles were less related to each other while the female grackles were more related to each other. Building on that, the average distance between closely related individuals of the male group was longer than the average distance of closely related females. But, the mantel correlograms for the males and females both lack a consistent trend. Overall, the results indicated suggest that the males are the dispersing sex while the females are potentially philopatric and that the average dispersal distance for the grackle is greater than 2000 meters, the size of the sampling range used in the experiment. These results will inform our long-term study on the relationship between behavioral flexibility and rapid geographic range expansion by elucidating which individuals are likely to experience similar conditions across their lives, and which are likely to face new conditions when they become breeders.
ContributorsSevchik, August L (Author) / Langergraber, Kevin (Thesis director) / Logan, Corina (Committee member) / College of Integrative Sciences and Arts (Contributor) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
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
The human gastrin receptor (CCKBR or CCK2R) is a class A G protein-coupled receptor (GPCR) found throughout the central nervous system, stomach, and a variety of cancer cells. CCK2R is implicated in the regulation of biological processes, including anxiety, satiety, arousal, analgesia, psychosis, and cancer cell growth and proliferation. While CCK2R is an attractive drug target, few drugs have managed to effectively target the receptor, and none have been brought to market. Contributory to this is the lack of high-resolution crystal structure capable of elucidating the binding regions of CCK2R to streamlining drug screening. While GPCRs are not amenable to traditional structural analysis methodologies, the advent of lipidic cubic phase (LCP) crystallography and serial femtosecond crystallography (SFX) at X-ray free electron lasers (XFELs), has extended the applicability of X-ray crystallography to these integral membrane proteins. LCP-SFX depends on optimizing the protein of interest for extraction, purification, and crystallization. Here we report our findings regarding the optimization of CCK2R suggesting the synergistic relationship between N-terminal truncations and the insertion of a fusion protein along ICL3, in addition to a 30-residue truncation of the C-terminus. Samples were expressed in Sf9 insect cells using a Bac-to-Bac baculovirus expression system, extracted using n-Dodecyl-β-D-Maltoside detergent, and purified via TALON immobilized metal-ion affinity chromatography. The constructs were characterized via SDS-PAGE, Western blot, and size exclusion chromatography. These findings demonstrate the improvements to CCK2R’s crystallographic amenability upon these modifications, however significant improvements must be made prior to crystallization trials. Future work will involve screening C-terminal truncations, thermostabilizing point mutations, and co-crystallizing ligands. Ideally this investigation will serve as a model for future CCK2R structural analysis and contribute to a heightened interest in CCK2R as a therapeutic target.
ContributorsStevens, Alexander Wade (Author) / Liu, Wei (Thesis director) / Chiu, Po-Lin (Committee member) / Mills, Jeremy (Committee member) / School of Human Evolution & Social Change (Contributor) / School of Molecular Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
When limited for iron, Escherichia coli secretes a siderophore, enterobactin, to solubilize and intake extracellular Fe3+ by a TonB-dependent high-affinity pathway. Consequently, E. coli tonB mutants grow poorly on a medium limited for iron. Upon longer incubation, however, faster growing colonies emerge and overcome this growth defect. The work presented in this paper reports and characterizes these faster growing colonies (revertants) in an attempt to dissect the mechanism by which they overcome the TonB deficiency. Genomic analysis revealed mutations in yejM, a putative inner-to-outer membrane cardiolipin transporter, which are responsible for the faster growth phenotype in a tonB mutant background. Further characterization of the revertants revealed that they display hypersensitivity to vancomycin, a large antibiotic that is normally precluded from entering E. coli cells, and leaked periplasmic proteins into the culture supernatant, indicating a compromised outer membrane permeability barrier. All phenotypes were reversed by supplying the wild type copy of yejM on a plasmid, suggesting that yejM mutations are solely responsible for the observed phenotypes. In the absence of wild type tonB, however, the deletion of all known of cardiolipin synthase genes (clsABC) did not produce the phenotype similar to mutations in the yejM gene, suggesting the absence of cardiolipin from the outer membrane per se is not responsible for the increased outer membrane permeability. These data show that a defect in lipid biogenesis and transport can compromise outer membrane permeability barrier to allow siderophore intake and that YejM may have additional roles other than transporting cardiolipin.
ContributorsQiu, Nan (Author) / Misra, Rajeev (Thesis director) / Bean, Heather (Committee member) / Yu, Julian (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
The dopamine 2 receptor (D2R) is a Class A GPCR which is essential for signaling in the nervous system, and has been implicated in numerous illnesses. While there are over 50 currently approved drugs which act on D2R, the structure has never been determined in detail. Although crystallography has historically been difficult with GPCRs, in recent years many structures have been solved using lipidic cubic phase (LCP) crystallization techniques. Sample preparation for LCP crystallization typically requires optimization of genetic constructs, recombinant expression, and purification techniques in order to produce a sample with sufficient stability and homogeneity. This study compares several genetic constructs utilizing different promoters, fusion proteins, fusion positions, and truncations in order to determine a high quality construct for LCP crystallization of
D2R. All constructs were expressed using the Bac-to-bac baculovirus expression system, then extracted with n-Dodecyl-β-D-Maltoside (DDM) and purified using metal affinity chromatography. Samples were then tested for quantity, purity, and homogeneity using SDS-PAGE, western blot, and size-exclusion chromatography. High quality samples were chosen based on insect cell expression levels, purification yield, and stability estimated by the levels of homomeric protein relative to aggregated protein. A final construct was chosen with which to continue future studies in optimization of thermal stability and crystallization conditions. Future work on this project is required to produce a sample amenable to crystallization. Screening of ligands for co-crystallization,
thermostabilizing point mutations, and potentially optimization of extraction and purification techniques prior to crystallization trials. Solving the D2R structure will lead to an increased understanding of its signaling mechanism and the mechanisms of currently approved drugs, while also providing a basis for more effective structure-based drug design.
D2R. All constructs were expressed using the Bac-to-bac baculovirus expression system, then extracted with n-Dodecyl-β-D-Maltoside (DDM) and purified using metal affinity chromatography. Samples were then tested for quantity, purity, and homogeneity using SDS-PAGE, western blot, and size-exclusion chromatography. High quality samples were chosen based on insect cell expression levels, purification yield, and stability estimated by the levels of homomeric protein relative to aggregated protein. A final construct was chosen with which to continue future studies in optimization of thermal stability and crystallization conditions. Future work on this project is required to produce a sample amenable to crystallization. Screening of ligands for co-crystallization,
thermostabilizing point mutations, and potentially optimization of extraction and purification techniques prior to crystallization trials. Solving the D2R structure will lead to an increased understanding of its signaling mechanism and the mechanisms of currently approved drugs, while also providing a basis for more effective structure-based drug design.
ContributorsErler, Maya Marie (Author) / Liu, Wei (Thesis director) / He, Ximin (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Gle1 is an mRNP export mediator with major activity localized to the nuclear pore complex in eukaryotic cells. The protein's high preservation across vast phylogenetic distances allows us to approximate research on the properties of yeast Gle1 (yGle1) with those of human Gle1 (hGle1). Research at Vanderbilt University in 2016, which provides the research basis of this thesis, suggests that the coiled-coil domain of yGle1 is best crystallized in dicationic aqueous conditions of pH ~8.0 and 10\u201420% PEG 8000. Further exploration of crystallizable microconditions revealed a favorability toward lower pH and lower PEG concentration. Following the discovery of the protein's native crystallography conditions, a comprehensive meta-analysis of scientific literature on Gle1 was conducted on the association of Gle1 mutations with neuron disease.
ContributorsGaetano, Philip Pasquale (Author) / Foy, Joseph (Thesis director) / Dawson, T. Renee (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12