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Title: Genetic and molecular control of oocyte function
Creator: Severance, Ashley L.
Date: 2018
Collection: Electronic Theses & Dissertations
Description: ABSTRACTGENETIC AND MOLECULAR CONTROL OF OOCYTE FUNCTIONByAshley L. SeveranceA high-quality oocyte will successfully accomplish three critical tasks: segregate chromosomes, reprogram its own genome and that of the incoming sperm to an embryonic state, and support the metabolism and early development of the embryo. This dissertation focuses on two critical determinants of oocyte quality, maintenance of the meiotic spindle and mRNA regulation during meiotic maturation. Chapter 3 of this...
Show moreABSTRACTGENETIC AND MOLECULAR CONTROL OF OOCYTE FUNCTIONByAshley L. SeveranceA high-quality oocyte will successfully accomplish three critical tasks: segregate chromosomes, reprogram its own genome and that of the incoming sperm to an embryonic state, and support the metabolism and early development of the embryo. This dissertation focuses on two critical determinants of oocyte quality, maintenance of the meiotic spindle and mRNA regulation during meiotic maturation. Chapter 3 of this dissertation focuses on the role of the translational repressor, EIF4EBP1, at the meiotic spindles. I show that blocking EIF4EBP1 phosphorylation, which is normally permissive of translation, disrupts the first meiotic spindle, and that inhibiting Polo-like kinase 1 (PLK1) affects EIF4EBP1 phosphorylation and spindle formation at both meiotic divisions. This exciting result suggests PLK1 as an important regulatory focus for controlling EIF4EBP1 and mRNA translation locally at the spindle, to enable the supply of essential proteins during meiotic maturation. Chapter 4 of this dissertation builds on the theme of understanding maternal mRNA regulation by characterizing the dynamic changes in the pool of mRNAs during oocyte maturation and addresses how this varies with oocyte quality. By comparing maturation changes in the transcriptome between two inbred strains (C57BL/6J and DBA/2J) and F1 hybrids between the two strains (BDF1), I discover differences in maternal mRNA regulation associated with superior BDF1 oocyte characteristics (i.e., oocyte hybrid vigor). This includes differences in regulating mRNAs related tomitochondrial physiology and histone production. I also show that many of the differencesbetween the three genotypes arise during maturation and thus in the absence of transcription,indicating differences in regulating mRNA degradation. Overall, a combination of processesunderlies the differential regulation of maternal mRNA in BDF1 oocytes compared to parentalstrains. A small amount of transgressive gene expression is seen, but the most prominentmechanisms responsible for differential mRNA regulation in BDF1 oocytes include “blending”(F1 expression levels intermediate between parental expression levels) and additivedominance. Interestingly, up to 25% of the mRNAs differ significantly between genotypes at theMII stage. This includes many proposed markers of oocyte quality, for which mRNAs differentialexpression between strains exceeded what was reported for differences associated with oocytequality. Because all three genotypes are fertile, this suggests that single markers may not bereliable indicators of oocyte quality. Overall, these results clearly show that the oocyte utilizescomplex regulatory mechanisms at both the meiotic spindles and throughout the ooplasm. Thisdissertation opens the door for many potential future directions to probe further intounderstanding these oocyte-specific adaptations. In the future, this research along with otherstudies could be useful to develop therapies to improve fertility outcomes in both agriculturalspecies and in humans.
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Title: CNGB1 mutation in papillon dogs : the identification, characterization and cure
Creator: Winkler, Paige A.
Date: 2015
Collection: Electronic Theses & Dissertations
Description: Progressive retinal atrophy (PRA) is an inherited retinal dystrophy that affects over 100 breeds of dogs. It is characterized by a bilateral retinal degeneration commonly resulting in blindness. Affected dogs typically present with loss of dim light vision, attenuation of retinal blood vessels and tapetal hyperreflectivity. The purpose of this study was to identify the underlying cause of PRA in Papillon dogs and to characterize the phenotype at the cellular and molecular levels. I identified...
Show moreProgressive retinal atrophy (PRA) is an inherited retinal dystrophy that affects over 100 breeds of dogs. It is characterized by a bilateral retinal degeneration commonly resulting in blindness. Affected dogs typically present with loss of dim light vision, attenuation of retinal blood vessels and tapetal hyperreflectivity. The purpose of this study was to identify the underlying cause of PRA in Papillon dogs and to characterize the phenotype at the cellular and molecular levels. I identified a mutation in the gene CNGB1 which accounts for 70% of the PRA in the Papillon dogs. The CNGB1 mutation involves a 6 base pair insertion and a 1 base pair deletion resulting in exon skipping and a premature stop codon cause by a frameshift. CNGB1 encodes the β-subunit of a cyclic nucleotide-gated ion (CNG) channel. CNGB1 has multiple splice variants expressed in rod photoreceptors, olfactory sensory neurons and other tissues. CNG channels are directly involved in rod phototransduction and olfactory transduction. The retinal phenotype of the CNGB1 affected dogs was characterized by in vivo and ex vivo analyses. Electroretinograms (ERGs) and behavioral vision testing were conducted to assess retinal function throughout the course of the disease. The CNGB1 affected dogs had decreased and abnormal rod function at the earliest age tested but cone function was preserved until 5.5 years of age. Histological analyses showed that the morphology of rod photoreceptors deteriorate slowly over the first ~1.5 years of life while cone photoreceptor morphology is preserved for longer.Adeno-associated viral (AAV) vector therapy was used to treat five CNGB1 affected dogs with a wild-type copy of canine CNGB1 cDNA. One eye was injected with a low titer (1x1012) of an AAV vector delivering CNGB1 cDNA, six eyes were injected with a higher titer (5x1012) and one eye was injected with a GFP-expressing construct as a vehicle and procedural control. All dogs treated with the AAV vector containing the wild-type copy of CNGB1 showed rescue of rod function that was maintained throughout the time course of the study (9 months).The CNGB1 affected dog olfactory phenotype was investigated using in vivo and ex vivo techniques. The olfactory epithelium and the olfactory bulbs were abnormal in the CNGB1 affected dogs when compared to control dogs. I developed a behavioral test that could assess olfactory function in the CNGB1 dogs. The CNGB1 affected dogs had decreased but not absent olfactory function. The detailed descriptions of the retinal and olfactory phenotypes, in addition to the successful gene replacement therapy trial, in the CNGB1 affected dogs have laid the ground work for future studies including working with clinicians to advance gene replacement therapy trials in human patients with mutations in the CNGB1 gene.
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Title: Dissecting the role of peroxisomes in modulating environmental stress response and photosynthesis
Creator: Li, Jiying
Date: 2015
Collection: Electronic Theses & Dissertations
Description: Peroxisomes are essential organelles that house a wide array of metabolic reactions important for plant growth and development. These organelles also interact with other organelles to support cellular functions. However, our knowledge regarding the role of peroxisomal proteins in various biological processes, including plant stress response and photosynthesis, is still incomplete. To address this question at the systems level, I exploited in silico analysis, mutant screens and in-depth...
Show morePeroxisomes are essential organelles that house a wide array of metabolic reactions important for plant growth and development. These organelles also interact with other organelles to support cellular functions. However, our knowledge regarding the role of peroxisomal proteins in various biological processes, including plant stress response and photosynthesis, is still incomplete. To address this question at the systems level, I exploited in silico analysis, mutant screens and in-depth physiological and biochemical characterizations. First, I used microarray data to generate a comprehensive view of transcript level changes for Arabidopsis peroxisomal genes during development and under abiotic and biotic stress conditions. Second, mutants of LON2 protease and the photorespiratory enzyme hydroxypyruvate reductase 1 (HPR1) were identified to have enhanced susceptibility to drought, suggesting the involvement of peroxisomal quality control and photorespiration in drought resistance. Third, I conducted a comprehensive peroxisomal mutant screen, in which 147 mutants of 104 Arabidopsis genes encoding peroxisomal proteins were subjected to an automated screening system, the Dynamic Environment Phenotype Imager (DEPI). This screen identified multiple peroxisomal proteins required for robust photosynthesis efficiency under dynamically changing light, including peroxisomal biogenesis and division proteins, photorespiratory proteins, and a NAD+ transporter protein PXN, which was found to be an additional player in photorespiration. Fourth, further characterization of the photorespiratory mutants provided insights into the molecular mechanisms regarding how the blocking of photorespiration alters photosynthetic efficiency. My data supported an integrated model for the events that occur in the photorespiration mutants, where metabolites and molecules resulting from the block of photorespiration inhibit triose phosphate isomerase (TPI) activity, compromise photosystem integrity, reduce photosystem subunit abundance, decrease proton efflux and diminish ATP synthase conductivity, induce cyclic electron flow (CEF) and activate energy dissipation. In summary, my work has provided significant insights into the connection between peroxisomal function and drought stress response and the links between photorespiration and photosynthesis. Knowledge gained from my dissertation research opens up new avenues to further investigate environmental stress response, photosynthesis, photorespiration and interorganellar communication.
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Title: Chromatin and transcriptional regulation in mouse macrophages
Creator: McAndrew, Michael
Date: 2017
Collection: Electronic Theses & Dissertations
Description: "Eukaryotic genomes must be extensively packaged into a DNA-protein complex called chromatin due to their large sizes and the spatial restrictions of the nucleus. Nucleosomes, the basic repeating unit of this complex, have long been viewed as a barrier to basic cellular processes including transcription, and recent studies suggest that chromatin architecture plays a critical role in the regulation of gene expression. We have used primary bone marrow-derived macrophages (BMDMs) as a model to...
Show more"Eukaryotic genomes must be extensively packaged into a DNA-protein complex called chromatin due to their large sizes and the spatial restrictions of the nucleus. Nucleosomes, the basic repeating unit of this complex, have long been viewed as a barrier to basic cellular processes including transcription, and recent studies suggest that chromatin architecture plays a critical role in the regulation of gene expression. We have used primary bone marrow-derived macrophages (BMDMs) as a model to investigate chromatin changes associated with inducible and cell-type specific gene expression in response to bacterial lipopolysaccharide (LPS). Macrophages are specialized cells of the innate immune system that arise during differentiation from multipotent hematopoietic stem and progenitor cells (HSPCs) through the coordinated action of lineage-specific transcription factors (TFs). These cells have unique functions in response to foreign threat, and previous genome-wide studies have identified macrophage-specific distal enhancers that play a key role in the pro-inflammatory response to LPS. Using a quantitative nucleosome occupancy assay, we have shown that nucleosomes are stably evicted from these enhancers under inducing conditions in BMDMs, and this depletion correlates with signal-induced TF binding and increased gene expression. Using a knockdown approach targeting BAF/PBAF chromatin remodeling complexes, we have shown that nucleosome remodelers are recruited to regulatory elements early during differentiation by lineage-specific TFs, and that disruption of this process results in increased nucleosome occupancy at these elements and prevents nucleosome eviction and gene induction in response to LPS. In order to more precisely determine how and when enhancers might be rendered accessible during differentiation, we further investigated chromatin structure in HSPCs. This led to the surprising finding that nucleosome occupancy may be universally low in these cells. We are now using a genome-wide extension of the quantitative nucleosome occupancy (GNO-seq, Global Nucleosome Occupancy-sequencing) to analyze changes in nucleosome occupancy associated with macrophage differentiation from HSPCs genome-wide. This research will provide crucial insights into the regulation of inducible gene expression, the role of remodelers in maintaining chromatin accessibility, and may demonstrate global differences in chromatin between cell types."--Pages ii-iii.
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Title: Cellular and genetic characterization of ocular melanosis in the Cairn terrier dog
Creator: Dawson-Baglien, Ethan
Date: 2017
Collection: Electronic Theses & Dissertations
Description: "Ocular melanosis (OM) is an inherited eye disease seen in the Cairn terrier dog breed. The disease is very common in Cairn terriers - although the exact number of dogs affected by the disease is not known, surveys of Cairn terrier breeders and owners frequently rank it near the top of health concerns in the breed. The disease progresses through several stages, starting off as a thickening and darkening of the iris. Eventually, dark brown-black pigment begins to appear in patches in abnormal...
Show more"Ocular melanosis (OM) is an inherited eye disease seen in the Cairn terrier dog breed. The disease is very common in Cairn terriers - although the exact number of dogs affected by the disease is not known, surveys of Cairn terrier breeders and owners frequently rank it near the top of health concerns in the breed. The disease progresses through several stages, starting off as a thickening and darkening of the iris. Eventually, dark brown-black pigment begins to appear in patches in abnormal areas of the eye, such as the sclera (the whites of the eye). These patches gradually grow and expand over time. Within the eye, pigmented material is shed into the anterior chamber of the eye (the fluid-filled space between the iris and the front of the eye). This pigmented material clogs up the eye's internal drainage pathways, and fluid builds up within the anterior chamber, leading to an increase in pressure within the eye. This increase in pressure can lead to painful glaucoma, and eventually blindness, in the dogs with the most severe cases of OM. The underlying causes of OM are not currently known. Two different methods were used to attempt to find out more about the disease - a cell culture method and a gene sequencing method. In the cell culture method, donated eyes from dogs with and without OM were used to isolate and grow uveal melanocytes - the pigmented cells of the eye which grow and migrate in OM. These cells were then tested using a variety of different cellular assays to determine how the melanocytes from affected eyes differed from those in unaffected eyes. The only tests where the melanocytes from the OM-affected dogs showed any difference from those in unaffected dogs were in pigmentation - the melanocytes from OM-affected dogs had much more pigment, and made new pigment more quickly, than those from unaffected dogs. Gene sequencing methods were also used to try to find where in the genome the mutation that causes OM was located. To determine the general location of the mutation, a whole-genome SNP array was used to test 94 dogs at 170k markers from all around the 2.8 billion base pairs of the canine genome, to see if any of the markers was associated with the disease. This identified a 7.5 million base-pair long region of chromosome 11 that was significantly associated with the disease. Next, the entire genome of 10 dogs was sequenced, 5 OM-affected and 5 unaffected, to look for the exact mutation causing OM. Analysis of the sequencing data failed to identify a likely causal variant, either within the identified region or in known genes related to pigmentation disorders. Finally, RNA sequencing was performed on eye tissues from 12 dogs; 7 OM-affected and 5 unaffected, to determine whether there were any differences in gene expression between the two populations. Six genes were identified that were expressed differently between the two populations that were in pathways known to be associated with cancer metastasis. Although a causal variation for OM has not yet been discovered, several promising new clues have been identified that can be followed up on, including the general location of the causal DNA mutation on chromosome 11, and a number of genes whose expression are altered in OM-affected dogs. Following these leads may finally allow us to identify the underlying cause of OM."--Pages ii-iii.
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Title: The role of sphingolipids in retinal vascular integrity
Creator: Kady, Nermin Mohammad Mohammad
Date: 2017
Collection: Electronic Theses & Dissertations
Description: "Diabetic retinopathy is a vision-threatening microvascular complication of diabetes mellitus. The increase in the prevalence of diabetes in the population will most likely lead to a higher incidence of diabetic retinopathy (DR), a devastating complication with limited treatment options. Years of investigations have yet to yield a fully understood mechanism for the causes of vascular dysfunction in DR. Dyslipidemia, as well as hyperglycemia, is a major metabolic insult of diabetes and is...
Show more"Diabetic retinopathy is a vision-threatening microvascular complication of diabetes mellitus. The increase in the prevalence of diabetes in the population will most likely lead to a higher incidence of diabetic retinopathy (DR), a devastating complication with limited treatment options. Years of investigations have yet to yield a fully understood mechanism for the causes of vascular dysfunction in DR. Dyslipidemia, as well as hyperglycemia, is a major metabolic insult of diabetes and is positively correlated with the development of DR. The objective of this study is to provide a mechanistic link between retinal dyslipidemia in diabetes and retinal vascular pathology in DR. Our studies revealed two main pathways of sphingolipid metabolism involved in the development of DR. The first pathway leads to disruption of blood retinal barrier (BRB) and thus increased vascular permeability-- an early sign of DR. The second pathway plays a role in retinal endothelial damage and its defective repair. First, we addressed the link between dyslipidemia and BRB using both in vitro and in vivo experiments. We demonstrated that diabetes induces down regulation of an essential retinal fatty acid elongase enzyme, Elongation of very long chain fatty acids-4 (ELOVL4), in the diabetic retina. Down regulation of ELOVL4 plays a crucial role in diabetes-induced blood retinal barrier dysfunction. Overexpression of ELOVL4 in retinal endothelial cells enhances barrier properties of retinal vasculature. Ceramides were found to co-localize with tight junction complexes. Lipidomic analysis of tight junction isolates revealed the presence of ELOVL4-produced VLC ceramides, notably, omega-linked acyl-ceramides that were previously identified only in the skin permeability barrier. Second, we investigated the effect of dyslipidemia on the function of both cell types involved in revascularization; human retinal endothelial cells (HRECs) and circulating angiogenic cells (CACs). We demonstrated that diabetes induces activation of acid sphingomyelinase (ASM), a key enzyme of sphingolipid metabolism in human retinal endothelial cells (HRECs) and human CD34+ CACs. Diabetes induced ASM upregulation has deleterious effect on both retinal endothelial cell and CD34+ CAC function. Diabetic HRECs with high ASM showed defective ability to form blood-vessel-like tubular structure and diabetic CD34+ CACs with high ASM showed defective incorporation into endothelial tubes formed by HRECs. Taken together, these findings indicate that modulation of sphingolipid metabolism to normalize retinal vascular dysfunction and improve retinal vascular repair can represent a novel therapeutic strategy for treating DR."--Pages ii-iii.
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Title: Dual role of PU.1 in enhancer priming in macrophages
Creator: Tagore, Mohita Malay
Date: 2017
Collection: Electronic Theses & Dissertations
Description: All multicellular organisms arise from a single-celled zygote by the precise execution of a gene expression program which ensures appropriate cell identity. This process is particularly challenging in eukaryotic cells since eukaryotic DNA is packaged by architectural proteins called histones into chromatin, which might act as a barrier to the transcriptional machinery. Macrophages are cells of the immune system which undergo rapid, large scale changes in gene expression in response to...
Show moreAll multicellular organisms arise from a single-celled zygote by the precise execution of a gene expression program which ensures appropriate cell identity. This process is particularly challenging in eukaryotic cells since eukaryotic DNA is packaged by architectural proteins called histones into chromatin, which might act as a barrier to the transcriptional machinery. Macrophages are cells of the immune system which undergo rapid, large scale changes in gene expression in response to bacterial or viral challenge. This makes macrophages an excellent model for studying cell-type specific as well as inducible gene expression. Studies at the genome-wide level have shown that distal regulatory elements like enhancers play an essential role in determining the macrophage inducible response to microbial challenge. Further, lineage-specific transcription factors like PU.1 and C/EBPβ are known to bind inducible enhancers prior to gene induction in resting macrophages. Earlier studies using genome-wide approaches indicate that PU.1 is able to interact with chromatin, thus functioning as a 'pioneer factor' in macrophages. However, not much is known about the mechanism by which PU.1 keeps enhancers accessible prior to gene induction in resting macrophages. Using bone-marrow derived primary mouse macrophage cells as well as PU.1 deficient cell lines, my work highlights the changes in chromatin associated with PU.1 binding during macrophage differentiation as well as in response to bacterial infection. Using a quantitative nucleosome occupancy assay, we reported that PU.1 binding correlates with low nucleosome occupancy at an inducible enhancer in resting macrophages. Further upon induction with an appropriate stimulus, nucleosomes are stably evicted from the distal enhancer and the corresponding gene can be induced. More importantly, my results suggest that lack of PU.1 binding renders regulatory regions (enhancers and promoters) of inducible genes susceptible to heterochromatin formation and silencing by Polycomb repressive complex 2 (PRC2) in differentiated macrophages. PRC2-mediated silencing is also associated with an increase in nucleosome occupancy at the target regions and the corresponding genes cannot be induced. Results obtained from this research will provide important insights into the role of lineage-specific transcription factors at regulatory elements both during normal development and disease.
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Title: A role for DeltaFosB in the regulation of parkin in brain regions containing differentially susceptible dopaminergic neurons
Creator: Patterson, Joseph Robert
Date: 2016
Collection: Electronic Theses & Dissertations
Description: The hallmark pathologies of Parkinson disease (PD) are the formation of Lewy bodies and the progressive loss of nigrostriatal dopamine (NSDA) neurons. In mice, the NSDA neurons are preferentially damaged through exposure to the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Another population of DA neurons that are initially damaged by MPTP, but are able to recover are the tuberoinfundibular DA (TIDA) neurons. Parkin is a product of the PARK2 gene, which is linked to...
Show moreThe hallmark pathologies of Parkinson disease (PD) are the formation of Lewy bodies and the progressive loss of nigrostriatal dopamine (NSDA) neurons. In mice, the NSDA neurons are preferentially damaged through exposure to the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Another population of DA neurons that are initially damaged by MPTP, but are able to recover are the tuberoinfundibular DA (TIDA) neurons. Parkin is a product of the PARK2 gene, which is linked to autosomal recessive or juvenile PD. Parkin has multiple functions in neurons and is predicted to protect against the neurotoxic effects of MPTP. Potential transcription factors of parkin were identified using TFSEARCH, PROMO, and Patch 1.0, and refined to 11 based the transcription factor being identified in all three programs, being known to be found in the brain, and known to respond to a type of stress that MPTP could cause. The candidate transcription factors were examined at 6 h after MPTP in regions containing the cell bodies of TIDA and NSDA neurons. From these candidates, only FosB and ΔFosB have expression patterns that mirror parkin.Further examination of the temporal expression and cellular localization of FosB and ΔFosB after acute neurotoxicant administration were examined. Regions containing the cell bodies of the TIDA (arcuate nucleus; ARC) and NSDA (substantia nigra; SN) neurons were dissected and processed for Western blot analysis. The results reveal that expression of FosB and ΔFosB correlates with parkin, increasing in the ARC and not in the SN. Furthermore, total FosB protein was localized to nuclei of NSDA and TIDA neurons, and expression of each FosB and ΔFosB examined in cytoplasmic and nuclear fractions derived from the ARC and SN. Though the number of DA neurons expressing total FosB does not change at 6 h post-MPTP, ΔFosB does increase in the nuclear fraction from the ARC.AAV-mediated expression vectors were used to increase ΔFosB in the NSDA and TIDA neurons, in both cases, parkin increased about 2-fold. The dominant negative protein ΔJunD, which lacks a DNA binding domain, predominantly dimerizes with the FosBs and inhibits their ability to act as transcription factors was injected into the ARC. The AAV-ΔJunD virus blocked the increase of parkin after MPTP in the TIDA neurons. Taken together, the results support the role of FosB and ΔFosB as transcription factors of parkin, since they are predicted to bind the Park2 promoter, their expression correlates with the differential expression of parkin, increases prior to parkin, are present in nuclei of TIDA neurons, ΔFosB is sufficient to drive parkin expression, and ΔJunD blocks the increase of parkin in the ARC in response to MPTP.
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Title: Investigating novel therapeutic targets for the treatment of Fragile X syndrome
Creator: Sethna, Ferzin
Date: 2014
Collection: Electronic Theses & Dissertations
Description: Fragile X syndrome (FXS) is the most common form of inherited mental retardation and a leading cause of autism. Most cases of FXS result from CGG repeat expansion in the 5' UTR of the FMR1 gene, which results in lack of expression of its protein product, FMRP. FMRP is an mRNA binding protein, which predominantly suppresses the translation of its targets. The absence of FMRP thus leads to excessive protein synthesis and altered synaptic signaling, which are believed to underlie the...
Show moreFragile X syndrome (FXS) is the most common form of inherited mental retardation and a leading cause of autism. Most cases of FXS result from CGG repeat expansion in the 5' UTR of the FMR1 gene, which results in lack of expression of its protein product, FMRP. FMRP is an mRNA binding protein, which predominantly suppresses the translation of its targets. The absence of FMRP thus leads to excessive protein synthesis and altered synaptic signaling, which are believed to underlie the pathophysiology of FXS. There is currently no cure for FXS and often multiple drugs are administered to manage the symptoms. This highlights the importance of understanding the molecular mechanisms that are altered in FXS brains, in order to develop better therapeutic targets. My study identifies that translation of the brain-specific type-1 adenylyl cyclase (AC1) mRNA is controlled by FMRP and that AC1 protein is overexpressed in the absence of FMRP. Using genetic knockdown and pharmacological inhibition of AC1, I show that reducing AC1 activity can rescue several cellular and behavioral phenotypes in the Fmr1 knockout mouse model. My research also reveals calmodulin inhibitor can rectify several cellular and behavioral phenotypes in Fmr1 KO mice. In summary this thesis describes the identification and validation of a novel therapeutic target and an FDA approved drug for treatment of FXS using the Fmr1 KO mouse model.
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Title: Determining the role of IRF6 in T cell development and functional commitment
Creator: Mansour, Tamer Ahmed
Date: 2014
Collection: Electronic Theses & Dissertations
Description: DETERMINING THE ROLE OF IRF6 IN T CELL DEVELOPMENT AND FUNCTIONAL COMMITMENT Interferon regulatory factor (IRF) is a protein family with nine members in mammals known to orchestrate and control homeostatic mechanisms of host defense. There are functional and/or developmental defects of immune cells in the knockouts of eight family members. Like other family members, IRF6 is involved in regulating the cell cycle but in keratinocytes and mammary epithelial cell with mutations...
Show moreDETERMINING THE ROLE OF IRF6 IN T CELL DEVELOPMENT AND FUNCTIONAL COMMITMENT Interferon regulatory factor (IRF) is a protein family with nine members in mammals known to orchestrate and control homeostatic mechanisms of host defense. There are functional and/or developmental defects of immune cells in the knockouts of eight family members. Like other family members, IRF6 is involved in regulating the cell cycle but in keratinocytes and mammary epithelial cell with mutations associated with squamous cell carcinomas. However, Irf6 is the only IRF known to be involved in morphogenesis. In humans, rare variants in IRF6 cause autosomal dominant orofacial clefting disorders. Common IRF6 variants contribute risk toward non-syndromic orofacial clefting. IRF6 is the only IRF family member with an as yet undetermined role in immunity. Here, we used publically available microarray data to uncover a dynamic expression pattern for Irf6 during hematopoietic development and functional commitment. We found that Irf6 is expressed early in hematopoiesis, especially in long term hematopoietic stem cells. Also we identified Irf6 expression in T cell lineage, including developing and functionally committed stages. Irf1, 2, 4, 8 are indispensable for a normal T cell development and differentiation. Genetic variants in IRF5, IRF7 and IRF8 are associated to autoimmune disorders of T cells including psoriasis, multiple sclerosis and systemic lupus erythematosis. Furthermore, protein complexes between IRF6/IRF5 and IRF6/IRF8 were described. These data together with DNA conservation among the IRF members and structural homology with IRF5 strongly suggests a role for Irf6 in the immune system, specifically in T-cell development and functional commitment. We utilized a mouse model to show that Irf6 was required for the regulation of thymocyte development. We found that Irf6 was expressed in the subcapsular region and medulla of the thymus. We further found that Irf6 regulated the distribution and proliferation of developing thymocytes. In addition, loss of Irf6 led to an increase in double negative cells with a concomitant increase in TCRγδ. Loss of Irf6 also led to a reduction in double positive cells with no corresponding reduction in single positive cell maturation. Also, we found that Irf6 dose is critical in development of both CD4+ and CD8+ cells in an age-dependent manner. These data suggest a novel gene function for Irf6 in thymocyte development and indicate further studies of IRF6 variants that might increase the risk of autoimmune disease. In the mouse, loss of Irf6 leads to perinatal lethality which hinders the ability to test the necessity of Irf6 in the functionally committed T helper (Th) subsets. We use a combination of in silico, in vivo and in vitro assays to determine the role of Irf6 in T cell differentiation. Using in silico analysis, we found and propose a model for Irf6 function in Th17/Treg balance. To test our hypothesis in vivo and overcome perinatal lethality, we employed an adaptive transfer of Irf6 knockout cells into lethally irradiated mice. We observed a 100% survival of chimeric mice receiving Irf6 knockout fetal liver, and mice receiving Irf6 knockout cells had no deficit in restoration of lymphocyte production. In addition, we used two in vitro models to assess the necessity of Irf6 in the commitment of T helper cells. Using a stromal-free culture we found that naive T cells lacking Irf6 could be differentiated into Th1, Th2, Th17 and Treg using a specific cytokine cocktail. We found no differences in cell frequency and mean fluorescence intensity of intracellular cytokines between wild type and Irf6 knockout cells. In vitro differentiation of dendritic cells showed significant increase of MHC-II expression after three days of culture. Irf6 might be involved in post-translational regulation of MHC-II. These data indicate that intrinsic Irf6 expression is not essential for T helper subset differentiation. However, a non-cell autonomous role for Irf6 in T cell differentiation through dendritic cells remains plausible.
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Title: Intestinal barrier function alters bone density
Creator: Raehtz, Sandra Ann
Date: 2016
Collection: Electronic Theses & Dissertations
Description: 1 in 2 women over 50 will experience a fracture due to osteoporosis. Current therapeutics work to prevent further bone loss, however, new therapies which can build bone or prevent bone loss are necessary to identify. The gut-bone axis is a promising target for the prevention of bone loss. Recent studies have shown that the probiotic Lactobacillus reuteri prevents bone loss during estrogen deficiency (OVX), type 1 diabetes (T1D) as well as increases bone density in healthy male mice. This...
Show more1 in 2 women over 50 will experience a fracture due to osteoporosis. Current therapeutics work to prevent further bone loss, however, new therapies which can build bone or prevent bone loss are necessary to identify. The gut-bone axis is a promising target for the prevention of bone loss. Recent studies have shown that the probiotic Lactobacillus reuteri prevents bone loss during estrogen deficiency (OVX), type 1 diabetes (T1D) as well as increases bone density in healthy male mice. This finding lead us to examine what changes occur in the intestine following estrogen deficiency. We found that ovariectomy (OVX) leads to decreased intestinal permeability which is prevented by estrogen supplementation. Additionally, OVX was found to decrease epithelial cell turnover as well as mucus production. To determine how treating the intestinal epithelial barrier can affect bone loss, we utilized a high molecular weight polymer, MDY, which is neither metabolized or absorbed to decrease intestinal inflammation and increase mucus in the intestine. MDY treatment in OVX mice prevented estrogen deficiency induced bone loss, highlighting the role of the intestinal barrier in bone density. In addition to characterizing how OVX alters intestine function, T1D was found to alter intestinal motility and permeability, both of which were correlated to bone loss. Treatment of diabetic mice with MDY prevented intestinal inflammation and increased bone density with respect to diabetic controls. Together our studies indicate an important role for the intestinal barrier in the health of bone. Understanding the role of the gut-bone axis can lead to the prevention of bone loss as well as the development of new bone building therapies.
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Title: Machine learning for the study of gene regulation and complex traits
Creator: Sonnenschein, Anne
Date: 2017
Collection: Electronic Theses & Dissertations
Description: "Functional elements are found in DNA outside of protein coding regions; an important class of these elements are 'enhancers', which govern when and where transcription occurs. Predicting the identity and function of potential enhancers based on DNA sequence remains a major goal of genomics. A number of features are associated with the enhancer state, but even combinations of these features in well-studied systems such as Drosophila have limited predictive accuracy. I have examined the...
Show more"Functional elements are found in DNA outside of protein coding regions; an important class of these elements are 'enhancers', which govern when and where transcription occurs. Predicting the identity and function of potential enhancers based on DNA sequence remains a major goal of genomics. A number of features are associated with the enhancer state, but even combinations of these features in well-studied systems such as Drosophila have limited predictive accuracy. I have examined the current limits of computational enhancer prediction, and analyzed which features are most useful for this task, by applying machine-learning methods to an extensive set of genomic features. Inferring the genetic underpinning of even well-characterized phenotypes is equally challenging, although similar analytical methods can be applied. Phenotypes are frequently defined based on a set of characteristic features; when images are used as specimens, these features are frequently based on morphometric landmarks, although computational pattern-recognition has been used as an alternative. I use Drosophila wing shape as a model for a complex phenotype, and use machine learning to predict underlying genotype using both traditional landmarks and features extracted using 'computer vision."--Page ii.
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Title: Roles of hfq-dependent srnas in e. amylovora regulation of virulence
Creator: Schachterle, Jeffrey Kent
Date: 2019
Collection: Electronic Theses & Dissertations
Description: Erwinia amylovora is the causative agent of fire blight disease of apple and pear trees, causing annual losses of over 100 million USD in the USA. E. amylovora cells are disseminated to new hosts by insects, wind, and rain, and then invade susceptible tissues and migrate systemically throughout the host, requiring coordinate regulation of several virulence factors, including production of the exopolysaccharides amylovoran and levan, biofilm formation, flagellar motility, and type III...
Show moreErwinia amylovora is the causative agent of fire blight disease of apple and pear trees, causing annual losses of over 100 million USD in the USA. E. amylovora cells are disseminated to new hosts by insects, wind, and rain, and then invade susceptible tissues and migrate systemically throughout the host, requiring coordinate regulation of several virulence factors, including production of the exopolysaccharides amylovoran and levan, biofilm formation, flagellar motility, and type III secretion. Complex regulatory mechanisms have evolved in E. amylovora that occur at the transcriptional, post-transcriptional, and post-translational levels to control these virulence factors. In my work, I analyze the role of small RNAs (sRNAs) as post-transcriptional regulators of virulence-associated traits in E. amylovora.The Hfq chaperone protein stabilizes sRNAs in the cell, allowing them to interact with and regulate mRNA targets. An hfq mutant differs from wild-type cells in several virulence-associated phenotypes including production of the exopolysaccharides amylovoran and levan, biofilm formation, flagellar motility, and type III secretion. E. amylovora encodes at least 40 Hfq-dependent sRNAs; in my work, I have systematically made deletion mutants of each sRNA singly, as well as constructed inducible expression vectors for each sRNA. Screening of this sRNA library has shown that several sRNAs contribute to regulation of each virulence phenotype, indicating complex regulation of the traits assessed. Of particular interest, the ArcZ sRNA regulates several of the virulence-associated traits we have assessed, and an arcZ deletion mutant loses virulence in both immature pear and apple shoot infection models.Flagellar motility, which enables E. amylovora cells to swim through flower nectar to invade natural openings in host flowers, is regulated by ArcZ. We have shown that ArcZ regulates motility by regulating the flagellar transcription factor FlhD at both the transcriptional and post-transcriptional levels. Because the ArcZ regulation of FlhD at the transcriptional and post-transcriptional levels has a contradiction in sign, we searched for additional layers of regulation between ArcZ and FlhD. We did so by conducting a transposon screen in the arcZ mutant background for suppressor mutants that restored flagellar motility. This screen yielded as the most common suppressor mutation the leucine responsive regulator protein (Lrp), a global transcription factor known for regulation of amino acid metabolism. We have found that Lrp not only acts as a regulator of flagellar motility between ArcZ and FlhD, but that it also reverses the regulatory effects of arcZ deletion on amylovoran and levan production, as well as biofilm formation. Our work shows that Lrp is a novel virulence regulator that plays an important role in regulating several virulence-associated traits in conjunction with the sRNA ArcZ.Transcriptomic comparison between the arcZ mutant and wild-type cells confirmed that ArcZ regulates several genes known to also be regulated by Lrp, and also indicated that ArcZ regulates several genes involved in mitigating the threat of reactive oxygen species, including genes encoding a catalase, a thiol-peroxidase, and a peroxiredoxin. We found that catalase makes the greatest contribution to diminishing the threat of exogenous hydrogen peroxide. Additional analysis suggests that ArcZ participates in regulation with an oxidative sensing transcription factor network that includes the transcription factors ArcA, Fnr, and Fur.This work shows that several sRNAs make small contributions to virulence trait regulation, and that a few sRNAs, like ArcZ, make major contributions to E. amylovora virulence. ArcZ regulates several virulence-associated traits through the global transcription factor Lrp, which we have found to be a novel virulence regulator. ArcZ also regulates genes involved in mitigating the threat of reactive oxygen species, which can protect E. amylovora cells from host defenses during infection. Thus, ArcZ plays an integral role in modulating phenotypic expression during fire blight disease progression that enables E. amylovora to successfully colonize and infect host plants. Mechanistic understanding of E. amylovora gene regulation moves us closer to understanding weaknesses that can be exploited for development of novel disease control strategies.
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Title: ARID1A MUTANT PATHOGENESIS OF THE ENDOMETRIAL EPITHELIUM
Creator: Reske, Jake Jordan
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Women’s health diseases represent an understudied, widespread medical concern with historically limited treatment options. Diseases of the endometrium, the innermost lining of the uterus, are a highly prevalent public burden. Endometriosis occurs in 1 in 10 women, and endometrial cancer is the most common gynecologic malignancy in the United States. Recent advances have revealed recurrent genetic causes of endometrial diseases, including gene mutations known to play a role in cancer...
Show moreWomen’s health diseases represent an understudied, widespread medical concern with historically limited treatment options. Diseases of the endometrium, the innermost lining of the uterus, are a highly prevalent public burden. Endometriosis occurs in 1 in 10 women, and endometrial cancer is the most common gynecologic malignancy in the United States. Recent advances have revealed recurrent genetic causes of endometrial diseases, including gene mutations known to play a role in cancer development. ARID1A is one such gene that is commonly mutated in endometrial diseases, and it encodes a protein involved in regulating DNA packaging and activity in the cell nucleus within a large complex known as SWI/SNF. The focus of this dissertation is to improve our understanding of how ARID1A mutations promote endometrial diseases at multiple biological levels, with a particular focus on how disrupted chromatin regulation affects physiologically relevant gene expression. In these works, genetic engineering techniques are leveraged in mice and human cell-based models supported by public and clinical data to establish the consequences of ARID1A mutations in the endometrium and how they relate to other common genetic alterations in these diseases. These studies have revealed that ARID1A is a tumor suppressor in the endometrial epithelium, such that ARID1A loss drives cellular invasion into nearby tissue. ARID1A mutations also promote invasive metastasis and squamous metaplasia in the context of aggressive TP53 mutations. At the level of chromatin, ARID1A and SWI/SNF directly regulate endometrial epithelial identity genes through both promoter and distal enhancer chromatin interactions. Mechanistically, ARID1A mutant invasion is driven by cell identity control regions known as super-enhancers that become hyperactivated, which can be reversed pharmacologically. Moreover, ARID1A physically and genomically interacts with other nuclear chromatin regulators to govern gene activation states through variant histone regulation. These works have contributed in multiple aspects toward deciphering how ARID1A mutations promote disease in the endometrium, including pre-clinical support for using epigenetic therapies to treat invasive ARID1A mutant endometrial conditions. Future efforts will aim to further identify and understand molecular and biochemical mechanisms linking ARID1A and SWI/SNF chromatin remodeling activity to regulation of gene expression. Ongoing work seeks to explain roles of ARID1A and SWI/SNF epigenetic regulation in normal physiological processes of the endometrium, such as hormone signaling across the menstrual cycle.
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Title: GENETIC INFLUENCES ON SOCIAL COGNITION, EXECUTIVE FUNCTION, AND ASSOCIATED NEURAL NETWORKS
Creator: Blanchett, Reid
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Fundamental cognitive domains include executive function and social cognition. Both social cognition and executive functioning can be studied using neuroimaging techniques that allow direct observations to be made about brain structure and function. These techniques can also be applied to the study of brain development, revealing how circuits involved in executive function and social cognition change during important developmental periods such as infancy. Along with providing a window into...
Show moreFundamental cognitive domains include executive function and social cognition. Both social cognition and executive functioning can be studied using neuroimaging techniques that allow direct observations to be made about brain structure and function. These techniques can also be applied to the study of brain development, revealing how circuits involved in executive function and social cognition change during important developmental periods such as infancy. Along with providing a window into brain maturation, neuroimaging can be used to study cases where cognitive domains are disrupted and make comparisons to learn about typical brain development and function. For my dissertation, I have explored these cognitive domains and associated neural circuits both in typically developing individuals and in individuals with Turner syndrome, a condition caused by the full or partial absence of the second sex chromosome. First, I used a classic twin design and demonstrated relatively low narrow-sense heritability estimates for neonatal resting-state functional connectivity phenotypes. I studied both between- and within- network connectivity in neonates and demonstrated that only 6 out of 36 phenotypes had heritability estimates greater than 0.10; no estimates were statistically significant. These within- and between-network phenotypes included networks heavily recruited for social cognition and executive functioning. I also showed statistically significant associations between neonate resting-state functional connectivity phenotypes and specific demographic and medical history variables. Second, I compared structural and functional connectivity between typically developing male and female infants and infants with Turner syndrome. I saw no differences between the three groups in integrity of the superior longitudinal fasciculus or reduced connectivity between the right precentral gyrus and brain regions in the occipital and parietal regions involved with social cognition, visuospatial reasoning, and executive function. Fronto-parietal connectivity and integrity of the superior longitudinal fasciculus are disrupted in older individuals with Turner syndrome and these results suggested that these changes emerge after the first year of life. I conducted a further exploratory analysis of 54 fiber tracts and showed significant group differences that primarily reflected masculinization of white matter microstructure in TS. Other differences may have arisen due to hemizygosity of the pseudoautosomal region. Finally, I developed a browser-based online testing platform targeting domains such as executive functioning and social cognition, which are often disrupted in Turner syndrome. I then validated the battery via administration to neurotypical males and females and to adult women with Turner syndrome, who performed more poorly on tests of executive function and visuospatial reasoning. Taken together, the results presented in this dissertation contribute greatly to our understanding of the role of genetics in social cognition, executive function, and their related neural networks. These results can be further utilized in longitudinal studies of brain development and in future cognitive testing research.
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Title: MOLECULAR MECHANISMS INVOLVING PPARY IN THE PLACENTAL PATHOPHYSIOLOGY OF PREECLAMPSIA
Creator: Grimaldi, Brooke A.
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Preeclampsia (PE) is a hypertensive disorder of pregnancy that effects 5-7% of all pregnancies and is the main cause of maternal-fetal morbidity and mortality worldwide. Despite significant advancements in obstetric and neonatal care, the prevalence of PE has remained steady over the past thirty years. There is no cure for PE other than placental and fetal delivery. The exact etiology of the PE syndrome remains unclear however, maternal vascular malperfusion and placental ischemia are...
Show morePreeclampsia (PE) is a hypertensive disorder of pregnancy that effects 5-7% of all pregnancies and is the main cause of maternal-fetal morbidity and mortality worldwide. Despite significant advancements in obstetric and neonatal care, the prevalence of PE has remained steady over the past thirty years. There is no cure for PE other than placental and fetal delivery. The exact etiology of the PE syndrome remains unclear however, maternal vascular malperfusion and placental ischemia are prominent features of the PE placenta that cause abnormal trophoblast differentiation and function. PE is considered a two-stage disease due to the ischemic-diseased placenta releasing altered secretion of placental proteins that negatively impact the maternal endothelium causing hypertension and end organ damage. The placental dysfunction is as well characterized by a reduction of the transcription factor, peroxisome proliferator activated receptor γ (PPARγ) which normally promotes trophoblast differentiation and healthy placental function. This dissertation has aimed to understand the link between PPARγ-driven trophoblast dysfunction and the imbalance of secreted proteins in PE. The restoration of these disrupted pathways by PPARγ actions in the placenta could offer potential therapeutic pathways to reverse the disease, extend pregnancy duration, and dampen maternal sequalae. This dissertation has utilized a collection of first trimester and term healthy and preeclamptic placentas in addition to immortalized cell lines to understand the effect of PPARγ activation by the drug, Rosiglitazone, during preeclamptic or in vitro ischemic conditions. These studies revealed several molecules that are regulated by PPARγ in the human placenta, including the anti-angiogenic soluble fms-like tyrosine kinase 1 (sFLT1) and the cytoprotective heme oxygenase (HO1). Both proteins were restored to normal levels in PE by treatment with the PPARγ activating drug, Rosiglitazone. Furthermore, PPARγ activation improved the anti-angiogenic environment in the PE placenta as shown by increasing the pro-angiogenic and growth factor proteins: placental growth factor, fibroblast growth factor 2, follistatin and heparin-binding epidermal growth factor. Placental activation of PPARγ further restored the angiogenic balance in PE through significant reductions in the anti-angiogenic proteins, angiopoietin-2 and soluble endoglin. Using an endothelial cell model representing the maternal response to the placental protein secretion, these works revealed improved angiogenesis in endothelial cells during culture with conditioned medium from Rosiglitazone-treated PE placentas. These studies collectively show the beneficial effects of placental activation of PPARγ to improve placental and vascular function in PE. Future works should aim to understand global changes from PPARγ regulation in the human placenta and focus on compounds that hold promise to be safely used during pregnancy with the goal to improve pregnancy outcomes.
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Title: A FRAMEWORK FOR BIOLOGICAL DATA INTEGRATION AND FEATURE SELECTION IN LARGE DATA SETS
Creator: Gonzalez-Reymundez, Agustin
Date: 2021
Collection: Electronic Theses & Dissertations
Description: The increasing volume of high-dimensional biological data (omics) has intensified the discovery of thousands of biomarkers across the different fundamental components of the cell (e.g., genome, transcriptome, proteome, epigenome) and allowed the characterization of complex phenotypes (e.g., metabolome, imaginome, phenome). However, the ability to integrate omics into informative results is constantly challenged by a seemingly ever-increasing volume of data. Furthermore, huge data sizes impose...
Show moreThe increasing volume of high-dimensional biological data (omics) has intensified the discovery of thousands of biomarkers across the different fundamental components of the cell (e.g., genome, transcriptome, proteome, epigenome) and allowed the characterization of complex phenotypes (e.g., metabolome, imaginome, phenome). However, the ability to integrate omics into informative results is constantly challenged by a seemingly ever-increasing volume of data. Furthermore, huge data sizes impose a tradeoff between how complex an omic integration algorithm can be and how much data it can handle (e.g., how fast can the algorithm be scaled to integrate large data sizes). In this dissertation, we explore statistical frameworks to face the challenges of modern omic data, including the integration of high-dimensional data of large sample sizes. We have developed a novel framework of competitive analytical performance compared with existing methods but suitable for omic data reaching biobank scales (i.e., hundreds of thousands of samples and variables). We implemented this method as an R package and showed its application on two traits of a complex molecular basis: cancer and regulation of energy intake and expenditure. In chapter one, we review the technologies and methods used to generate and integrate omic data. Chapter two describes our novel method and software of omic integration, shows examples in synthetic data, and evaluates its computational and analytical performance. Chapter three presents an application of our method to reveal a novel pan-cancer classification of tumors beyond the tissue of origin, regulated by distinct sets of molecular signatures. In chapter four, we present an application of our method to integrate phenomics data and identify patterns of energy balance regulated by genomic variation. Finally, in chapter five, we offer general conclusions to the entire thesis.
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Title: The role of switch region DNA and protein factors in immunoglobulin class switch recombination
Creator: Masani, Shahnaz Firdaus
Date: 2015
Collection: Electronic Theses & Dissertations
Description: THE ROLE OF SWITCH REGION DNA AND PROTEIN FACTORS IN IMMUNOGLOBULIN CLASS SWITCH RECOMBINATIONByShahnaz Firdaus MasaniImmunoglobulin class switch recombination (CSR) is a recombination event that changes the heavy chain constant region of an antibody while maintaining the same variable region. The process of CSR alters the effector function of the antibody without altering antigen specificity, which ensures efficient pathogen clearance. Defects in CSR can result in hyper-IgM syndrome,...
Show moreTHE ROLE OF SWITCH REGION DNA AND PROTEIN FACTORS IN IMMUNOGLOBULIN CLASS SWITCH RECOMBINATIONByShahnaz Firdaus MasaniImmunoglobulin class switch recombination (CSR) is a recombination event that changes the heavy chain constant region of an antibody while maintaining the same variable region. The process of CSR alters the effector function of the antibody without altering antigen specificity, which ensures efficient pathogen clearance. Defects in CSR can result in hyper-IgM syndrome, autoimmune diseases and chromosomal translocations that result in lymphoid malignancies. Thus, understanding the mechanism of CSR will give us better insight into these diseases. The work presented in this dissertation aims to improve our understanding of the molecular mechanism of CSR.CSR occurs via a cut and paste mechanism that involves the generation and repair of DNA double stranded breaks (DSBs) within intronic regions known as switch regions. The first part of this thesis aims to identify sequence features within switch regions that are important for CSR. We show that WGCW (W=A/T) motifs, which occur at a high frequency in switch regions, are important determinants of CSR efficiency. The next study focuses on elucidating the mechanism of DSB formation during CSR. It is known that uracils within switch regions are recognized and excised by Uracil DNA Glycosylase to result in abasic sites. AP endonucleases then create a nick at abasic sites and two closely spaced nicks on both DNA strands can act as DSBs. However the identity of the AP endonuclease that creates DSBs during CSR was unclear. Our work shows that APE1, the major AP endonuclease in mammalian cells, is essential for CSR.Finally, we focused on understanding the mechanism of DSB repair by studying the role of DNA ligases in CSR. Mammals have three DNA Ligases, each of which are conventionally thought to have distinct, non-overlapping functions. By creating cells that lack one or two DNA ligases we were able to better understand their role in CSR as well as in other aspects of DNA metabolism. Our results show a remarkable level of functional redundancy between the three DNA ligases. We show that DNA Ligase I, previously thought to be essential for joining Okazaki fragments during DNA replication, is dispensable for cell viability, a number or DNA repair pathways as well as for CSR. In addition we also constructed and characterized a cell line with only one DNA ligase, Ligase III. These cells are viable and show no increased hypersensitivity to a number of DNA damaging agents. Our results show a previously unanticipated level of redundancy between DNA ligases in CSR as well as in DNA replication and repair. These studies have improved our understanding of the molecular mechanism of CSR. In addition the creation of cell lines lacking APE1 and DNA ligases will provide important tools for the study of DNA repair and replication.
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Title: A nitrogen-responsive small peptide signaling mechanism modulates plant root system architecture
Creator: Lay-Pruitt, Katerina Sibala
Date: 2021
Collection: Electronic Theses & Dissertations
Description: The plant root system changes dynamically in response to environmental cues. Plants utilize their root system for uptake of essential mineral nutrients that are heterogeneously distributed in the soil environment. Nutrient-dependent modulation of root system architecture (RSA) traits such as primary root growth, lateral root emergence, and the angles at which these roots grow allows for optimization of nutrient acquisition. Among signaling pathways by which plants may sense the availability...
Show moreThe plant root system changes dynamically in response to environmental cues. Plants utilize their root system for uptake of essential mineral nutrients that are heterogeneously distributed in the soil environment. Nutrient-dependent modulation of root system architecture (RSA) traits such as primary root growth, lateral root emergence, and the angles at which these roots grow allows for optimization of nutrient acquisition. Among signaling pathways by which plants may sense the availability of nutrients from the environment, small signaling peptide (SSP) pathways play important roles in optimizing root functions. These SSP pathways may further regulate molecular processes underlying RSA, such as the biosynthesis and transport of the major plant growth hormone, auxin. Characterization of these nutrient-responsive SSP pathways is thus of great importance and critical for understanding plant development in nutrient-poor environments. For my dissertation, I have identified and characterized a nitrogen (N)-responsive SSP pathway modulating root gravitropic response and lateral root development. Co-regulation of these RSA components by this module is proposed to prevent root outgrowth into N-poor regions and drive deeper root growth towards mobile nitrate (NO3-) resources stratified deeper in the soil profile. First, I show that a signaling pathway involving the CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) family of peptides and the CLAVATA1 (CLV1) receptor kinase, which is involved in N-dependent repression of lateral root emergence, also enhances root gravitropic response under N-limited conditions. Transcriptomic profiling of a clv1 mutant and CLE3 overexpressing lines identified Arabidopsis thaliana CENTRORADIALIS (ATC), a mobile protein previously characterized for its role in flowering regulation, as a downstream target of CLE-CLV1 signaling. Loss of ATC function significantly weakens root gravitropic response and has a moderate impact on lateral root emergence under low NO3- availability. ATC promoter activity and protein localization are also detected throughout the phloem and in the root columella cells, which are major centers for gravity sensing. Second, I demonstrate the relevance of ATC function on the molecular processes underlying root gravitropic response. While mutation in ATC does not impact gravity sensing via amyloplast sedimentation, it does inhibit the asymmetric transport of auxin needed for gravitropic bending. I determine that this occurs via the significant reduction of the PIN3 auxin efflux transporter in the vasculature and root tip of atc mutant lines. Lastly, I examine how the known roles of ATC in floral development could be implicated in root developmental processes. ATC binds to phosphatidic acid and phosphatidylserine, which is contrary to the binding capacity of its homolog FLOWERING LOCUS T (FT) to phosphatidylcholine and may contribute to its activity in N-limited environments. I also investigate the interaction of ATC and the transcription factor FD in the transcriptional regulation of PIN3. Although FD appears to have an impact on root gravitropic response, FD inhibits the expression of PIN3, suggesting potentially complex control of this gene via floral regulatory components. Taken together, the results presented in this dissertation contribute greatly to our understanding of how plant root architecture alters in response to N. These results can be further utilized in plant engineering strategies to regulate root growth in nutrient-limited soils.
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Title: Heterogeneous Thalamic Reticular Nucleus Neurons and Their Functional Role in Thalamocortical Processing
Creator: Harding-Jackson, Laura
Date: 2021
Collection: Electronic Theses & Dissertations
Description: The thalamic reticular nucleus (TRN) is an integral regulator of information flow between the thalamus and cortex. The TRN receives synaptic inputs from both cortical and thalamic regions and based upon this information it selectively inhibits thalamic activity. TRN neurons produce action potentials in two distinct modes: a fast, transient burst discharge from a hyperpolarized state, and a prolonged, tonic discharge from a relatively depolarized state. While previous studies have...
Show moreThe thalamic reticular nucleus (TRN) is an integral regulator of information flow between the thalamus and cortex. The TRN receives synaptic inputs from both cortical and thalamic regions and based upon this information it selectively inhibits thalamic activity. TRN neurons produce action potentials in two distinct modes: a fast, transient burst discharge from a hyperpolarized state, and a prolonged, tonic discharge from a relatively depolarized state. While previous studies have characterized burst discharge as a transient high frequency discharge (> 250 Hz), these electrophysiological studies reveal a highly variable range of burst frequencies (4- 342 Hz). In these studies, I aim to discover the mechanisms underlying these highly variable burst frequencies, as well as their functional role in thalamocortical processing.In chapter two, I found that bursts from TRN neurons with relatively higher frequency discharge (>100 Hz) contain more action potentials per burst. These neurons also have higher input resistances, broader action potentials, higher action potential thresholds, and larger somas. The amplitude of the T-type calcium channel-mediated low-threshold spike, which underlies the burst discharge, is positively correlated with both the burst discharge frequency and the number of action potentials per burst. I next investigated whether small conductance calcium-activated potassium channels (SK channels) could mediate the differences in burst firing rate and action potential number. Blocking SK channels increased the frequency and duration of the burst but did not increase the amplitude of the underlying T-type calcium current. Prior studies suggest that T-type calcium channels are distributed along the dendrites in TRN neurons with high frequency burst discharge. In chapter three, I examine the distribution of dendritic calcium activity within the lower frequency bursting neurons. While the calcium signal was lower in these neurons all along the dendrites, the calcium signal was evenly distributed across proximal, intermediate, and distal dendritic regions. Investigation of SK channel activity revealed significant location-specific effects. In lower frequency bursting neurons, SK channels had the greatest influence at proximal and distal locations. In higher frequency bursting neurons, SK channels had the greatest influence at proximal and intermediate dendritic locations. Heterogeneous TRN burst discharge frequencies may represent a diverse cell population with unique dendritic ion channel composition and distribution. These results may improve our understanding of the mechanisms of TRN neuron afferent synaptic integration as well as modulation of thalamocortical inhibition. In chapter four I investigate whether intrinsic properties of TRN neurons are altered in the Fmr1-KO mouse model of Fragile X Syndrome (FXS). Individuals with FXS experience a variety of comorbidities that could involve TRN function, such as altered sensory perceptions, sleep disorders, and epilepsy. Analysis of intrinsic cellular properties revealed no differences in TRN neuron properties. Further investigation of synaptic plasticity, which is an abnormal finding in several other brain regions in FXS, also revealed no pathology. These findings suggest that TRN dysfunction does not contribute to FXS pathology.
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