Theses and Dissertations from UMD
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New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a give thesis/dissertation in DRUM
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Item Investigation of progerin expression in non-Hutchinson-Gilford Progeria Syndrome individuals(2023) Yu, Reynold; Cao, Kan; Mount, Steve; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Hutchinson-Gilford Progerin Syndrome (HGPS) is a premature aging disease caused by a point mutation in the LMNA gene, which encodes A-type lamins. This mutation activates a cryptic splice donor in exon 11 and leads to the production of a toxic lamin variant called progerin. Interestingly, small amounts of progerin have also been found in cells and tissues of normal individuals. Here we examine the expression of progerin in publicly available RNA-seq data from normal individuals of the GTEx project. Among the 30 available tissues, progerin expression in normal individuals is highest in sun-exposed skin samples, and its expression in different tissues of the same donor is correlated. In addition, telomere shortening is significantly correlated with progerin expression. Transcriptome-wide correlation analyses suggest that the level of progerin expression is highly correlated with switches in gene isoform expression patterns, perhaps reflecting widespread isoform shifts in these samples. Differential expression analyses show that progerin expression is correlated with significant changes in the level of transcripts from genes involved in splicing regulation and a significant reduction of mitochondrial transcripts. Interestingly, 5’ splice sites whose use is correlated (either positively or negatively) with progerin expression have significantly altered frequencies of consensus trinucleotides within the core 5’ splice site. Furthermore, introns whose alternative splicing is correlated with progerin have reduced GC content. Together, our study suggests that progerin expression in normal individuals is part of a global shift in splicing patterns and provides insight into the mechanism behind these changes.Item INVESTIGATION OF ACCELERATED SKIN AGING AND PEROXISOMAL ABNORMALITIES IN HUTCHINSON-GILFORD PROGERIA SYNDROME(2022) Mao, Xiaojing; Cao, Kan; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Hutchinson-Gilford progeria syndrome (HGPS) is a detrimental premature aging disease caused by a point mutation in human LMNA gene. This mutation results in the abnormal accumulation of a truncated pre-lamin A protein called progerin. Among the drastically accelerated signs of aging in HGPS patients, severe skin phenotypes such as alopecia and sclerotic skins always develop with the disease progression. In this dissertation, I study the HGPS molecular mechanisms focusing on early skin development by differentiating patient-derived induced pluripotent stem cells (iPSCs) to a keratinocyte lineage. Interestingly, HGPS iPSCs showed an accelerated commitment to the keratinocyte lineage than the normal control. To study potential signaling pathways that accelerated skin development in HGPS, I investigated the WNT pathway components during HGPS iPSCs-keratinocytes induction. Surprisingly, despite the unaffected β-catenin activity, the expression of a critical WNT transcription factor LEF1 was diminished from an early stage in HGPS iPSCs-keratinocytes differentiation. Chromatin immunoprecipitation (ChIP) experiment further revealed strong bindings of LEF1 to early-stage epithelial development markers K8 and K18 and that the LEF1 silencing by siRNA down-regulates the K8/K18 transcription. During the iPSCs-keratinocytes differentiation, correction of HGPS mutation by Adenine base editing (ABE), while in a partial level, rescued the phenotypes for accelerated keratinocyte lineage-commitment. ABE also reduced the cell death in HGPS iPSCs-derived keratinocytes. These findings brought new insight into the molecular basis and therapeutic application for the skin abnormalities in HGPS. One important feature in both HGPS and normal aging is the elevated levels of Reactive Oxygen Species (ROS), which are generated from metabolic pathways to cause oxidative damage to macromolecules within the cells. Although peroxisomal bioreactions can generate free radicals as their byproducts, many metabolic enzymes within the peroxisomes play critical roles as ROS scavengers, particularly catalase. In this dissertation, I observed impaired peroxisomes-targeting protein trafficking, which suggested that the poorly assembled peroxisomes might cause high oxidative stress, contributing to the premature senescent phenotype in HGPS. I also investigated the ROS clearance efficiency by peroxisomal enzymes and found a significantly decreased catalase expression in HGPS. Furthermore, I evaluated the effects of two promising HGPS-treatment drugs Methylene Blue and RAD001 (Everolimus, a rapamycin analog), on catalase in HGPS fibroblasts. I found that both drugs effectively reduced cellular ROS levels. As a well-known antioxidant, MB did not affect catalase expression or activity. Interestingly, the RAD001 treatment significantly upregulated catalase activity in HGPS cells. This is the first characterization of peroxisomal function in HGPS and provides new insights into the cellular aspects of HGPS and the ongoing clinical trial.Item INVESTIGATION OF DEFECTIVE CELL SIGNALING CASCADE INVOLVED IN THE OSTEOGENESIS IN HUTCHINSON-GILFORD PROGERIA SYNDROME(2018) Choi, Ji Young; Cao, Kan; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Human bone homeostasis is maintained through constant bone remodeling, which balances bone formation by osteoblasts and bone resorption by osteoclasts. Patients with Hutchinson-Gilford progeria syndrome (HGPS) have low bone mass that manifests in a high risk of fractures and an atypical skeletal geometry, suggesting impaired bone remodeling. HGPS is a premature aging disease caused by truncated lamin A that is permanently farnesylated. The mutant lamin A is referred as progerin. Several previous clinical reports discussed abnormal skeletal development of the children with HGPS, but the molecular mechanistic study on defective osteogenesis of HGPS stem cells need to be further elucidated. The major aim of my dissertation research is to investigate dysfunction in stem cell differentiation due to aberrant cell signaling in osteoprogenitor cells that express progerin. To achieve this aim, the study demonstrates both in vitro and in vivo models of HGPS to support defective mechanism of the canonical WNT/β-catenin pathway, seemingly at the level of efficiency of nuclear import of β-catenin and impaired osteoblast differentiation. Restoring β-catenin activity rescues osteoblast differentiation and significantly improves bone mass. In particular, HGPS patient-derived induced pluripotent stem cells (iPSCs)-osteoprogenitors and primary mesenchymal stem cells (MSCs) expressing the HGPS mutant progerin display defects in osteoblast differentiation, characterized by deficits in alkaline phosphatase activity and mineralizing capacity. Mechanistic investigation reveals that canonical WNT/β-catenin pathway, a major signaling cascade involved in skeletal homeostasis, is impaired by progerin, causing a reduction in nuclear active β-catenin protein levels and reciprocal aberrant cytoplasmic accumulation which causes reduced transcriptional activity for osteogenesis. Non-farnesylation of progerin in MSCs attains higher level of active β-catenin protein expression and consequently increasing the signaling, enhancing mineralization capacity and ameliorating the defective osteogenesis. Moreover, in vivo analysis of the Zmpste24-/- HGPS mouse model demonstrates that treatment with a sclerostin-neutralizing antibody (SclAb), which targets an antagonist of canonical WNT/β-catenin signaling pathway, fully rescues the low bone mass phenotype to wild-type levels. This study implicates β-catenin signaling cascade as a therapeutic target for restoring defective skeletal microarchitecture in HGPS. Given the fundamental nature of WNT/β-catenin signaling to stem cell renewal and lineage allocation, the findings from this dissertation may provide broader inferences for the treatment options in HGPS.