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Gene Therapy For Amyotrophic Lateral Sclerosis: An Aav Mediated Rnai Approach For Autosomal Dominant C9orf72 Associated Als, Gabriela Toro Mar 2019

Gene Therapy For Amyotrophic Lateral Sclerosis: An Aav Mediated Rnai Approach For Autosomal Dominant C9orf72 Associated Als, Gabriela Toro

GSBS Dissertations and Theses

Amyotrophic lateral sclerosis (ALS) is a terminal neurodegenerative disease that affects motor neurons causing progressive muscle weakness and respiratory failure. In 2011, the presence of a hexanucleotide repeat expansion within chromosome 9 open reading frame 72(C9ORF72) was identified in ALS patient samples, becoming the major known genetic cause for ALS and frontotemporal dementia (FTD). Carriers of this mutation present reduced levels of C9ORF72 mRNA, RNA foci produced by the aggregating expansion and toxic dipeptides generated through repeat-associated non-ATG translation. These findings have led to multiple hypotheses on the pathogenesis of C9ORF72: 1) Haploinsufficiency, 2) RNA gain-of-function, 3) RAN Translation ...


The Mechanistic Role And Therapeutic Potential Of Microrna-122 In Alcoholic Liver Disease: A Dissertation, Abhishek Satishchandran Apr 2016

The Mechanistic Role And Therapeutic Potential Of Microrna-122 In Alcoholic Liver Disease: A Dissertation, Abhishek Satishchandran

GSBS Dissertations and Theses

Chronic alcohol use results in accelerated liver injury, leading to alcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma. However, due to the complex nature of this disease process, a central, druggable mechanism has remained elusive. microRNAs are potent post-transcriptional regulators of gene expression. A single miRNA has the ability to regulate hundreds of pathways simultaneously, defining cellular fate and function. microRNA-122 (miR-122), the most abundant miRNA in hepatocytes, has a demonstrated role as an tumor suppressor, regulator of hepatocyte metabolism, and hepatic differentiation.

In this dissertation I demonstrate the role of miR-122 on alcoholic liver disease (ALD) pathogenesis over four parts. In ...


Single-Molecule Imaging Reveals That Argonaute Re-Shapes The Properties Of Its Nucleic Acid Guides: A Dissertation, William E. Salomon Dec 2015

Single-Molecule Imaging Reveals That Argonaute Re-Shapes The Properties Of Its Nucleic Acid Guides: A Dissertation, William E. Salomon

GSBS Dissertations and Theses

Small RNA silencing pathways regulate development, viral defense, and genomic integrity in all kingdoms of life. An Argonaute (Ago) protein, guided by a tightly bound, small RNA or DNA, lies at the core of these pathways. Argonaute uses its small RNA or DNA to find its target sequences, which it either cleaves or stably binds, acting as a binding scaffold for other proteins. We used Co-localization Single-Molecule Spectroscopy (CoSMoS) to analyze target binding and cleavage by Ago and its guide. We find that both eukaryotic and prokaryotic Argonaute proteins re-shape the fundamental properties of RNA:RNA, RNA:DNA, and DNA ...


Subtle Controllers: Micrornas Drive Pancreatic Tumorigenesis And Progression: A Dissertation, Brian J. Quattrochi Apr 2015

Subtle Controllers: Micrornas Drive Pancreatic Tumorigenesis And Progression: A Dissertation, Brian J. Quattrochi

GSBS Dissertations and Theses

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal malignancies in the United States, with an average five-year survival rate of just 6.7%. One unifying aspect of PDAC is mutational activation of the KRAS oncogene, which occurs in over 90% of PDAC. Therefore, inhibiting KRAS function is likely an effective therapeutic strategy for this disease, and current research in our lab and others is focused on identifying downstream effectors of KRAS signaling that may be therapeutic targets. miRNAs are powerful regulators of gene expression that can behave as oncogenes or tumor suppressors. Dysregulation of miRNA expression is commonly observed ...


Microrna Markers Of Acetaminophen Toxicity: A Master's Thesis, Jeanine Ward Jul 2012

Microrna Markers Of Acetaminophen Toxicity: A Master's Thesis, Jeanine Ward

GSBS Dissertations and Theses

Background
To investigate plasma microRNA (miRNA) profiles indicative of hepatotoxicity in the setting of lethal acetaminophen (APAP) toxicity in mice.

Methods
Using plasma from APAP poisoned mice, either lethally (500 mg/kg) or sublethally (150 mg/kg) dosed, we screened commercially available murine microRNA libraries (SABiosciences, Qiagen Sciences, MD) to evaluate for unique miRNA profiles between these two dosing parameters.

Results
We distinguished numerous, unique plasma miRNAs both up- and down-regulated in lethally compared to sublethally dosed mice. Of note, many of the greatest up- and down-regulated miRNAs, included, but were not limited to, 574-5p, 466g, 466f-3p, 375, 29c, and ...


Understanding Small Rna Formation In Drosophila Melanogaster: A Dissertation, Elif Sarinay Cenik Jul 2012

Understanding Small Rna Formation In Drosophila Melanogaster: A Dissertation, Elif Sarinay Cenik

GSBS Dissertations and Theses

Drosophila Dicer-2 generates small interfering RNAs (siRNAs) from long double-stranded RNA (dsRNA), whereas Dicer-1 produces microRNAs from premicroRNA. My thesis focuses on the functional characteristics of two Drosophila Dicers that makes them specific for their biological substrates. We found that RNA binding protein partners of Dicers and two small molecules, ATP and phosphate are key in regulating Drosophila Dicers’ specificity. Without any additional factor, recombinant Dicer-2 cleaves pre-miRNA, but its product is shorter than the authentic miRNA. However, the protein R2D2 and inorganic phosphate block pre-miRNA processing by Dicer-2. In contrast, Dicer-1 is inherently capable of processing the substrates of ...


Analysis Of Integrin Α6Β4 Function In Breast Carcinoma: A Dissertation, Kristin D. Gerson Apr 2012

Analysis Of Integrin Α6Β4 Function In Breast Carcinoma: A Dissertation, Kristin D. Gerson

GSBS Dissertations and Theses

The development and survival of multicellular organisms depends upon the ability of cells to move. Embryogenesis, immune surveillance, wound healing, and metastatic disease are all processes that necessitate effective cellular locomotion. Central to the process of cell motility is the family of integrins, transmembrane cell surface receptors that mediate stable adhesions between cells and their extracellular environment. Many human diseases are associated with aberrant integrin function. Carcinoma cells in particular can hijack integrins, harnessing their mechanical and signaling potential to propagate cell invasion and metastatic disease, one example being integrin α6β4. This integrin, often referred to simply as β4, is ...


Dissecting Somatic Cell Reprogramming By Micrornas And Small Molecules: A Dissertation, Zhonghan Li Mar 2012

Dissecting Somatic Cell Reprogramming By Micrornas And Small Molecules: A Dissertation, Zhonghan Li

GSBS Dissertations and Theses

Somatic cells could be reprogrammed into an ES-like state called induced pluripotent stem cells (iPSCs) by expression of four transcriptional factors: Oct4, Sox2, Klf4 and cMyc. iPSCs have full potentials to generate cells of all lineages and have become a valuable tool to understand human development and disease pathogenesis. However, reprogramming process suffers from extremely low efficiency and the molecular mechanism remains poorly understood.

This dissertation is focused on studying the role of small non-coding RNAs (microRNAs) and kinases during the reprogramming process in order to understand how it is regulated and why only a small percentage of cells could ...


Cooperativity In Mammalian Rna Silencing: A Dissertation, Jennifer A. Broderick Jul 2011

Cooperativity In Mammalian Rna Silencing: A Dissertation, Jennifer A. Broderick

GSBS Dissertations and Theses

Argonaute proteins are the core component of an RNA silencing complex. The human genome encodes four Argonaute paralogs –Ago1, Ago2, Ago3 and Ago4– proteins that are guided to target mRNAs by microRNAs. More than 500 miRNAs are conserved between mammals, and each microRNA can repress hundreds of genes, regulating almost every cellular process. We still do not fully understand the molecular mechanisms by which miRNAs regulate gene expression. Although we understand many aspects of microRNA biogenesis and formation of the RNA-induced silencing complex, much less is known about the subsequent steps leading to target mRNA regulation.

Mammalian microRNAs rarely have ...


The Role Of Mir-21 And Mir-31 In Cellular Responses Mediated By Tgf-Β: A Dissertation, Charisa L. Cottonham May 2011

The Role Of Mir-21 And Mir-31 In Cellular Responses Mediated By Tgf-Β: A Dissertation, Charisa L. Cottonham

GSBS Dissertations and Theses

The function of transforming growth factor β (TGF-β) in cancer is notoriously complex. Initially TGF-β limits tumorigenesis, but at later stages in tumor progression TGF-β promotes the malignant spread of tumor cells. Past studies to understand the pro-metastasis utility of TGF-β centered upon its ability to regulate protein-coding genes. Recently, a small class of non-coding RNAs known as microRNAs (miRNAs) emerged as novel posttranscriptional regulators of gene expression. The significance of miRNA function in cellular processes from embryonic development to the maintenance of homeostasis in adult tissues is becoming increasingly clear. Also apparent is the strong association between aberrant miRNA ...


Cooperating Events In Core Binding Factor Leukemia Development: A Dissertation, Dmitri Madera Mar 2011

Cooperating Events In Core Binding Factor Leukemia Development: A Dissertation, Dmitri Madera

GSBS Dissertations and Theses

Leukemia is a hematopoietic cancer that is characterized by the abnormal differentiation and proliferation of hematopoietic cells. It is ranked 7th by death rate among cancer types in USA, even though it is not one of the top 10 cancers by incidence (USCS, 2010). This indicates an urgent need for more effective treatment strategies. In order to design the new ways of prevention and treatment of leukemia, it is important to understand the molecular mechanisms involved in development of the disease.

In this study, we investigated mechanisms involved in the development of acute myeloid leukemia (AML) that is associated ...


Endogenous Small Rnas In The Drosophila Soma: A Dissertation, Megha Ghildiyal Mar 2010

Endogenous Small Rnas In The Drosophila Soma: A Dissertation, Megha Ghildiyal

GSBS Dissertations and Theses

Since the discovery in 1993 of the first small silencing RNA, a dizzying number of small RNAs have been identified, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). These classes differ in their biogenesis, modes of target regulation and in the biological pathways they regulate.

Historically, siRNAs were believed to arise only from exogenous double-stranded RNA triggers in organisms lacking RNA-dependent RNA polymerases. However, the discovery of endogenous siRNAs in flies expanded the biological significance of siRNAs beyond viral defense. By high throughput sequencing we identified Drosophila endosiRNAs as 21 nt small RNAs, bearing a 2´-O ...


Functional Analysis Of Microrna-10b In Breast Carcinoma: A Dissertation, Charlotte M. Harwood Moriarty May 2009

Functional Analysis Of Microrna-10b In Breast Carcinoma: A Dissertation, Charlotte M. Harwood Moriarty

GSBS Dissertations and Theses

MicroRNAs (miRNAs) represent a class of small noncoding RNAs that regulate gene expression. Recent studies have shown that miRNAs are mis-expressed in various human cancers and that some miRNAs have the potential to act as tumor suppressors or oncogenes. MiR-10b is one miRNA that has been shown to be deregulated in breast cancer. However, current findings regarding miR-10b’s role in breast cancer are controversial. MiR-10b was originally reported to be downregulated in breast cancer compared to normal breast tissue. Subsequently, miR-10b was argued to be upregulated in metastatic breast cancer cell lines, acting as a potent pro-metastatic agent via ...


Delineating The C. Elegans Microrna Regulatory Network: A Dissertation, Natalia Julia Martinez Apr 2009

Delineating The C. Elegans Microrna Regulatory Network: A Dissertation, Natalia Julia Martinez

GSBS Dissertations and Theses

Metazoan genomes contain thousands of protein-coding and non-coding RNA genes, most of which are differentially expressed, i.e., at different locations, at different times during development, or in response to environmental signals. Differential gene expression is achieved through complex regulatory networks that are controlled in part by two types of trans-regulators: transcription factors (TFs) and microRNAs (miRNAs). TFs bind to cis-regulatory DNA elements that are often located in or near their target genes, while microRNAs hybridize to cis-regulatory RNA elements mostly located in the 3’ untranslated region (3’UTR) of their target mRNAs.

My work in the ...


Differentially Expressed Micrornas Act As Inhibitors Of Bdnf In Prefrontal Cortex - Implications For Schizophrenia: A Dissertation, Nikolaos Mellios Mar 2009

Differentially Expressed Micrornas Act As Inhibitors Of Bdnf In Prefrontal Cortex - Implications For Schizophrenia: A Dissertation, Nikolaos Mellios

GSBS Dissertations and Theses

During my thesis work I studied the expression and potential function of brain expressed microRNAs (miRNAs) in human prefrontal cortex (PFC). Initially, I used combinatorial computational analysis and microarray data to identify miRNAs that are predicted with high probability to target the human Brain Derived Neurotrophic Factor (BDNF) 3’ Untranslated Region (3’UTR) and are expressed in moderate to high levels in adult human prefrontal cortex. A subset of 10 miRNAs segregating into 5 different miRNA families (miR-30a-d, miR-103/107, miR-16/195, miR-191 and miR-495) met the above criteria. I then designed a protocol to detect these miRNAs with Locked ...


Small Rna Sorting In Drosophila Produces Chemically Distinct Functional Rna-Protein Complexes: A Dissertation, Michael D. Horwich Jun 2008

Small Rna Sorting In Drosophila Produces Chemically Distinct Functional Rna-Protein Complexes: A Dissertation, Michael D. Horwich

GSBS Dissertations and Theses

Small interfering RNAs (siRNAs), microRNAs (miRNAs), and piRNAs (piRNA) are conserved classes of small single-stranded ~21-30 nucleotide (nt) RNA guides that repress eukaryotic gene expression using distinct RNA Induced Silencing Complexes (RISCs). At its core, RISC is composed of a single-stranded small RNA guide bound to a member of the Argonaute protein family, which together bind and repress complementary target RNA. miRNAs target protein coding mRNAs—a function essential for normal development and broadly involved in pathways of human disease; small interfering RNAs (siRNA) defend against viruses, but can also be engineered to direct experimental or therapeutic gene silencing; piwi ...


Dissecting Small Rna Loading Pathway In Drosophila Melanogaster: A Dissertation, Tingting Du Jan 2008

Dissecting Small Rna Loading Pathway In Drosophila Melanogaster: A Dissertation, Tingting Du

GSBS Dissertations and Theses

In the preceding chapters, I have discussed my doctoral research on studying the siRNA loading pathway in Drosophila using both biochemical and genetic approaches. We established a gel shift system to identify the intermediate complexes formed during siRNA loading. We detected at least three complexes, named complex B, RISC loading complex (RLC) and RISC. Using kinetic modeling, we determined that the siRNA enters complex B and RLC early during assembly when it remains double-stranded, and then matures in RISC to generate Argonaute bearing only the single-stranded guide. We further characterized the three complexes. We showed that complex B comprises Dcr-1 ...


Understanding Assembly Of Ago2 Risc: The Rnai Enzyme: A Dissertation, Christian B. Matranga Sep 2007

Understanding Assembly Of Ago2 Risc: The Rnai Enzyme: A Dissertation, Christian B. Matranga

GSBS Dissertations and Theses

In 1990, Richard Jorgensen’s lab initiated a study to test if they could create a more vivid color petunia (Napoli et al. 1990). Their plan was to transform plants with the chalcone synthase transgene––the predicted rate limiting factor in the production of purple pigmentation. Much to their surprise, the transgenic plants, as well as their progeny, displayed a great reduction in pigmentation. This loss of endogenous function was termed “cosuppression” and it was thought that sequence-specific repression resulted from over-expression of the homologous transgene sequence. In 1998, Andrew Fire and Craig Mello described a phenomenon in which double ...


Interaction Of A Mammalian Virus With Host Rna Silencing Pathways: A Dissertation, Bradford Michael Stadler Mar 2007

Interaction Of A Mammalian Virus With Host Rna Silencing Pathways: A Dissertation, Bradford Michael Stadler

GSBS Dissertations and Theses

In the complex relationships of mammalian viruses with their hosts, it is currently unclear as to what role RNA silencing pathways play during the course of infection. RNA silencing-based immunity is the cornerstone of plant and invertebrate defense against viral pathogens, and examples of host defense mechanisms and numerous viral counterdefense mechanisms exist. Recent studies indicate that RNA silencing might also play an active role in the context of a mammalian virus infection. We show here that a mammalian virus, human adenovirus, interacts with RNA silencing pathways during infection, as the virus produces microRNAs (miRNAs) and regulates the expression of ...