Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 290-308).

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Includes bibliographical references (p. 167-181).

Ribosomal protein genes are crucial for ribosome biogenesis. The ribosome itself is responsible for protein synthesis and hence cellular growth and development. Intertwining network of proteins in conjugation with cellular environment such as nutrition and growth factors collectively regulate expression of the ribosomal protein genes. DNA microarray analysis has implicated the role of 26S proteasome in transcriptional regulation of the ribosomal protein genes tying protein degradation to protein synthesis pathway. To determine the mechanism as to how the 26S proteasome promotes transcription of the ribosomal protein genes a series of experiments were performed. The results reveal that the 19S subcomplex of the 26S proteasome is recruited to the promoters of the ribosomal protein genes in a TOR (Target of Rapamycin)-dependent manner. TOR signals environmental cues and controls the expression of the ribosomal protein genes. Thus recruited 19S proteasome subcomplex promotes transcriptional initiation via facilitation of the recruitment of co-activator NuA4 (Nucleasome acetyltransferase of histone H4) complex to activator Rap1. NuA4 enhances PIC (Pre-initiation complex) formation at the core promoter, but it is not clearly understood how does it do so. Researches have identified two different forms of TBP: TAF (TBP associated factor)-dependent form of TBP and TAF-independent form of TBP. This work shows that impaired association of NuA4 interferes with TFIID recruitment, but recruits TAF-independent form of TBP to the core promoter. This recruitment of TBP is dependent on SAGA (Spt-Ada-Gcn5-acetyltransferase). Like ribosomal protein genes, antisense transcription is also enhanced by TAFs. However, it remains unknown whether NuA4 also promotes TAF-regulated antisense transcription. The results illustrate that like ribosomal protein genes, transcription of GAL10 antisense is also promoted by NuA4 HAT (histone acetyl transferase). NuA4 HAT is recruited to the 3’-end of the GAL10 coding sequence, acetylates histone H4 and promotes GAL10 antisense transcription. This work also reveals the roles of other chromatin regulatory factors in controlling antisense transcription. Collectively, these results significantly advance our current understanding of the regulatory mechanisms of ribosomal protein genes’ expression and antisense transcription. The ribosome and antisense are involved in virtually all the biological processes. Aberrant expression of the ribosomal protein genes and antisense transcripts are associated with numerous human disorders including cancers and cardiovascular diseases. Therefore, analyses of their regulatory processes provide valuable information toward understanding the etiology of numerous human diseases with potential therapeutic interventions.

abstract: The need of organs for transplantation has become an increasing medical need due to a limited donor organ supply. Many organs fail within 10 years due to acute and chronic rejection. Acute or antibody mediated rejection leads to decreased long term graft survival and increases the need for a repeat transplant. In prior work, reducing endothelial heparan sulfation and blockade of chemokine-glycosaminoglycan (GAG) interaction with Myxomavirus-derived protein, M-T7, reduced aortic and renal graft vascular inflammation and rejection. Conditional endothelial Ndst1 deficiency and inhibition of chemokine-GAG interaction reduces early allograft damage and suggest new therapeutic options for graft rejection. Here acute renal rejection was examined in grafts with conditional endothelial N-deacetylase-N-sulfotransferase-1 knockout (Ndst1-/-) and in wildtype (WT) C57Bl6/J grafts treated with saline, M-T7, antisense oligonucleotides (ASO) for Ndst1 or a scrambled ASO control. Viruses have a highly adaptive ability to evade hosts defense and immune response. The immunomodulatory proteins derived from viruses provide potential therapeutic uses to alleviate this need for organs. The Myxoma virus derived protein M-T7 is a promising therapeutic for reducing kidney transplant rejection. Orthotopic transplantations in mice are extremely difficult and costly because they require a highly trained microsurgeon. This kidney to kidney subcapsular and subcutaneous transplant model is a practical and simpler method that requires fewer mice, one kidney can be used for transplants in 6 or more mice and there is much lower morbidity, pain and mortality. Heterotopic transplantation of allografts is a simple model for preliminary testing of treatments for early inflammation, ischemia, and graft rejection. Subcapsular kidney transplantation provides a first step approach to test virus-derived proteins as potential treatments to reduce transplant rejection and inflammation. This project reports on a broadly applicable platform on which to rapidly and conveniently test new treatments for transplant rejection. This finding will significantly lower the barrier to entry for labs which are interested in translating their laboratory findings to animal models of organ transplantation which is a complex surgical procedure, and thus accelerate the bench-to-bedside translation of novel, putative treatments for transplant rejection as an initial screening tool. / Dissertation/Thesis / Masters Thesis Molecular and Cellular Biology 2020

Au cours de la dernière décennie, les avancées du séquençage à haut débit ont permis de caractériser un grand nombre d’ARN non codant et d’établir l’existence de transcrits “antisens” pour de nombreux gènes chez les mammifères. Cependant leur rôle dans le contrôle de l’expression des gènes “sens” auxquels ils sont associés est encore très mal connu. Mes travaux ont porté sur la caractérisation de certains aspects du mécanisme d’action des longs ARN non codants. Ils reposent sur le développement d’une approche de constructions indicatrices fluorescentes dont l’expression est suivie par cytométrie en flux en présence ou non d’ARN “antisens”. Cette approche a le potentiel de mettre en évidence une régulation même si elle n’est présente que dans une sous population cellulaire. Une première série d’expériences a été réalisée en expression transitoire pour bénéficier d’un contexte chromatinien simplifié. Mais dans ce cas les silencing observés sont aussi actifs sur une construction contrôle, indiquant la mise en place d’une réponse non spécifique de séquence qui évoque la réponse de type interféron. Cependant, l’expression globale des gènes cellulaire n’est pas significativement affectée, indiquant une certaine spécificité de la réponse. Parmi les voies testées ni la kinase PKR, ni la RNaseL ou la voie de l’interférence par l’ARN ne peuvent rendre compte du silencing observé. Une des caractéristiques de cette régulation est qu’elle n’affecte pas les gènes intégrés au génome mais uniquement les gènes exprimés à partir d’une construction épisomale ce qui évoque des caractéristiques souhaitables pour un mécanisme antiviral. Cependant l’ampleur de cette réponse non spécifique empêche toute étude plus approfondie d’un mécanisme spécifique s’il existe. Mes travaux se sont alors portés sur l’étude de ces mêmes constructions en clone stable dans deux contextes différents pour l’expression de l’ARN antisens ; en cis ou en trans. Dans le cas de l’expression en trans, un ARN antisens sans séquence régulatrice particulière ne permet pas la mise en place d’un silencing. Cette observation est en accord avec le faible nombre de longs ARN antisens connus pour agir en trans dans la nature. Par contre l’expression en cis d’un ARN antisens peut conduire à un silencing spécifique. Cette organisation dans laquelle les gènes « sens » et « antisens » sont situés sur le même fragment d’ADN correspond à celle majoritairement observée pour les longs ARNs antisens dans la nature (cisNAT, cis Natural Antisense Transcripts). Cependant, mes travaux montrent que le silencing observé n’est pas stable dans le temps et disparaît dès lors que la transcription antisens cesse, indiquant l’absence d’une mémoire épigénétique. Un tel mode de régulation est compatible avec une interférence transcriptionnelle dans laquelle la transcription et non le produit ARN est la cause du silencing. Par ailleurs, j’ai observé un certain nombre de cas de co-régulation du transcrit sens et antisens ce qui traduit la possibilité d’activer en cis la transcription du gène cible par le promoteur de son ARN antisens. Ce phénomène est probablement facilité par la petite taille de nos constructions, mais cette dualité de réponse est en accord avec l’absence de corrélation (positive ou négative) entre l’expression des gènes et de leur transcrits antisens. L’ensemble de mes travaux montrent la faible capacité d’un ARN antisens à induire un silencing. L’approche développée doit donc permettre de rechercher des co-activateurs du silencing, par exemple en introduisant des sites de recrutement de complexes modificateurs de la chromatine. / During the last decade next generation sequencing has allowed the characterization of a large number of non-coding RNA and to establish that a majority of mammalian genes were also transcribed in the opposite orientation. However the functional significance of this antisense transcription is currently unclear.My work focused on the characterization of the regulatory potential of long non-coding RNA. It relied on the use of fluorescent reporter constructs, the expression of which in the presence or absence of antisense RNA is analyzed by flow cytometry. . This approach has the potential to uncover a regulation mechanism even if it takes place only in a subpopulation of cells.A first series of experiments has been realized by transient expression assays in order to benefit from a simplified chromatin context. However in this case the silencing associated with antisense transcripts is also active on control constructs, indicating that at least part of the response is not sequence specific suggesting the involvement of an interferon-type response. However, cellular gene expression is not significantly affected indicating some level of specificity. Among the investigated pathways, neither the PKR kinase, nor RNaseL or RNA interference pathway can account for the observed silencing. One of this regulation attributes is that it does not affect genes integrated in the genome but only genes expressed from episomes, a selectivity which would seem appropriate for an antiviral mechanism. Nevertheless the extent of this non-specific response impedes any further study on a specific mechanism, if it operates.My work then focused on the study of these reporter constructs after integration in the genome, antisense RNA being expressed in cis or in trans.In the case of trans expression, an antisense RNA devoid of any specific regulatory sequence does not allow the setting of a silencing. This observation is consistent with the low number of long antisense RNA known to act in trans in nature.On the other side, the cis expression of an antisense RNA can lead to a specific silencing. This organization in which “sense” and “antisense” genes are located in the same DNA fragment matches with the ones mostly observed for long antisense RNA in nature (cisNAT, cis Natural Antisense Transcripts). However, my work shows that the observed silencing is not stable over time and the effects terminate once antisense transcription stops, which indicates the absence of an epigenetic memory. This mode of regulation is compatible with a transcriptional interference in which transcription – and not its RNA product - is causing the silencing. Besides, I observed a certain number of sense and antisense transcript co-regulation cases highlighting the possibility to activate the transcription of the target gene by the promoter of its antisense RNA. This phenomenon is probably facilitated by the small size of our constructs, but this duality of response is in agreement with the lack of correlation (either positive or negative) between the expression of genes and their antisense transcripts.This study shows the limited capacity of an antisense RNA to induce a silencing. The developed approach should allow the search for silencing co-activators, for instance by introducing chromatin remodeling complexes recruitment sites.

Algal blooms caused by toxigenic cyanobacterial species are an increasing economic burden globally, as high anthropogenic inputs of nitrogen and phosphorous, coupled with rising levels of atmospheric CO2, promote eutrophication and enhance bloom proliferation. Of the freshwater bloom forming species, Microcystis aeruginosa has garnered the most attention due to the production of toxic secondary metabolites known as microcystins. These cyclic peptides are potent eukaryotic protein phosphatase 1 and 2A inhibitors, and can induce hepatic damage if concentration levels reach above the World Health Organization level of 1 µg/L. Current mitigation strategies of water column disruption or by use of broad acting chemicals, are limited in their range and may cause unwanted off target effects to the surrounding biota. Antisense oligonucleotides are short single-stranded DNA polymers that hybridize with transcribed mRNA, and suppress translation of protein products through steric hindrance of ribosomes, or by RNAse H degradation of the DNA/RNA bound complex. While antisense oligonucleotide applications have proven successful in the pharmaceutical industry, their potential remains largely unexplored in environmental contexts. For this reason, we investigated the knockdown of microcystin synthetase gene cluster mcyE in M. aeruginosa. We found that ionic charge neutralization coupled with heat shock were effective chemical competence based methods for delivery, mcyE transcript abundance in cells treated with phosphodiester linked antisense oligonucleotides significantly decreased in RT-qPCR analysis, and production of intracellular microcystin significantly decreased over a 24 hour period (-1.9 fg/cell). This work demonstrates a novel proof of concept for the potential use of exogenous antisense oligonucleotides to target M. aeruginosa in harmful algal bloom occurrences.

Multiple Sclerosis (MS) is a chronic, inflammatory disease of the central nervous system. MS is caused by the immune-mediated destruction of myelin and oligodendrocytes, resulting in demyelination and neurodegeneration. The microRNA miR-145-5p has been demonstrated to be upregulated in MS lesions. Our lab has previously shown that dysregulation of miR-145-5p can interfere with oligodendrocyte differentiation in mice and that knockout of miR-145-5p protects mice from experimental autoimmune encephalomyelitis (EAE), a model for MS. The objective of this study is to determine if inhibition of miR-145-5p with an antisense oligonucleotide (ASO) is sufficient to protect mice from EAE. Female mice were induced with EAE and then treated with a control or miR-145 ASO at the onset of disease. We evaluated disease progression by monitoring clinical severity, and evaluating molecular and structural characteristics of EAE by RT-qPCR, histology, immunohistochemistry and electron microscopy. We have shown that the miR-145 ASO reduced miR-145-5p expression in the lumbar spinal cord, spleen and thymus following EAE induction. Treatment with the miR-145-5p ASO resulted in improved clinical severity of EAE, reduced neuroinflammation and increased myelination. Inhibition of miR-145-5p may represent a novel treatment for MS.

No description available.

No description available.

<p> Cationic polyelectrolytes have attracted growing attention in the field of non-viral oligonucleotides (ONs) deliveries because of their ability to bind ONs by electrostatic interactions for efficient cellular uptake. However the formation of electrostatic polymer/ONs complexes and their biological effects are still poorly understood. The relationships between polymer structure and complexation performance have not been well established. The objectives of this research are to synthesize and characterize well-defined and well-controlled cationic polyelectrolytes and to evaluate the effects of polyelectrolyte chain properties on ONs complexation. Poly(2-(dimethylamino) ethyl methacrylate) (polyDMAEMA) and its derivatives are used as the polymer candidate. A fluorescein-labeled oligonucleotide, 5 '-FGCGGAGCGTGGCAGG-3' (F: fluorescein), is used as the oligonucleotide candidate.</p> <p> Low-molecular-weight cationic polyDMAEMA samples having narrow molecular weight distribution were synthesized by living anionic polymerization (LAP) and atom transfer radical polymerization (ATRP) methods. Fully charged polyDMAEMA quats were prepared by sequential quaternization of polyDMAEMA samples, as well as by direct ATRP of the quaternized DMAEMA monomer. An aqueous GPC calibration method was first developed for the characterization of these cationic polyelectrolytes. It was found that the type of counter-ion has little effect on the hydrodynamic volume of polyDMAEMA quat. Therefore the dimethyl sulfate salt of polyDMAEMA provided a reliable calibration standard for other types of quaternized DMAEMA homopolymers.</p> <p> Cationic block copolymers of polyDMAEMA with 2-hydroxyethyl methacrylate (HEMA) and polyethylene glycol (PEG) were also prepared by ATRP. It was found that the order of monomer addition, solvent type, temperature, and molecular weight of macroinitiator have significant effects on the living feature of the polymerization. Well-controlled block copolymers were obtained when polyHEMA was used as the macro initiator.</p> <p> The complexation capability of the prepared polyelectrolytes with oligonucleotides (15 mer) was evaluated by a fluorescence technique. It was found that the complexation performance depends on polymer molecular weight, charge density, and counter-ion type, as well as polymer concentration and block composition. The polymer sample that has double molecular weight of the ONs gave the optimal complexation performance.</p> / Thesis / Master of Applied Science (MASc)