Thesis (Ph.D.) -- University of Rhode Island, 2008. / Typescript. Includes bibliographical references (leaves 125-134).

In the HIV viral integration procedure, 3’-processing of the viral DNA by the integrase enzyme is an essential first step which is followed by the integration of viral DNA into the host genome. In 3’-processing, the integrase cleaves the backbone of the DNA substrate on the 3’ end of a conserved CA dinucleotide motif and inserts a helix between the two DNA strands, forcing them apart (Hare, S., 2012). Our study confirms that the presence of a G-amino group is crucial for 3’-processing. Substituting inosine for G in the CA step removes this amino group and results in loss of enzyme activity. Further work showed that the presence of a terminal duplex segment is not required for 3’-processing. Additional substrate modifications are studied in order to evaluate the actual importance of the CA step.

Despite years of research, an effective therapy for treatment of ischemic stroke has yet to be found. Survivors of stroke may suffer debilitating and permanent motor dysfunction for the remainder of their lives. Current treatments are limited to physical therapy and tissue plasminogen factor (tPA), a thrombolytic medication with a time- window of efficacy up to only three hours after symptom onset. Clinical studies and animal models have shown that partial recovery of motor function occurs with or without pharmacological interventions due to adaptive plasticity and reorganization in the brain. The precise mechanisms, though unclear, have become a major focus of stroke research. In the following study, we investigated inosine, a naturally occurring purine nucleoside that stimulates axonal growth, as a potential long-term stroke treatment. Following controlled cortical ischemia in the motor cortex of rhesus monkeys, recovery of dominant hand function was monitored through NHP Upper Extremity Motor Dysfunction Scale ratings for two weeks post-operation and through performance on two motor tasks, the Hand Dexterity Task (HDT) and the Digit Coordination Task (DCT). Results of cage- side assessment ratings demonstrated a trend towards greater recovery in the group treated with inosine for functional strength in the dominant hand on 12-14 days after surgery. The suggested trend is enough evidence to pursue research on the use of inosine as a therapeutic agent in post-stroke functional recovery.

RNA editing by adenosine deamination is catalyzed by members of an enzyme family known as adenosine deaminases that act on RNA (ADARs). ADARs can change the structure of RNA by changing an AU base-pair to an IU mismatch. This frequently modifies the function of the encoded protein, and an emerging theme associated with A-to-I mRNA editing is that tissues often regulate the ratio of proteins expressed from edited and unedited mRNAs to fine-tune cellular responses and functions. In mammals, pre-mRNA of receptor proteins involved in neurotransmission, including serotonin receptors and glutamate receptors, are edited. Currently, only a limited number of human ADAR substrates are known, whereas indirect evidence suggests a substantial fraction of all pre-mRNAs being affected. To identify RNAs containing inosine residues, this study used a multi step approach; including (1) inosine-specific base cleavage and RNase T1 digestion, (2) purification of polyA-tailed mRNA, (3) RT w/ T7-polydT primer, (4) probe synthesis and microarray analysis. Using this method it is possible to identify novel targets of A to I editing. Approximately 100 genes showed a significant decrease in two arrays. Future analysis of these targets should reveal the biomedical significance of A-to-I editing.

Thesis (M.S.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains vii, 51 p. : ill. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--Virginia Commonwealth University, 2008. / Prepared for: School of Pharmacy. Bibliography: leaves 170-178. Available online via the internet.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2003. / Vita. Bibliography: 69-74.

No description available.

Les éléments Alu sont les retrotransposons les plus prolifiques chez l’humain avec plus d’1 million de copies occupant plus de 10% du génome. Afin de contrecarrer l’expansion des rétro-éléments, les organismes ont développés différents mécanismes pour préserver l’intégrité de leurs génomes. Le plus proéminent, également utilisé pour lutter contre la réinsertion d’ADN viral dans le génome hôte, est l’édition de l’ARN. Chez les mammifères, la plus courante est la déamination de l’adénine en inosine catalysée par la famille de protéine ADAR dont Les principales cibles sont les éléments Alu chez l’humain. L’édition des éléments Alu conduit à leur séquestration dans le noyau des cellules, mute leurs promoteurs internes, cible de l’ARN polymérase III (POLIII), et leurs queues poly-A, prévenant ainsi leur future rétrotransposition. Dans la première partie de cette étude, l’analyse de données de séquençage haut-débit révèle que ~40% des éléments Alu sont reconnus par POLIII, qu’ils sont présents en tant que petits ARN dans le cytoplasme et le noyau des cellules, que certain d’entre eux sont associés à la chromatine, et que la transcription des éléments Alu est un phénomène courant dans les tissus somatiques qui concorde avec l’expression d’éléments LINE1 fonctionnels. Ceci suggère que la rétrotransposition peut être un mécanisme normal dans la plupart des tissus humains. Enfin, l’analyse de l’expression des éléments Alu et LINE1 chez la souris montre que la transcription de rétrotransposons n’est pas spécifique de l’humain. Dans la seconde partie de cette étude, une nouvelle méthode a été développée pour explorer l’impact de l’édition de l’ARN sur le transcriptome en identifiant les ARN édités par séquençage haut-débit. Dans un premier temps, un anticorps ciblant ADAR a été utilisé pour extraire les ARN associés aux protéines de l’édition. Cette méthode n’étant pas suffisamment efficace, une autre stratégie, qui extrait directement les ARN contenant de l’inosine, a été développée : dans un premier temps, l’ARN est fixé à des billes magnétiques par leurs extrémités 3’, ensuite, les billes sont traitées au glyoxal/acide borique et à la RNAse T1 pour libérer la région 5’ des ARN contenant une ou plusieurs inosines, et enfin, les ARN libérés sont séquencés par séquençage haut débit. En utilisant cette méthode, 1822 sites d’éditions ont été identifiés dans l’ARN de cerveau de souris, incluant 28 nouveaux sites présents dans des séquences codantes qui conduisent à des mutations non-synonymes des futures protéines. Des sites d’éditions ont aussi été observés pour la première fois dans les ARN ribosomaux, les snoRNA et les snRNA. / The Alu repeats comprise more than 10% of the human genome. They spread in the genome by retrotransposition. As a response to this invasion, organisms developed mechanisms to preserve the integrity of their genome, such as RNA editing. The most abundant type of editing in mammals is A-to-I editing where the ADAR proteins transform adenosine into inosine and targets mainly Alu elements in human. Editing of the Alu elements leads to their sequestration in the nucleus and mutates their internal POLIII promoter and their poly-A tail, thus preventing their subsequent transposition. In the first part of this study, we challenged the view that Alu elements are dormant occupant of the genome by characterizing their activity. Deep-sequencing data analyses revealed that ~40% of Alu elements can bind POLIII, they present a definite localization in the cell and associate with chromatin and polysomes, and that Alu elements transcription is a widespread phenomenon in normal tissues which correlates with functional LINE1 elements expression. This suggested that Alu element retrotransposition may be a natural mechanism in most normal human tissues. Further analyses showed that SINE and LINE expression in somatic tissues was not exclusive to human but also occurs in mouse. Finally, attempts were made to identify tissue specific insertions in the human genome resulting from retrotransposition events. In the second part of this study, a new method was developed to understand the full impact of RNA editing on transcriptomes by characterizing the edited RNA in a high-throughput fashion. First, immunoprecipitation was attempted to pull-down RNA associated with the editing enzymes ADARs. Since this method was inefficient, another approach purifying directly the edited RNA was developed. First, the RNA was sequestered on magnetic beads. Then an inosine specific cleavage based on RNAseT1 treatment of RNA protected with glyoxal and borate allowed the separation of the edited RNA from the total RNA. Finally, deep sequencing was used to identify edited RNA. 1,822 editing sites were found in mouse brain RNA by this method, including 28 new editing sites modifying the coding sequences of genes and editing in rRNA, snoRNA and snRNA which were never observed before.

The enzyme inosine triphosphate pyrophosphatase (ITPase) is responsible for controlling the levels of the by-products guanosine monophosphate (GMP) and adenosine monophosphate (AMP) through their precursor inosine monophosphate (IMP). ). Human ITPase consists of a 194-amino acid homodimer which relies upon either an Mg2+ ion or a Mn2+ ion for catalytic activity, and orthologs of this protein have been found in many different organisms. The purpose of this project was to try out methods learned throughout the education and to use this knowledge to gather new data about the human protein inosine triphosphate pyrophosphatase (ITPase). The protein was expressed in BL21/DE3 cells from a pre-made vector. Experiments performed during this project include secondary- and tertiary stability measurements, tryptophan fluorescence spectra, binding curve and thermic stability to ITPase with ANS and methotrexate. The Tm-value of human ITPase was examined with Trp-Fluorescence, ANS-fluorescence and Near-UV and Far-UV circular dichroism (CD). The stability of ITPase monitored by Near-UV as well as Far-UV coincides, indicating that secondary- and tertiary-unfolding occur simultaneously without any intermediates. The results of Trp-fluorescence showed that the tryptophans were already exposed and thus it did not yield a reliable result. The binding properties of ANS and MTX to ITPase were also examined.