Les membres de la famille des 5-phosphatases d’inositols polyphosphates et de phosphoinositides sont des enzymes caractérisées par la présence de deux domaines catalytiques conservés qui hydrolysent un phosphate en position 5 sur un noyau inositol. SKIP (Skeletal Muscle and Kidney enriched Inositol Phosphatase), également appelée Pps (Putative PI 5-phosphatase) est un des derniers membres de la famille des 5-phosphatases à avoir été découvert à ce jour. Cette enzyme hydrolyse majoritairement le phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) et le phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). Les phosphoinositides (PtdIns) représentent environ 10% des lipides membranaires et sont impliqués dans de nombreuses cascades de signalisation cellulaire conduisant, entre autres, à la prolifération, l’apoptose, la différenciation, la sécrétion, le trafic vésiculaire et la mobilité cellulaire. Des études de surexpression de SKIP en cellules tendent à montrer que cette protéine pourrait jouer un rôle de régulateur négatif dans la formation du cytosquelette d’actine et/ou dans la voie de signalisation de l’insuline. Afin d’étudier in vivo la fonction de la protéine SKIP chez la souris, nous avons décidé de générer des souris transgéniques surexprimant cette protéine de manière conditionnelle. Dans ce but, nous avons infecté des embryons murins par des lentivirus porteurs d’un transgène SKIP et avons obtenu, après réimplantation des embryons infectés dans des femelles pseudogestantes, deux lignées de souris transgéniques. Celles-ci ont ensuite été croisées avec des souris exprimant la recombinase Cre de manière ubiquitaire afin de pouvoir activer la transcription de SKIP dans l’ensemble des organes. Des expériences de Western blot, de dosage d’activité 5-phosphatase ainsi que des PCR en temps réel sont venus confirmer la présence de la protéine transgénique et de son activité catalytique. L’ensemble des expériences qui ont été menées du point de vue phénotypique tend à montrer que dans notre modèle, la surexpression de SKIP ne provoque aucune anomalie évidente du point de vue anatomique, glycémique ou immunologique. Toutefois, des expériences concernant la physiologie rénale ont été réalisées sur base des résultats d’immunohistochimie et nous ont permis de détecter une anomalie dans les mécanismes de réabsorption d’eau ainsi que dans l’expression et la phosphorylation des canaux hydriques AQP2.

The efficiency of retrovirus-mediated gene transfer can be dramatically enhanced by inducing flocculation of viruses. Addition of oppositely charged polymers to virus stocks resulted in the formation of virus-polymer complexes. The complexes specifically incorporated virus particles and only few other proteins, were not cytotoxic, did not reduce the stability of the viruses, and were large enough to sediment, delivering the viruses to the cells more rapidly than by simple diffusion. Increases in the rate of transport of viruses correlated with increases in the rate of transduction, as the polymers did not affect the efficiency of post-binding steps of transduction. The formation of virus-polymer complexes also permitted concentrating viruses and purifying the stocks from inhibitors of transduction. Pelleting of the complexes followed by resuspension of the pellet in a reduced volume of fresh cell culture medium resulted in substantial enhancement of transduction. Purified virus stocks could be used in smaller quantities than unprocessed stocks to achieve a given level of gene transfer and reduced uncertainties about the relationship between the amount of virus used and the number of genes transferred. When using high concentrations of purified viruses, the efficiency of gene transfer was dependent on the number of envelope proteins displayed on the surface of each virus particle. Viruses with a low number of envelope proteins transduced cells more efficiently than did viruses with a high number of envelope proteins, and allowed more integrations of the transgene per target cell. In contrast, when the number of envelope proteins per virus particle was high, transduction appeared to be limited by a reduction in availability of functional receptors for viruses pseudotyped with the same envelope. Taken together, this novel method for processing retrovirus stocks and a better understanding of major limitations of transduction should simplify efforts to predict the outcome of retrovirus transduction protocols and should help to increase the likelihood that human gene therapy protocols will succeed.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Feb. 2, 2010). Includes bibliographical references.

Retroviruses infect a wide range of vertebrates. The understanding of the deep history and host distribution of retroviruses remains far from complete. Retroviruses can be integrated into their host genomes and occasionally become vertically inherited genomic loci. These integrated retroviruses, known as endogenous retroviruses (ERVs), provide "molecular fossils" for past retroviral infections and are useful for studying the deep history and ecology of retroviruses. ERVs are highly abundant in vertebrate genomes. However, endogenous foamy viruses and lentiviruses appear to be extremely rare. The primary focus of the research presented here is to discover and analyze novel endogenous foamy viruses and lentiviruses in animal genomes. Foamy virus has been thought to exclusively infect three placental mammal superorders (Laurasiatheria, Euarchontoglires, and Xenarthra). The discovery of endogenous foamy viral elements (CoeEFV) in the genome of the coelacanth (Latimeria chalumnae) extends the host range of foamy viruses to fish lineages (Appendix A). I demonstrate that foamy viruses have likely codiverged with their vertebrate hosts for more than 407 million years. The discovery of CoeEFV provides evidence for an ancient marine origin of retroviruses. Endogenous foamy virus-like elements (PSFVaye) were also identified within the genome of a Malagasy lemur, the aye-aye (Daubentonia madagascariensis) (Appendix B). Phylogenetic analysis shows that PSFVaye is divergent from all currently known simian foamy viruses, suggesting a potentially ancient association between foamy viruses and primate species. Another novel endogenous foamy virus (CaEFV) was identified in the genome of the Cape golden mole (Chrysochloris asiatica). The discovery of CaEFV reveals foamy virus infection in the placental mammal superorder Afrotheria and the long-term cospeciation between foamy viruses and placental mammals (Appendix C). Lentivirus has been thought to have a relatively recent origin. Endogenous lentivirus insertions (MELV) were discovered within the genomes of some species of the Weasel family (Mustelidae) (Appendix D). I verified the presence of MELV insertions in the genomes of several species of the Lutrinae and Mustelinae subfamilies but not the Martinae subfamily, which suggests that the lentiviral invasion likely occurred between 8.8 and 11.8 million years ago. Phylogenetic analysis suggests MELV might represent a novel lentiviral group. The discovery of MELV extends the host range of lentiviruses to the Caniformia. Endogenous lentiviruses (GvaELV) were also identified in the genome of the Sunda flying lemur (Galeopterus variegatus) (Appendix E). Phylogenetic analysis shows that GvaELV is a sister group of all known lentiviruses. The discovery of GvaELV might give a clue to the early evolution of lentiviral genome architecture. In summary, the discoveries and analyses of these novel ERVs provide important insights into the deep history and ecology of foamy viruses and lentiviruses as well as the retroviruses as a whole.

Cancer is an uncontrolled growth of the body's own cells. While cancer rates increase with age, this disease afflicts both young and old. Traditional cancer therapy has had three major facets: 1) chemotherapy, which can non-specifically damage healthy tissue, 2) radiation, which can make some types of cancer more likely in the future, and 3) surgery, which can be physically traumatic and is not effective in removing unseen microtumors or circulating metastases. Immunotherapy, by its very nature, is drastically different. Immunotherapy seeks to employ cells or molecules from the immune system, in their original or a modified form, to augment, assist or replace missing elements of the native functioning immune system. Our immunotherapeutic approach has been to develop novel chimeric antigen receptors (CAR) and deliver the engineered transgene into hematopoietic stem cells (HSC). We have developed a novel single chain TCR (scTCR) in which the TCR V-alpha and V-beta segments are joined by a flexible linker. In addition to our scTCR we developed a single chain antibody molecule (scFv) to increase avidity to the tumor antigen and avoid the potential limitation of MHC restriction. Our lab has previously developed a signaling cassette based on the CD3 zeta chain, CD28 and p56Lck proteins which are prominent in the T-cell signaling pathway. The single chain specificities are linked to the signaling cassette that we have shown to function in T-cells. With specificity and signaling coupled, the chimeric antigen receptor can be transduced into hematopoietic stem cells (HSC) via a lentivirus vector. This adoptive immunotherapy can potentially eliminate malignant cells or supplement traditional therapies by providing engineered specificity and a useful method to transfer and expand tumor specific T-cells. We show in this study that the CAR can be delivered effectively to HSC and that the introduced transgene is expressed in multiple cell lineages. We also have developed a novel method of increasing lentiviral transduction efficiency. Both transduced fraction of cells and overall expression can be increased by proper timing and coordination of cell growth, cell cycle phase, vector addition and treatment with heat shock.

Lentiviral gene therapy has held great promise for treating a wide range of neurological disorders due to its ability to stably integrate into the genome of nondividing cells like neurons, in addition to dividing cells. The nervous system is a complex and highly heterogeneous system, and while a therapeutic intervention may have beneficial effects in one population of cells it may have severe side effects in another. For this reason, specific targeting of lentiviral vectors is crucial for their ultimate utility for research and clinical research use. Two different approaches for focusing the targeting of lentiviral vectors were employed in these studies. The first method involved assessing the effects of vector production strategies on the resulting virus’s tropism both in vivo and in vitro. The changes in vector transduction were determined via flow cytometry on cells in culture and immunohistochemistry following brain injections. Results from these experiments suggest that while the production conditions do impact the vectors efficacy, there is not a distinct effect on their tropism. A unique characteristic of retroviral and lentiviral vectors is their capacity for being pseudotyped, conferring a new tropism on the vector. Native tropisms are generally not specific beyond very broad cell types, which may not be sufficient for all applications. In this case, chimeric targeting molecules can provide an even more refined targeting profile compared to native pseudotypes. The second approach utilizes novel chimeric glycoproteins made from nerve growth factor and the vesicular stomatitis virus glycoprotein. These chimeras are designed to pseudotype lentiviral vectors to target nociceptive sensory neurons for a variety of disorders. While these chimeras were successfully produced as protein, they were misfolded and sequestered in the endoplasmic reticulum and therefore unavailable to produce lentivirus. While neither strategy was completely successful, they do provide interesting information for the design and creation of lentiviral vectors. This research shows that small differences in the steps followed as part of a lentivirus production protocol can greatly impact the resulting vectors efficacy. It also shows that while VSV has been used to create chimeric glycoproteins, not all targeting molecules are suitable for this purpose.

Gliomas are the commonest primary tumours of the brain and glioblastoma multiforme (GBM) represents more than 50% of this group. GBM remains a neurosurgical conundrum since patients often succumb to the disease within one year. Surgery followed by radiation and medical regimens over the years have had minimal impact on the prognosis of patients with this cancer and hence, alternative and novel therapeutic modalities are required if the survival of patients with this disease is to be significantly improved. The ATM gene, which is mutated in the disease ataxia-telangiectasia (A-T), is implicated in response to radiation-induced DNA damage, leading to profound radiosensitivity. By reducing the levels of ATM in the radioresistant GBM cells through antisense or RNA interference (RNAi) technology delivered by lentiviruses, malignant GBM tumour cells were successfully sensitised to radiation treatment. In conjunction with surgery, this strategy will provide an enhanced therapeutic intervention especially in the case of GBM where the tumour is untreatable. In this thesis, analysis of the D-3-Phosphoglycerate dehydrogenase promoter in a GBM cell line as well as the development of a novel rat model for GBM using a bioluminescent F98 cell line will also be presented.

A cohort study was conducted in order to measure the effect of the chronic indurative lymphocytic mastitis caused by the South African strain ofMaedi visna virus (SA-OMVV) on the pre-weaning growth of lambs born of naturally infected and uninfected ewes kept under similar conditions. 50 naturally infected ewes and 40 controls from an MVV-free source were purchased and kept separately. All ewes were of the same breed - the Dorper¬and 3 to 4 years old. From the adaptation period, through mating, pregnancy and lactation periods they were monitored for MVV antibodies and managed under similar conditions. The lambs were weighed at birth and thereafter every two weeks until the age of 90 days, when they were weaned. The ewes were slaughtered, their udders examined histologically and the lesions were assessed by counting typical lymphocytic follicles. Although the observed values indicated a correlation between the number of follicles in the udder and the reduction in the growth rate of the lambs, this was not statistically significant. Similarly, despite higher counts of lymphoid follicles in the udder of sero-positive ewes as compared to sero-negatives and the observed lower ewe productivity indexes (EPI) in infected ewes, no statistically significant differences were found in the EPI of ewes in different follicle categories. The present study was a first attempt to evaluate the effect of the SA-OMVV infection on sheep productivity in South Africa. / Dissertation (MSc)--University of Pretoria, 2000. / Production Animal Studies / Unrestricted

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