No description available.

No description available.

No description available.

Programmed cell death is a widespread phenomenon in the developing nervous system. During early development, neurons are initially produced in excess and up to 70% of them are eliminated in later stages of development, during a period of synapse formation with their targets. However, the mechanisms that initiate the death of neurons are not clear. In the avian ciliary ganglion, neurons go through the period of target-dependent cell loss between E8 and E14; however, almost all neurons in the ganglion are prevented from dying by the chronic in ovo treatment with α7-nAChRs specific antagonists, α- bungarotoxin or MLA. Since α7-nAChRs are implicated in the cell death of ciliary ganglion neurons, I tested whether the activation of these receptors directly on the ciliary ganglion neurons facilitates cell death by inducing large increases in intracellular Ca2+. I found that the ciliary ganglion neurons are heterogeneous with respect to their surface α7-nAChR density and, as a result, activation of these receptors by nicotine leads to large increases in [Ca2+]i in some neurons but not in others. Furthermore, immature E8 neurons exhibit slower rates of Ca2+ decay after nicotine stimulation than E13 neurons, suggesting that E8 neurons do not clear [Ca2+]i efficiently and could be more susceptible to Ca2+ overload. Expressing the αbtx that is tethered to the cell membrane via the glycosylphosphatidylinositol anchor (GPIαbtx) in the ciliary ganglion neurons inhibits the increases in [Ca2+]i induced by nicotine through α7-nAChRs specifically. This cellautonomous inhibition of α7-nAChRs prevents cell death of ciliary and choroid neurons. For this to happen, GPIαbtx must be expressed in neurons; the expression of this construct in the surrounding non-neural tissue does not prevent neuronal loss in the ciliary ganglion. Later in development, α7-nAChRs are prevented from inducing cell death by the chicken PSCA molecule that is significantly upregulated in the ciliary ganglion between E8 and E15. The chicken PSCA is neuronal specific molecule that belongs to the Ly-6/neurotoxin superfamily that includes αbtx and lynx1 and compared to other tissues, it is highly expressed in the ciliary ganglion. The expression of the PSCA mRNA in tissues correlates with the expression of α7-nAChR mRNA, suggesting that PSCA modulates the signaling via these receptors. In fact, overexpressing the PSCA in the ciliary ganglion neurons prevents nicotine-induced increases in [Ca2+]i through α7- nAChRs. Misexpressing the PSCA in E8 ciliary ganglion prevents choroid but not ciliary neurons from dying. Therefore afferent inputs can induce cell death by activation of α7- nAChRs in the developing ciliary ganglion by increasing the [Ca2+]i over the threshold for cell death. Upregulation of endogenous prototoxins, such as PSCA, opposes the large increases in [Ca2+]i via α7-nAChRs and prevents these channels from facilitating cell death after the final numbers of neurons have been established. These results indicate that the control of cell death is more complex than originally proposed by the neurotrophic hypothesis and present the mechanism by which cell death in the developing ciliary ganglion is regulated, thus, further highlighting the importance of non-traditional roles of α7-nAChRs during the development of the nervous system.

Après intégration dans le génome cellulaire de l’hôte, l’expression des rétrovirus dépend d’éléments agissant en cis localisés dans la longue répétition terminale 5’ (LTR5’) et la région leader, de facteurs de transcription cellulaires et viraux agissant en trans ainsi que de l’organisation chromatinienne du provirus intégré. Notre laboratoire a précédemment identifié dans le génome du rétrovirus HIV-1 (Human Immunodeficiency Virus type 1) une région intragénique importante (nt 4079-6026, où nt +1 est le début de U3 dans le LTR5’) composée du fragment 5103, du site hypersensible aux nucléases SH7 et du fragment 5105. Lors de ce travail, nous avons caractérisé physiquement et fonctionnellement différents sites de liaison pour des facteurs de transcription cellulaires localisés dans la région intragénique du virus HIV-1, dont trois sites de liaison pour le facteur inductible AP-1, dans des expériences de retard de migration sur gel et de transfection transitoire. Nous avons montré l’importance de ces trois sites AP-1 pour la réplication virale au niveau transcriptionnel dans des expériences d’infection et d’immunoprécipitation de la chromatine. De plus, nous avons caractérisé l’activité transcriptionnelle associée à la région intragénique du virus HIV-1. D’autre part, la structure nucléosomale du provirus intégré et les modifications épigénétiques associées jouent un rôle crucial pour l’expression des rétrovirus. La répression transcriptionnelle du rétrovirus oncogène BLV (Bovine Leukemia Virus) lui permet d’échapper au système immunitaire de son hôte bovin et favorise ainsi l’apparition de tumeurs. Dans ce contexte, nous avons montré que la méthylation de l’ADN au niveau du promoteur viral permet le maintient de la latence transcriptionnelle. En effet, la méthylation des dinucleotides CpGs localisés dans le LTR5‘ empêche le recrutement in vivo des facteurs de transcription activateurs CREB/CREM/ATF. Nous avons également montré que l’activation transcriptionnelle de l’expression du BLV par la combinaison PMA/ionomycine s’accompagne d’un remodelage chromatinien rapide mais transitoire au niveau du promoteur viral par des expériences de marquage indirect des extrémités et d’immunoprécipitation de chromatine. Nous avons ensuite démontré l’importance du site de liaison pour le facteur de transcription PU.1 et de la E-box 4 qui lie USF-1/-2, tous deux localisés dans la région dont l’accessibilité aux nucléases s’accroît après traitement des cellules, pour l’activation transcriptionnelle de l’expression virale par cette combinaison d’inducteurs. En conclusion, notre travail devrait permettre une meilleure compréhension des mécanismes transcriptionnels et épigénétiques régulant l’expression des rétrovirus HIV-1 et BLV.

Thesis (Ph. D.)--West Virginia University, 1999. / Title from document title page. Document formatted into pages; contains vi, 119 p. : ill. Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [154]-164).

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2009. / Committee Chair: Le Doux, Joseph; Committee Member: Bellamkonda, Ravi; Committee Member: Lyon, Andrew; Committee Member: Prausnitz, Mark; Committee Member: Spencer, Trent. Part of the SMARTech Electronic Thesis and Dissertation Collection.

No description available.

The Jaagsiekte Sheep Retrovirus (JSRV), an exogenous type B/D-retrovirus with about 10-15 endogenous counterparts in all normal sheep genomes, causes Jaagsiekte (JS) or ovine pulmonary adenocarcinoma (OPA), a contagious lung cancer of sheep. This sheep lung cancer has been identified as the best natural out-bred model that can be used to study human epithelial tumours. The close similarity between the pathology of the sheep disease and Human Bronchiolo-alveolar carcinoma are highly suggestive that the human disease could have a similar aetiology and mechanism to the sheep disease. However, in the case of sheep at the time of the study there was a need for an assay that could be used to screen for infected sheep. The isolation, cloning and subsequent sequencing of the first full-length exogenous and endogenous forms of JSRV contributed greatly towards JSRV research. Until recently the diagnosis of OPA was based mostly on clinical presentation with confirmation by micro and macro examination of the affected lungs by expert pathologists. In the absence of a specific humoral response no serology-based tests were available to diagnose the disease early in live animals. Control and management of the disease was primarily by regular flock inspections and prompt culling of the suspected cases. The objective of this research project was therefore to assess and investigate the serological and molecular diagnosis of JSRV. In an attempt to develop a serology based assay three proteins were identified as candidate diagnostic antigens, the group specific antigen JSRV p26, the transmembrane and the orf-X proteins. Genes coding for all three proteins were isolated, cloned and expressed. The JSRV p26 was sufficiently purified and its potential as a diagnostic antigen was evaluated in both a Western blot and ELISA. Our studies confirmed that there were no circulating antibodies to the JSRV capsid protein. Evidence suggested that the immune response was localised to the lungs. Lung lavage samples were therefore collected from infected and normal sheep and analysed for the presence of JSRV p26 antibodies using an in-house JSp26 peroxidase conjugate in an antigen capture assay. This assay lacked sensitivity but the results indicated that there was a specific localised immune response to JSRV in the lungs of OPA affected sheep. This was confirmed with an in-house antigen capture assay that we developed. JS antigen was detected in the lung and nasal fluid of affected sheep, but not in equivalent samples from normal sheep. Three molecular assays were investigated for their sensitivity and specificity, the LTR-gag PCR, U3/LTR hemi-nested PCR and the PCR that covered the V1/V2 region. The U3/LTR hemi-nested assay was 2 logs more sensitive than the LTR-gag PCR. However, it detected the endogenous JSRV5.9A1 loci at higher concentrations. This was overcome by designing a more specific primer P3M for the first step of the U3/LTR hemi-nested PCR and the use of the AmpliTaq Gold DNA polymerase. This assay proved to be both sensitive and specific enough to screen for the infectious exogenous JSRV in peripheral blood samples from individual sheep. It is now possible to use this assay to selectively eradicate the disease from a flock through a selective culling programme. Furthermore, the assay could be made quantitative by the inclusion of concentration standards. / Thesis (M.Med.)-University of KwaZulu-Natal, 2005.