Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 136 pages. Includes Vita. Includes bibliographical references.

Regulatory T cells (Tregs), interleukin 17-producing T helper cells (Th17 cells), and other immune cells play important roles in the maintenance of pregnancy, and their function is impacted by HIV infection. I hypothesized that FIV-infection may likewise alter placental T cell gene expression causing aberrant immune function and compromised pregnancy. The purpose of this project was to evaluate the expression placental immunomodulators related to Treg and Th17 cell function in the FIV-infected cat model. Gene expression was quantified in placenta and serum using quantitative PCR and ELISA, respectively. Abnormal expression of cytokines was frequently associated with infection and fetal nonviability, resulting in discordant relationships between cytokine pairs and the nuclear transcriptional regulators FoxP3 and RORã. The expression of IL-6 in the periphery mirrored that of the placenta, indicating a potential serological means to predict pregnancy outcome. In conclusion, aberrant placental immunomodulation accompanied failed pregnancy in the FIV-infected cat model.

La dystrophie musculaire de Duchenne est une maladie génétique liée au chromosome X qui atteint un garçon sur 3 500. Cette maladie est caractérisée par l'absence de dystrophine à la surface des fibres musculaires. Sans cette protéine, les fibres se brisent plus fréquemment et une faiblesse musculaire progressive apparait. Les patients décèdent généralement au début de la vingtaine. Il n'y a présentement aucun traitement pour cette pathologie. La greffe de cellules myogéniques est une thérapie possible, mais se heurte à un rejet par le système immunitaire du patient. Pour contourner ce problème, il est possible de développer une thérapie génique ex vivo, basée sur la greffe de cellules autologues modifiées génétiquement. Malheureusement, aucune technique efficace de modification génétique des cellules n'était disponible il y a quatre ans. Nous avons testé deux nouvelles techniques de modification génétique. Une première est non virale et la seconde utilise les lentivirus. La première consiste à transfecter un plasmide d'expression de la dystrophine par Nucléofection. Pour intégrer les séquences, un second plasmide, codant pour l'intégrase PhiC31, est aussi introduit dans les cellules. Cette technique nous a permis de stabiliser des plasmides allant de 7 kb à 21 kb, ce qui en fait les plus grosses séquences jamais stabilisées dans des cellules de culture primaire humaine. Cette expression a pu être détectée dans les fibres musculaires après une greffe. Nous avons aussi utilisé des lentivirus pour effectuer une modification génétique des cellules. Ce vecteur viral est très efficace pour introduire des cassettes d'expression pour des versions tronquées de la dystrophine. L'expression de cette dystrophine est détectable in vitro, mais aussi in vivo après la transplantation. De plus, une cassette servant à faire le saut d'exon thérapeutique a aussi été introduite dans des cellules myogéniques et a permis de faire exprimer une dystrophine presque complète par des cellules issues de patients DMD. Cette expression a aussi été détectée dans des modèles murins. Ces travaux constituent une preuve de principe de la faisabilité d'une thérapie génique ex vivo pour la DMD. Plusieurs améliorations restent à apporter, mais il semble que ces travaux laissent croire qu'un essai clinique sera réalisable. / Duchenne muscular dystrophy (DMD) is a severe X-linked muscle genetic illness that afflicts one boy per 3 500. Cell therapy is a possible cure for this illness that usually kills patients around age 25. Transplantation of the heterologus myogenic cells is, however, restricted by the immune rejection by the patient. Ex vivo gene therapy offers an evasion to this problem. Introduction of the therapeutic gene into the patient’s own myogenic precursor cells, followed by transplantation is the base of this therapeutic. Four years ago, no efficient procedure to stably modify myogenic cells was available. New gene introduction techniques were thus tested in the present thesis. The first one is a non-viral method. We used a new transfection technology (Nucleofection) to introduce plasmid DNA coding for dystrophin with success. To stabilize the expression, human myogenic cells were co-nucleofected with a PhiC31 expressing plasmid. This integrase was capable of stabilising expression plasmids ranging from 7 kb to 21 kb. This very large sequence was the largest plasmid ever stabilised into human primary cultured cells. The presence of full-length dystrophin protein was detected in vitro and confirmed in vivo, after the transplantation of the myogenic precursor. Another technique was used: the lentiviral vectors. These viral vectors were designed to deliver an expression cassette for a truncated version of the dystrophin gene. The viral vector was efficient at modifying the cells. The expression was shown in vitro and in vivo after the transplantation of the modified cells. The lentiviral vectors were also essayed to deliver a U7 exon skipping cassette into DMD cells. It was then possible to demonstrate that this introduction led to the expression of a quasi normal dystrophin protein in vitro. The expression was also shown in vivo after the transplantation into SCID mice model. A non-viral approach combining nucleofection and the PhiC31 integrase may eventually permit safe auto-transplantation of genetically modified cells. The utilisation of lentiviral vectors also provided evidences that an ex vivo gene therapy is possible for DMD. We believe these results are paving the way to an eventual clinical trial for ex vivo gene therapy.

Artrite Encefalite Caprina é uma enfermidade infecciosa, multissistêmica, causada pelo vírus da Artrite Encefalite Caprina, que pertence dentre os Lentivírus de Pequenos Ruminantes. Sobre o aspecto reprodutivo na transmissão, o objetivo do presente estudo consiste em avaliar experimentalmente a transmissibilidade do lentivírus caprino em cabras e suas crias pela placenta e sêmen. Para avaliar a influência da transmissão via placentária, foram utilizadas cinco fêmeas com CAEV inseminadas artificialmente com sêmen de bode livre de CAEV. Para avaliar a influência da transmissão pelo sêmen, foram utilizadas seis fêmeas livres de CAEV inseminadas artificialmente com sêmen de bode livre de CAEV. O CAEV-Cork foi adicionado ao sêmen fresco com a finalidade de se obter título infectante com carga viral em 105 TCID50/mL. Como grupo controle, duas cabras livres de CAEV foram inseminadas artificialmente com sêmen de mesmo bode sem o inóculo viral; e outras duas cabras com CAEV inseminadas com a carga viral. As fêmeas foram monitoradas durante a gestação até 15 dias pós-parto e as crias separadas das mães foram monitoradas até 12 meses de idade, empregando-se as técnicas de IDGA, cELISA e nested-PCR. As fêmeas com CAEV apresentaram resultados positivos em IDGA (87,10%), cELISA (88,71%) e nested-PCR (25,81%). As crias apresentaram resultados negativos em ambos os testes de IDGA e cELISA, embora na técnica de nested-PCR, 7,14% das amostras apresentaram banda positiva. Das seis fêmeas livres de CAEV, quatro (66,67%) apresentaram soroconversão com 18,46% das amostras positivas ao IDGA, 49,23% das amostras positivas ao cELISA e nenhuma para nested-PCR. Anticorpos anti-CAEV foram detectados 30 dias pós IA. Quanto às crias, 5,26% e 11,28% das amostras apresentaram resultado positivo para IDGA e nested-PCR, respectivamente. Nenhuma amostra apresentou positividade para cELISA. O grupo controle livre de CAEV tiveram resultados negativos para as três técnicas, incluindo suas crias; enquanto que as fêmeas com CAEV inseminadas com o inóculo viral apresentaram 61,90% das amostras positivas para IDGA, 100% para cELISA e nenhuma amostra para nested-PCR. Suas crias obtiveram no total somente um resultado positivo para IDGA e outro para nested- PCR. A positividade encontrada em nested-PCR nas crias pode ter um significado particular de identificar animais infectados porém soronegativos, como em situações de soroconversão tardia. Entretanto, não é possível assumir a transmissão do CAEV para crias de forma insofismável, embora não é descartada a possibilidade de infecção das crias pelo sêmen e placenta com soroconversão tardia. Contudo é possível a transmissão do CAEV por IA com sêmen infectado em fêmeas livres de CAEV. A carga viral utilizada no estudo foi capaz de infectar as fêmeas. Com relação aos testes utilizados, o cELISA detectou a soroconversão mais cedo que o IDGA. A técnica de nested-PCR falhou em detectar a infecção antes da soroconversão nas fêmeas. Este estudo proporcionou maiores informações sobre a transmissão do lentivírus caprino sob o aspecto reprodutivo, com a utilização de três diferentes métodos diagnósticos. / Caprine Arthritis Encephalitis is an infectious, multisystemic disease caused by Caprine Arthritis Encephalitis virus, which belongs among the Small Ruminant Lentiviruses. Regarding the reproductive behavior in the transmission, the objective of the present study consists of experimentally evaluate the transmissibility of caprine lentivirus in goats and their offspring through the placenta and semen. To evaluate the influence of placentary transmission, five CAEV females were artificially inseminated with semen from CAEV-free buck. In order to evaluate the influence of transmission by semen, six CAEV-free females were artificially inseminated with semen from CAEV-free buck. The CAEV-Cork was added to fresh semen to obtain infecting titres for viral load of 105 TCID50/mL. As control group, two CAEV-free goats were artificially inseminated with semen from same buck without viral inoculum; and other two CAEV goats inseminated with the viral inoculum. The females were monitored during gestation until 15 days post-partum and the offspring, separated from their mothers, were monitored until 12 months old, using AGID, cELISA and nested-PCR techniques. The CAEV females presented positive results in AGID (87,10%), cELISA (88,71%) and nested-PCR (25,81%). The offspring presented negative results in both AGID and cELISA tests, however in nested-PCR technique, 7,14% of samples presented positive band. From the six CAEV-free females, four (66,67%) seroconverted with 18,46% positive samples at AGID, 49,23% at cELISA and none at nested-PCR. Anti-CAEV antibodies were detected 30 days after AI. Regarding to their offspring, 5,26% and 11,28% of samples presented positive results at AGID and nested-PCR, respectively. No sample was positive at cELISA. The CAEV-free females from control group had negative results at the three techniques, including their offspring; while the CAEV females inseminated with the viral inoculum have shown 61,90% of positive samples at AGID, 100% at cELISA and none at nested-PCR. Their offspring have only one sample positive at AGID and another at nested-PCR. The positivity found in offspring at nested-PCR may have a particular meaning in identify infected but seronegative animals, as in late seroconversion situations. However it is not possible to assume the CAEV transmission to offspring indisputably, though is not discarded the possibility of infection through semen and placenta in offspring with delayed seroconversion. Nevertheless it is possible the CAEV transmission by AI with infected semen in CAEV-free females. The viral load used in the present study was able to infect the females. Regarding the tests used, the cELISA detected seroconversion earlier than the AGID. The nested-PCR technique failed in detecting the infection before seroconversion in females. This study provided more information about the caprine lentivirus transmission concerning the reproductive aspect, using three different diagnostic methods.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Notre laboratoire a développé un prototype de vaccin unique contre le VIH-1 / SIDA. C'est un un lentivecteur ADN non-intégratif qui a été testé dans une étude pilote utilisant des modèles animaux. L'étude a montré la protection de tous les macaques (6/6) vaccinés et la réponse était composée de cellules effectrices (EM) et des cellules T mémoire centrale (CM). Plus important encore, elle contenait également des cellules antigène spécifique à haute capacité de prolifération contenant des cellules T mémoire de type cellule souche (TSCM). Durant le travail de cette thèse, le génome vaccinal a été encore amélioré en commutant son enveloppe dotée de tropisme CXCR4 contre des enveloppes à tropisme CCR5 de virus de clade B (WARO) obtenu à partir d'un patient infecté de façon chronique et de trois souches de VIH-1 de Clade C transmetteur foundateur (T/F) de patients Zambiens. Une deuxième amélioration du vaccin a été réalisée en modifiant le génome afin qu’il puisse incorporer des adjuvants moléculaires capables d'améliorer d’avantage son immunogénicité.Etant donné que le lentivirus humain VIH-1 a développé plusieurs stratégies complexes pour persister, l’autre partie de la thèse a été consacrée à développer un outil pour comprendre la latence dans les cellules T CD4 + de la mémoire infectée. Les cellules latentes ont des génomes d'ADN viral intégrés non exprimés. Un des principaux mécanismes de cette latence est l'absence de transactivation du promoteur LTR par Tat. Les développements récents de la thérapie antivirale hautement active (HAART) efficace pour contrôler les cellules infectées circulantes et dans les tissus reste inefficaces contre les cellules du réservoir composé de cellules infectées latentes. Un des obstacles pour ce type d'études est l'absence de prototypes de lentivirus de primates appropriés incapables de d’effectuer la latence pour s’en servir comme modèle d'infection extrême dans l'évaluation. Nous avons émis l'hypothèse qu'un génome SHIV réplicatifdont l’expression est sous le contrôle de LTR du CAEV, Tat-indépendant doté de promoteur constitutif constituera un outil précieux pour de telles études. Nous avons conçu des LTRs chimères de CAEV portant les séquences d'attachement de celles du SIV à leurs extrémités et nous les ont utilisés pour contrôler l’expression du génome complet de SHIV-KU2. La construction résultante est SHIV-YCC qui devrait générer un virus qui ne n’effectue pas de latence en absence de Tat. Nous avons observé que les cellules transfectées avec le génome SHIV-YCC produisent des protéines SHIV qui s’assemblent en particules infectieuses excrétées des cellules. Les virions sont capables d'infecter les lymphocytes T CD4 + cibles tant dans les PBMC primaires que dans les lignées cellulaires. Le passage en série du virus dans les PBMC de macaques augmente la réplication et l'infectiosité du virus. SHIV-YCC est le premier lentivirus chimérique réplicatif de primates qui exprime de manière constitutive toutes les protéines virales. Ce nouveau modèle offre la possibilité d'étudier les événements précoces par lesquels le provirus subit une latence, en particulier lorsque le gène de l'enveloppe sera remplacé par celui du T / F CCR5 tropique VIH-1. / Our lab has previously described the generation of a unique vaccine prototype against HIV-1/AIDS. It is a non-integrative DNA lentivector vaccine tested in pilot studies in animal models of HIV vaccine. The non-human primate study showed protection of all 6/6 macaques and immune response correlates were composed of a variety of effector (EM) and central memory (CM) T cells. More importantly, they also contained high proliferating antigen specific cells containing a type of stem cell-like memory T cells (TSCM). In this thesis the vaccine was enhanced further by switching the CXCR4 envelope of the vaccine to CCR5 tropic envelopes such as the clade B WARO obtained from a chronically infected patient and a series of three transmitted/founder (T/F) HIV Clade C strains from Zambia. To improve further the vaccine we developed new strategies to incorporate molecular adjuvants able to enhance and sustain the newly elicited immune responses.Since the human lentivirus HIV-1 has developed multiple complex strategies to persist, the focus of the next part of my thesis was to develop a tool to ease and better understand the underlying mechanisms of latency in infected memory CD4+ T cells. Latently-infected cells have non-expressed integrated viral DNA genomes. One of the main mechanisms of this latency is absence of Tat transactivation of the LTR promoter. The recent focus post development of efficient highly active antiviral therapy (HAART), is the cure of the reservoir of latently infected cells. One of the obstacles for this type of studies is the lack of proper primate lentivirus prototypes incapable of undergoing latency as extreme infection model in the evaluation. We hypothesized that a replication-competent SHIV genome driven by the Tat-independent constitutive-expression LTRs of CAEV will be a valuable tool for such studies. We designed chimeric CAEV LTRs bearing the attachment sequences of SIV at their extremities and used them to drive the complete genome of SHIV-KU2. The resulting construct is SHIV-YCC which is expected to generate virus that will not undergo latency due to absence of Tat. We found that cells transfected with SHIV-YCC genome produce SHIV proteins that are assembled into infectious particles released out of the cells. Virions are able to infect target CD4+ T cells both in primary PBMCs and cell lines. Passaged virus in macaques PBMCs increased virus replication and infectivity. SHIV-YCC is the first chimeric primate replication-competent lentivirus that constitutively expresses all viral proteins. This new model offers the possibility of studying the early events by which provirus undergoes latency particularly when the envelop gene will be replaced with that of the T/F CCR5 tropic HIV-1.

Le syndrome d'immunodéficience acquise (SIDA) est une maladie provoquée chez l'homme par le virus de l'immunodéficience humaine (VIH), un lentivirus à ARN monocaténaire qui infecte les cellules humaines qui expriment les CD4 à leur surface. Depuis son apparition en 1982 chez l’homme, il y a eu environ 80 millions d'individus infectés dans le monde et près de la moitié d'entre eux sont déjà décédés. Aucun vaccin n'existe actuellement mais l'espérance de vie d’un grand nombre de patients est maintenant prolongée grâce au développement et la disponibilité d'un traitement antirétroviral hautement actif (HAART en anglais). En raison de la complexité des interactions hôte/pathogène liées à l'infection par le VIH-1 chez l'homme et les modèles primates non-humains actuels, le développement d’un modèle plus simple est nécessaire pour étudier et mieux comprendre les mécanismes sous-jacents de l'augmentation de la pathogenèse du VIH-1 chez l’humain. Dans ce but, un virus chimérique CAL-HIV-R1 a été construit dans notre laboratoire en échangeant les longues séquences répétées terminales (LTR) du VIH par celles du CAEV, un lentivirus caprin. Parce que ces LTR de CAEV ont un promoteur constitutif qui est indépendant du trans-activateur de la transcription, ce virus chimérique ne devrait pas subir de latence dans les cellules T CD4+ mémoire. Pour rendre son efficacité réplicative plus performante, cette chimère a subi plusieurs passages successifs sur des cellules humaines en culture. En plus de la présence de son récepteur primaire, la protéine CD4, le VIH doit interagir avec une seconde molécule co-réceptrice pour entrer dans la cellule hôte. Des clones moléculaires infectieux contenant des génomes proviraux complets de plusieurs isolats de VIH-1 ont été reçus de la banque de produits "NIH AIDS Reagent Program Repository". Trois d'entre eux, à savoir pNL4-3, p89.6 et WARO, ont été utilisés pour produire des stocks de virus après transfection des cellules de la lignée humaine HEK-293T et utilisés pour infecter d’autres lignées cellulaires telles que : 1) des cellules GHOST, utilisées pour examiner le tropisme des virus en fonction de leur utilisation des co-récepteurs et qui sont respectivement X4, X4/R5 et R5; 2) la lignée cellulaire M8166, utilisée comme cellules indicatrices du fait de ses propriétés fusogéniques, et qui sert à examiner les capacités de réplication et enfin, 3) la lignée cellulaire TZM-bl utilisée pour évaluer le titre infectieux des virus. Par ailleurs, un vaccin basé sur un vecteur ADN lentiviral chimérique, le CAL-SHIV-IN-, a été développé au laboratoire et testé chez des macaques. Dans le cadre de cette étude, un test de séro-neutralisation a été réalisé sur des échantillons de sérum des macaques vaccinés avec ce vecteur, et des animaux témoins, pour examiner la présence d'anticorps pouvant neutraliser le virus. Bien que des anticorps furent présents aucune capacité neutralisante n'a pu être détectée. / Acquired Immuno-Deficiency Syndrome (AIDS) is a disease caused by immunodeficiency viruses in human (HIV-1) and some animal species. The virus is a small enveloped particle that has a single-strand RNA genome and belongs to the lentivirus genus that belongs to the Retroviridae family. In human the virus infects and replicates mainly in cells that express the CD4 on their surface. Since its apparition in human in 1982 the virus has infected around 80 million individuals worldwide and caused the death of nearly half of them. No vaccine exists but life expectancy of near half of HIV-1-infected individuals has been now prolonged due to extensive highly active antiretroviral therapy (HAART). Because of the complexity of the host/pathogen interactions that are associated with HIV-1 infection in human and non-human primate models, a simple model system is strongly needed to ease the studies aiming at better understanding the underlying mechanisms of increased pathogenesis of HIV-1 in human. A chimeric virus CAL-HIV-R1 was created in our laboratory by exchanging the long terminal repeats (LTRs) of HIV with those of CAEV, a caprine lentivirus. Because these CAEV LTRs have a constitutive promoter, which is independent of the trans-activator of transcription, we expect that this chimeric virus should not undergo latency in memory CD4+ T cells. To increase the potency of this chimera, serial passages on cultured human cells were performed. Besides its primary receptor, CD4, HIV needs to interact with another molecule as a co-receptor. Several infectious molecular clones of HIV-1 isolates pDNAs containing the complete proviral genomes were received from the NIH AIDS Reagent Program Repository. Three of these, namely pNL4-3, p89.6 and WARO, were used to produce virus stocks following transfection in the human HEK-293T cell line and used to infect a variety of cell lines such as: 1) GHOST cells that were used to examine the tropism for the co-receptor that were X4, X4/R5 and R5 respectively; 2) M8166 a fusogenic indicator cell line to evaluate the replication competency, 3) TZM-bl to determine the infectivity titers of the viruses by scoring the blue cells enabled by infections. A vaccine based on a chimeric DNA vector, CAL-SHIV-IN-, has been developed in our laboratory and tested in macaques. A sero-neutralization assay was performed on sera of macaques, which had been vaccinated with this vector and challenged in parallel with control animals with a pathogenic virus. This assay was used to verify the presence of neutralizing antibodies, but, unfortunately none could be detected.

Artrite Encefalite Caprina é uma enfermidade infecciosa, multissistêmica, causada pelo vírus da Artrite Encefalite Caprina, que pertence dentre os Lentivírus de Pequenos Ruminantes. Sobre o aspecto reprodutivo na transmissão, o objetivo do presente estudo consiste em avaliar experimentalmente a transmissibilidade do lentivírus caprino em cabras e suas crias pela placenta e sêmen. Para avaliar a influência da transmissão via placentária, foram utilizadas cinco fêmeas com CAEV inseminadas artificialmente com sêmen de bode livre de CAEV. Para avaliar a influência da transmissão pelo sêmen, foram utilizadas seis fêmeas livres de CAEV inseminadas artificialmente com sêmen de bode livre de CAEV. O CAEV-Cork foi adicionado ao sêmen fresco com a finalidade de se obter título infectante com carga viral em 105 TCID50/mL. Como grupo controle, duas cabras livres de CAEV foram inseminadas artificialmente com sêmen de mesmo bode sem o inóculo viral; e outras duas cabras com CAEV inseminadas com a carga viral. As fêmeas foram monitoradas durante a gestação até 15 dias pós-parto e as crias separadas das mães foram monitoradas até 12 meses de idade, empregando-se as técnicas de IDGA, cELISA e nested-PCR. As fêmeas com CAEV apresentaram resultados positivos em IDGA (87,10%), cELISA (88,71%) e nested-PCR (25,81%). As crias apresentaram resultados negativos em ambos os testes de IDGA e cELISA, embora na técnica de nested-PCR, 7,14% das amostras apresentaram banda positiva. Das seis fêmeas livres de CAEV, quatro (66,67%) apresentaram soroconversão com 18,46% das amostras positivas ao IDGA, 49,23% das amostras positivas ao cELISA e nenhuma para nested-PCR. Anticorpos anti-CAEV foram detectados 30 dias pós IA. Quanto às crias, 5,26% e 11,28% das amostras apresentaram resultado positivo para IDGA e nested-PCR, respectivamente. Nenhuma amostra apresentou positividade para cELISA. O grupo controle livre de CAEV tiveram resultados negativos para as três técnicas, incluindo suas crias; enquanto que as fêmeas com CAEV inseminadas com o inóculo viral apresentaram 61,90% das amostras positivas para IDGA, 100% para cELISA e nenhuma amostra para nested-PCR. Suas crias obtiveram no total somente um resultado positivo para IDGA e outro para nested- PCR. A positividade encontrada em nested-PCR nas crias pode ter um significado particular de identificar animais infectados porém soronegativos, como em situações de soroconversão tardia. Entretanto, não é possível assumir a transmissão do CAEV para crias de forma insofismável, embora não é descartada a possibilidade de infecção das crias pelo sêmen e placenta com soroconversão tardia. Contudo é possível a transmissão do CAEV por IA com sêmen infectado em fêmeas livres de CAEV. A carga viral utilizada no estudo foi capaz de infectar as fêmeas. Com relação aos testes utilizados, o cELISA detectou a soroconversão mais cedo que o IDGA. A técnica de nested-PCR falhou em detectar a infecção antes da soroconversão nas fêmeas. Este estudo proporcionou maiores informações sobre a transmissão do lentivírus caprino sob o aspecto reprodutivo, com a utilização de três diferentes métodos diagnósticos. / Caprine Arthritis Encephalitis is an infectious, multisystemic disease caused by Caprine Arthritis Encephalitis virus, which belongs among the Small Ruminant Lentiviruses. Regarding the reproductive behavior in the transmission, the objective of the present study consists of experimentally evaluate the transmissibility of caprine lentivirus in goats and their offspring through the placenta and semen. To evaluate the influence of placentary transmission, five CAEV females were artificially inseminated with semen from CAEV-free buck. In order to evaluate the influence of transmission by semen, six CAEV-free females were artificially inseminated with semen from CAEV-free buck. The CAEV-Cork was added to fresh semen to obtain infecting titres for viral load of 105 TCID50/mL. As control group, two CAEV-free goats were artificially inseminated with semen from same buck without viral inoculum; and other two CAEV goats inseminated with the viral inoculum. The females were monitored during gestation until 15 days post-partum and the offspring, separated from their mothers, were monitored until 12 months old, using AGID, cELISA and nested-PCR techniques. The CAEV females presented positive results in AGID (87,10%), cELISA (88,71%) and nested-PCR (25,81%). The offspring presented negative results in both AGID and cELISA tests, however in nested-PCR technique, 7,14% of samples presented positive band. From the six CAEV-free females, four (66,67%) seroconverted with 18,46% positive samples at AGID, 49,23% at cELISA and none at nested-PCR. Anti-CAEV antibodies were detected 30 days after AI. Regarding to their offspring, 5,26% and 11,28% of samples presented positive results at AGID and nested-PCR, respectively. No sample was positive at cELISA. The CAEV-free females from control group had negative results at the three techniques, including their offspring; while the CAEV females inseminated with the viral inoculum have shown 61,90% of positive samples at AGID, 100% at cELISA and none at nested-PCR. Their offspring have only one sample positive at AGID and another at nested-PCR. The positivity found in offspring at nested-PCR may have a particular meaning in identify infected but seronegative animals, as in late seroconversion situations. However it is not possible to assume the CAEV transmission to offspring indisputably, though is not discarded the possibility of infection through semen and placenta in offspring with delayed seroconversion. Nevertheless it is possible the CAEV transmission by AI with infected semen in CAEV-free females. The viral load used in the present study was able to infect the females. Regarding the tests used, the cELISA detected seroconversion earlier than the AGID. The nested-PCR technique failed in detecting the infection before seroconversion in females. This study provided more information about the caprine lentivirus transmission concerning the reproductive aspect, using three different diagnostic methods.

Lysyl oxidase like-2 (LOXL2) was found to be present extracellularly in primary human gingival fibroblast cells. This project has been primarily focused on investigating our hypothesis that LOXL2 may play a critical role in regulating cell proliferation and collagen accumulation in primary human gingival fibroblast cells, which may contribute to the development of fibrotic changes in human gingival tissue. LOXL2 shRNA lentivirus reduced the LOXL2 mRNA and protein expression by 90 – 95%. Knockdown of LOXL2 or inhibition of LOXL2 enzymatic activity strongly inhibited both basal and CCN2/CTGF-stimulated collagen accumulation (p<0.05). Proliferation assays demonstrated a marked decrease in cell proliferation in both the short and long term in LOXL2 shRNA knockdown cells with minimal or no stimulation of cell apoptosis. Pharmacologic inhibition of LOXL2 enzyme activity reduced basal and CCN2/CTGF-stimulated cell proliferation (40% and 50%) in short term cultures. Furthermore, there was 15-20% inhibition seen in long term assays. Recombinant active LOXL2 significantly increased collagen accumulation and cell proliferation (p<0.05). Thereby, our investigation in vitro by loss and gain of function experiments confirmed that LOXL2 is critically required for both gingival fibroblast proliferation and for collagen accumulation in the presence or absence of CCN2/CTGF. LOXL2 stimulation is critical for both proliferation and collagen accumulation in primary human gingival fibroblasts. Lastly, we found that the presence of LOXL2 extracellularly and LOXL2 may regulate cell proliferation by enhancing the phosphorylation of PDGFR.

Le tabac est consommé par environ un milliard de personnes. D'après l'Organisation Mondiale de la Santé, le tabagisme est la première cause évitable de mortalité dans le monde, provocant six millions de morts par an. La nicotine est le composant neuro-actif principal dans le tabac, et exerce ses effets neurologiques via une activation directe des récepteurs nicotiniques de l’acétylcholine (nAChR). Ces récepteurs transmembranaires sont composés de sous-unités alpha, ou alpha plus beta, créant une variété de canaux ioniques ligand-dépendants activés par le neurotransmetteur ACh. Les études génétiques chez l’homme ont mis en évidence des variants dans le cluster génomique CHRNA5-CHRNA3-CHRNB4, codant pour les sous-unités α5, α3 et β4, comme facteurs influençant le tabagisme. Cette thèse a étudié le rôle des nAChRs contenant la sous-unité β4 (β4*) dans l’addiction à la nicotine. En collaboration, nous avons montré que les souris déficientes pour la sous-unité β4 (β4 KO), sont moins sensibles aux effets récompensant et aversifs de la nicotine. En générant un lentivirus exprimant la séquence murine d'ADN complémentaire de β4, j’ai pu restaurer son expression dans des régions d’intérêt du cerveau, sur un fond génétique β4KO. Ceci a permis de mettre en évidence le rôle du réseau habénulo-interpedonculaire dans la contribution des β4* nAChRs à la consommation de nicotine. Ceci a également démontré le rôle modulateur de ces récepteurs dans les réponses de la voie mésolimbique à la nicotine, voie centrale dans l'effet renforçant des drogues. / Tobacco is consumed by an estimated 1 billion people world-wide. The World Health Organization names tobacco consumption the primary cause of preventable morbidity and mortality, causing six million deaths per year. Nicotine is the principal neuro-active compound in tobacco, and exerts neurological effects by binding to nicotinic acetylcholine receptors (nAChRs). These transmembrane receptors are composed of alpha or alpha plus beta subunits, forming a diverse variety of ligand-gated ion channels endogenously activated by ACh. Human genetic studies have highlighted variants in the CHRNA5-CHRNA3-CHRNB4 genomic cluster, coding for subunits α5, α3 and β4, as altering smoking behaviours. The present thesis investigated the role of β4-containing (β4*) nAChRs in nicotine addiction. In collaboration, we showed that β4 knockout (KO) mice are less sensitive to nicotine reward and nicotine aversion. Generating a lentivirus for the expression of mouse β4 nAChR subunit complementary DNA, I was able to restore receptor expression to brain regions of interest on a KO background, locating the role of β4* nAChR in nicotine reward and aversion to the habenulo-interpedunular pathway. This also demonstrated the receptor’s modulation of nicotinic responses of the mesolimbic system, central hub of drug reinforcement.