Die Entwicklung von Dickdarmkrebs wird durch eine Reihe von Lebens- und Essgewohnheiten sowie Umweltfaktoren begünstigt. Den letzteren beiden sind Substanzen zuzurechnen, die bei der Zubereitung der Nahrung entstehen und mit ihr aufgenommen werden. Zu diesen Verbindungen gehört das 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridin (PhIP) aus der Substanzklasse der heterozyklischen aromatischen Amine. Es entsteht bei der Erhitzung zahlreicher proteinhaltiger Nahrungsmittel und die Zielorgane in Nagerstudien stimmen mit der Häufung von Krebsinzidenzen in westlichen Industrienationen überein. Dieser Zusammenhang konnte jedoch bis heute nicht endgültig bewiesen werden. Fütterungsversuche mit Ratten wurden mit Konzentrationen der Substanz durchgeführt, die weit über der menschlichen Exposition liegen. Durch das Verfüttern einer humanrelevanten Dosis PhIP sollte geklärt werden, ob auch geringe Konzentrationen dickdarmkrebstypische Mutationen, präneoplastische Läsionen oder Tumore induzierten. Die mit humanrelevanten Dosen gefütterten Tiere wiesen weniger Läsionen als die Hoch-Dosis-PhIP-Gruppe auf, in der allerdings keinerlei maligne Tumoren des Dickdarms auftraten. Hinweise auf dickdarmkrebstypische Mutationen fanden sich ebenfalls in beiden Gruppen, wobei hier keine Dosisabhängigkeit beobachtet werden konnte. Die Sequenzierung ergab ein deutlich von Literaturdaten abweichendes Spektrum. In Bezug auf das verwendete Tiermodell wurden erhebliche Abweichungen in der Empfindlichkeit der Tiere gegenüber der Substanz im Vergleich zu ähnlichen Studien festgestellt. Beide Fütterungsgruppen zeigten deutlich weniger Läsionen; als mögliche Gründe wurden Unterschiede in der Futterzusammensetzung und –zubereitung sowie in der Tierhaltung und –herkunft ausgemacht. Es konnte erstmalig ein Zusammenhang zwischen PhIP in niedrigen Dosen in der Nahrung und der Induktion von Entzündungen gezeigt werden. Diese waren sowohl makroskopisch als auch histologisch sichtbar, der genaue Mechanismus ihrer Entstehung ist jedoch unbekannt.<br><br> Die zusammenfassende Betrachtung aller Ergebnisse lässt vermuten, dass PhIP allein über lange Zeiträume aber in geringen Dosen verabreicht nicht für die hohe Zahl an Krebserkrankungen in westlichen Industrienationen ursächlich ist. / The development of colon cancer is associated with several nutritional, life style, and environmental factors. Among the environmental factors probably involved are substances formed during food processing and taken up with food. One of these substances is the heterocyclic aromatic amine (HAA) 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which is formed during the heating of proteinaceous food such as meat and fish. In rodent studies the target organs for HAA-derived cancer development are identical with human organs showing high tumor incidences in western countries. Whether there is an association between exposure to PhIP and high tumor incidences in humans is still uncertain. The amount of PhIP administred to rodents in several studies was far above the levels of human exposure towards HAA. Thus, the aim of this study was to elucidate whether low concentrations of the substance are able to induce finger-print colon cancer gene mutations, preneoplastic lesions or tumors in rats. Animals fed with high amounts of PhIP developed fewer lesions than animals fed with a human-relevant concentration of PhIP. However none of the groups developed tumors of the colon. Both groups showed finger-print mutations for colon cancer, but not in a dose-dependent manner. Sequencing showed that the mutations were different from the known mutation spectum of PhIP. The susceptibility of the F344 rats to PhIP used in this study differed from that in previous feeding studies, with both groups showing much less lesions of the colon. Differences in composition and processing of the animal diets as well as animal maintenance and –origin may explain this discrepancy. For the first time an association between low doses of PhIP in the diet and induction of inflammation was shown. Signs of inflammation were observed macroscopically as well as in histological slices, but the mechanism of its induction remains to be clarified.<br><br> Taken together the results suggest that a chronical exposure to low doses of PhIP alone is not sufficient to explain the high incidences of colon cancer in western countries.

Familial adenomatous polyposis (FAP) is a condition caused by germline mutations in tumor suppressor gene APC, inherited in autosomal dominant manner. Patients with FAP develop hundreds to thousands of adenomatous colorectal polyps with extremely high risk of malignant reversal into adenocarcinoma of colon and/or rectum. The aim of this thesis is to re-analyze a cohort of highly suspected FAP probands from years 1993-2004 whose diagnosis previously failed to be confirmed by at that time commonly used methods of molecular diagnostics. Next generation sequencing on MiSeq and NextSeq platforms (Illumina®) was performed on 78 samples of probands' DNA, isolated from peripheral blood, using gene panel CZECANCA version 1.2 (Czech Cancer Panel for Clinical Application). The panel enables sequencing of exons and exon-intron junctions of 226 genes linked to hereditary cancer predispositions, newly also including the diagnostically important promoter 1B region of APC. Pathogenic variant in the APC gene was detected in 18 % of re-analyzed probands, 11 % of probands carry pathogenic variants in other genes associated with colorectal polyps. Additional 13 % of probands are carriers of a variants of unknown clinical significance. NGS gene panel CZECANCA enabled diagnosis confirmation or re-evaluation of 22 FAP...

O comércio de vegetais minimamente processados (VMP) tem crescido substancialmente nos últimos anos devido a sua conveniência, frescor e aparente salubridade. No entanto, o processamento mínimo não reduz as populações de microrganismos patogênicos para níveis seguros. A irradiação de alimentos é utilizada para estender a vida de prateleira e inativar patógenos presentes nos alimentos. Seu uso combinado com o processamento mínimo poderia aumentar a segurança e qualidade dos VMP. Dois diferentes métodos de detecção de alimentos irradiados, um biológico, o DEFT/APC, e outro bioquímico, o teste do cometa, foram aplicados a VMP com o objetivo de testar sua aplicabilidade na detecção do tratamento por radiação. O DEFT/APC é um método de varredura microbiológico baseado no uso da técnica de epifluorescência direta em filtro (DEFT) e da contagem padrão em placas (APC). O teste do cometa detecta o dano no DNA devido, por exemplo, a radiação ionizante. Amostras de acelga, agrião, alface, catalônia, couve, escarola, espinafre e repolho do comércio varejista foram irradiadas com 0,5kGy e 1,0kGy utilizando um irradiador de 60Co. O processamento por irradiação garantiu a redução de pelo menos dois ciclos logarítmicos nas populações de microrganismos aeróbios e psicrotróficos. Em geral, com o aumento das doses de radiação, as contagens DEFT se mantiveram similares independentemente do processamento por irradiação, enquanto as contagens APC diminuíram gradualmente. A diferença das duas contagens aumentou gradualmente com o incremento da dose em todas as amostras. Uma diferença entre o valor de DEFT e do APC maior a 2,0 log seria indicativa de que o VMP foi tratado por irradiação. O teste do cometa permitiu distinguir amostras não irradiadas das irradiadas, que mostraram diferentes tipos de cometas decorrentes da fragmentação do DNA. Tanto o método DEFT/APC quanto o teste do cometa foram satisfatoriamente utilizados como métodos de varredura para a detecção do tratamento por irradiação. / Marketing of minimally processed vegetables (MPV) are gaining impetus due to its convenience, freshness and apparent healthy. However, minimal processing does not reduce pathogenic microorganisms to safe levels. Food irradiation is used to extend the shelf life and inactivation of food-borne pathogens. Its combination with minimal processing could improve the safety and quality of MPV. Two different food irradiation detection methods, a biological, the DEFT/APC, and another biochemical, the DNA Comet Assay were applied to MPV in order to test its applicability to detect irradiation treatment. DEFT/APC is a microbiological screening method based on the use of the direct epifluorescent filter technique (DEFT) and the aerobic plate count (APC). DNA Comet Assay detects DNA damage due to ionizing radiation. Samples of lettuce, chard, watercress, dandelion, kale, chicory, spinach, cabbage from retail market were irradiated 0.5kGy and 1.0kGy using a 60Co facility. Irradiation treatment guaranteed at least 2 log cycle reduction for aerobic and psychrotroph microorganisms. In general, with increasing radiation doses, DEFT counts remained similar independent of irradiation processing while APC counts decreased gradually. The difference of the two counts gradually increased with dose increment in all samples. It could be suggested that a DEFT/APC difference over 2.0 log would be a criteria to judge if a MPV was treated by irradiation. DNA Comet Assay allowed distinguishing non-irradiated samples from irradiated ones, which showed different types of comets owing to DNA fragmentation. Both DEFT/APC method and DNA Comet Assay would be satisfactorily used as a screening method for indicating irradiation processing.

O comércio de vegetais minimamente processados (VMP) tem crescido substancialmente nos últimos anos devido a sua conveniência, frescor e aparente salubridade. No entanto, o processamento mínimo não reduz as populações de microrganismos patogênicos para níveis seguros. A irradiação de alimentos é utilizada para estender a vida de prateleira e inativar patógenos presentes nos alimentos. Seu uso combinado com o processamento mínimo poderia aumentar a segurança e qualidade dos VMP. Dois diferentes métodos de detecção de alimentos irradiados, um biológico, o DEFT/APC, e outro bioquímico, o teste do cometa, foram aplicados a VMP com o objetivo de testar sua aplicabilidade na detecção do tratamento por radiação. O DEFT/APC é um método de varredura microbiológico baseado no uso da técnica de epifluorescência direta em filtro (DEFT) e da contagem padrão em placas (APC). O teste do cometa detecta o dano no DNA devido, por exemplo, a radiação ionizante. Amostras de acelga, agrião, alface, catalônia, couve, escarola, espinafre e repolho do comércio varejista foram irradiadas com 0,5kGy e 1,0kGy utilizando um irradiador de 60Co. O processamento por irradiação garantiu a redução de pelo menos dois ciclos logarítmicos nas populações de microrganismos aeróbios e psicrotróficos. Em geral, com o aumento das doses de radiação, as contagens DEFT se mantiveram similares independentemente do processamento por irradiação, enquanto as contagens APC diminuíram gradualmente. A diferença das duas contagens aumentou gradualmente com o incremento da dose em todas as amostras. Uma diferença entre o valor de DEFT e do APC maior a 2,0 log seria indicativa de que o VMP foi tratado por irradiação. O teste do cometa permitiu distinguir amostras não irradiadas das irradiadas, que mostraram diferentes tipos de cometas decorrentes da fragmentação do DNA. Tanto o método DEFT/APC quanto o teste do cometa foram satisfatoriamente utilizados como métodos de varredura para a detecção do tratamento por irradiação. / Marketing of minimally processed vegetables (MPV) are gaining impetus due to its convenience, freshness and apparent healthy. However, minimal processing does not reduce pathogenic microorganisms to safe levels. Food irradiation is used to extend the shelf life and inactivation of food-borne pathogens. Its combination with minimal processing could improve the safety and quality of MPV. Two different food irradiation detection methods, a biological, the DEFT/APC, and another biochemical, the DNA Comet Assay were applied to MPV in order to test its applicability to detect irradiation treatment. DEFT/APC is a microbiological screening method based on the use of the direct epifluorescent filter technique (DEFT) and the aerobic plate count (APC). DNA Comet Assay detects DNA damage due to ionizing radiation. Samples of lettuce, chard, watercress, dandelion, kale, chicory, spinach, cabbage from retail market were irradiated 0.5kGy and 1.0kGy using a 60Co facility. Irradiation treatment guaranteed at least 2 log cycle reduction for aerobic and psychrotroph microorganisms. In general, with increasing radiation doses, DEFT counts remained similar independent of irradiation processing while APC counts decreased gradually. The difference of the two counts gradually increased with dose increment in all samples. It could be suggested that a DEFT/APC difference over 2.0 log would be a criteria to judge if a MPV was treated by irradiation. DNA Comet Assay allowed distinguishing non-irradiated samples from irradiated ones, which showed different types of comets owing to DNA fragmentation. Both DEFT/APC method and DNA Comet Assay would be satisfactorily used as a screening method for indicating irradiation processing.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Le VIH-1 manipule les cellules présentatrices d’antigènes (APC) qui orchestrent les réponses immunes innées et adaptatrices, pour se propager dans l’hôte et établir le réservoir viral. Au laboratoire, nous étudions le rôle de l’autophagie dans les interactions entre les cellules dendritiques (DC) et le VIH-1 et la présentation des antigènes viraux. Dans divers modèles, la macroautophagie et l’autophagie médiée par les chaperonnes (CMA) semblent en effet être impliquées dans l’apprêtement d’antigènes sur les molécules du CMH. Ainsi, nous avons montré, dans une étude précédente, que la macroautophagie participait à la dégradation du VIH entrant dans les DC, conduisant à l’activation de lymphocytes T (LT) CD4+ spécifiques du VIH-1.Bien que sa réplication y soit limitée, le VIH-1 peut également infecter productivement les DC. J’ai donc voulu vérifier si les protéines virales néosynthétisées du virus peuvent constituer une source additionnelle d’antigènes. J’ai montré que, de façon remarquable, dans les DC infectées, des antigènes endogènes du VIH-1 peuvent être présentés par les molécules du CMH-II aux LT CD4 spécifiques. En utilisant différents outils, comme des inhibiteurs de l’autophagie ou des shRNA, j’ai montré que ni la macroautophagie ni la CMA ne contribuent significativement à l’apprêtement d’épitopes de la protéine virale Gag néosynthétisée sur les molécules du CMH-II. En parallèle, j’ai utilisé une protéine de fusion, Gag-LC3, pour acheminer spécifiquement Gag dans les autophagosomes (LC3+) des DC. Dans ce contexte, les drogues qui inhibent la macroautophagie réduisent drastiquement la présentation d’épitopes de Gag aux LT CD4. De façon remarquable, la présence de Gag dans les autophagosomes conduit à la génération d’épitopes antigéniques qui, dans le contexte infectieux, ne sont pas apprêtés sur les molécules de CMH-II par la voie endogène. Ainsi, diriger des protéines du VIH dans les autophagosomes conduirait à des variations dans le répertoire des antigènes endogènes présentés sur les molécules de CMH-II. Pour évaluer l’impact de l’autophagie sur la réplication du VIH dans les DC, j’ai ensuite analysé si la protéine Gag néosynthétisée pouvait être dégradée dans les autophagosomes. Dans les DC infectées, contrairement aux observations déjà décrites dans les macrophages, Gag ne colocalise ni avec les vésicules autophagiques LC3+, ni avec p62, une protéines adaptatrice impliquée dans le ciblage des protéines dans les autophagosomes. Ces résultats suggèrent que, dans ce contexte, les virions nouvellement produits ne sont pas acheminés et dégradés dans les autophagosomes. La protéine de fusion Gag-LC3 est utilisée dans ces expériences comme contrôle positif de colocalisation. Pour déterminer si mes observations pouvaient révéler un mécanisme d’échappement développé par VIH-1, j’ai utilisé différentes souches virales mutantes, modulé le flux autophagique avec des drogues et des ligands TLR, et exprimé Gag dans les DC en l’absence d’autres protéines virales. Dans l'ensemble, mon travail suggère que le VIH-1 ne manipule pas la macroautophagie dans les DC productivement infectées. En outre, la modulation de l’autophagie dans les DC (à l'aide de shRNA) n'a aucune incidence sur la réplication du VIH-1 et sur sa propagation.Mes travaux mettent en lumière la complexité des interactions entre l’autophagie et le VIH-1 dans les DC. Contrairement à ce qui a été observé lors des étapes d’entrée du virus, le virus ne semble pas être acheminé dans les autophagosomes une fois les DC infectées, et l’autophagie ne participe pas à l’apprêtement des antigènes néosynthétisés sur les molécules de CMH II. Cependant, les DC infectées activent de façon efficace les LT CD4 spécifiques du virus. Forcer l’acheminement d’antigènes du VIH dans les autophagosomes augmente fortement cette activation, et semble conduire à une diversification du répertoire des épitopes présentés sur les molécules de CMH-II par la voie endogène. / HIV-1 manipulates antigen-presenting cells (APC) such as dendritic cells (DC), witch orchestrate innate and adaptive immune responses, in order to propagate in the host and to establish viral reservoirs. We are studying the role of autophagic processes in DC/HIV-1 interactions with a focus on antigen presentation. We have previously shown that macroautophagy in DC participates in the degradation of incoming HIV-1 particles leading to activation of HIV-1-specific (HS) CD4 T cells. HIV-1 can also productively infect DC. I thus first asked whether neo-synthetized viral proteins might represent an additional source of HIV-1 antigens. Remarkably, I have shown using infected monocyte derived DC that de novo expression of Gag leads to the activation of HS CD4 T cells, highlighting that this antigen is endogenously processed in order to be presented into MHC-II molecules. Since macroautophagy and chaperon-mediated autophagy (CMA) are known to be involved in this process for other viral antigens and model antigens, I then dissected the role of these two pathways. Using several tools including inhibitors and shRNA, I demonstrated that in HIV-1-infected DC neither macroautophagy nor CMA contribute significantly to the processing of HIV-1-Gag epitopes into MHC-II molecules. I also used a Gag-LC3 fusion protein to specifically channel Gag into LC3+ autophagic vesicles in DC. In this context, inhibiting autophagy dramatically reduced the presentation of HIV-1-Gag epitopes to CD4+ T cells. Strikingly, channelling Gag into autophagosomes generated epitopes that were not processed endogenously in the context of HIV-1 infection. Thus specifically directing HIV-1 proteins toward autophagosomes might influence the repertoire of MHC II-restricted HIV-1 antigens. I further analyzed whether autophagy could affect HIV-1 replication in infected DC. In these cells, in contrast to what has been described in macrophages, Gag did not colocalize with LC3 or with the autophagic adaptor p62, suggesting that newly-produced HIV-1 particles are not sequestrated into autophagosomes. The Gag-LC3 fusion protein was used here as a positive control of colocalization. To determine whether my findings might reveal a DC-specific escape mechanism developed by HIV-1, I used various HIV-1 mutants, enhanced autophagic flux using drugs or TLR ligands, and expressed Gag in the absence of other HIV-1 proteins. Overall, my work suggests that HIV-1 does not manipulate autophagy in productively-infected DC. Moreover, modulating autophagy in DC (using shRNA) does not impact HIV-1 replication and propagation. Finally, my work highlights the complexity of the interactions between the autophagic process and HIV-1 replication in DC. Unlike during viral entry, HIV-1 does not seem to be targeted into autophagosomes after viral replication in infected DC, and autophagy does not contribute significantly to the processing of endogenous viral antigens. Nonetheless HIV-1-infected DC efficiently activates HS CD4 T cells, and targeting HIV antigens into autophagosomes greatly enhances this activation and might broaden the repertoire of MHC-II-restricted antigen. Further dissection of the various routes of endogenous HIV antigen processing would aid in the development of innovative vaccines.

Le Syndrome Dysgénésique et Respiratoire Porcin (PRRS) est la maladie infectieuse endémique la plus couteuse en élevage porcin dont l'agent responsable est un Arterivirus, le PRRSV, qui présente une grande diversité génétique. L'infection par le PRRSV est fréquemment associée à l'infection par les virus influenza. La vaccination est une méthode de lutte adaptée contre ces virus. Dans le cas du PRRSV, les vaccins les plus utilisés sont des virus vivants modifiés (MLV) qui induisent une immunité protectrice peu efficace contre les variants viraux. Dans le cas du virus influenza, les vaccins inactivés utilisés présentent la même insuffisance.Dans ce travail de thèse, j'ai évalué des stratégies vaccinales visant à induire une immunité efficace contre des variants viraux, en utilisant des antigènes conservés entre souches, adressés aux cellules présentatrices d'antigènes (APC), et j'ai analysé l'effet de différentes voies et modes d'administration.Dans le cas du virus grippal, le ciblage d'antigènes conservés (HA2, M2e, NP) au CD11c a permis d'augmenter la réponse T uniquement lors d'administration par voie intramusculaire (IM) et fut sans effet sur la réponse anticorps. La vaccination par voie intradermique s'est traduit par une exacerbation de la pathologie lors d'une épreuve virale, alors que la vaccination par voie IM a réduit les symptômes, la durée d'excrétion virale en corrélation avec une meilleure réponse anticorps anti-HA2 et M2e.Dans le cas du virus PRRSV qui fut mon sujet principal d'étude, j'ai cherché à optimiser des réponses lymphocytaires T IFNγ en employant une stratégie vaccinale ADN codant des antigènes contenant des épitopes T conservés entre souches, ciblés aux APC. En effet, alors que les mutations virales conduisent à un échappement aux anticorps neutralisants, la réponse lymphocytaire T IFNγ a été proposée impliquée dans la protection croisée. J'ai montré que l'immunogénicité optimale de vaccins ADN PRRSV, conduisant à la réponse T la plus large, est obtenue par l'administration intradermique associée aux nanoparticules de PLGA (NP), suivi d'une électroporation (EP), par rapport à EP seul ou délivrance intradermique ou transcutanée avec des patches à micro-aiguilles résorbables. Cette immunogénicité optimale est associée à une bonne transfection des cellules de la peau, à une accumulation de cellules inflammatoires, et à une mobilisation des cellules dendritiques. J'ai ensuite utilisé ce mode d'administration EP+NP pour immuniser des porcs avec des plasmides codant des antigènes conservés du PRRSV adressés ou non aux APC via CD11c ou XCR1. Les porcs ont été immunisés soit avec des injections répétées d'ADN seul soit en prime-boost ADN-MLV. Le régime ADN-MLV s'est montré supérieur pour l'induction de réponse B et T à celui de l'ADN ou du MLV seuls, et le ciblage aux APC a nettement augmenté la réponse anticorps mais pas la réponse T IFNγ. Dans une expérience suivante à visée d'application sur le terrain, j'ai utilisé le régime ADN-MLV (sans NP cette fois), délivré avec EP ou avec jet sous pression (PJ). Dans ces conditions, la primo-vaccination avec ADN n'a pas significativement augmenté la réponse T IFNγ induite par le MLV, mais elle a clairement augmenté la réponse anticorps avec un bénéfice du ciblage des APC. L'immuno-potentialisation induite par la primo-vaccination ADN n'a pas conduit à l'amélioration de la protection contre une épreuve avec un virus hétérologue et a montré que cette protection n'est au final pas corrélée avec la réponse lymphocytaire T IFNγ et opère en l'absence d'anticorps neutralisants détectables. Enfin, l'ensemble de ce travail montre que l'effet du ciblage des APC chez le porc est influencé par la voie d'administration et par le régime d'administration comme le prime-boost ADN-MLV. / The Porcine Reproductive and Respiratory Syndrome (PRRS) is the most damaging infectious disease in pigs worldwide. The etiologic agent is an Arterivirus, the PRRSV, which presents a large genetic diversity. PRRSV infection is frequently associated with influenza virus co-infection. Vaccination is a highly suitable way to control these viruses. In the case of PRRSV, the most effective commercial vaccines are modified live vaccines (MLV) which induce only a partial protection against heterologous strains. In the case of the influenza virus, the available inactivated vaccines show the same weakness.With the goal to control emerging influenza and PRRSV variants, I evaluated vaccine strategies involving conserved viral antigens between strains which were targeted to antigen-presenting cells (APC) and delivered by different routes and methods.In the case of influenza virus, the targeting of conserved antigens (HA2, M2e and NP) to CD11c led to increased IFNγ T cell responses only when vaccines were delivered by the intramuscular (IM) route and had no effect on the humoral response. The intradermal route exacerbated disease following challenge whereas the IM route reduced the symptoms, the duration of viral excretion in correlation with higher anti-HA2 and anti-M2e antibody responses.In the case of PRRSV, which was my main subject, I sought to optimize the IFNγ T cell responses by using DNA vaccines encoding antigens with conserved T-epitopes between strains, and targeted to APC. Indeed, whereas viral mutants escape neutralizing antibodies, it has been proposed that the IFNγ T cell responses are instrumental for cross-protection. I showed that the broadest T cell responses were induced by DNA vaccines combined to nanoparticles PLGA (NP) injected by the intradermal route, followed by electroporation (EP) compared with EP-only, intradermal route-only or transcutaneous dissolvable microneedles. This optimal immunogenicity was associated with a high transfection level of skin cells, an accumulation of inflammatory cells, and dendritic cells mobilisation. Next I used the EP+NP method to immunize pigs with plasmids encoding conserved PRRSV antigens targeted or not to APC via CD11c or XCR1. Pigs were immunized either with repeated injections of DNA alone or with a prime-boost DNA-MLV. The DNA-MLV regimen induced improved humoral and IFNγ T cell responses compared to DNA alone or MLV alone and the APC-targeting significantly increased the humoral response but not the IFNγ T cell response. Finally, I evaluated the DNA-MLV regimen efficacy, with an applied perspective, using naked DNA without NP and delivered by EP or by a convenient needle free injection technology (PJ). In these conditions, the DNA prime did not significantly increase the IFNγ T cell response induced by the MLV, but clearly increased the humoral response with a benefit of the APC-targeting. However, the immune potentiation induced by the DNA prime did not lead to an improved protection following a heterologous challenge. The heterologous protection was not correlated to the measured humoral and IFNγ T cell responses, and neutralizing antibodies were undetectable. Thus cross-protective effectors have not been sufficiently activated by our DNA-MLV strategy and the immune correlates of protection against heterologous PRRSV are still to be identified to develop cross-protective vaccines. Finally, this work shows that the effect of APC-targeting in pigs is influenced by delivery routes and methods and by vaccine regimen such as the prime-boost DNA-MLV.

<p>Within individual cells, the opposing processes of proliferation and apoptosis are precisely regulated. When this regulatory balance is interrupted, cells may become abnormal or even transformed. Understanding how to reverse or avoid these detrimental transformative processes begins with an intimate knowledge of the processes governing the cell cycle and apoptosis. Cell proliferation is governed by the cell cycle machinery. The cell cycle is driven by Cyclin-dependent kinase (Cdk) activity, which is dependent on the availability of specific Cyclin binding partners. The amount of available Cyclin is tightly controlled by a ubiquitin ligase protein complex called the anaphase promoting complex/cyclosome (APC/C.) This complex mediates the timely ubiquitylation and degradation of cell cycle regulators in order to control mitotic exit, the G1/S transition and to respond to signals emanating from spindle assembly checkpoint. </p><p>Given the importance of the APC/C, cells develop many ways to regulate APC/C activity. Post-translational modifications of the APC/C have been shown to alter its functionality, and many pseudosubstrate-based inhibitors have been discovered. Moreover, inhibitors such as Emi1 and Emi2, have been showed to inhibit the APC/C through their own intrinsic ubiquitin E3 ligase activities. Utilizing the <italic>Xenopus</italic> egg extract system, our laboratory has previously demonstrated that the RING domain-containing ubiquitin E3 ligase Xnf7 can inhibit Xenopus APC/C activity. In the thesis, we have identified TRIM39 as an Xnf7-related human regulator of the APC/C. Our study showed that TRIM39 restrains the ability of the APC/C to ubiquitylate Cyclin B in vitro and attenuates the degradation of Cyclin B and geminin when TRIM39 is incubated in cell lysates. Notably, it has been reported that TRIM39 activity is responsible for the accumulation of the Bax-interacting protein (and activator) MOAP-1 following etoposide-induced DNA damage. Our data indicated that MOAP-1 is a novel APC/C substrate, and that the ligase activity of TRIM39 appears to be essential for preventing its degradation. We further demonstrated that decreased levels of the APC/C activator Cdh1 induces MOAP-1 protein accumulation, thereby promoting DNA damage-induced apoptosis in 293T, PC3 and H1299 cells. This study illustrates a potential function for the APC/C in DNA damage induced apoptosis and also demonstrates that TRIM39 regulates both the cell cycle and apoptosis via APC/C inhibition.</p><p>To extend our observations regarding the role for TRIM39 in APC/C regulation, we investigated effects on the cell cycle via real-time imaging microscopy. We found cells arrest at G1/S in TRIM39 depleted RPE cells, a cell line which is commonly used for cell cycle analysis. This arrest phenotype is not observed in 293T, PC3 and H1299 cells which bear mutant p53 alleles. Further analysis showed that TRIM39 depleted RPE cells upregulate many genes that function downstream of p53 activity, such as the cdk inhibitor p21--thus, arresting cells at G1/S and reducing proliferation. The reduced growth can be rescued by p53 knockdown. Mechanistically, TRIM39 interacts with p53 and promotes destruction of p53 by ubiquitylation. This ubiquitylation is independent of the activity of the most intensively studied p53-directed E3 ligase, MDM2; depletion of both MDM2 and TRIM39 has a synergistic effect on p53 accumulation. This elevated p53 leads to more apoptosis in cancer cells bearing wildtype p53. Consequently, TRIM39 depletion might be employed as a combination treatment with MDM2 inhibitor, such as nutlin-3a, to stimulate tumor cell death.</p><p>In the thesis, we have found TRIM39 inhibits both the APC/C and p53. Both are essential regulators of cell cycle and apoptosis. Moreover, we have determined that the inhibitory activity of TRIM39 requires its E3 ligase activity. Future experiments will be directed towards investigating how TRIM39 protein stability and ligase activity are regulated to understand more fully the physiological situations in which TRIM39 is able to exert its ability to modulate the cell cycle and apoptosis. I will also discuss some preliminary data regarding changes in TRIM39 ligase activity induced by Chk1 and changes in TRIM39 protein abundance regulated by polo-like kinase 1(Plk1). Chk1 and Plk1 are essential kinases for cell cycle checkpoint and progression. Connecting Chk1 and Plk1 to TRIM39 may provide a more thorough understanding of TRIM39's ability to control the APC/C inhibition and p53 ubiquitylation in response to cell cycle or cell damage cues. Since the APC/C and p53 both can regulate cell cycle and apoptosis, further investigations into the involvement of TRIM39 in the life-or-death decision will be of great interest.</p> / Dissertation

The paralogous C-terminal binding proteins (CtBP) 1 and 2 are evolutionarily conserved transcriptional coregulators that target and disrupt the expression of several genes essential for multiple cellular processes critical to regulating tumor formation. CtBP’s ability to govern the transcription of genes necessary for apoptosis, tumor suppression, invasion/migration and EMT gives rise to its oncogenic activities. Both isoforms of CtBP are found to be overexpressed in cancers including colorectal, pancreatic, ovarian, and breast, with higher levels correlating to lower overall median survival. Although multiple lines of evidence suggest CtBP plays a role in tumorigenesis, it has never been formally characterized as an oncogene. For this reason, the goal of this dissertation was to design a set of experiments to determine the transforming ability of CtBP2 in vitro using both murine and human fibroblast and in vivo using the Apcmin/+ mouse model of cancer. Specifically, we demonstrate that overexpression of CtBP2 alone can drive transformation of NIH3T3 cells leading to loss of contact inhibition, increased x invasion/migration, and anchorage independent growth. In addition, CtBP2 was found to cooperate with the large T-antigen (LT) component of the simian virus 40 (SV40) to lead to transformation of murine embryonic fibroblasts (MEFs) and with both LT and small T-antigen (ST) to induce migration/invasion and anchorage-independent growth in BJ human foreskin fibroblasts. To confirm the role of Ctbp2 in a mouse tumor model with Ctbp overexpression, we bred Apcmin/+ mice to Ctbp2 heterozygous (Ctbp2+/-) mice, which otherwise live normal lifespans. CtBP is a known target of the APC tumor suppressor and is thus stabilized in APC mutated human colon cancers and is found in high levels in Apcmin/+ polyps. Remarkably, removing an allele of Ctbp2 doubled the median survival of Apcmin/+ mice (P <0.001) and reduced polyp formation to near undetectable levels. These data suggest the importance of CtBP2 in driving cellular transformation and identify it as a potential target for prevention or therapy in APC mutant backgrounds.

Accurate chromosome segregation is crucial as mis-segregation results in aneuploidy, which can lead to severe diseases such as cancer. The spindle checkpoint monitors sister-chromatid attachment and inhibits the onset of anaphase until all chromosomes are correctly bi-oriented on the mitotic spindle. The spindle checkpoint machinery of S.pombe is composed of many proteins, one of which is the kinase Mph1 (Mps1p-like pombe homolog). It previously has been shown that Mph1 is essential for the spindle checkpoint but not whether this is due to its kinase activity. In this study we determined the role of Mph1 kinase activity in the spindle checkpoint. To do so a kinase-dead version of Mph1, which had no detectable kinase activity, was analysed. Using this kinase-dead allele we showed that lack of Mph1 kinase activity abolished the spindle checkpoint and led to chromosome missegregation. As a result of these two defects cell viability of cells lacking Mph1 kinase activity was severely impaired. These results led to the question of how Mph1 kinase activity regulates the spindle checkpoint. Spindle checkpoint signalling is thought to mainly take place at two sites, at the kinetochore and at the anaphase promoting complex (APC). The APC is an E3 ubiquitin ligase that drives cells into anaphase by targeting the separase inhibitor securin and cyclin B for degradation by the 26 S proteasome. Upon activation of the spindle checkpoint the APC is inhibited by the mitotic checkpoint complex (MCC) composed of Slp1, Mad2 and Mad3. In this study we wanted to test whether the regulatory role of Mph1 kinase in the spindle checkpoint is via MCC binding to the APC. Using the kinase-dead version of Mph1 we showed that Mad2 and Mad3 binding to the APC is severely impaired in the absence of Mph1 kinase activity. This result led to the hypothesis that Mph1 might regulate Mad2 and Mad3 binding Using kinase assays Mad2 and Mad3 were identified as in vitro substrates of Mph1 and phosphorylation sites in Mad2 and Mad3 were determined by mass spectrometry. Phosphorylation mutants of Mad2 and Mad3 showed spindle checkpoint defects, indicating that they are important Mph1 substrates.