Unlike most RNA and DNA containing viruses, which induce cytogenetic damage at random sites throughout the human genome, the highly oncogenic adenovirus type 12 is also capable of inducing damage at specific chromosomal sites. Infection of human embryonic retinal or kidney cells with Ad12 results in the induction of heterochromatic gaps at specific (17q21-22, 1p36, 1q21, and 1q42-43) and random sites in the cellular chromosome. Previous work by Durnam et al. (1986) demonstrated that the viral early region 1 (E1) is sufficient for the induction of damage at band 17q21-22. The objective of the present study was to 1) identify the Ad12 E1 gene product(s) required for the induction of aberrations in human diploid cells, and 2) to determine whether the same or different functions are involved in the induction of damage at specific and random sites. To this end, adenovirus type 12/adenovirus type 5 recombinants with hybrid E1 sequences as well as viruses with mutations in the Ad12 E1B genes were used to map the Ad12 E1 function(s) required for the induction of chromosomal aberrations. The results of this study indicate that the expression of E1A proteins is not sufficient for this effect. On the other hand, mutations within the E1B 55Kd protein but not the E1B 19Kd protein were found to affect the ability of the virus to induce both specific and random damage (Schramayr et al., 1990). / Thesis / Master of Science (MS)

The potential of human adenovirus (Ad) to serve as a vector for expression of heterologous genes was evaluated. An experimental gene, consisting of sequences coding for the glycoprotein of vesicular stomatitis virus (VSV) attached to the promoter and polyadenylation signal of the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene, was inserted into early region 3 of adenovirus, in both orientations. The TK promoter was functional in both orientations. The TK promoter was functional in both orientations and responded to trans-activation by HSV infection. Abundant expression of VSV G however depended on the presence of a second transcript. This transcript was present only in the recombinant carrying the insertion in the orientation parallel to the E3 promoter (AdG12) and was initiated upstream of the insertion, within Ad sequences. The potential of Ad recombinants to serve as vaccine vectors was investigated using the recombinant AdG12. Antibody against VSV G was induced in cows, pigs, and dogs in response to infection with AdG12. Protection of mice, immunized with AdG12, against a lethal challenge with VSV demonstrated the biological effectiveness of this immune response. / Thesis / Master of Science (MS)

Transformation by human adenovirus type 5 requires the cooperation of gene products from both the E1a and E1B early transcription units. Our major goal was to better understand the individual roles that the E1B proteins play in the transformation process. In order to determine the specific contribution made by the two major E1B proteins, 19K and 58K, mutants were constructed which were defective in the synthesis of each protein. Analysis with these mutants suggested that 58K appeared to be necessary for efficient plaque formation on human HeLa cells whereas 19K was not required. Mutants which failed to produce 19K or made a truncated 19K product displayed the cyt/deg phenotype characterized by production of large plaques and degradation of DNA These properties were not apparent with point mutants at methionine 120 or serine 164 of 19K or with mutants defective for 58K production. All E1B mutants produce E1A at levels comparable to wild type adenovirus 5, suggesting that neither E1B protein affects the regulation of E1A expression. Of interest was the observation that in combination with E1A, both 19K and 58K were able to induce transformation of baby rat kidney cells. However, the efficiency of transformation was greatly increased if both these E1B products were present. It seems likely that the mechanism of transformation involving each of these E1B proteins utilizes different pathways, but these pathways appear to be additive. / Thesis / Master of Science (MS)

The repair of DNA damage is vital to the health and survival of organisms and their cells. In humans, there exist several disorders that involve the inefficient processing or repair of DNA damage. Cellular sensitivity to DNA-damaging agents is a hallmark of repair-deficient syndromes which are often associated with an increased risk of cancer. In this work, I have investigated the repair of cisplatin-damaged DNA by utilizing host cell reactivation and cellular capacity assays that assess DNA repair using adenovirus (Ad) as a probe. Cisplatin is a widely used chemotherapeutic drug that induces both intrastrand and interstrand crosslinks in DNA. The host cell reactivation (HCR) assay examines the ability) of host cells to repair and hence, replicate cisplatin-damaged Ad DNA. This assay is believed to primarily be a measure of bulk nucleotide excision DNA repair. The cellular capacity assay examines the ability of cisplatin-damaged cells to support the replication of undamaged Ad DNA, and is thought to reflect the repair of the active cellular genes necessary for Ad replication. The repair of cisplatin-damaged DNA was studied in three human genetic syndromes -Roberts syndrome (RS), xeroderma pigmentosum (XP) and Li-Fraumeni syndrome (LFS). Fanconi's anemia (FA) cells were also used as a contml strain. RS is characterized by growth retardation, limb reductions and craniofacial abnormalities. Cell; from a subset of RS patients, termed RS+, are hypersensitive to several DNA-damaging agents, and it has been suggested that this hypersensitivity may result from a deficiency in the DNA repair capacity of these cells. (XP patients are sensitive to ultra violet light and are prone to the development of skin cancers. Cells from these patients are deficient in the nucleotide excision repair (NER) pathway responsible for repair of UV -induced lesions.) Patients with FA have a variety of congenital abnormalities, including a high susceptibility to leukemia. FA cells are sensitive to DNAcrosslinking agents such as mitomycin C (MMC) and cisplatin. Using the HCR and cellular capacity assays, deficiencies in DNA repair were detected in the XP and FA fibroblasts but not in the RS+ fibroblasts when compared to normal strains. The NERdeficient XP cells showed a significant reduction in both HCR of cisplatin-damaged Ad and in their capacity to support Ad replication following cellular cisplatin damage suggesting that cisplatin damage is repaired at least in part by the NER pathway. The normal HCR and capacity response of the RS+ cells compared to the XP cells suggests that the hypersensitivity of RS+ cells to DNA damage is not due to a deficiency in NER. The FA cells had normal HCR of cisplatin-damaged Ad but were significantly reduced in their capacity to support Ad replication following cisplatin treatment which was attributed to a defici,~ncy in the repair of DNA interstrand crosslinks. RS+ cells were not reduced in their capc.city to support Ad DNA replication, suggesting that the RS+ cellular hypersensitivity doe; not result from a deficiency in interstrand crosslink repair as seen with FA cells. LFS is a cancer prone syndrome that involves mutations in the p53 tumour suppressor gene. It was found that HCR of cisplatin-damaged Ad was normal in both p53-heterozygous and -hemizygous LFS cells, whereas the NER-deficient XP cells had significantly reduced HCR. The capacity of cisplatin-damaged, p53-heterozygous LFS fibroblasts was significantly reduced compared to normal cells. This suggests that although the LFS fibroblasts appear to have normal bulk NER, as shown by HCR, they appear to be deficier in the repair of the actively transcribed cellular genes necessary for viral replication. These results suggest a role for p53 in the repair of cisplatin damage of active genes. / Thesis / Master of Science (MS)

The requirement for identical inverted terminal repeats (ITRs) for viral viability and the role of internal viral sequences in the specification of the sequences of the termini were investigated. The viral strains used in this study were a variant Ad2 strain Ad2 (mac) and the wild type Ad5 strain which was very similar to the former one in sequence except at the extreme end of the terminal repeat. A hybrid virus (sub54), obtained by recombination between Ad2 (mac) and Ad5, derived the left 41-51% of its genome from Ad2 (mac) and the right 59-49% from Ad5. The identity of the termini was determined by Southern blotting analysis using 32p end labeled oligocleoxynucleotides. Analysis of the sub54 isolate indicated that both Ad2 (mac) and Ad5 ITRs were present. Plaque purification of sub54 demonstrated that viruses with non identical terminal sequences were viable and allowed their characterization. This analysis also indicated that Ad5 ITRs are converted to Ad2 (mac) ITRs possibly as a result of repair of the ends to yield viruses with identical termini. A model involving replication and emphasizing the importance of panhandle formation as a replicative intermediate is proposed. These results also indicated a possible role of the internal sequences of adenovirus in the selection and maintenance of serotype specific ITRs. The preference for Ad2 (mac) termini observed during repair of the ends of sub54 may be related to the origin of the genes coding for the adenoviral polymerase and/or the terminal protein both of which were derived from Ad2 (mac). Further investigation would be required to determine whether these replicative proteins are actually involved in ITR conversion. Transformation of Escherichia coli with a DNA preparation from sub54 infected rat embryo cells resulted in the isolation of the plasmid pFG154. This plasmid contained the entire adenovirus genome with an Ad2 (mac) ITR at the "left" terminus covalently linked to an Ad5 ITR at the "right". Analysis of the viral progeny generated upon transfection of mammalian cells with pFG154 indicated that the Ad2 (mac) ITRs were very efficiently converted to Ad5 termini. These results, although apparently contradictory to those initially obtained from the plaque purification of sub54, may be explained by an ITR repair model which is specific for infectious circles. / Thesis / Master of Science (MS)

Immune responses during lung infections must be tightly regulated in order to permit pathogen eradication while maintaining organ function. Although mechanisms involve complex networks of cytokines, the interferon (IFN) response has been shown to be an important driver of lung inflammation. Type I IFNs consist of a group of structurally similar cytokines that are produced during virus infection and are an integral part in regulating the immune response. However, in response to certain stimuli, type I IFNs have also been found to be central in the initiation of lung inflammatory responses by inducing the recruitment and activation of immune cells and thus may contribute to disease severity. Another cytokine that has been associated with chronic lung inflammation is the gp130 cytokine, Oncostatin M. Transient pulmonary overexpression of Oncostatin M by Adenovirus vector (AdOSM) induces lung inflammation biased toward Th2 cytokines associated with eosinophilia and alternatively activated (AA/M2) macrophage accumulation. Here we demonstrated that C57Bl/6 mice deficient of the type I interferon receptor (IFNAR1-/-), were less responsive against a suboptimal dose of AdOSM at day 7 post infection compared to AdOSM-treated wild-type. We observed a significant reduction in OSM mRNA and protein levels in AdOSM-treated IFNAR1-/- mice, compared to treated wild-type, which resulted in significant attenuation in OSM-induced inflammatory cell infiltration, epithelial hyperplasia, and alveolar wall thickening. Furthermore, IL-6 overexpression (as a comparator gp130 cytokine), induced lymphocyte accumulation in IFNAR1-/- mice, but at significantly lower levels than AdIL-6-treated wild-type. These results demonstrate that cross talk between IFNAR and gp130 cytokine signaling were required for maximal AdOSM- and AdIL-6-mediated pulmonary inflammation. We also observed that IFNAR1 deficiency directly and negatively regulated OSM-mediated responses in vitro. OSM-induced pSTAT3 levels were consistently lower in murine and human IFNAR1-deficient fibroblasts, compared to OSM-stimulated wild-type cells. This was associated with diminished OSM-induced IL-6 and MCP-1 production from IFNAR1-/- fibroblasts. Furthermore, we found that the combination of OSM and IFN-α led to increased IL-6 production from C57Bl/6 and BALB/c-derived Mouse Lung Fibroblasts (MLFs) then when either cytokine was used alone suggesting that these two cytokines can work in concert. Our findings are the first to suggest that IFNAR signaling participates in OSM-mediated responses in vitro and is required for maximal AdOSM-induced pulmonary inflammation in vivo. / Thesis / Master of Science (MSc)

Les adénovirus ont une double nature, soit comme pathogène omniprésent qui peuvent occasionnellement causer des maladies soit comme vecteurs utilisés de transfert de gène. À nos connaissances, les 30 premières minutes depuis la liaison au récepteur jusqu'à l'arrivée au pore nucléaire sont identiques pour le pathogène comme pour le vecteur. L'objectif de ma thèse était de comprendre les mécanismes impliqués dans la liaison au récepteur, l'internalisation, l'échappement et le trafic endosomal vers le MTOC. J'ai d'abord étudié le mécanisme impliqué dans l'hémagglutination des virus à tropisme pour CAR et à tropisme pour SA. J'ai identifié la présence de CAR sur les érythrocytes humains et montré qu'il était le principal responsable de l'agglutination induite par les virus à tropisme pour CAR. De plus, j'ai montré que la présence de CAR sur les érythrocytes pouvait piéger le virus dans le sang et ainsi empêcher l'infection au niveau du foie. Dans un deuxième temps, j'ai participé à la caractérisation du rôle de la protéine VI et la translocation du virus au MTOC. Nous avons montré que Nedd4 était impliqué dans le ciblage du virus au MTOC via l'ubiquitination de la protéine VI. Enfin, j'ai travaillé sur le neurotropisme de CAV-2 et caractérisé sa localisa tion subcellulaire au niveau des synapses. J'ai montré qu'une partie de CAR était localisée dans des radeaux lipidiques à la synapse et que CAV-2 entrait via la voie de recyclage des vésicules synaptiques. / Adenoviruses have a dual nature as ubiquitous pathogens that occasionally cause life-threatening disease and their use as gene transfer vectors. To the best of our current knowledge, the first 30 min from binding to nuclear pore docking of both wild-type virus and vector are identical. The goal of my thesis is to understand different mechanisms involved in receptor binding, internalization, endosomal escape and trafficking to the MTOC. First I studied the mechanism involved in hemagglutination of CAR-tropic and SA-tropic viruses. I identified the presence of CAR on human erythrocytes and showed that it was the main responsible for the agglutination mediated by CAR-tropic viruses. Moreover, I show that CAR on erythrocytes can sequester virus in the bloodstream and block liver infection. In a second part I participated to the characterization of the role of the protein VI and the translocation of HAd to the MTOC. We showed that Nedd4 was involved in the targeting of the virus to MTOC through ubiquitination of this protein VI. Finally, I worked on the neurotropism of CAV-2 and characterize its subcellular localization at the synapse. I showed that a part of CAR was localized in lipid raft at the synapse and enter through the synaptic vesicle-recycling pathway.

L'objectif de ce travail a consisté à (i) déterminer l'occurrence et (ii) à étudier le comportement de différents micro-organismes pathogènes pour l'Homme reconnus comme responsables d'épidémies d'origine hydrique dans des ressources (eaux de surface et eaux de nappes) et des eaux traitées de plusieurs collectivités situées le long de la Moselle. Trois agents microbiologiques aux propriétés morphologiques et intrinsèques très différentes ont été étudiés: des bactéries (C. jejuni et C. coli), des virus entériques (adénovirus humains dont les adénovirus entériques), des parasites (C. parvum, C. hominis, C. meleagridis). Après avoir mis en place différentes méthodologies permettant leur détection, leur quantification et la révélation de leur viabilité et/ou leur infectiosité, nous avons montré que l'eau de la Moselle était largement contaminée par ces pathogènes à des concentrations variables selon la période de l'année. Ainsi par exemple, il a été retrouvé des bactéries C. jejuni plus fréquemment en été que pendant la période hivernale alors que l'inverse a été observé pour les adénovirus humains. Les résultats d'occurrence au niveau des nappes alluviales étudiées ont montré que malgré une protection liée à la filtration naturelle de l'eau, elles pouvaient être contaminées par des campylobacters viables et des adénovirus entériques. Concernant les eaux traitées, plusieurs signatures génomiques liées à ces deux derniers agents (bactérien et viral) ont été révélées par des outils de biologie moléculaire sans qu'un risque en lien avec la viabilité et l'infectiosité puisse être établi. Dans ces eaux, les expérimentations d'inactivation ont clairement montré la grande sensibilité de ces deux agents au traitement chloré mais pas celle des parasites Cryptosporidium qui s'avèrent être les pathogènes les plus résistants quel que soit le milieu ou la température d'incubation / The aim of this study was (i) to determine the occurrence and (ii) to study the behavior of different microorganisms known to cause human waterborne outbreaks in raw waters (surface water and groundwater) and treated water of several reservoirs along the Moselle river. Three pathogens were studied: bacteria (C. jejuni and C. coli), enteric viruses (human adenoviruses which enteric adenoviruses), parasites (C. parvum, C. hominis, C. meleagridis). Because of these micro-organisms have different properties such as size, various methodologies were used for their detection, quantification and viability and/or infectivity. In the study of occurrence we showed that the Moselle river was heavily contaminated by these pathogens in varying concentrations depending on time of the year. For example it was found bacteria C. jejuni more frequently in summer than during winter while the reverse was observed for human adenoviruses. The results obtained in groundwater showed that despite a protection linked to the natural filtration of water, they could be contaminated with viable campylobacters and enteric adenoviruses. In the treated water, several genomic signatures associated with these two agents (bacterial and viral) were revealed by molecular biology tools without any risk related to the viability and infectivity can be established. In these waters, the inactivation experiments clearly showed the high sensitivity of these two agents to chlorination but not the parasite Cryptosporidium, which known to be the most resistant pathogens, whatever the type of water or incubation temperature

Angiogenesis, the formation of new blood vessels from pre-existing ones, is a crucial step in many pathologies, including tumor growth and metastasis. We developed an adenovirus vector allowing the 16K hPRL expression, an antiangiogenic factor, in situ. We show 16K hPRL inhibits oxygen-induced retinopathy in mice. Then, using the 16K-Ad vector, we investigated the ability of 16K hPRL to prevent metastatic spread through inhibition of angiogenesis. We show that 16K hPRL administered via adenovirus-mediated gene transfer inhibits tumor growth in a subcutaneous B16-F10 mouse melanoma model by reducing the size and width of tumor vessels. We also show, for the first time, that 16K hPRL considerably reduces the establishment of B16-F10 metastases in an experimental lung metastasis model. These results highlight a potential role for 16K hPRL in anticancer therapy for both primary tumors and metastases. In parallel, we have sought to identify in human 16K PRL (16K hPRL) a peptide that might be responsible for its antiangiogenic activity. Although the 16K fragments of the other three human PRL/GH-family members are also potently antiangiogenic, the sequence similarity of these fragments is low (around 35% similarity between all mammalian PRL/GH sequences). This led us to seek a peculiar common structural feature rather than a similar sequence. We demonstrate that all these fragments possess a 14-amino-acid sequence having the characteristics of a tilted peptide. We show for the first time that tilted peptides exert antiangiogenic activity. The tilted peptides of hPRL and hGH induce endothelial cell apoptosis, inhibit endothelial cell proliferation, and inhibit capillary formation both in vitro and in vivo. These antiangiogenic effects are abolished when the peptides hydrophobicity gradient is altered by mutation. We further demonstrate for the first time that the well-known tilted peptides of SIV gp32 and Alzheimers beta-amyloid peptide are also angiogenesis inhibitors. Taken together these results point to a potential new role for tilted peptides in regulating angiogenesis. Langiogenèse, la formation de nouveaux vaisseaux à partir de vaisseaux préexistants, joue un rôle important dans de nombreuses pathologies incluant la croissance tumorale et la dissémination des métastases. Nous avons développé un vecteur adénoviral (16K-Ad) nous permettant de produire le facteur antiangiogène hPRL 16K directement in situ. Une première analyse permettant détudier les effets de la hPRL 16K produite par lapproche adénovirale a été réalisée in vivo. Nous montrons tout dabord que la hPRL 16K produite est capable de prévenir la néovascularisation rétinienne dans un modèle murin de rétinopathie. Par la suite, en utilisant le vecteur 16K-Ad, nous montrons que la hPRL 16K peut inhiber le développement tumoral dans un modèle murin développant des tumeurs formées par les cellules de mélanome B16-F10 dans le tissu sous-cutané. Cette inhibition de la croissance tumorale est corrélée avec une diminution de la taille des vaisseaux. Nous montrons aussi, pour la première fois, que la hPRL 16K peut considérablement réduire létablissement des métastases B16-F10 dans un modèle expérimental de métastases se développant dans le poumon. Ces résultats soulignent le rôle potentiel de la hPRL 16K dans la thérapie anticancéreuse dirigée contre les tumeurs primaires et les métastases. Parallèlement à ces travaux, nous nous sommes attachés à identifier une région responsable de lactivité de la hPRL 16K. Partant du fait que les fragments 16K des trois autres membres de la famille humaine PRL/GH sont aussi de puissants facteurs antiangiogènes, malgré que leur similarité de séquence soit faible (environ 35% de similarité entre les séquences de la PRL et de la GH), nous avons recherché une caractéristique structurale commune partagée par ces différents fragments. Nous avons identifié un domaine susceptible d'adopter une structure en peptide oblique dans les séquences protéiques des fragments de 16 kDa de la famille PRL/GH. Leffet antiangiogène des peptides obliques de la hPRL 16K et de la hGH 16K est montré dans des expériences menées in vitro et in vivo. Nous montrons également leffet antiangiogène des peptides obliques du peptide de fusion gp 32 du virus SIV et du peptide b-amyloïde.

Gene therapy has received much attention the last decade as a method to correct a number of disorders arising from a defective gene. Gene therapy can be defined as the introduction of a functional genetic element into a cell for a therapeutic purpose. This is a very broad term and gene therapy can be applied to a wide range of diseases from genetic diseases such as cystic fibrosis to infectious diseases or even acquired genetic diseases such as cancers. Adenoviruses (Ad) are the second most common vector for gene therapy in clinical trials today, and these vectors are mostly based on serotype 2 or 5 (Ad2 and Ad5). It has been shown that Ad2 and Ad5 use a receptor that is often downregulated in malignant cells and they also suffer from shortcomings because of the high levels of pre-existing immunity against these serotypes in the society. Hence, new and improved vectors serving as alternatives to these serotypes need to be developed to make gene therapy a successful treatment option. The work presented herein is devoted to analyse what alternative adenovirus vectors could be used for treatment of glioma brain tumours. A number of different adenovirus serotypes were screened for their ability to infect human glioma tumour cells in vitro. Established cell lines as well as low-passage glioma cells from different donors were used. Adenovirus serotype 11p (Ad11p) proved to be a promising vector candidate because of its capacity to efficiently infect the glioma cells and its low prevalence in the society. The complete genome of this serotype was sequenced to further develop this as an alternative adenovirus vector. Furthermore, a number of cell lines were produced to generate E1 deleted Ad11p vectors. Other promising vector candidates were Ad16 and a chimpanzee adenovirus called CV23. Ad16 was the most efficient human serotype to infect human low-passage glioma cells and the prevalence for this serotype is also very low. The overall most efficient virus was surprisingly the non-human CV23 virus. This adenovirus has no prevalence in humans, but efficiently infects human cells in vitro. The first analysis was made on established glioma cell lines and was followed up by using low passage glioma cells from a number of different patients. The glioma cells were analysed when subjected to <20 passages (low passage) and then again at >40 passages (high passage). The cells at a higher passage number were significantly more permissive to Ad5 than the cells analysed at a low passage number. This could in part explain why some of the promising in vitro data for Ad5 have shown a limited success in vivo. In contrast, CV23 infected the low and high passage gliomas equally. This indicates that CV23 uses an internalisation mechanism subjected to less variation than the mechanism used by Ad5. We further characterised the receptor interaction of CV23 and found that none of the previously known primary receptors for adenoviruses were of importance for binding. We found that bovine serum albumin present in the growth medium was responsible for the high binding capacity to cells. Binding is a criterion for the first step of the infection, but not necessarily a good correlate to the infection capacity. CV23 infected human cells efficiently also in the absence of bovine serum albumin.