This master thesis has examined a user’s capabilities to utilize the Swedish national web archive Kulturarw³ for research purposes. The aim was also to identify any potential areas of improvement in the user’s capabilities working with Kulturarw³. The research questions are: 1. How does Kulturarw³ operate? 2. What are the main factors which affect Kulturarw³ structure and function? 3. What capabilities exist for researchers and students to utilize Kulturarw³ for their research? Are there any potential areas of improvement to the web archives user capabilities? The author has analyzed the web archive altogether using institutional theory in organization studies. The analysis has been loosely structured after Staffan Furusten’s model of the outside world in using institutional theory in organization studies. The purpose of this is to explain why the web archive looks the way it does today. An understanding of the web archive will better illuminate why any potential areas of improvement identified may or may not be possible for KW3 to implement. The author has conducted email interviews, in-person interviews as well as digital interviews with the staff responsible for working with Kulturarw³ at the Swedish National Library, Kungliga biblioteket. A draft of guidelines concerning Kulturarw³ from Kungliga biblioteket and a video-interview at Internetmuseum with one of the the founders of the web archive has also been used as source-material for this master thesis. The author concluded that Kulturarw³ is a national web archive with a long history. Its functions and limitations are complex. Kulturarw³s operation has changed greatly throughout its lifetime because of the surrounding environment. Several main factors which affect Kulturarw³ were identified. Several Swedish laws, international charters and initiatives, collaborations between and relations to other web archives, use of open-source software and digitalization’s impact on Kulturarw³ is discussed in detail. Kulturarw³'s long history of archiving the Swedish web makes it a valuable and plentiful source for research. Its collections and functions should be sufficient for anyone to conduct qualitative research. Yet at the current moment, the web archive is too inaccessible to live up to user’s expectations. That makes it an unviable option for research purposes. Unfortunately, there is not a lot Kulturarw³ can currently change to make it more assessable. The lack of information readily available also hinders users from using the web archive at max efficiency. There is a lot of opportunities for KB to better inform its users of its value and capabilities. An increased collaboration with Swedish research institutions would also benefit both researchers and the web archive in the long run.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Mast cells play a critical role in both acute and chronic inflammation and mature in peripheral tissues from bone marrow-derived progenitors that circulate in the blood as immature precursors. Mast cell progenitors are likely to encounter the serum-borne bioactive sphingolipid metabolite, sphingosine-1-phosphate (S1P), during migration to target tissues. Mast cells developed from human cord blood-derived progenitors cultured with stem cell factor (SCF) alone express intragranular tryptase (MCT), the phenotype predominant in the lung. S1P accelerated the development of cord blood-derived mast cells (CB-MCs) and strikingly increased the numbers of mast cells expressing chymase. These mast cells have functional FcepsilonRI, and similar to skin mast cells that express both tryptase and chymase (MCTC), also express CD88, the receptor for C5a, and are activated by anaphylatoxin C5a and the secretagogue compound 48/80. S1P induced release of IL-6, a cytokine known to promote development of functionally mature MCTC, from cord blood cultures containing adherent macrophages, and from highly purified macrophages, but not from macrophage-depleted CB-MCs. In contrast, S1P stimulated secretion of the chemokine, monocyte chemoattractant protein 1 (MCP-1/CCL2), from these macrophage-depleted and purified CB-MCs.

Les stratégies de traitement biologiques ciblant le TNF-α se sont avérées efficaces pour réduire l'inflammation et les symptômes cliniques dans plusieurs maladies inflammatoires chroniques et sont maintenant couramment utilisées pour les patients qui ne répondent pas aux AINS au cours de la spondyloarthrite (SpA). Cependant, 30 à 40% des patients ne répondent pas aux anti-TNF, et il est actuellement impossible de prédire la réponse des patients à ces biomédicaments. Pour améliorer les résultats cliniques, nous avons besoin d’une part d’une meilleure compréhension des mécanismes d’action des anti-TNF sur le système immunitaire, et d’autre part de biomarqueurs permettant de prédire la réponse à ces biomédicaments afin de guider la décision thérapeutique. Mon projet de doctorat a porté sur deux objectifs complémentaires: (i) l'objectif principal était de progresser dans notre compréhension des mécanismes pathogéniques impliqués dans la SpA axiale et de définir de quelle façon les anti-TNF-α affectent les réponses immunitaires des patients, (ii) de développer des biomarqueurs pour prédire la réponse thérapeutique aux inhibiteurs du TNF. En collaboration avec l'équipe du Pr. Dougados à l'Hôpital Cochin, nous avons recruté deux cohortes indépendantes de patients SpA ayant une maladie active et pour lesquels nous avons collecté des échantillons de sang avant l'initiation du traitement par anti-TNF puis 1 semaine et 3 mois après le début du traitement. Les réponses immunitaires de ces patients ont été analysées à l'aide de tests hautement standardisés réalisés ex-vivo sur sang circulant. Ces tests "TruCulture" se présentent sous forme de seringues, dans lesquelles 1 ml de sang total est mis à incuber avec un stimulus spécifique ; 20 stimuli différents ont été testé et validé avant et après traitement dans les deux cohortes de patients. Nous avons observé une réduction très significative de la sécrétion de IL-1ra, IL-1β, IL-8, and MIP-1β en réponse à des stimuli microbiens et à des agonistes des TLR dans les échantillons de sang prélevés 7 jours et/ou 3 mois après le début du traitement. Pour identifier les bases moléculaires de l’action des inhibiteurs du TNF nous avons analysé l'expression des gènes dans ces différentes conditions de stimulation. L'analyse bioinformatique quantitative de l'expression des gènes (QuSAGE) a révélé que les gènes les plus modulés par le traitement anti-TNF étaient NF-KB et les gènes cibles de NF-kB, y compris le TNF lui-même et l’IL1B. Nos données suggèrent que les inhibiteurs du TNF agissent principalement en perturbant une boucle autorégulatrice pilotée par NF-kB. Afin d'identifier les signatures immunologiques de réponse aux anti-TNF avant le début du traitement, nous avons corrélé les réponses immunitaires chez les patients analysés au temps 0 à la réponse thérapeutique aux anti-TNF mesurée à 3 mois. Nos résultats suggèrent que les patients atteints de SpA et exprimant des niveaux inférieurs de PAX5 et des niveaux supérieurs de SPP1 en réponse à la stimulation avec SEB avant l'initiation de la thérapie anti-TNF ont les meilleures réponses thérapeutiques. Notre recherche montre que les tests TruCulture sont un outil efficace pour étudier les fonctions immunitaires chez les patients atteints de SpA et que les effets du traitement anti-TNF peuvent être mesurés lorsque les cellules immunitaires sont stimulées. En terme de recherche translationnelle, nous avons identifié des molécules qui pourront être utilisés comme biomarqueurs pour aider les cliniciens à prédire les réponses thérapeutiques aux traitements anti TNF / The introduction of anti-TNF therapy has proven effective to reduce inflammation and clinical symptoms in several chronic inflammatory diseases. However, 30-40% of patients do not respond to TNF blockers and it is currently not possible to predict responsiveness of patients to anti-TNF therapy. Furthermore, their impact on the immune system is incompletely understood. The goals of my PhD project were (i) to define the impact of anti-TNF therapy on immune responses to microbial challenges and stimuli targeting specific immune pathways in spondyloarthritis (SpA) patients, and (ii) to identify immunological correlates associated with therapeutic responses to TNF-blockers.Using a set of whole-blood, syringe-based assays to perform ex vivo stimulation while preserving physiological cellular interactions (TruCulture assays), we have performed a pilot study in SpA patients and investigated immune responses to 20 different stimuli before and 3 months after initiation of anti-TNF therapy. These findings were validated in a replication cohort, also assessing the effects of anti-TNF agents after only one week of treatment. We observed a highly significant reduction of the secretion of IL-1ra, IL-1β, IL-8 and MIP-1β in response to selected stimuli after 3 months of treatment compared to the baseline. Interestingly, these changes were already detectable after a single injection of an anti-TNF agent. To gain insight into the molecular mechanism of TNF blockers, we profiled gene expression in the stimulation cultures from all patients. Quantitative set analysis for gene expression (QuSAGE) revealed that the gene modules most affected by anti-TNF therapy are NF-kB transcription factors and inhibitors and NF-kB target genes, including TNF itself and IL1B. Our data suggest that TNF-blockers primarily act by disrupting an autoregulatory loop driven by NF-kB. We also tested whether there is a correlation between the responses of immune cells to specific stimuli and the clinical response to TNF-blockers. The decision tree model that we trained and validated suggests that SpA patients who expressed lower levels of PAX5 and higher levels of SPP1 in response to SEB stimulation before initiation of anti-TNF therapy had the best therapeutic responses. Our study shows that TruCulture assays are an efficient and robust tool to monitor immune functions in SpA patients and that the effects of anti-TNF therapy can be measured when immune cells are challenged, but not at steady state. Our data also indicate that analyzing immune responses in patients before therapy is a promising strategy to develop biomarkers for prediction of therapeutic responses to TNF-blockers

NRH quinone oxidoreductase 2 (NQO2) is regarded as a mammalian Phase I detoxifying enzyme responsible for reducing quinones to hydroquinones. NQO2 is highly expressed in different types of cancer such as breast and prostate cancer suggesting its participatory role in the progression of these diseases. A potential reason for this is that NQO2 has the ability to modulate the stability of cyclin D1 and activity of NF-ÃŽÂoB and it has been shown that inhibition of NQO2, either genetically or pharmacologically, can alter the pattern of proliferation of cancer cells. However, the biological roles of NQO2 in cancer progression are still ambiguous and need further investigation. A panel of seven ovarian cancer cell lines (OVCs) were screened for the presence and functionality of NQO2. SKOV-3 and TOV-112D cells expressing comparatively the highest and lowest levels of NQO2 were stably transduced to silence and overexpress NQO2 respectively. Pharmacological inhibition was achieved using resveratrol or a series of novel 4-aminoquinolines synthesised in-house. Cell proliferation was monitored by cell counting and clonogenic assays. Flow cytometric analysis was used to determine cell cycle distribution and levels of ROS following modulation of NQO2 function. The expression of cell cycle regulatory markers was determined by Western blot. The contributory roles of NQO2 in determining the cytotoxicity of Adriamycin (ADR) towards OVCs was investigated using MTT assay together with evaluation of P-gp expression and basal ROS levels. In the OVCs panel, NQO2 protein levels and enzymatic activity showed an excellent correlation; with activity varying 36-fold between the cell lines. The sensitivity of OVCs to CB1954 was significantly increased when combined with the NRH-like co-factor, EP0152R. This supports the notion that NQO2 mediates the toxicity of CB1954, which is further confirmed by the strong correlation between cellular NQO2 activity and the responsiveness of the OVC cell lines to CB1954. Hydrazone quinolines showed the highest inhibitiory potency against NQO2 in SKOV-3 when compared to the typical and in-house synthesised quinolines inhibitors. NQO2-overexpressing TOV-112D cells showed more aggressive growth pattern and higher capacity to form colonies than wild-type cells. This was consistently associated with an enhancement in the progression of cells through cell cycle phases and significant reduction in Rb expression. A reduction in ROS levels in NQO2-OE cells may also explain this enhancement in cell growth. Overexpressing NQO2 also resulted in destabilisation of CDK4 and cyclin D1 with significant reduction in their expression levels, and concomitant increase in p-cyclin D1 (Thr286). The involvement of NQO2 in controlling cyclin D1 turnover is also confirmed in SKOV-3 cells when genetic silencing of NQO2 was accompanied by significant reduction in p-cyclin D1 and subsequent stabilisation of cyclin D1 levels. In spite of this, no alterations in the growth pattern of SKOV-3 cells were observed highlighting the impact of cell type on the variations in cellular responses. The role of NQO2 in determining the toxicity of ADR treatment was not proved in OVC cells. This was despite that modulation of NQO2 levels caused significant changes in P-gp expression. The intracellular basal levels of ROS was found to affect the responsiveness of OVCs to ADR as demonstrated when treating SKOV-3 with resveratrol was accompanied by significant increase in ROS levels and concomitant enhancement in the cells’ response to ADR. In conclusion, NQO2 can profoundly alter the proliferation characteristics of OVCs and is a potential therapeutic target for the treatment of this disease. However, the biological functions of NQO2 and its contributory roles in particular pathways are varied among different types of cancer -in other words- are highly dependent on cancer type.

In the gastrointestinal tract, probiotics have been shown to promote host immunity and to regulate immune signaling pathways. This study used Caco-2 cell line to examine the effects of a Lactobacillus rhamnosus isolate from “amabere amaruranu” a Kenyan traditional cultured milk, on the production inflammatory cytokines in enterocytes. Live Lactobacillus rhamnosus (MRS6AN), its cytoplasmic fraction (CF), filtered spent broth (FSB) or heat inactivated FSB (HIB) were used as treatments on differentiated Caco-2 cell monolayer in transwells. Cytokine content in the cell lysates, apical and basolateral supernatants were determined using ELISA. Caco-2 cell lysate treatments showed significantly increased anti-inflammatory TGF-β (ng/ml) levels on average about 100x more compared to the increase in pro-inflammatory IL-8 (pg/ml) levels. These levels were significantly reduced after inhibition of NF-κB. In conclusion, live Lactobacillus rhamnosus, its CF, FSB or HIB seemed to modulate the production of inflammatory cytokines in enterocytes partly via the NF-κB signaling pathway.

Nanoparticles, while included in a growing number of consumer products, may pose risks to human health due to heavy metal leaching and/or the production of reactive oxygen species following exposures. Subcellular mechanisms of action triggered as a result of exposure to various nanoparticles are still largely unexplored. In this work, an effort to elucidate such toxicological parameters was accomplished by evaluating oxidative stress generation, changes in gene and protein expression, and cell cycle status after low-dose exposures to a variety of metal and carbon-based nanomaterials in primary human dermal cells. Additionally, mitigation of nanoparticle toxicity via microencapsulation was investigated to assess the feasibility of utilizing nanomaterials in dermally implantable biosensor applications. Cellular immune and inflammatory processes were measured via qPCR and immunoblotting, which revealed gene and protein expression modulation along the NF-kappaB pathway after a variety of nanoparticle exposures. The role of immunoregulatory transcription factor NF-kappaB was examined in an oxidative stress context in cells exposed to a panel of nanoparticles, whereby glutathione conversion and modulation of oxidative stress proteins in normal and NF-kappaB knockdown human dermal fibroblasts were monitored. Results revealed decreased antioxidant response and corresponding increased levels of oxidative stress and cell death in exposed normal cells, compared to NF-kappaB incompetent cells. However, reactive oxygen species production was not an absolute precursor to DNA damage, which was measured by the comet assay, gamma-H2AX expression, and flow cytometry. Protein analysis revealed that map kinase p38, rather than p53, was involved in the halting of the cell cycle in S-phase after ZnO exposures, which caused DNA double strand breaks. Microencapsulation of fluorescent quantum dot nanoparticles, specifically, was utilized as a method to improve system functionality and surrounding cellular viability for the purpose of a dermal analyte detection assay. In vitro results indicated a functional localization of nanoparticles, as well as cessation of cellular uptake. Subsequently, cellular metabolism was unaffected over the range of time and concentrations tested in comparison to unencapsulated quantum dot treatments, indicating the usefulness of this technique in developing nanoparticle-driven biomedical applications.

Le syndrome de Bartter de type III résulte de mutations du gène CLCNKB codant le canal chlorure CLC-KB. Un grand nombre de mutations a été répertorié, mais peu d'entre elles ont été caractérisées fonctionnellement. Devant ce manque de données, nous nous sommes fixés comme objectif de procéder à une analyse fonctionnelle des mutations de CLCNKB pour tenter d'approfondir davantage les mécanismes de régulation de CLC-KB. Ce travail a nécessité l'emploi de trois systèmes d'expression hétérologue, les ovocytes de Xenopus laevis et les lignées cellulaires rénales HEK293T et MDCK, afin de procéder à des analyses électrophysiologiques et des expériences de biologie cellulaire. Nous démontrons que toutes les mutations étudiées altèrent l'expression membranaire de la protéine et que la conductance résiduelle est proportionnée à cette expression. Ainsi, la réduction des courants provient d'une réduction du nombre de canaux et non d'une altération majeure de la conduction ou de la régulation. Nous rapportons également que certaines mutations modifient la sensibilité au pH et au calcium extracellulaires du CLC-KB. Cette altération peut avoir un impact pathologique majeur. En conclusion, cette étude fonctionnelle montre le rôle essentiel du CLC-KB dans le maintien de la balance sodée et laisse entrevoir un espoir thérapeutique à long terme de restauration des anomalies précisément identifiées. Enfin, l'analyse de mutations est un outil extrêmement puissant pour les études structure-fonction. Associée à la recherche de nouveaux partenaires, elle devrait permettre de découvrir les régulations physiologiques de ce canal, pour l'heure presqu'inconnues.

Le virus T lymphotrope humain de type 1 (HTLV-1, Human T cell Leukemia Virus type 1) est l'agent responsable de la leucémie à cellules T de l'adulte, une prolifération maligne de lymphocytes T CD4+. L'activation constitutive de la voie NF-kB dans les lymphocytes T exprimant la protéine virale Tax s'est révélée primordiale pour la prolifération et la transformation induites par HTLV-1. Selon le modèle classique, Tax agit à deux niveaux de la voie NF-kB. Dans le cytoplasme, Tax active constitutivement le complexe IKK (IKB Kinase) en se liant à sa sous-unité régulatrice NEMO/IKKy. Dans le noyau, Tax interagit directement avec les dimères NF-kB dans des corps nucléaires Tax. L'ubiquitinylation et la SUMOylation de Tax ont été initialement décrites comme nécessaires pour l'activation de la phase cytoplasmique et de la phase nucléaire respectivement. Cependant, les mécanismes régulateurs des modifications post-traductionnelles de Tax restent difficiles à identifier car il n'a pas été possible d'étudier séparément l'ubiquitinylation et la SUMOylation de Tax.Au laboratoire, nous avons généré et caractérisé fonctionnellement un nouveau mutant de Tax qui nous a permis de découpler les rôles de l'ubiquitinylation et de la SUMOylation de Tax. Tax- P79AQ81A est ubiquitinylé de façon quantitativement similaire à Tax mais présente une forte réduction (80%) de SUMOylation. De plus, Tax-P79AQ81A ne forme pas de corps nucléaires. Néanmoins, ces deux défauts ne semblent pas préjudiciables pour la capacité du mutant à activer la voie NF-KB non seulement dans des lignées cellulaires mais également dans des lymphocytes T CD4+ primaires. En parallèle, nous avons montré que les corps nucléaires Tax sont rarement présents dans des lymphocytes T chroniquement infectés par HTLV-1, renforçant l'idée que ces structures ne sont pas requises pour l'activation de la voie NF-KB et probablement pas pour les autres fonctions de Tax. Enfin, nous avons démontré que les capacités d'activation de la voie NF-KB de différents mutants de Tax sont fortement corrélées à leur niveau d'ubiquitinylation mais pas de SUMOylation, confirmant que l'ubiquitinylation de Tax est la modification essentielle pour l'activation de la voie NF-KB.Le virus HTLV-2 ne possède pas les propriétés transformantes du virus HTLV-1 et les propriétés de la protéine Tax2 comparées à celles de Tax1 pourraient être à l'origine des différences de pathogénicité entre les deux virus. Notre étude a révélé que, de façon surprenante, l'activation de la voie NF-KB par la protéine Tax2 est non seulement indépendante de la SUMOylation et de la formation des corps nucléaires comme pour Tax1, mais également indépendante d'une quelconque ubiquitinylation, suggérant des mécanismes différents d'activation du complexe IKK parTax1 et Tax2.Nos études, aussi bien de la protéine Tax1 que de la protéine Tax2, nous ont donc permis de revisiter le modèle actuel d'activation de la voie NF-kB en démontrant l'impact mineur de la SUMOylation et en révélant une différence majeure en ce qui concerne le rôle de l'ubiquitinylation, distinguant les virus HTLV-1 et HTLV-2

Large signalosome assembly is a prerequisite for NF-κB signaling upon engagement of various immunoreceptors. Adaptor proteins containing protein-protein interaction domains oligomerise in response to such stimuli in order to propagate signaling. Each immunoreceptor uses distinct adaptors, as well as common ones, to achieve that. The main characteristic shared by these proteins is their ability to undergo poly-ubiquitinylation in a non-degradative manner, leading to optimal NF-κB activation. In this work, we aimed to identify novel deubiquitinylating enzymes that control ubiquitinylation status. That is how USP34 came up to be a negative regulator of NF-κB signaling in TCR-activated Jurkat cells, a T lymphocyte cell line. Our data suggest a model whereby USP34 prevents excessive NF-κB activation by acting rather late, directly or indirectly on the NF-κB:IκBα dimers, downstream of IKK, altering transcription factor DNA binding affinity. In parallel, studies of the endocellular membrane microenvironment that hosts mature signalosomes in response to TCR-, TNFR- and CD40 ligation led to the identification of an ER-residing protein, Metadherin (MTDH), which seems to globally integrate signaling before forwarding it to downstream pathway components able to activate IKK.