The success of many emerging biotechnologies depends upon the ability to tune cell function to mimic conditions found in vivo. Cells exhibit complex interactions with their surrounding environment known and the extracellular matrix (ECM). These interactions control many cell functions such as proliferation, differentiation and cell death. ECM components span the meso-, micro- and nano-length scales. Successful biotechnologies therefore must also exhibit patterning over these length scales. The objective of this study is to fabricate and analyze cell response to micro and nanopatterned polymer substrates. Experiments examined cell alignment and proliferation to various substrates. The substrates used featured micropatterned grooves and holes, micropatterned carbon nanotubes, and combinations of microgrooves and nanogrooves. Results showed significant interactions between cell alignment and the patterned topography for all substrate types, while cell proliferation showed no significant dependence on these topographic parameters.

This thesis reports an investigation from the “bedside” back to the “bench”. That is, from the economic evaluation of a medical intervention to basic research and development of a contractility assay. The underlying theme of this thesis is cellular contractility, which was stimulated from our laboratory’s work in the microvascular complications of Diabetic Retinopathy (DR). The health economic perspective of this thesis evaluates the cost effectiveness and cost utility of DR prognosis using the prog-DR test. This novel prognostic test developed in our laboratory relies on the contractile response of blood vessels to detect subjects with high risk of developing DR. Markov modeling based on information in the literature was used to estimate the outcomes of a hypothetical population. The costs, health and utility outcomes of DR were compared to the potential outcomes if the prog-DR test was used. The model show that the prog-DR test can improve the health of the hypothetical population as measured in the number of life years (LY), sight years (SY) and quality-adjusted life years (QALY). The prog-DR test was more cost effective than the benchmark of annual or bi-annual screening and the incremental cost effectiveness ratio (ICER) appears to be at an acceptable level. Scenario and sensitivity analysis also show that the cost effectiveness of the prog-DR test can be improved by (i) better blood glucose management post prog-DR test, (ii) targeted screening (as opposed to population-wide screening) and (iii) reduced costs of both screening and management of DM and DR. The physiological perspective of the thesis aimed to develop a contractility assay for DR that was based on a 3D scaffold, which was affordable, easy to make and mimicked the three dimensional physiological environment of blood vessels. The contractility assay was developed using a 3D, hollow scaffold (PE-PAH capsule) and involved (i) the selection of the optimal core material, (ii) optimisation of the manufacturing process, (iii) characterisation of the scaffold and (iv) ensuring that cells can be grown on it. The cyto-biocompatibility of the candidate polyelectrolyte Poly(Sodium 4-Styrene Sulfonate) (PSS) and Poly(Allylamine Hydrochloride) (PAH) in the thin films format were investigated using three different cell lines and the effects of these thin films were also compared to titanium and titanium nitride thin films. In essence, PSS and PAH are not cytotoxic and was used to develop the contractile scaffold, PE-PAH capsule. This scaffold is relative elastic and the contractile force exerted by the 3T3-L1 cells was calculated based on the deformation of the PE-PAH capsule. The contractility assay was sufficiently sensitive to detect the nano-Newton magnitude of force developed by individual cells and discriminated the change in force due to disruption of the F-actin cytoskeleton by forskolin and cytochalasin D.

Cellular immune responses to Hepatitis C (HCV) epitopes are crucial for successful host response to HCV infection. We investigated a platform to assess specific and global immune responses in HCV infection. We identified 57 HCV peptides from literature (24 of CD4+, 33 of CD8+ specificity) and tested them in two peptide pools to assess specific response in non-transplanted and post-liver transplant (LT) patients. Robust interferon-gamma (IFN) response to CD4+ peptide and mitogen stimulation was seen in sustained virological clearance. IFN response to the CD4+ peptide pool could differentiate between SVR and NR with 82% accuracy. In patients with recurrent HCV post-LT, HCV-specific responses were attenuated, but global immune responses were preserved. Significantly lower specific (CD4+) and global immune responses (mitogen response) were observed in patients with advanced allograft disease (fibrosis score>2). Quantiferon-HCV may identify patients likely to respond to anti-HCV treatment, as well as post-LT patients with aggressive HCV recurrence. / Experimental Surgery

No description available.

The control of cell adhesion to synthetic polymers is a key factor in tissue engineering, resting on the ability to direct specific cell types to adhere and proliferate in order to stimulate tissue reconstruction. But often the surface properties are compromised for the sake of the bulk properties, leading to surfaces that do not support sufficiently the level of bioactivity required and accordingly the polymeric biomaterial will fail clinically. Laser treatment offers a unique means of enhancing the osteoblast cell response of the surface of a polymeric biomaterial, whilst keeping the already sufficient bulk properties intact. To this end, infra-red (IR) and ultraviolet (UV) lasers have been employed to modify the wettability characteristics of nylon 6,6, as wetting is often the primary factor dictating the adhesion and bonding potential of materials, as a route to enhancing the surface in terms of osteoblast cell response. What is more, modifying wettability characteristics in this way is a highly attractive means of estimating the biofunctionality of a polymer. IR (CO2) and UV (F2 and KrF excimer) lasers were employed to carry out two different processes: laser whole area irradiative processing and laser-induced patterning. With both CO2 and the excimer lasers changes in the wettability characteristics could be effected with subsequent enhancement of osteoblast cell response. This was also the case with both laser-induced patterning and laser whole area irradiative processing. Essentially, an approach has been established whereby the osteoblast cell response on the surfaces of laser treated nylon 6,6 can be predicted through the laser-induced wettability characteristics modification, particularly for the laser whole area irradiative processed nylon 6,6. This ultimately allows one to determine the osteoblast cell response of the laser surface treated nylon 6,6 surfaces directly from the laser operating parameters. In concurrence with established wetting theory the laser whole area irradiative processing of the nylon 6,6 surfaces caused increased surface roughness, increased surface oxygen content, increased polar component, γP , and increased total surface energy, γT ; thereby generating surfaces displaying reduced contact angle, θ, making the nylon 6,6 surfaces more hydrophilic. The laser-induced patterned samples differed from current theory insofar as the nylon 6,6 surfaces became less hydrophilic due to an increase in θ despite an increase in surface roughness, an increase in surface oxygen content, an increase in γP and an increase in γT . This phenomena can be explained by the transition in wetting regimes from a Wenzel regime to a mixed-state wetting regime. Nevertheless, collation of the wettability characteristics results revealed that θ was a strong correlative decreasing function of both γP and γT , indicating that surface energy played a large role in determining the wetting nature of the nylon 6,6. It was found that for all laser whole area irradiative processed nylon 6,6 surfaces the osteoblast cell response was an increasing correlative and therefore predictive function of θ and was a decreasing function of γP . To an extent, the surface oxygen content and surface roughness could be used indirectly to foretell the osteoblast cell response of the nylon 6,6 surfaces. This is on account of the CO2 and KrF excimer laser whole area irradiative processing bringing about increased surface toxicity, which above a certain level hindered the osteoblast cell response. For the laser-induced patterned nylon 6,6 samples there did not appear to be any particular correlative trend between the modified surface parameters and osteoblast cell response. This can be accounted for by the transition in wetting regimes. Another important factor is that cell morphologies were modulated over all samples which suggests that varying surface parameters on account of laser surface treatment gave rise to variations in cell signaling. It was determined that θ, γP and γT all had very strong correlative relationships with the cytotoxicity. The cytotoxicity reduced upon an increase in θ until a minimum constant was achieved, whereas the cytotoxicity remained constant at low γP and γT until a point at which the cytotoxicity began to increase. These results are noteworthy as they allow one to deduce that, with constant cytotoxicity levels, the osteoblast cell response appeared to be modulated by the wettability characteristics. But once the cytotoxicity increased, the toxicity began to dominate and so negated the identified positive wettability characteristic correlations with osteoblast cell response. Practically, the surface roughness and surface oxygen content could be implemented indirectly to estimate the cytotoxicity. Increase in cytotoxicity was the result of the laser processing with higher fluences generating excessive melting. As a result of this, it is possible to deduce that there was a maximum threshold fluence, beyond which the toxicity of the nylon 6,6 began to dominate, giving rise to a less enhanced osteoblast cell response. On account of the correlative trends which have been identified between the laser surface treatment, wettability characteristics and osteoblast cell response of nylon 6,6 it is likely for one to have the ability to estimate the osteoblast cell response in vitro. This is significant as it indicates that laser surface modification of polymeric materials could have tremendous potential for application within the field of regenerative medicine.

Gene therapy has emerged as a field to modulate cell functions by introducing genes of interest to target cells. An emerging focus in this field is to employ non-viral vectors to deliver immunosuppressive cytokines to dendritic cells (DCs) to attenuate damaging immune responses. DCs serve as potential targets for suppression of T cell responses. In this work, we investigated the ability of polycation-modified polymeric particles complexed with interleukin-10 (IL-10) gene to modulate DCs. The delivery systems (designated as PSO10H6 and PLGAO10H6) were formed by coating cationic peptide O10H6 (O: ornithine; H: histidine) on the polystyrene (PS) and poly (lactic-co-glycolic acid) (PLGA) particulates. A mouse IL-10 encoding plasmid (pIL-10) was loaded on the surface of PSO10H6 and PLGAO10H6 via ionic interactions. Physical characterization of these particles revealed stable colloidal dispersions (diameters: 297.2±14nm in PLGAO10H6-pIL-10 and 126.0±8nm in PSO10H6-pIL-10). DNA molecules carried by PSO10H6 and PLGAO10H6 were protected from serum digestion. Results from in vitro gene transfection studies showed two-fold enhancement of IL-10 expression in bone marrow-derived DCs transfected with PSO10H6-pIL-10 and PLGAO10H6-pIL-10 compared to untransfected DCs. Their suppressive functions were evaluated in an in vitro mixed lymphocyte model. Results indicated that PSO10H6-pIL-10 and PLGAO10H6-pIL-10 modified DCs elicited weakest proliferation of allogeneic bulk T cells as well as CD4 and CD8 T cells among all the delivery modes. Using cell-embedded Matrigel as a surrogate graft, we showed that IL-10 gene-modified DCs suppressed host cell infiltration in vivo. These data suggested PSO10H6-pIL-10 and PLGAO10H6-pIL-10 deliver an overriding suppressive signal to T cells. Further studies revealed T cells stimulated by the IL-10 gene-modified DCs exhibited characteristics of regulatory T (Treg) cells, as evident by up-regulation of a Treg cell marker forkhead-type transcription factor 3 (Foxp3). This result was concomitant with an increase in of transforming growth factor beta (TGF-beta) production. <br>Taken together, this work demonstrated that PSO10H6 and PLGAO10H6 are effective in delivering pIL-10 to modulate DCs to suppress T cell responses. Collectively, the results raise the prospects of using PSO10H6 and PLGAO10H6 as vectors to deliver immunosuppressive genes to modulate T cell responses in vivo. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences / Pharmaceutics / PhD / Dissertation

M.tb, the causative agent of pulmonary tuberculosis (TB) remains one of the leading causes of infectious disease-based death worldwide. BCG, the only clinically approved TB vaccine, has been in use for almost a century to vaccinate against TB. Despite its success in protecting against disseminated forms of TB, it is unable to provide protection against pulmonary M.tb infection. Although there have been many recent efforts to enhance or replace BCG, our lack of understanding towards host immunity against M.tb has substantially hindered this goal. One aspect of pulmonary M.tb infection that remains poorly understood is the induction of Th1 immunity, which is substantially delayed in comparison to other pulmonary infections. This allows the bacteria to establish an infectious foothold within the host and impairs the ability of the host to clear the infection. Given the importance of the innate immune response in the induction of adaptive immunity, this delay in the establishment of Th1 immunity following pulmonary M.tb infection is likely due to a defect in the early innate immune response. However, the specific roles of this immune compartment in regards to T cell activation following pulmonary M.tb infection is still not well understood. As such, the scope of this thesis is to gain an increased understanding towards the role of the innate immune compartment in the generation of Th1 responses. Such insights will allow us to develop new strategies to improve upon future and existing TB vaccine design. / Thesis / Doctor of Philosophy (PhD)

Les arbovirus sont un groupe de virus transmis par des vecteurs arthropodes contaminant chaque année plusieurs centaines de millions de personnes à travers le monde. De nombreux facteurs environnementaux, comme les xénobiotiques peuvent influencer la propagation des infections virales, la susceptibilité de l'hôte et la sévérité de l'infection. Parmi eux, le cadmium, métal toxique est connu pour moduler la résistance de l'hôte lors des infections par les arbovirus. Ces travaux de thèse se sont portés sur l'étude de l'alphavirus Sindbis et du flavivirus Zika infectant des cellules épithéliales humaines. Les résultats de cette thèse ont permis de démontrer que les cellules A549 étaient permissives à la souche épidémique de 2013 du virus Zika. Le virus se réplique efficacement et induit une apoptose de type mitochondriale dans les cellules A549. La réponse immunitaire innée des cellules a été caractérisée et le rôle des interférons de type I a été mis en avant. Ces résultats peuvent contribuer à une meilleure compréhension des mécanismes de pathogénicité de ce virus chez l'homme. Par la suite, les effets d'une exposition au cadmium lors des infections par les virus Sindbis et Zika ont été évalués. L'infection par le virus Zika n'a pas été modulée par l'exposition au cadmium in vitro. Mais de façon surprenante dans le cas de l'infection des cellules HEK 293, le cadmium a protégé les cellules des effets cytopathiques induits par le virus Sindbis. En présence de cadmium, l'apoptose induite par le virus a été inhibée et la réplication du virus Sindbis a été réduite. / Arboviruses are a group of viruses transmitted by arthropod vectors infecting hundreds of millions of people each year worldwide. Many environmental factors including xenobiotics can influence the spread, the susceptibility and the outcome of viral infections. Among them, cadmium, a toxic metal is known to affect the host resistance against arboviral infection. In this thesis, our work has been focused on studying the infection capability of the Sindbis alphavirus and the Zika flavivirus in human epithelial cells. The results demonstrated that A549 cells was permissive to the 2013 epidemic strain of Zika virus. The virus replicates efficiently leading to mitochondrial apoptosis. The innate immune response was characterized and the crucial role of type I interferon was highlighted. These results may contribute to a better understanding of Zika virus pathogenesis in humans. Thereafter, the effects of cadmium exposure on Sindbis virus and Zika virus infection was evaluated. Zika virus infection was not modulated by cadmium in vitro. Interestingly, cadmium protected the HEK 293 cells from the cytopathic effect induced by Sindbis virus. Cadmium exposure inhibited the apoptosis and reduced the viral replication.

Le lupus érythémateux disséminé (LED) est une maladie auto-immune systémique caractérisée par une inflammation provoquant des lésions dans de nombreux organes tels que les reins, les poumons ou la peau. Dans cette pathologie, une activation excessive du système immunitaire conduit à la production d’auto-anticorps dirigés, le plus souvent, contre du matériel nucléaire. La différenciation des lymphocytes B (LB) en cellules productrices d’anticorps requiert une communication entre les LT et les LB. Ce dialogue est régulé par de nombreux acteurs cellulaires et moléculaires afin de permettre la mise en place d’une réponse humorale efficace en cas d’infections, mais aussi de prévenir le développement de maladies auto-immunes. Mon projet de thèse a consisté à étudier l’implication de deux de ces acteurs, l’un favorisant la différenciation des LB en plasmocytes, à savoir, les cellules T folliculaires auxiliaires (TFH) et le second régulant négativement l’activation lymphocytaire, le récepteur co-inhibiteur BTLA (pour B and T Lymphocyte Attenuator) dans le LED chez l’Homme. Au cours de cette étude, nous avons d’une part amélioré les connaissances concernant les sous-populations de TFH circulantes humaines, en décrivant que parmi les cellules TFH CXCR3-CCR6- sont retrouvées des cellules aux propriétés suppressives. De plus, nous avons suggéré que la contraction des TFH1 (CXCR3+CCR6-) au profit des TFH2 (CXCR3-CCR6-), observées chez les patients lupiques, pourrait être le reflet d’une migration des TFH1 vers les organes inflammés. D’autre part, nous avons mis en évidence un défaut fonctionnel de BTLA dans les LT CD4+ de patients lupiques. Ce défaut, restauré en normalisant le métabolisme lipidique des LT CD4+, semble associé à la sévérité de la pathologie. En parallèle de ces observations, nous avons démontré un défaut d’expression de BTLA sur les LB et les LT CD4+ régulateurs de patients lupiques. L’ensemble de nos données sont prometteuses et ouvrent de nouvelles perspectives thérapeutiques pour le traitement du LED. / Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by lesions in several organs such as kidneys, lungs and skin for instance. In this pathology, an excessive activation of the immune system leads to the production of autoantibodies targeting mainly nuclear antigens. B cell differentiation into antibody-secreting cells requires a close collaboration between T and B cells. This cross-talk is regulated by various cellular and molecular factors in order to mount an efficient humoral response in case of infection, but also to prevent autoimmune disease development. The aim of my thesis was to study two regulating factors of the B cell response, one promoting the B cell differentiation into plasma cells, i.e the follicular helper T cells (TFH) and the other one inhibiting lymphocyte activation, i.e a co-inhibitory receptor called BTLA (« B and T Lymphocyte Attenuator ») in human SLE. In this study, we first improved our knowledge concerning human circulating TFH cells, by describing among the CXCR3-CCR6- TFH cell subset, a population with suppressive capacities. Moreover, we suggested that the decreased frequency of TFH1 in lupus patients’ blood could be explained by the migration of these cells into inflamed tissues. We also highlighted a BTLA functional deficiency in lupus CD4+ T cells. This deficiency, which can be restored by normalizing the lipid metabolism, seems to be associated to disease severity. Furthermore, we described an altered expression of BTLA in lupus B cells and regulatory T cells. Altogether, our data show promising results and suggest new potential therapeutic strategies for lupus treatment.

L’activation des lymphocytes T (LT) CD8 naïfs mène à leur différenciation en deux sous-populations d'effecteurs, les SLEC (short-lived effector cells) et MPEC (memory precursor effector cells). Après contrôle de l’infection, les SLEC meurent par apoptose tandis que les MPEC deviennent des cellules mémoires qui protègent contre la réinfection. Peu de choses sont connues sur le rôle des mécanismes post-traductionnels, tel que la déubiquitination lors de la différenciation des LT CD8+. La déubiquitinase (DUB) BAP1 joue un rôle clé dans la différenciation thymique et dans le maintien des populations de LT matures. Elle interagit avec plusieurs partenaires comme YY1 et EZH2 dans des cellules autres que les LT. Certains de ces partenaires ont des rôles importants dans la biologie des LT CD8 notamment en contexte infectieux suggérant que BAP1 régule la réponse des LT CD8+ lors d’une infection. Afin de tester cela, des LT CD8 OT-I spécifiques pour le peptide ovalbumine dans lesquelles BAP1 a été surexprimé ont été transférés dans des souris infectées avec la bactérie Listeria monocytogenes codant pour l’ovalbumine (LM-OVA). Nos résultats au pic de la réponse démontrent un défaut de l’expansion clonale des LT CD8+, de la différenciation en SLEC et une augmentation de la différenciation en MPEC. Nous observons aussi une augmentation de la différenciation en LT centrale mémoire (TCM) au stade mémoire. Finalement, nous évaluerons aussi l’impact de la délétion de Bap1 dans la réponse des LT CD8+. Cela contribuera à une meilleure compréhension du rôle de l’ubiquitination dans la biologie des LT CD8+ dont l’importance est centrale dans la réponse face aux infections et au cancer. / Following antigen recognition, naive CD8+ T cells expand massively and differentiate into effector cells. After pathogen clearance, the short-lived effector cells (SLECs) die while memory precursor effector cells (MPECS) persist and differentiate into memory T cells to confer long-term protection against reinfection. The transcriptional network controlling the SLEC/MPEC differentiation is well characterized but little is known about the role of posttranslational modifications, such as deubiquitination, in this process. The deubiquitinase BAP1 interacts with multiple partners including YY1 and EZH2 that are important for CD8+ T cell response. BAP1 has been shown to participate in thymic differentiation and in the maintenance of mature peripheral T cells. However, the function of BAP1 during CD8+ T cell response to infection is unknow. To address this, we overexpressed BAP1 wild type (WT) in ovalbumin-specific (OT-I) CD8+ T cells by retroviral transduction and analysed their response after adoptive transfer into mice infected with Listeria monocytogenes encoding ovalbumin. The overexpression of BAP1 WT severely reduced CD8+ T cell expansion, SLEC differentiation and functionality. It also induces enhanced MPEC differentiation. In fact, we observed an increase in central memory CD8 T cell (TCM) differentiation 30 days following infection. Finally, we confirmed the presence of key partners of BAP1 complex in activated and naïve CD8+ T cells. As next steps, we will analyse the impact of BAP1-deficiency in CD8+ T cell response to infection. This will contribute to a better understanding of the role of deubiquitination in CD8+ T cell response