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Dendritic cells (DCs) are the sentinels of the immune system. They mature at the encounter of the appropriate stimuli or danger signals, which induce them to perform pro-inflammatory antigen presentation to naïve and memory T cells, resulting in inflammation. Remaining in an immature state however, DCs acquire a tolerogenic phenotype. When activated by TLR ligands, DCs undergo metabolic re-programming and switch to TBK1/IKKe/AKT-induced glycolysis at the early activation phase, which is sustained due to nitric oxide (NO)-mediated inhibition of mitochondrial metabolism at the later activation phase. Targeting DC activation in the view of promoting less activated or tolerogenic DCs could be an approach to reduce or abrogate inflammation in settings such as solid organ transplant rejection or in autoimmune diseases such as systemic lupus erythematosus (SLE). In this thesis, we present data pertaining to three different approaches for targeting DC activation including 1) the use of ethyl p / Microbiology and Immunology

The focus of this research was to investigate the use of an immortalized bovine mammary epithelial cell line as a starting material for xenogeneic transplantation into the mammary glands of immunocompetent recipients. PSG-5 cells (a clonal derivative of the MAC-T cell line engineered to express ovine IGF-I ) were transplanted into the cleared mammary fat pads of recipient mice. Following transplantation, spheroidal cell structures were observed in the cleared mammary fat pads of immunocompetent control mice and in mice exposed to PSG-5 cells during fetal development. Spheroidal cell structures were not observed in glands that had not received cell transplants. The success of this xenogeneic transplantation prompted the development of a MAC-T cell clone expressing a bacterial β-galactosidase (βMAC-T's) for use as a histological marker protein. Studies were then performed to determine the most appropriate age and location for cell transplantation into ovine recipients. Between three and 12 weeks of age, parenchymal volume in the mammary glands collected from e\ves in this study (n=4/age group: 3,6,9, and 12 weeks) increased nearly lO-fold (3.8cm³ to 34.5cm³ ). Total gland volume increased approximately 5-fold (67.3cm³ to 316.2cm³ ). Based on these determinations of parenchymal and glandular volumes, we determined that transplantation should begin with lambs at about three weeks of age. This data provides a starting point to begin trials using βMAC-T cells, which have been engineered to express a histological marker protein, for transplantation into intact ovine and murine mammary glands. / Master of Science

Organ transplantation is one of the most important and effective solutions to save end-stage patients, who have one or more critical organ failures. However, the inadequate organs for transplantation to meet the demands has been the major issue. Even worse, the lack of accurate non-invasive assessment methods wastes 20% of donor organs every year. Currently, the most frequently used organ assessment methods are visual inspections and biopsy. Yet both methods are subjective: the assessment accuracy depends on the evaluator's experience. Moreover, repeating biopsies will potentially damage the organs. To reduce the waste of donor organs, online non-invasive and quantitative organ assessment methods are in great needs. Organ viability assessment is a challenging issue due to four reasons: 1) there are no universally accepted guidelines or procedures for surgeons to quantitatively assess the organ viability; 2) there is no easy-deployed and non-invasive biological in situ data to correlate with organ viability; 3) the organs viability is difficult to model because of heterogeneity among organs; 4) both visual inspection and biopsy can be applied only at present time, and how to forecast the viability of similar-but-non-identical organs at a future time is still in shadow. Motivated by the challenges, the overall objective of this dissertation is to develop online non-invasive and quantitative assessment methods to predict and forecast the organ viability. As a result, four data-driven modeling research tasks are investigated to achieve the overall objective: 1) Quantitative and qualitative models are used to jointly predict the number of dead cells and the liver viability based on features extracted from biopsy images. This method can quantitatively assess the organ viability, which could be used to validate the biopsy results from pathologists to increase the evaluation accuracy. 2) A multitask learning logistic regression model is applied to assess liver viability by using principal component analysis to extract infrared image features to quantify the correlation between liver viability and spatial infrared imaging data. This non-invasive online assessment method can evaluate the organ viability without physical contact to reduce the risk of damaging the organs. 3) A spatial-temporal smooth variable selection method is conducted to improve the liver viability prediction accuracy by considering both spatial and temporal effects from the infrared images without feature engineering. In addition, it provides medical interpretation based on variable selection to highlight the most significant regions on the liver resulting in viability loss. 4) A multitask general path model is implemented to forecast the heterogeneous kidney viability based on limited historical data by learning the viability loss paths of each kidney during preservation. The generality of this method is validated by tissue deformation forecasting in needle biopsy process to potentially improve the biopsy accuracy. In summary, the proposed data-driven methods can predict and forecast the organ viability without damaging the organ. As a result, the increased utilization rate of donor organs will benefit more end-stage patients by dramatically extending their life spans. / Doctor of Philosophy / Organ transplantation is the ultimate solution to save end-stage patients with one or more organ failures. However, the inadequate organs for transplantation to meet the demands has been the major issue. Even worse, the lack of accurate and non-invasive viability assessment methods wastes 20% of donor organs every year. Currently, the most frequently used organ assessment methods are visual inspections and biopsy. Yet both methods are subjective: the assessment accuracy depends on the personal experience of evaluator. Moreover, repeating biopsies will potentially damage the organs. As a result, online non-invasive and quantitative organ assessment methods are in great needs. It is extremely important because such methods will increase the organ utilization rate by saving more discarded organs with transplantation potential. The overall objective of this dissertation is to advance the knowledge on modeling organ viability by developing online non-invasive and quantitative methods to predict and forecast the viability of heterogeneous organs in transplantation. After an introduction in Chapter 1, four research tasks are investigated. In Chapter 2, quantitative and qualitative models jointly predicting porcine liver viability are proposed based on features from biopsy images to validate the biopsy results. In Chapter 3, a multi-task learning logistic regression model is proposed to assess the cross-liver viability by correlating liver viability with spatial infrared data validated by porcine livers. In Chapter 4, a spatial-temporal smooth variable selection is proposed to predict liver viability by considering both spatial and temporal correlations in modeling without feature engineering, which is also validated by porcine livers. In addition, the variable selection results provide medical interpretations by capturing the significant regions on the liver in predicting viability. In Chapter 5, a multitask general path model is proposed to forecast kidney viability validated by porcine kidney. This forecasting method is generalized to apply to needle biopsy tissue deformation case study with the objective to improve the needle insertion accuracy. Finally, I summarize the research contribution and discuss future research directions in Chapter 6. The proposed data-driven methods can predict and forecast organ viability without damaging the organ. As a result, the increased utilization rate of donor organs will benefit more patients by dramatically extending their life spans and bringing them back to normal daily activities.

A method for preserving transplant organs for extended periods of time has been developed in the transplant organ preservation cooler. The preservation cooler enhances organ viability by maintaining a temperature controlled organ bath and pumping perfusate through the transplant organ. The emphasis on the transplant organ preservation cooler is to provide a simple and portable system which will be powered by boiled off oxygen from a liquid oxygen source. The design of the preservation cooler pump and temperature control system are presented. Results of tests proving the successful operation of the preservation cooler prototype are also presented. / Master of Science

No / There is growing interest in the use of mesenchymal stem cells (MSC) for immune therapy. Clinical trials that use MSC for treatment of therapy resistant graft versus host disease, Crohn's disease and organ transplantation have initiated. Nevertheless, the immunomodulatory effects of MSC are only partly understood. Clinical trials that are supported by basic research will lead to better understanding of the potential of MSC for immunomodulatory applications and to optimization of such therapies. In this manuscript we review some recent literature on the mechanisms of immunomodulation by MSC in vitro and animal models, present new data on the secretion of pro-inflammatory and anti-inflammatory cytokines, chemokines and prostaglandins by MSC under resting and inflammatory conditions and discuss the hopes and expectations of MSC-based immune therapy.

La transplantation de cellules souches hématopoïétiques (CSH) est un traitement couramment utilisé pour traiter plusieurs types de maladies hématologiques telles que les leucémies. Par contre, une limite importante de ce type de traitement est la quantité restreinte de CSH disponibles pour la transplantation. Il importe donc de trouver des moyens pour expandre efficacement ces cellules ex vivo tout en préservant leurs propriétés. Le gène HOXB4 est présentement un candidat très prometteur pour atteindre cet objectif. Il a en effet été montré que HOXB4 est capable d’expandre les CSH in vivo et in vitro sans mener au développement de leucémie. Le gène HOXC4, qui appartient au même paralogue est aussi en mesure d’expandre les cellules hématopoïétiques primitives suggérant un rôle commun pour les gènes HOX du paralogue 4 dans l’autorenouvellement des CSH. Le gène HOXA4 est dix fois plus exprimé que le gène HOXB4 dans des CSH du foie fœtal au moment de leur principale expansion. De plus, les CSH mutantes pour Hoxa4, contrairement aux CSH mutantes pour Hoxb4, sont incapables de reconstituer un receveur irradié lorsqu’elles sont transplantées en condition de compétition. HOXA4 pourrait donc jouer un rôle plus important que les autres gènes du paralogue 4 pour l’expansion des CSH au niveau physiologique. Nous avons donc posé l’hypothèse que HOXA4 est capable d’expandre des CSH de façon plus importante que HOXB4. Les résultats obtenues dans le cadre de ce projet de recherche ont montré que la surexpression de HOXA4 était capable d’expandre les CSH et les progéniteurs hématopoïétiques primitifs dans le même ordre que ce qui est connu pour HOXB4. Des cultures et des essais de transplantation en situation de compétition ont confirmé la capacité égale des CSH surexprimant HOXA4 et HOXB4 de proliférer et de reconstituer les receveurs irradiés à long terme. Par contre, nous avons observé une meilleure reconstitution périphérique à court terme par les CSH HOXA4+ par rapport aux CSH HOXB4+, associée à une meilleure reconstitution lymphoïde. Nous avons aussi comparé les niveaux d’expression de gènes cibles potentiels dans des CSH surexprimant HOXA4 ou HOXB4 et observer que plusieurs gènes importants pour la fonction des CSH était régulé positivement suite à leur surexpression, notamment plusieurs gènes impliqués dans les voies de signalisation Notch et Wnt, tels que des récepteurs et ligands. Les gènes HOX du paralogue 4 pourraient donc réguler la communication entre les CSH et leur microenvironnement via ces voies de signalisation majeures et ainsi réguler leur autorenouvellement. La modulation de différents gènes codant pour des facteurs de transcription et des molécules impliquées dans la pluripotence suggère également que HOXA4 et HOXB4 utilisent des mécanismes intrinsèques et extrinsèques pour réguler leur potentiel d’autorenouvellement. Ces connaissances pourront ainsi être utilisées pour optimiser les protocoles d’expansion ex vivo des CSH dans un but thérapeutique. / Transplantation of hematopoietic stem cells (HSC) is a treatment commonly used to treat several types of hematological diseases such as leukemia. However, a major limitation of this type of treatment is the limited number of HSC available for transplantation. It is therefore important to develop ways to expand these cells ex vivo. The HOXB4 gene is a promising candidate for achieving this goal. It has indeed been shown that HOXB4 is able to expand HSC in vivo and in vitro without inducing leukemia. HOXC4, which belongs to the same paralog group, is also able to expand primitive hematopoietic cell suggesting a common role for paralog 4 HOX genes in the self-renewal of HSC. HOXA4 is ten times more expressed in fetal liver HSC during their primary expansion. Furthermore, Hoxa4 mutant HSC, unlike Hoxb4 mutant HSC, are unable to reconstitute an irradiated recipient when transplanted in competition. Therefore, HOXA4 could play a more important role than other paralog 4 genes for HSC expansion at the physiological level and we hypothesized that HOXA4 can expand HSC more efficiently than HOXB4. The results obtained during this research project showed that the overexpression of HOXA4 expand HSC and primitive hematopoietic progenitors in the same order as HOXB4. Direct competitive culture and transplantation assays confirmed the equal capacity of HSC overexpressing HOXA4 and HOXB4 to proliferate and engraft at long-term. However, we observed a better short-term peripheral reconstitution by HOXA4+ HSC compared to HOXB4+ HSC, which was associated with a better lymphoid reconstitution. We also compared the expression levels of potential target genes in HSC overexpressing HOXA4 or HOXB4 and observed that many genes important for HSC function were upregulated following their overexpression, including several genes involved in the Notch and Wnt signaling pathway. These included both receptors as well as ligands, indicating that HOX4 genes might regulate the communication of primitive HSCs with their environment through these major signaling pathways and promote self-renewal. In addition, modulation of genes coding for transcription factors and molecules known for their function in pluripotency suggest that HOXA4 and HOXB4 have both intrinsic and extrinsic potential to control self renewal potential. This knowledge can then be further explored and used to optimize ex vivo HSC expansion protocols for clinical purposes.

Le couple franco-brésilien (composé d’un homme français et d’une femme brésilienne) est un exemple de la nécessité des constructions stratégiques de la vie conjugale et familiale selon les normes contemporaines du mariage. Le modèle de la transplantation de la femme brésilienne en France est un des moyens explicatifs du processus de socialisation conjugale et familiale de ce couple. La conjugalité est étudiée selon une démarche idéale-typique, prenant en considération les facteurs géographiques et culturels de la femme brésilienne, où la question du stéréotype est au centre de l’objet d’étude. La distance culturelle, fondatrice de cette forme de mixité conjugale, nous permet d’analyser la négociation à l’œuvre entre l’homme français et la femme brésilienne pour parvenir à l’épanouissement conjugal et familial, selon les normes établies. / The French-Brazilian couple (composed of a French man and a Brazilian woman) is an example of the necessity of the strategic constructions of the married and family life according to the contemporary norms of marriage. The model of the transplantation of the Brazilian woman in France is one way of explaining the process of conjugal and family socialization of this couple. Conjugality is studied according to an ideal-typical approach with the issue of stereotypes at the center of the object of study. Geographical and cultural factors of the Brazilian woman are taken into consideration. Cultural distance, which is at the foundation of this type of conjugal mixity, allows us to analyze the negotiation that takes place between the French man and the Brazilian woman, to reach conjugal and family fulfillment, according to the established norms.

Doctor of Philosophy / Department of Animal Sciences and Industry / Duane Davis / Umbilical cords matrix stem cells (UCs) have been characterized most thoroughly in humans (HUCs) and are considered to have great promise for regenerative medicine and cell-based therapy. Although UCs were first identified in pigs the description of porcine UCs (PUCs) is limited. Here we reported some standard mesenchymal stem cell characteristics for PUCs. Development of knowledge about PUCs is useful because the pig is a valuable biomedical model for humans and the species is an important human food source. PUCs were isolated from Wharton’s jelly using an explant technique. They attached on the plastic and showed fibroblast-like morphology. Immunophenotype analysis showed they are positive for CD44, CD90 and CD105 and negative for CD31, CD45 and SLA-DR. Under specific in vitro conditions, PUCs were differentiated to adipocytes, chondrocytes and osteocytes. The growth curve of PUCs exhibited a lag phase, log phase and doubling time of 24, 60 and 13.8 hour respectively. Engraftment potential of allogeneic PUCs administered orally and intraperitoneally (IP) was evaluated. Newborn, 1-day, 1-week, 2-week and 3-week old pigs were administered a dose of fluorescently labeled PUCs (1.1x107 cells/kg body weight) and their tissue incorporation were evaluated using confocal microscopy with confirmation by PCR to detect SRY gene, the Y-chromosome gene of male PUCs in female recipients. One week after PUCs administration, they were found mostly in the gastrointestinal tract and abdominal organs after either oral or intraperitoneal transplantation. The intestinal mucosa layer around the base of villi and intestinal crypts was the main location. PUCs were also detected in thoracic organs, muscle and bone marrow. Additionally, PKH26-labeled fibroblasts labeled were detected in recipient intestine 1 week after IP injection. Donor cells were not found in blood at one week post transplantation. When recipients were sacrificed at 6 h after IP injection PKH26-labeled PUCs were found mostly in omentum and diaphragm by PCR. It is likely these are the primary sites for donor cells in the peritoneal cavity to enter the circulation. Fluorescent in situ hybridization (FISH), using an SRY probe and PCR, demonstrated the PUCs isolated from recipient intestines by enzymatic digestion. Therefore, transplanted PUCs were recovered from the intestinal mucosa and were viable and able to proliferate in vitro.