Thesis (PhD)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Diabetes mellitus is amongst the leading causes of morbidity and mortality in the world, affecting young, adult and old people. Beta cell replacement therapy for insulin delivery remains the ultimate remedy for diabetes. However, insufficient donor pancreas and the use of immunosuppressive drugs prevent the wide-spread of this therapy. Other avenues of self generated beta cells within the organ itself need to be explored. Therefore, understanding the chronobiology of cellular mechanisms in the lineage of beta cell induced neogenesis is a valuable tool in improving beta cell replacement in patients with diabetes. The aim of this study was to induce recapitulation of the morpho-genetic sequence of endocrine cells development in the pancreas of rats after the pancreatic duct ligation (PDL) procedure. Serial sections of PDL tissues of the pancreas were obtained from 78 Sprague- Dawley rats and were assessed morphologically. The immunofluorescent tissues were statistically analysed using a computerized morphometry technique. The protein expression indices of Caspase3, Insulin, Pdx1, Ngn3, NeuroD and Pax6 were quantified. The efficiency levels of coexpression of these homeodomain proteins separately with insulin were defined by the ratio of the mean value of insulin expression to the mean value of their respective protein expression. The morphological changes were characterized by the appearance of granulated acinar cells at 6 hours post-PDL and the proliferation of endocrine tissues from 84 hours through to 120 hours. The morpho-immunofluorescent evaluation showed the highest immunoreactivity of Caspase3 and Pdx1 at 6 hours, Ngn3 at 36 hours, Pax6 and insulin at 84 hours while NeuroD expression was at 120 hours. The immunohistofluorescent analysis showed that caspase3 and Pdx1 were the first to be expressed at 6 hours while the insulin and NeuroD expression appeared later at 84 hours and 120 hours, respectively. However, Pax6 expression was continuous across time periods post-PDL, while Ngn3 expression showed a peak at 36 hours. The efficiency (highest and earliest expression) of co-expression of all these homeodomain proteins with insulin was restricted between 12 hours and 24 hours. The optimal efficiency was at 12 hours by Ngn3 with insulin. A good efficiency was shown for Pdx1 with insulin, NeuroD with insulin and Pax6 with insulin at 12 hours and 24 hours, respectively. A low efficiency was observed for insulin and caspase3 co-expression at 24 hours. This study suggests that for transplantation, PDL tissues harvested at an early time post-PDL (between 12 and 24 hours) could yield a higher success rate; the study also provides evidence for a connection between morphological changes in the PDL pancreas and the protein synthesis necessary for the lineage of endocrine cell development. / AFRIKAANSE OPSOMMING: Diabetes Mellitus resorteer onder die vernaamste oorsake van morbiditeit en mortaliteit wêreldwyd, en tuister jongmense, volwassenes en bejaardes. Daar bestaan egter ‘n wêreldwye tekort aan skenkerorgane met immuun-onderdrukingsterapie as ondersteuningsbehandeling. Beta-sel vervangingsterapie, vir die voorsiening van insulien, bly daarom die voorkeur behandeling vir die siekte wat noodsaak dat die wetenskap kyk na alternatiewe behandelingsregimens wat meganismes rondom orgaanregenerasie insluit. Begrip van die chronobiologie van die sellulêre meganismes betrokke rondom beta-sel ontwikkeling mag waardevolle lig werp op die neogenese van beta-selle wat gevolglik daartoe mag lei dat beta-sel vervanging as ‘n moontlike behandelingsterapie oorweeg mag word vir pasiënte met suikersiekte. Die oogmerk van hierdie studie is om die rekapitulasie van die morfo-genetiese volgorde van die endokriene pankreas na afbinding van die pankreasbuis te bepaal. Pankreasbuis afbinding is op 78 Sprague-Dawley laboratorium rotte onder algemene narkose uitgevoer, die pankreas is na voorafbepaalde tydsvakke verwyder en in histologiese seriesnitte gesny. Snitte is immunositochemiese gekleur en morfometries assesseer. Die afskeidingsindeks vir selboodskappers vir Caspase3, Insulien, Pdx1, Ngn3, NeuroD en Pax6 is kwantifiseer. Die gelyktydige afskeiding van elk van bogenoemde boodskappers tesame met insulien is omskryf as ‘n verhouding tot mekaar en in terme van dié van insulien. Die morfologiese verandering in die weefsel bespeur is gekenmerk deur die verskyn van gegranuleerde asinêre selle ses (6) ure na buisafbinding en die proliferasie van endokriene weefsel vanaf vier-en-tagtig (84) ure deurlopend tot een-honderd-en-twintig (120) ure. Die morfo-immunofluoresserende evaluering toon dat Caspase3 en Pdx1 by 6 uur die hoogste is, die van Ngn3 by 36 ure, Pax6 en insulien by 84 ure en NeuroD by 120 ure. Verder toon die analise dat Caspase3 en Pdx1 rondom 6 ure hul verskyning gemaak het terwyl dié van insulien en NeuroD eers rondom 84 tot 120 uur verskyn het. Die verskyning van Pax6 het deurlopend regoor al die tydsduurtes verskyn en Ngn3 het rondom 36 uur sy hoogste vlak bereik. Die gelyktydige uitdrukking van homeodomein proteïene tesame met insulien het slegs tussen die tydperke van 12 en 24 ure plaasgevind. Die uitdrukking van Pdx1 met insulien, NeuroD met insulien en Pax6 met insulien het almal tussen 12 en 24 ure plaasgevind. Caspase3 tesame met insulien is slegs by die 24 uur tydsperiode bespeur. Vir die oorplant van pankreas weefsel wat aan buisafbinding onderwerp is suggereer hierdie studie dat die geskikste tyd vir die oes van endokriene weefsel liewer vroeër (12 to 24 ure) as later uitgevoer behoort te word. Verder wil dit voorkom of hierdie tydsperiode ook die hoogste seltelling lewer. Die studie lewer waardevolle inligting oor die verwantskap tussen die morfologiese veranderings wat na buisafbinding plaasvind en die proteïen sintese wat sel-opvolgontwikkeling bevorder.

Eph kinases are the largest family of cell surface receptor tyrosine kinases. The ligands of Ephs, ephrins (EFNs), are also cell surface molecules. Ephs interact with EFNs and the receptors and ligands transmit signals in both directions, i.e., from Ephs to EFNs and from EFNs to Ephs. Ephs and EFNs are widely involved in various developmental, physiological pathophysiological processes. Our group and others have reported the roles of Ephs/EFNs in the immune system. To further investigate the function of EphBs/EFNBs in T cell development and responses, we generated EFNB1, EFNB2, EphB4 conditional gene knockout (KO) mice and EFNB1/2 double KO mice. In the projects using EFNB1 and EFNB2 knockout mice, we specifically deleted EFNB1 or EFNB2 in T cells. The mice had normal size and cellularity of the thymus and spleen as well as normal T cell subpopulations in these organs. The bone marrow progenitors from KO mice and WT mice repopulated the host lymphoid organs to similar extents. The activation and proliferation of KO T cells was comparable to that of control mice. Naïve KO CD4 cells differentiated into Th1, Th2, Th17 and Treg cells similar to naïve control CD4 cells. In EFNB2 KO mice, we observed a significant relative increase of CD4CD8 double negative thymocytes in the thymus. Flowcytometry analysis revealed that there was a moderate increase in the DN3 subpopulation in the thymus. This suggests that EFNB2 is involved in thymocyte development. Our results indicate that the functions of EFNB1 and EFNB2 in the T cell compartment could be compensated by each other or by other members of the EFN family, and that such redundancy safeguards the pivotal roles of EFNB1 and EFNB2 in T cell development and function. In the project using EFNB1/B2 double knockout (dKO) model, we revealed a novel regulatory function of EFNb1 and EFNb2 in stabilizing IL-7Rα expression on the T cell surface. IL-7 plays important roles in thymocyte development, T cell homeostasis and survival. IL-7Rα undergoes internalization upon IL-7 binding. In the dKO mice, we observed reduced IL-7Rα expression in thymocytes and T cells. Moreover, the IL-7Rα internalization was accelerated in dKO CD4 cells upon IL-7 stimulation. In T cell lymphoma cell line, EL4, over-expression of either EFNB1 or EFNB2 retarded the internalization of IL-7Rα. We further demonstrated compromised IL-7 signaling and homeostatic proliferation of dKO T cells. Mechanism study using fluorescence resonance energy transfer and immunoprecipitation demonstrated that physical interaction of EFNB1 and EFNB2 with IL-7Rα was likely responsible for the retarded IL-7Rα internalization. In the last project, using medullary thymic epithelial cell (mTEC)-specific EphB4 knockout mice, we investigated T cell development and function after EphB4 deletion in mTEC. EphB4 KO mice demonstrated normal thymic weight and cellularity. T cell development and function were not influenced by the EphB4 deletion. Lastly, the KO mice developed normal delayed type hypersensitivity. Overall, our results suggest that comprehensive cross interaction between Eph and EFN family members could compensate function of a given deleted member in the T cell development, and only simultaneous deletion of multiple EFNBs will reveal their true function in the immune system. In fact, such redundancy signifies vital roles of Ephs and EFNs in the immune system. / Kinases Eph est la plus grande famille de tyrosines kinases récepteurs Éphrines (EFN) est un ligand de Ephs. Eph et EFN sont toutes les molécules de surface cellulaire. L’interaction entre Ephs et EFNs permet de transmettre des signaux dans les deux directions (c.-à-d. partir de Ephs à EFNs, et de EFNs à Ephs.) Eph et EFNs sont largement impliqués dans divers processus développementaux, physiologiques et physiopathologiques. Notre groupe et d'autres groupes ont rapporté les rôles de Ephs / EFNs dans le système immunitaire. Pour approfondir la fonction de EphBs / EFNBs dans le développement des lymphocytes T et des réponses immunitaires, nous avons généré des souris EFNB1, EFNB2, et EphB4 knock-out conditionnel (KO) et des souris EFNB1 / 2 doubles KO. Dans les projets qui utilisent EFNB1 et EFNB2 comme souris knock-out, nous avons spécifiquement supprimé EFNB1 ou EFNB2 dans les cellules T. Les souris présentaient une taille normale, la cellularité du thymus et de la rate, ainsi que des sous-populations de cellules T étaient normales dans ces organes. Les progéniteurs de la moelle osseuse de souris KO et les souris WT ont repeuplé les organes lymphoïdes de l’hôte à des degrés similaires. L'activation et la prolifération des cellules KO T étaient comparables à celles des souris témoins. Les cellules CD4 naïves KO différenciées en Th1, Th2, Th17 et Treg étaient similaires aux cellules CD4 naïves de souris contrôle. Chez les souris KO EFNB2, nous avons observé une augmentation relative importante des thymocytes CD4CD8 : les double négatifs dans le thymus. L'analyse par cytométrie en flux a révélé qu'il y avait une augmentation modérée de la sous-population DN3 dans le thymus. Les résultats suggèrent qu’EFNB2 est impliqué dans le développement des thymocytes. Nos résultats indiquent que les fonctions de EFNB1 et EFNB2 dans le compartiment des cellules T pourraient être compensées entre eux ou par d'autres EFNB. La redondance des fonctions suggèrent le contrôle critique d’EFNB1 et EFNB2 dans le développement des cellules T. Dans le projet, en utilisant EFNB1/B2 (modèle double KO) (dKO), nous avons observé une fonction de régulation de EFNB1 et EFNB2. dans la stabilisation de l’expression l'IL-7R α , à la surface des cellules T, IL-7 joue un rôle important dans le développement des thymocytes, l'homéostasie des lymphocytes T , et leur survie. IL-7R α subit une internalisation i contraignante de IL-7. Chez les souris DKO, nous avons observé une perte d’expression de l’ IL-7Rα dans les thymocytes et les cellules T. En outre, l’ internalisation IL-7Rα a été accélérée dans les cellules CD4 dKO, suite à la stimulation IL-7. Dans la lignée cellulaire de lymphome T, EL4, la surexpression de EFNB1 ou EFNB2 retarde l'internalisation de l'IL-7Rα. Nous avons aussi démontré les signalisations compromises de l’ IL-7 et de la prolifération homéostatique des cellules T dKO. Les études du méchanisme qui utilisent la fluorescence de transfert d'énergie par résonance et immunoprécipitation ont montré que l'interaction physique de EFNB1 et EFNB2 avec IL-7R était probablement responsable du retard de l’ internalisation IL-7Rα. Dans le dernier projet, nous avons étudié le développement des cellules T et la fonction des cellules épithéliales médullaires du thymus (mTEC), chez les souris knock-out EphB4. Les souris KO EphB4 ont démontré un poids et une cellularité qui sont normaux. La fonction et le développement de cellules T ne sont pas influencés par la suppression de l’ EphB4. Enfin, les souris KO ont développé une hypersensibilité de type retardée normale. Dans l'ensemble, nos résultats suggèrent que l'interaction globale de croisement entre Eph et les membres de la famille EFN pourrir compenser la fonction d'un membre supprimé. Seule la suppression simultanée de plusieurs EFNBs va révéler leur vraie fonction dans le système immunitaire. En fait, une telle redondance montre les rôles vitaux d’Ephs et EFNS dans le système immunitaire.

The chromatin structure, consisting of DNA and histones, changes dynamically during the cell cycle and cell differentiation. DNA can only be transcribed and replicated when it is packaged loosely, whereas tight packaging allows for more efficient storage. Chromatin remodelling is therefore one of the tools of gene expression control. The chromatin remodelling factors recognise chromatin with varying specificity and have an effect on the interaction between DNA and the histones. One of these factors is the Smarca5 protein. This study investigates the role of Smarca5; its goal is to create a mouse model with the ability to trigger Smarca5 overproduction in specific tissues. This model will be used to study the effect of a high, unregulated dose of Smarca5 on the physiological function of the protein. Previous studies have shown that non-physiological expression of a chromatin-remodelling factor can lead to malignant transformation. Our model can help to understand this process. Another goal of this study is to investigate some phenotype aspects of the mouse model with conditional deletion of Smarca5 in T and B cells, in particular the effects of this deletion on progenitor cell differentiation. Our results show that Smarca5 has an important role in lymphocyte development, and we have observed that...

Les membres de la famille Homeobox jouent un rôle critique dans le développement hématopoïétique normal. L'expression ectopique des gènes Homeobox provoque des désordres dans l'hématopoïèse et le développement de leucémies. L'oncogène TLX3 s'exprime de manière ectopique exclusivement dans les Leucémies Aiguës Lymphoblastiques T (LAL-T), avec un blocage des thymocytes à un stade de différentiation précoce cortical CD4+CD8+ DP. De nombreuses études ont investigué les mécanismes d'action des oncogènes TLX1/3, mais plusieurs questions restent en suspens. Durant ma thèse, j'ai étudié l'impact de l'expression ectopique de l'oncogène TLX3 sur le développement lymphocytaire T et les mécanismes de transformation leucémique associés. L'expression de TLX3 a provoqué le blocage des thymocytes à un stade DN2 avec une immortalisation des clones preleucémiques. Les souris transplantées avec les cellules TLX3 ont développé des tumeurs similaires aux LAL-T. Les analyses de ChIP-Seq et d'expression génique ont identifié un recrutement de TLX3 sur les enhancers spécifiques aux cellules T par le motif de fixation Ets/Runx1. Nos résultats suggèrent que la fixation de TLX3 sur les éléments cis-régulateurs peut contribuer à la transformation maligne des thymocytes en perturbant les réseaux transcriptionnels responsables de l'oncogenèse LAL-T. / It is now well established that members of the homeobox gene family play a critical role in normal hematopoietic cell development and that their unbalanced or ectopic expression can lead to characteristic perturbations in haemopoiesis and the onset of leukaemia. TLX3 expression in human haematologic malignancies is exclusive to T-ALL, where it is almost universally associated with transformation of early cortical CD4+CD8+ DP thymocytes. Multiple studies intensively investigated the mechanisms by which TLX1/3 oncogenes could promote complex tumor development, but many questions remain still unclear. During my thesis I investigated the impact of ectopic TLX3 expression on T cell development, and the initiating mechanisms of T-cell transformation leading to leukemia onset. Forced expression of TLX3 disrupted the thymic develoment at DN2-like stage giving rise to immortalized preleukemic clones. Following the transfer into immunodeficient mice TLX3 preleukemic cells initiated malignant cell transformation resulting into leukemia-like disease. Applying a combination of ChIP sequencing and gene expression profiling, we identified TLX3 recruitment onto T-cell specific enhancers via interaction with Ets1/Runx1 composite motif sites as preferential molecular events in the initial steps of TLX3-induced transformation. Thus our findings suggest that the genome-wide binding properties of TLX3 on cis-regulatory elements may contribute to its ability to promote thymocyte preleukaemic state via perturbation of transcriptional regulatory networks responsible for T-ALL oncogenesis.

Der (Pro)Renin Rezeptor (PRR) ist ein hoch konservierter Transmembranrezeptor, der ursprünglich beschrieben wurde Renin und Prorenin zu binden. Durch Bindung an Renin und Prorenin beeinflusst der PRR das Renin-Angiotensin-Systems und induziert eine MAP-Kinase-Signaltransduktion. Teile des PRR sind assoziiert mit der vakuolären H+-ATPase (vATPase), welche wichtig für die Azidifizierung zellulärer Organellen ist. Kürzlich wurde eine neue Funktion des PRR für den WNT/β-catenin Signalweg beschrieben. Hier dient der PRR als Verbindungsglied zwischen den WNT Rezeptoren und der vATPase. Die Mechanismen der Funktionen des PRR sind noch nicht verstanden, aber es wird angenommen, dass der PRR in die Regulation verschiedenster zellulärer Mechanismen involviert ist. Es gibt bis jetzt keine biochemische und strukturelle Charakterisierung des PRR. In der vorliegenden Arbeit wurden strukturelle Studien mit verschiedenen Konstrukten des extrazellulären Teils des PRR durchgeführt. Alle PRR Konstrukte (hsPRR (170-303), hsPRR (101-257) und hsPRR (166-257)) zeigten eine alpha-helikale Faltung und konnten nicht an Renin oder Prorenin binden. Der hsPRR (101-257) liegt in einem Konzentrations- und pH-abhängigen Monomer-/Oligomerequilibrium vor, während der hsPRR (166-257) als Monomer/Dimerequilibrium vorkommt. Diese Daten bilden die Grundlage für weitere strukturelle und funktionelle Untersuchungen. Konditionelle KO Mäuse sind eine exzellente Methode, um die physiologische Rolle des PRR in vivo zu untersuchen. Eines der wichtigsten Proteine des Wnt/β-catenin Signaltransduktionsweges, β-catenin, ist fundamental für die T-Zell Entwicklung. Aus diesem Grund wurde untersucht, ob die Deletion des PRR in T-Zellen ebenfalls zu einem Verlust von T-Zellen und zu einer Entwicklungsstörung führt. Die Ergebnisse zeigen, dass der PRR wichtig für eine vollständige T-Zellentwicklung ist und unterstützen die Hypothese, dass der PRR eine Rolle für Wnt/β-catenin Singaltransduktion in T-Zellen spielt. / The (pro)renin receptor (PRR) is an evolutionary conserved transmembrane receptor that was first discovered to bind renin and prorenin. Upon binding, PRR was shown to influence the activity of the renin-angiotensin-system (RAS) and to induce MAP kinase signalling. It was previously shown that a truncated, transmembrane part of PRR was associated to vacuolar H+-ATPase (vATPase), a proton pump which is important for acidification. Recently, a new function of PRR in the WNT/β-catenin signalling pathway was described. Here, the PRR was shown to be an adaptor between WNT receptors and the vATPase. The precise mechanisms by which PRR functions, are still elucidative but the PRR is supposed to regulate various cellular processes. Currently, no biochemical characterization or structural analysis is available for PRR. In order to gain understanding of the function of the PRR, structural studies were performed with several truncated proteins of the extracellular part of the PRR. All PRR proteins (hsPRR170-303, hsPRR 101-257 or hsPRR 166-257) showed an overall alpha helical folding and did not bind renin or prorenin. The oligomeric assembly of the proteins was investigated. The hsPRR (101-257) was shown to be in a concentration and pH dependent monomer/oligomer equilibrium, whereas hsPRR (166-257) is only present in a monomer/dimer equililibrium. These data are the basics for further structural and functional studies. Additionally, conditional KO animals are an excellent tool to investigate the physiological role of the PRR in vivo. As the major mediator of the Wnt/β-catenin signaling pathway, β-catenin, is crucial for T cell maturation, a conditionel deletion of PRR in T cells was analyzed. PRR deletion resulted in a loss of mature T cells. Moreover, a defect in T cell maturation in the thymus was determined. Our data showed that PRR is critical for proper T cell development and support the hypothesis that PRR contributes to Wnt/β-catenin signaling in T cells.

Eph kinases are the largest family of cell surface receptor tyrosine kinases. The ligands of Ephs, ephrins (EFNs), are also cell surface molecules. Ephs interact with EFNs and the receptors and ligands transmit signals in both directions, i.e., from Ephs to EFNs and from EFNs to Ephs. Ephs and EFNs are widely involved in various developmental, physiological pathophysiological processes. Our group and others have reported the roles of Ephs/EFNs in the immune system. To further investigate the function of EphBs/EFNBs in T cell development and responses, we generated EFNB1, EFNB2, EphB4 conditional gene knockout (KO) mice and EFNB1/2 double KO mice. In the projects using EFNB1 and EFNB2 knockout mice, we specifically deleted EFNB1 or EFNB2 in T cells. The mice had normal size and cellularity of the thymus and spleen as well as normal T cell subpopulations in these organs. The bone marrow progenitors from KO mice and WT mice repopulated the host lymphoid organs to similar extents. The activation and proliferation of KO T cells was comparable to that of control mice. Naïve KO CD4 cells differentiated into Th1, Th2, Th17 and Treg cells similar to naïve control CD4 cells. In EFNB2 KO mice, we observed a significant relative increase of CD4CD8 double negative thymocytes in the thymus. Flowcytometry analysis revealed that there was a moderate increase in the DN3 subpopulation in the thymus. This suggests that EFNB2 is involved in thymocyte development. Our results indicate that the functions of EFNB1 and EFNB2 in the T cell compartment could be compensated by each other or by other members of the EFN family, and that such redundancy safeguards the pivotal roles of EFNB1 and EFNB2 in T cell development and function. In the project using EFNB1/B2 double knockout (dKO) model, we revealed a novel regulatory function of EFNb1 and EFNb2 in stabilizing IL-7Rα expression on the T cell surface. IL-7 plays important roles in thymocyte development, T cell homeostasis and survival. IL-7Rα undergoes internalization upon IL-7 binding. In the dKO mice, we observed reduced IL-7Rα expression in thymocytes and T cells. Moreover, the IL-7Rα internalization was accelerated in dKO CD4 cells upon IL-7 stimulation. In T cell lymphoma cell line, EL4, over-expression of either EFNB1 or EFNB2 retarded the internalization of IL-7Rα. We further demonstrated compromised IL-7 signaling and homeostatic proliferation of dKO T cells. Mechanism study using fluorescence resonance energy transfer and immunoprecipitation demonstrated that physical interaction of EFNB1 and EFNB2 with IL-7Rα was likely responsible for the retarded IL-7Rα internalization. In the last project, using medullary thymic epithelial cell (mTEC)-specific EphB4 knockout mice, we investigated T cell development and function after EphB4 deletion in mTEC. EphB4 KO mice demonstrated normal thymic weight and cellularity. T cell development and function were not influenced by the EphB4 deletion. Lastly, the KO mice developed normal delayed type hypersensitivity. Overall, our results suggest that comprehensive cross interaction between Eph and EFN family members could compensate function of a given deleted member in the T cell development, and only simultaneous deletion of multiple EFNBs will reveal their true function in the immune system. In fact, such redundancy signifies vital roles of Ephs and EFNs in the immune system. / Kinases Eph est la plus grande famille de tyrosines kinases récepteurs Éphrines (EFN) est un ligand de Ephs. Eph et EFN sont toutes les molécules de surface cellulaire. L’interaction entre Ephs et EFNs permet de transmettre des signaux dans les deux directions (c.-à-d. partir de Ephs à EFNs, et de EFNs à Ephs.) Eph et EFNs sont largement impliqués dans divers processus développementaux, physiologiques et physiopathologiques. Notre groupe et d'autres groupes ont rapporté les rôles de Ephs / EFNs dans le système immunitaire. Pour approfondir la fonction de EphBs / EFNBs dans le développement des lymphocytes T et des réponses immunitaires, nous avons généré des souris EFNB1, EFNB2, et EphB4 knock-out conditionnel (KO) et des souris EFNB1 / 2 doubles KO. Dans les projets qui utilisent EFNB1 et EFNB2 comme souris knock-out, nous avons spécifiquement supprimé EFNB1 ou EFNB2 dans les cellules T. Les souris présentaient une taille normale, la cellularité du thymus et de la rate, ainsi que des sous-populations de cellules T étaient normales dans ces organes. Les progéniteurs de la moelle osseuse de souris KO et les souris WT ont repeuplé les organes lymphoïdes de l’hôte à des degrés similaires. L'activation et la prolifération des cellules KO T étaient comparables à celles des souris témoins. Les cellules CD4 naïves KO différenciées en Th1, Th2, Th17 et Treg étaient similaires aux cellules CD4 naïves de souris contrôle. Chez les souris KO EFNB2, nous avons observé une augmentation relative importante des thymocytes CD4CD8 : les double négatifs dans le thymus. L'analyse par cytométrie en flux a révélé qu'il y avait une augmentation modérée de la sous-population DN3 dans le thymus. Les résultats suggèrent qu’EFNB2 est impliqué dans le développement des thymocytes. Nos résultats indiquent que les fonctions de EFNB1 et EFNB2 dans le compartiment des cellules T pourraient être compensées entre eux ou par d'autres EFNB. La redondance des fonctions suggèrent le contrôle critique d’EFNB1 et EFNB2 dans le développement des cellules T. Dans le projet, en utilisant EFNB1/B2 (modèle double KO) (dKO), nous avons observé une fonction de régulation de EFNB1 et EFNB2. dans la stabilisation de l’expression l'IL-7R α , à la surface des cellules T, IL-7 joue un rôle important dans le développement des thymocytes, l'homéostasie des lymphocytes T , et leur survie. IL-7R α subit une internalisation i contraignante de IL-7. Chez les souris DKO, nous avons observé une perte d’expression de l’ IL-7Rα dans les thymocytes et les cellules T. En outre, l’ internalisation IL-7Rα a été accélérée dans les cellules CD4 dKO, suite à la stimulation IL-7. Dans la lignée cellulaire de lymphome T, EL4, la surexpression de EFNB1 ou EFNB2 retarde l'internalisation de l'IL-7Rα. Nous avons aussi démontré les signalisations compromises de l’ IL-7 et de la prolifération homéostatique des cellules T dKO. Les études du méchanisme qui utilisent la fluorescence de transfert d'énergie par résonance et immunoprécipitation ont montré que l'interaction physique de EFNB1 et EFNB2 avec IL-7R était probablement responsable du retard de l’ internalisation IL-7Rα. Dans le dernier projet, nous avons étudié le développement des cellules T et la fonction des cellules épithéliales médullaires du thymus (mTEC), chez les souris knock-out EphB4. Les souris KO EphB4 ont démontré un poids et une cellularité qui sont normaux. La fonction et le développement de cellules T ne sont pas influencés par la suppression de l’ EphB4. Enfin, les souris KO ont développé une hypersensibilité de type retardée normale. Dans l'ensemble, nos résultats suggèrent que l'interaction globale de croisement entre Eph et les membres de la famille EFN pourrir compenser la fonction d'un membre supprimé. Seule la suppression simultanée de plusieurs EFNBs va révéler leur vraie fonction dans le système immunitaire. En fait, une telle redondance montre les rôles vitaux d’Ephs et EFNS dans le système immunitaire.

The absolute clarity of the lens of the eye is vital in the visual system. The unique structural and physiological properties of the lens are tightly integrated with highly ordered protein content to allow the lens to remain transparent. Consequently, any alteration or disturbance of these highly ordered proteins can affect the optical properties of the lens. In humans, cataracts are the major cause of blindness, yet the exact aetiology of cataract formation (cataractogenesis) is not fully understood. The purpose of the current research was to investigate whether deregulation of the Ca²⁺-dependent enzyme, calpains, following changes in lens Ca²⁺ homeostasis, is a key mechanism leading to undesired cleavage of a number of proteins that are linked with maintaining lens transparency and contributing to cataractogenesis. An ovine lens culture (in vitro) system and the heritable ovine cataract (in vivo) model were used to test the research hypothesis. The Ca²⁺ ionophore, ionomycin, was used to induce a Ca²⁺ overload and in vitro opacification during lens culture. Opacity in the lens was graded by a computer image analysis program. Protein profile (SDS-PAGE, 2-DE and Western detection), calpain activity (casein zymography), lens structure (microscopic view) and cytotoxicity level (LDH leakage assay) were analysed in Ca²⁺-induced opaque lenses. The involvement of calpain during opacification was further examined by applying synthetic exogenous calpain inhibitors to the in vitro system. Two novel exogenous calpain inhibitors were also assessed for their therapeutic potential in preventing the progression of cataracts in the in vivo cataract model by topical administration of the inhibitor direct to the sheep's eye over a 11 week period. HPLC was used to detect the penetration of these compounds into ocular tissues. Sustained Ca²⁺ influx into cultured lenses caused dense opacification. The opacity was characterised by formation of a turbid fraction and cell death in the outer cortex of the ovine lens. There was increased calpain autolysis associated with the progress of opacification, indicating increased calpain activity. Major degradation of the cytoskeletal proteins, spectrin and vimentin, was observed whilst there was limited degradation of the lens structural soluble proteins, crystallins, in response to a Ca²⁺ flux. Lens proteins were protected from degradation by adding synthetic calpain inhibitors to the culture medium. Topical administration of novel anti-calpain molecules failed to retard the progression of cataractogenesis in the ovine inherited cataract model. Further investigation of drug penetration showed that efficacy of inhibitory compounds was limited by permeability of these molecules across the cornea and the ability of the molecules to reach and penetrate into the lens. The ovine lens Ca²⁺-induced opacification (OLCO) model in this thesis has provided a model to understand the role of Ca²⁺ homeostasis in lens transparency. With sustained intracellular Ca²⁺ level, the degradation of cytoskeletal elements is highly correlated with calpain activity. Cataractogenesis is the pathological response to the loss of lens Ca²⁺ homeostasis in this model. The current results support the hypothesis that the deregulation of calpain activity is a trigger for a series of cascading events, leading to death of the cells in the lens.

Abstract Hematopoietic cells, i.e., erythrocytes, platelets and white blood cells, differentiate from hematopoietic stem cells in a process that is similar in vertebrates. Hematopoiesis is regulated by molecules expressed by both the hematopoietic stem and progenitor cells and the surrounding microenvironments. Knowledge of these molecules is important since many of the genes involved in normal hematopoiesis are mutated in leukemia. Furthermore, this information can be utilized in more efficient isolation and expansion of hematopoietic cells in vitro. However, these molecules are not yet sufficiently characterized. Transmembrane immunoglobulin and mucin domain (TIM) genes form a known family of immunoregulators. In mammals, TIM-4 is expressed by antigen presenting cells, while TIM-1, TIM-2 and TIM-3 are expressed by T cells, in which they regulate differentiation of TH cells. The role of TIM molecules in hematopoiesis has not yet been investigated. The aim of this thesis work was to identify and analyze novel molecules involved in embryonic hematopoiesis using chicken and mouse as model organisms. This was carried out by generating a cDNA library of hematopoietic stem and progenitor cells from embryonic chicken para-aortic region. Both previously known and novel candidate genes were identified from the library. Among them, we found homologs to tim genes. Their expression and role in hematopoiesis was studied further. TIM-2 expression was shown to be tightly governed during B cell development. It is expressed by common lymphoid progenitors and highly proliferative large-pro and large pre-B cells during both fetal liver and adult bone marrow hematopoiesis. In mouse, tim-4 expression was restricted to fetal liver CD45+F4/80+ cells. Furthermore, two distinct populations were identified: F4/80hiTIM-4hi and F4/80loTIM-4lo. The results suggest that the F4/80hiTIM-4hi cells are yolk sac-derived macrophages and the F4/80loTIM-4lo cells myeloid progenitors. This work shows for the first time that TIM family molecules are expressed during hematopoiesis. TIM-2- and TIM-4 are expressed by specific cell types during hematopoietic cell development, and in the future they may be utilized as markers in isolation of hematopoietic progenitor cells. / Tiivistelmä Verisolut eli punasolut, verihiutaleet ja immuunipuolustuksessa tärkeät valkosolut kehittyvät alkion veren kantasoluista prosessissa, joka on kaikissa selkärankaisissa samankaltainen. Veren kanta- ja esisolujen sekä ympäröivän mikroympäristön tuottamat molekyylit säätelevät hematopoieesia eli verisolujen kehitystä. Näiden molekyylien tunteminen on tärkeää, sillä useat normaalia verisolujen kehitystä säätelevät geenit ovat osallisena myös verisyöpien synnyssä. Lisäksi tätä tietoa on mahdollista hyödyntää verisolujen tehokkaammassa eristämisessä ja kasvattamisessa hoitoja varten. Immuunipuolustuksen solut, kuten syöjäsolut eli makrofagit ja T-solut, ilmentävät TIM-molekyylejä (Transmembrane Immunoglobulin and Mucin). Ne toimivat immunologisen vasteen säätelyssä sekä solusyönnissä, mutta niiden roolia verisolujen kehittymisessä ei ole selvitetty aikaisemmin. Tässä väitöstutkimuksessa etsittiin uusia hematopoieesiin vaikuttavia geenejä käyttäen mallieläiminä sekä kanaa että hiirtä. Tutkimuksessa luotiin geenikirjasto kanan alkion para-aortaalisen alueen veren kanta- ja esisoluista. Kirjastosta tunnistettiin useita ennalta tiedettyjä sekä uusia verisolujen kehitykseen vaikuttavia geenejä. Tutkimuksessa analysoitiin tarkemmin kirjastosta löytyneiden TIM-geeniperheen jäsenten ilmentymistä ja roolia verisolujen kehityksessä. Tutkimuksessa osoitettiin, että TIM-2 proteiinin ilmentymistä säädellään tarkasti B-solujen kehityksen aikana. Lymfosyyttien yhteiset esisolut sekä suuret pro-B- ja pre-B-solut ilmentävät TIM-2 proteiinia B-solukehityksen aikana sekä alkion maksassa että aikuisen luuytimessä. Hiiren alkiossa tim-4 geenin ilmentyminen oli rajoittunut maksaan, jossa erottui kaksi erillistä solupopulaatiota: F4/80hiTIM-4hi ja F4/80loTIM-4lo. Tutkimuksen tulokset viittaavat siihen, että maksan F4/80hiTIM-4hi solut ovat ruskuaispussista lähtöisin olevia syöjäsoluja ja F4/80loTIM-4lo solut myeloidisen linjan esisoluja. Tämä tutkimus on ensimmäinen osoitus TIM-molekyylien ilmentymisestä kehittyvissä verisoluissa. Havaitsimme, että TIM-2 ja TIM-4-molekyylejä ekspressoidaan tietyissä soluissa verisolujen erilaistumisen aikana, joten tulevaisuudessa niitä on mahdollista käyttää merkkiproteiineina hematopoieettisten solujen esiasteita eristettäessä.

Mast cells contribute to the activities of innate and adaptive branches of the immune system. They participate in pro-inflammatory responses to a wide range of pathogens, such as parasites, bacteria, and other foreign agents. These beneficial properties are in contrast to the contribution of mast cells to certain pathologies, such as asthma, allergy, autoimmune disorders, anaphylaxis, and systemic mastocytosis. Thorough knowledge of mast cell biology in health and disease is critical for the development of new therapeutic approaches. However, molecular mechanisms that control mast cell development and function are still incompletely defined. Our preliminary data indicate that the transcription factor C/EBP is a key player in mast cell biology. Here, using in vitro and in vivo models, we determine how C/EBP regulates the commitment of hematopoietic progenitors towards mast cells, and modulates mast cells function. These efforts provide novel insights to the role of C/EBP in hematopoiesis, and contribute to a better understanding of the mechanisms governing mast cell biology. Key words Mast cells, C/EBP, transcription factors, bone marrow-derived mast cell cultures, mast cell development, Cebpg conditional knockout mice

<p>    </p> <p>When mammalian eggs are fertilized, they undergo an egg-to-embryo transition during which different egg activation events take place. Egg activation events include the establishment of blocks to polyspermy, which prevent multiple sperm from fertilizing an egg. One of these blocks to polyspermy occurs at the level of the egg plasma membrane (the membrane block to polyspermy). Previous work in our lab provides evidence that the mammalian membrane block to polyspermy is mediated by sperm-induced calcium signaling and the egg’s actomyosin cytoskeleton (McAvey et al., 2002). This thesis research builds upon this foundation, testing hypotheses about two specific effector molecules, one involved in calcium signaling and one with the actin cytoskeleton, and also developing the use of an actin probe for live-cell imaging, with the goal of imaging actin dynamics in eggs undergoing fertilization. Specifically, we examined the calcium effector molecule Ca2+/Calmodulin-dependent-protein kinase IIg (<strong>CaMKII</strong>g), based on previous studies showing that CaMKII plays a role in the membrane block (Gardner et al., 2007) and that the g isoform of CaMKII is necessary and sufficient for eggs to complete meiosis (Backs et al., 2010). We tested the hypothesis that CaMKIIg would mediate the membrane block to polyspermy but found that egg activation driven by expression of a constitutively active form of CaMKIIg was not sufficient to establish the membrane block. Our studies of the actin cytoskeleton focused on the Arp2/3 complex as a candidate. We tested the hypothesis that Arp2/3, which mediates actin filament branching, was involved in membrane block establishment, building on the finding that disruption of actin with the drug cytochalasin D impairs the membrane block (McAvey et al., 2022). These studies used the Arp2/3 inhibitor CK666, predicting that we would see increased sperm incorporation in CK666-treated eggs. However, an assay of sperm incorporation over time indicated that Arp2/3 may not play a significant role in the membrane block to polyspermy, although follow-up studies will be beneficial. Lastly, the actin probe SiR- Actin was assessed for use on oocytes undergoing live-cell imaging during meiosis I and II. Oocytes were treated with differing concentrations of SiR-Actin and live cell imaged while maturing through meiosis I or completing meiosis II. Higher doses and longer exposure to SiR- Actin caused abnormalities in oocytes during meiosis I but not in eggs completing meiosis II. Together, this work sets the stage of a range of future studies into the mammalian membrane block to polyspermy. </p>