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No description available.

No description available.

Endometriosis affects 5-10% of women and is characterized by the growth of endometrial tissue outside of the uterus. Treatment for endometriosis primarily focuses on symptom relief, is short term with severe side effects and often leads to recurrence of the condition. Establishing new blood supply is a fundamental requirement for endometriosis lesions growth. This has led to the idea that antiangiogenic therapy may be a successful approach for inhibiting endometriosis. Recent evidence indicates that endothelial progenitor cells (EPCs) contribute to neoangiogenesis of endometriotic lesions. These EPCs are recruited to the lesion site by stromal cell-derived factor-1 (SDF-1). We hypothesize that SDF-1 is central to the neoangiogenesis and survival of endometriotic lesions and that administration of SDF-1 blocking antibody will inhibit lesion growth by inhibiting angiogenesis in a murine model of endometriosis. Immunohistochemistry for SDF-1 and CD34 was performed on human endometriosis and normal endometrial samples. Quantification of SDF-1 and EPCs was performed in the blood of endometriosis patients and controls using ELISA and flow cytometry, respectively. A new mouse model of endometriosis was developed using BALB/c-Rag2-/-/IL2rg-/- mice to investigate role of SDF-1 in neoangiogenesis. Either SDF-1 blocking antibody or an isotype control was administered on a weekly basis for four weeks. Weekly samples of peripheral blood from mice were analyzed for SDF-1, other cytokines of interest and EPCs. Mice were euthanized at seven weeks to observe lesion growth and blood vessel development. Our results indicate overabundance of SDF-1 and CD34+ progenitor cells in human endometriotic lesions compared to eutopic endometrium. In the mouse model, SDF-1 and circulating EPC levels decreased from pre-treatment levels after one week, and remained constant over the course of the treatment in both SDF-1 blocking antibody and isotype control groups. In the SDF-1 blocking group, reduced vascularity of lesions, identified by immunofluorescence staining for CD31, was revealed compared to isotype controls. These findings suggest that SDF-1 may be responsible for CD34+ progenitor cell recruitment to the neoangiogenic sites in endometriosis. Blocking of SDF-1 reduces neovascularization of human endometriotic lesions in a mouse model. Further studies on blocking SDF-1 in combination with other antiangiogenic agents are needed. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2011-12-21 19:34:43.054

Phytoöstrogene sind in den vergangenen Jahren zunehmend in den Fokus der Wissenschaft gerückt, weil sie eine potentielle Alternative zur klassischen Hormonersatztherapie darstellen. Diese ist aufgrund von zum Teil drastischen Nebenwirkungen für den Einsatz von klimakterischen Beschwerden umstritten. In dieser Arbeit wird die dosisabhängige Wirkung des Phytoöstrogens Genistein an ausgewählten östrogenselektiven Organen näher untersucht. Die Versuche wurden am Modell der langzeitovarektomierten Maus durchgeführt. Es wurden 70 ovarektomierte Tiere in sieben Gruppen aufgeteilt und untersucht. Fünf der sieben Gruppen erhielten genisteinhaltiges Futter in verschiedenen Konzentrationen. Die anderen beiden Gruppen erhielten Estradiol als Zusatz oder sojafreie Diät und dienten jeweils als Positiv- und Negativkontrollgruppe. Zusätzlich gab es eine nicht ovarektomierte intakte Kontrollgruppe, die ebenfalls sojafreies Futter erhielt. Während des dreimonatigen Versuchszeitraums wurden regelmäßig Gewicht und Futterverbrauch der Tiere gemessen. Nach Ende des Versuchs wurden die Feuchtgewichte von Uterus und Herz bestimmt sowie die Genexpression am linken Ventrikel von IGF-1, ERα und Myocardin mittels PCR analysiert. Darüber hinaus wurde am Tibia-Knochen per pQCT die Messung der Spongiosadichte, des polaren Widerstandsmoments und des prozentualen Anteils der Trabekel an der Spongiosaquerschnittsfläche durchgeführt. Die Ergebnisse zeigen, dass Genistein direkt am Herz wirkt, indem es das relative Herzgewicht und die Genexpression am Herz erhöht. Genistein beeinflusst auch das Körpergewicht und das relative Gewicht des Uterus und die untersuchten Knochenparameter dosisabhängig. Genistein kann in höherer Dosierung am Uterus proliferierend wirken, jedoch nach derzeitigem Kenntnisstand weniger stark als der klassische Hormonersatztherapie-Wirkstoff E2. Genistein kann zukünftig nur dann eine Therapiealternative zur klassischen Hormonersatztherapie darstellen, wenn es gelingt, eine Dosis zu finden, bei der Genistein die gewünschten Wirkungen entfaltet, gleichzeitig aber die unerwünschte proliferierende Wirkung an Brust und Uterus sicher ausgeschlossen werden kann. Im Modell der ovarektomierten Maus scheint eine Dosis von 1 g/kg Genistein im Futter ein vielversprechender Ansatzpunkt für weitere Untersuchungen zu sein.

Metallothioneins (MTs) are small, non-enzymatic proteins that are involved in cellular detoxification and metal homeostasis because of their high cysteine content. MTs have also been identified as one of the vast number of adaptive responses to mitochondrial respiratory chain (RC) deficiencies. Aside from this, numerous other studies have linked MTs to several mitochondrion-linked components, including reactive oxygen species (ROS) and oxidative stress, apoptosis, glutathione, energy metabolism and nuclear- and mitochondrial DNA transcription regulation. However, most of the reports concerning the putative link between MTs and mitochondria are from in vitro studies and relatively little supportive in vivo evidence has been reported. Information on the involvement of MTs with respiratory chain function is especially limited. Is was therefore the aim of this study to investigate the involvement of MTs in mitochondrial respiration and respiratory chain enzyme function by using an MT knockout (MTKO) mouse model, which was treated with the irreversible complex I inhibiting reagent, rotenone. The aim was achieved by implementing three objectives: firstly, the RC function was investigated as a complete working unit; secondly, the functional and structural properties of single units (enzymes) of the RC were investigated utilising enzyme activity assays and BN- PAGE/western blot analysis; and thirdly, the possible effect of MTs on mtDNA copy number was investigated. While some tendencies of variation in RC enzyme activity and expression were identified, no significant effect on the overall mitochondrial respiratory function, or any significant differences in the relative mtDNA copy number of MTKO mice were observed. Thus it is concluded, while MTs have in this study revealed relatively small changes in respiratory chain function, which may still prove to have biological ignificance in vivo, the exact nature of the putative role of MTs in mitochondrial respiration or oxidative phosphorylation remains undefined. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2012.

Metallothioneins (MTs) are small, non-enzymatic proteins that are involved in cellular detoxification and metal homeostasis because of their high cysteine content. MTs have also been identified as one of the vast number of adaptive responses to mitochondrial respiratory chain (RC) deficiencies. Aside from this, numerous other studies have linked MTs to several mitochondrion-linked components, including reactive oxygen species (ROS) and oxidative stress, apoptosis, glutathione, energy metabolism and nuclear- and mitochondrial DNA transcription regulation. However, most of the reports concerning the putative link between MTs and mitochondria are from in vitro studies and relatively little supportive in vivo evidence has been reported. Information on the involvement of MTs with respiratory chain function is especially limited. Is was therefore the aim of this study to investigate the involvement of MTs in mitochondrial respiration and respiratory chain enzyme function by using an MT knockout (MTKO) mouse model, which was treated with the irreversible complex I inhibiting reagent, rotenone. The aim was achieved by implementing three objectives: firstly, the RC function was investigated as a complete working unit; secondly, the functional and structural properties of single units (enzymes) of the RC were investigated utilising enzyme activity assays and BN- PAGE/western blot analysis; and thirdly, the possible effect of MTs on mtDNA copy number was investigated. While some tendencies of variation in RC enzyme activity and expression were identified, no significant effect on the overall mitochondrial respiratory function, or any significant differences in the relative mtDNA copy number of MTKO mice were observed. Thus it is concluded, while MTs have in this study revealed relatively small changes in respiratory chain function, which may still prove to have biological ignificance in vivo, the exact nature of the putative role of MTs in mitochondrial respiration or oxidative phosphorylation remains undefined. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2012.

LKB1 is the second most commonly altered tumour suppressor gene in lung adenocarcinoma, the most prevalent form of lung cancer. LKB1 is a "master kinase" that has been shown to phosphorylate up to 13 downstream targets. We hypothesised that LKB1 loss is associated with an increased dependency on alternative, targetable pathways. The overall aims of this project were to better understand the role of LKB1 loss in lung cancer and to identify novel approaches to selectively target LKB1 mutated cells. We generated isogenic cells with or without LKB1 and used these to study the effect of LKB1 on cell proliferation. Importantly, we used a range of models including 2D culture, 3D spheroids and, sub-cutaneous and orthotopic xenograft models. To understand the role of LKB1 loss in lung cancer, the effect of LKB1 on mRNA expression was analysed using whole genome RNA Sequencing. To identify novel approaches to selectively target LKB1 mutated cells, we used biological screening methods and also investigated the effect of several metabolic inhibitors. We found that loss of LKB1 expression had no effect on cell proliferation in 2D culture, but was associated with increased growth in 3D spheroids, sub-cutaneous and orthotopic xenografts, as well as greater metastasis in a lung orthotopic model. Gene ontology analysis of the transcriptome identified that genes associated with cAMP signalling and cytoskeletal organisation were differentially expressed between LKB1 deficient and proficient cells. We confirmed that cAMP signalling was increased in LKB1 deficient cells, though there was no difference in sensitivity between LKB1 deficient and proficient cells to cAMP signalling modulators. The bioactive small molecule screen showed that LKB1 deficient cells underwent apoptosis more slowly and therefore, were less sensitive to many compounds, compared with LKB1 proficient cells. Screening in 3D spheroids was a novel approach that we used to identify microtubule inhibitors as potentially selective compounds acting in LKB1 deficient cells. Our RNASeq data suggests that there was a metabolic shift from oxidative phosphorylation to aerobic glycolysis in LKB1 deficient cells, although this did not affect sensitivity to complex I inhibitors. Importantly, LKB1 deficient cells were more sensitive to glucose and glutamine deprivation which suggests that targeting these metabolic pathways may hold the greatest promise to selectively inhibit proliferation in LKB1 mutated cells.

La Granulomatose Septique Chronique (CGD) est une maladie génétique rare de l’immunodéficience innée affectant les cellules phagocytaires (neutrophiles, macrophages). Elle est causée par des mutations dans les sous-unités du complexe NADPH oxydase formé du cytochrome b558 membranaire (NOX2 associé à p22phox) et de facteurs cytosoliques (p47phox, p67phox et p40phox). La déficience de ce complexe enzymatique va conduire à l’absence de formation de formes réactives de l’oxygène (FRO) microbicides et donc à l’apparition d’infections graves et récurrentes très tôt dans l’enfance. La chimioprophylaxie à vie permet de protéger ces patients mais peut être responsable d’effets indésirables. La seule thérapie curative est la transplantation de moelle osseuse mais tous les patients ne peuvent en bénéficier, et la thérapie génique n’est pas encore envisageable. Il y a donc un manque réel de nouvelles thérapies pour cette maladie. Cependant pour développer de nouveaux traitements, il faut disposer de modèles physiopathologiques pertinents. Or, les modèles existants sont imparfaits ou manquants. Le but de notre travail est donc de produire des modèles cellulaires et animaux de la CGD pour développer dans un second temps, une nouvelle approche thérapeutique basée sur l’utilisation de protéoliposomes.Grâce à leurs propriétés de pluripotence et d’auto-renouvellement à l’infini, les cellules souches pluripotentes induites (iPS) sont un outil puissant pour la modélisation physiopathologique. Ainsi, à partir de fibroblastes de patients atteints de CGD reprogrammés en cellules iPS, nous avons mis au point un protocole efficace de différenciation hématopoïétique in vitro en neutrophiles et macrophages. Nous avons montré que ces cellules phagocytaires sont matures et reproduisent parfaitement le phénotype déficient en FRO des patients CGD. Nous avons donc obtenu des modèles cellulaires pertinent modélisant trois formes génétiques de CGD, la CGD liée à l’X et deux formes autosomiques récessives, CGDAR22 et CGDAR47.Nous avons ensuite réalisé la preuve du concept de l’efficacité de protéoliposomes thérapeutiques sur les macrophages modélisés de la forme CGDX, la forme génétique la plus fréquente (70 % des cas) due à l’absence du cytochrome b558 membranaire (NOX2/p22phox). Grâce à une collaboration avec la start-up Synthelis SAS, des liposomes contenant le cytochrome b558 au niveau de la membrane lipidique ont été produits dans un système d’expression acellulaire basé sur l’utilisation d’extraits d’Escherichia coli. Ces liposomes NOX2/p22phox sont capables de reconstituer une enzyme NADPH oxydase fonctionnelle in vitro et de délivrer le cytochrome b558 à la membrane plasmique des macrophages CGDX qui présentent alors une restauration de l’activité NADPH oxydase avec la production de FRO.Enfin, nous nous sommes proposés de générer des souris dites « humanisées » par transplantation de cellules souches hématopoïétiques CD34+ capables de prise de greffe et de reconstitution hématopoïétique dans des souris immunodéficientes. A partir de cellules iPS saines, nous avons réussi à produire des cellules hématopoïétiques CD34+ possédant un potentiel hématopoïétique in vitro. Cependant, malgré des résultats encourageants, aucune prise de greffe in vivo n’a pu être réellement confirmée à ce jour.Pour conclure, nous avons donc montré au cours de ce projet, la production de modèles cellulaires de trois formes génétiques de CGD à partir de cellules iPS. Puis le modèle de macrophages CGDX nous a permis de faire la preuve de l’efficacité d’une nouvelle thérapie in vitro, une « enzymothérapie substitutive liposomale », qui pourrait à terme, offrir une alternative thérapeutique pour le traitement des infections aigües pulmonaires des patients CGD réfractaires aux traitements antibiotiques et antifongiques conventionnels. / Chronic Granulomatous Disease (CGD) is a rare inherited pathology of the innate immune system that affects the phagocytic cells (neutrophils, macrophages). This disease is caused by mutations in the subunits of the NADPH oxidase complex composed of the membrane cytochrome b558 (NOX2 associated with p22phox) and the cytosolic components (p47phox, p67phox et p40phox). Dysfunction in this enzymatic complex leads to the absence of microbicidal reactive oxygen species (ROS) and therefore to the development of recurrent and life-threatening infections in early childhood. Life-long prophylaxis is used to protect these patients but it may be responsible for side effects. Bone marrow transplantation is the only curative treatment but it can not be proposed to all the patients. In addition, gene therapy is not possible up to now. So there is a real lack of new therapies for this disease. However, to develop new therapeutic approaches, relevant physiopathological models must be available. Actually, existing models are imperfect or missing. Thus, the goal of our work is to produce cellular and animal models of CGD to develop a new proteoliposome-based therapy.Induced pluripotent stem cells (iPSCs) are a powerful tool for physiopathologic modeling due to their pluripotency and self-renewal properties. Using CGD patient-specific iPSCs regrogrammed from fibroblasts, we developped an efficient protocol for in vitro hematopoietic differentiation into neutrophils and macrophages. We showed that the phagocytic cells produced are mature and reproduce the ROS-deficient phenotype found in CGD patients. Thus, we obtained relevant cellular models for three genetic forms of CGD: X-linked CGD and the two autosomal recessive forms AR22CGD and AR47CGD.Then, we demonstrated the proof-of-concept of the efficacy of therapeutic proteoliposomes on X-CGD iPS-derived macrophages. Indeed, X-CGD is the main form of the disease (70% of cases) and is caused by the absence of the membrane cytochrome b558 (NOX2/p22phox). Thanks to a collaboration with the start-up Synthelis SAS, liposomes integrating the cytochrome b558 into lipid bilayers were produced in an E. coli-based cell-free protein expression system. These NOX2/p22phox liposomes were able to reconstitute a functional NADPH oxidase enzyme in vitro and to deliver the cytochrome b558 at the plasma membrane of X-CGD macrophages, leading to restore the NADPH oxidase activity with a ROS production.Finally, we proposed to generate « humanized » mice models with a human immune system after transplantation of CD34+ hematopoietic stem cells able to engraft and reconstitute long-term hematopoiesis in immunodeficient mice. Using healthy iPSCs, we successfuly produced CD34+ hematopoietic cells with in vitro hematopoietic potential. However, no in vivo engraftment was really confirmed yet.In conclusion, during this project, we produced cellular models of three genetic forms of CGD using patient-specific iPSCs. Then, X-CGD macrophages were used to demonstrate in vitro the efficacy of a new therapy. This « liposomal replacement enzymotherapy » could, in the future, represents a curative alternative against life-threatening lung infections refractory to conventional antibiotic and antifungal therapy.

Largely as a consequence of the ongoing obesity epidemic, research into adipose tissue biology has increased substantially in recent years. Worldwide, the number of people classed as overweight or obese is growing, and this represents a major public health concern. Adipose tissue is broadly divided into two types; white and brown. Whilst white adipose tissue (WAT) functions to store and mobilise triglycerides, brown adipose tissue burns chemical energy to generate heat. WAT is further divided into visceral “bad” fat and subcutaneous “good” fat depots, and it is an increase in the former that is linked to obesity-associated diseases. As well as adipocytes, several other cell types including haematopoietic and endothelial are found within adipose tissue, and comprise the stromal vascular fraction (SVF). Adipocyte precursor cells (APCs) also reside within the SVF and are essential for the maintenance and expansion of adipose tissue. The protein encoded by the Wilms’ tumour 1 (Wt1) gene is predominantly known to function as a transcription factor, but also has a role in post-transcriptional processing. Deletion of Wt1 in adult mice results in a considerable loss of fat tissue. Moreover, recent work has revealed that a proportion of the APCs from all visceral WAT depots express Wt1, therefore revealing heterogeneity within the APC population. Additionally, visceral WAT depots are encapsulated by a WT1 expressing mesothelial layer, which has its origins in the lateral plate mesoderm (LPM), and can give rise to mature adipocytes. Lineage tracing has demonstrated that a significant proportion of the mature adipocytes in all adult visceral WAT depots (but not subcutaneous) are derived from cells that express Wt1 in late gestation. These findings uncovered key ontogenetic differences between visceral and subcutaneous WAT and led us to ask whether Wt1 functions in visceral adipose tissue biology. Preliminary work has shown that adipocytes derived from Wt1 expressing (Wt1+) precursor cells have fewer, larger lipid droplets than those derived from non-Wt1 expressing (Wt1-) precursors. In this thesis, this heterogeneity is explored further using a Wt1GFP/+ knock-in mouse. When Wt1+ and Wt1- APCs are cultured separately, the Wt1+ population differentiate into adipocytes more readily. Moreover, the Wt1+ APCs are more proliferative than the Wt1-. Preliminary results also suggest that the Wt1+ APCs may secrete a factor(s) that causes the Wt1- APCs to exhibit improved adipogenic differentiation, a result that is supported by data from comparative transcriptomic analysis. Finally, the percentage of APCs decreases when mice are fed a high fat diet. Interestingly, this decrease is more pronounced for the Wt1+ population. Therefore, it appears that as well as exhibiting differing behaviours in vitro, the Wt1+ and Wt1- populations respond differently to physiologically relevant conditions in vivo. Whilst the LPM is a major source of visceral WAT, the origin of subcutaneous WAT is currently unknown. Here, the Prx1-Cre and Prx1-CreERT2 mouse lines are used to investigate this. It is shown that the majority of subcutaneous WAT adipocytes and APCs are labelled by Prx1-Cre, however this is not the case for most of the visceral WAT depots. The exception to this is the pericardial (heart fat) depot, in which approximately 70% of the adipocytes and 40% of the APCs are labelled. Moreover, a proportion of the Prx1-Cre labelled pericardial APCs also express Wt1, therefore suggesting additional heterogeneity. Preliminary results show that this heterogeneity may have functional consequences, at least in vitro. Additionally, lineage tracing studies suggest that the somatic LPM may be one source of subcutaneous WAT and pericardial visceral WAT Finally, it is shown that the conditional deletion of Wt1 in the Prx1-Cre lineage results in abnormal diaphragm development. Congenital diaphragmatic hernia (CDH) is severe birth defect, the etiology of which is not well understood. Here, a new model of CDH has been developed, and the cellular and molecular mechanisms responsible for the defect in this model are investigated.