Nicotinic acid (NA) has been clinically used for over 50 years to regulate lipid plasma levels. It is the only drug in current clinical use that significantly raises HDL cholesterol and reduces inflammatory markers. However, mechanistic understanding into its wide range of actions remains unclear. The recent identification of the Gi-coupled protein receptor HCAR2, for which NA is a potent agonist, provides intriguing insight due to its anti-lipolytic action and restricted, yet specific, expression in adipose tissue and immune cells. The HCAR2 gene is 96% homologous to HCAR3, but the HCAR3 receptor shares neither the specificity for NA, nor the range of functional effects. Moreover, the close homology makes it difficult to separate the genetic variability and regulation of the two genes. To this end, I resequenced HCAR2 and HCAR3 in a selected population to characterize the variability of the two genes and to inform the subsequent design of specific genotyping assays. The Oxford Biobank, which is a random population-based collection of 30-50 year old men and women in Oxfordshire with a wide range of collected phenotypes, was used to explore genetic associations. A preliminary trend with HDL and rs7314976 in HCAR2 motivated the further search associations. However after increasing the sample size, the HDL association did not reach significance. When looking at inflammatory phenotypes, a 20% lower level of systemic hsCRP was found in males with a promoter region variant in HCAR3 (N=1808, p=0.007 for rs55718746). Replication of this finding in two relevant cohorts (NPHS-II, N=2185 and Whitehall, N=4228) resulted in conflicting findings. After optimising the specific detection of both HCAR2 and HCAR3 transcripts, I characterized gene expression in human AT biopsies. This revealed an 18% increase in HCAR2 expression in the female abdominal depot (N=106, p<0.0001) and a reduction in abdominal HCAR2 in both males (β=-0.37, p<0.001, N=107) and females (β=-0.251, p=0.005, N=106) with increasing adiposity. The rs55718746 variant in HCAR3 was also seen to influence expression of both HCAR2 (N=182, p=0.018 in the abdominal depot) and HCAR3 (N=198, p=0.005) but surprisingly in opposite directions, establishing it as the first cis-eQTL for this genomic region. Finally, I used human adipocyte in vitro culture systems to setup a pilot to study the anti-inflammatory effects of NA. The gene expression of HCAR2 and HCAR3 increased significantly with adipocyte differentiation in vitro. NA led to a drop in IL-6 transcript abundance in two out of three of the in vitro differentiated human adipocytes. In conclusion, genetic variability in HCAR2 and HCAR3 shows weak associations with cardiovascular disease risk phenotypes relating to their respective pathways. The relevance of HCAR2 and HCAR3 gene expression and the role of the receptor in the control of inflammation will require further studies.

No description available.

No description available.

No description available.

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by consistently high blood glucose, resulting from a combination of insulin resistance and reduced capacity of β-cells to secret insulin. While the exact causes of T2DM is yet unknown, obesity is known to be a major risk factor as well as co-morbidity for T2DM. As the global prevalence of obesity continues to increase, the association between obesity and T2DM warrants further study. Traditionally, mathematical models to study T2DM were mostly empirical and thus fail to capture the dynamic relationship between glucose and insulin. More recently, mechanism-based population models to describe glucose-insulin homeostasis with a physiological basis were proposed and offered a substantial improvement over existing empirical models in terms of predictive ability. The primary objectives of this thesis are (i) examining the predictive usefulness of semi-mechanistic models in T2DM by applying an existing population model to clinical data, and (ii) exploring the relationship between obesity and T2DM and describe it mathematically in a novel semi-mechanistic model to explain changes to the glucose-insulin homeostasis and disease progression of T2DM. Through the use of non-linear mixed effects modelling, the primary mechanism of action of an antidiabetic drug has been correctly identified using the integrated glucose-insulin model, reinforcing the predictive potential of semi-mechanistic models in T2DM. A novel semi-mechanistic model has been developed that incorporated a relationship between weight change and insulin sensitivity to describe glucose, insulin and glycated hemoglobin simultaneously in a clinical setting. This model was also successfully adapted in a pre-clinical setting and was able to describe the pathogenesis of T2DM in rats, transitioning from healthy to severely diabetic. This work has shown that a previously unutilized biomarker was found to be significant in affecting glucose homeostasis and disease progression in T2DM, and that pharmacometric models accounting for the effects of obesity in T2DM would offer a more complete physiological understanding of the disease.

Κύριος σκοπός τη παρούσης μελέτης ήταν η εκτίμηση της επίδρασης της οξείας και χρόνιας εντατικής άσκησης και του χρόνιου αρνητικού ενεργειακού ισοζυγίου στα επίπεδα αντιπονεκτίνης, ρεζιστίνης και βισφατίνης. Επιπλέον, μελετήθηκε η συσχέτιση των επιπέδων των ανωτέρω λιποκινών με τα επίπεδα κορτιζόλης και ινσουλίνης, καθώς και με τις παραμέτρους έντασης της άσκησης. Ως μοντέλο ενεργειακής και μεταβολικής ομοιοστασίας χρησιμοποιήθηκαν αθλήτριες Ρυθμικής Γυμναστικής (RGs) υψηλού επιπέδου πρωταθλητισμού, Οι RGs υποβάλλονται σε χρόνια έντονη ψυχολογική και σωματική καταπόνηση, ενώ, στην προσπάθεια για επίτευξη και διατήρηση λεπτού σωματότυπου, υιοθετούν αυστηρές διαιτητικές συνήθειες και χρόνιο αρνητικό ενεργειακό ισοζύγιο. Υλικό και Μέθοδοι Η αδυναμία συλλογής δειγμάτων αίματος από αθλητές κορυφαίου επιπέδου πρωταθλητισμού επέβαλε τη συλλογή και επεξεργασία σιέλου για την πραγματοποίηση των ορμονικών προσδιορισμών. Ως εκ τούτου, προτού ξεκινήσει η υλοποίηση του πρωτοκόλλου της μελέτης (διαμόρφωση πληθυσμού και συλλογή δειγμάτων), σχεδιάστηκε και εκπονήθηκε μια μελέτη με σκοπό την ανίχνευση και τη μέτρηση των συγκεντρώσεών των λιποκινών αντιπονεκτίνη, βισφατίνη και ρεζιστίνη στο σίελο και τη συσχέτιση των συγκεντρώσεων τους στο σίελο με τις αντίστοιχες συγκεντρώσεις στο αίμα. Προσδιορισμός των συγκεντρώσεων λιποκινών στο σίελο Τα επίπεδα των λιποκινών αντιπονεκτίνη, ρεζιστίνη και βισφατίνη προσδιορίσθηκαν στο σάλιο και τον ορρό 50 υγιών άτομα (33 γυναίκες και 17 άνδρες), με ταυτόχρονη μέτρηση των ανθρωπομετρικών χαρακτηριστικών (ύψος και βάρος σώματος, υπολογισμός ΒΜΙ, ποσοστό λιπώδους μάζας) . Η επίδραση της άσκησης και του αρνητικού ενεργειακού ισοζυγίου στην ορμονική ομοιοστασία του λιπώδους ιστού Στη μελέτη συμπεριελήφθησαν 51 RGs υψηλού επιπέδου από 8 Ευρωπαϊκές χώρες, οι οποίες συμμετείχαν στη διοργάνωση «Κύπελλο Καλαμάτας 2010» τον Απρίλιο του 2010 στην Καλαμάτα. Επίσης, η μελέτη συμπεριέλαβε 27 υγιείς γυναίκες, μη αθλήτριες και χωρίς σημαντική διαφορά ως προς την ηλικία σε σύγκριση με τον πληθυσμό των αθλητριών (matched for age) ως πληθυσμό ελέγχου (μάρτυρες). Το πρωτόκολλο της μελέτης περιελάμβανε μη επεμβατικές κλινικές και εργαστηριακές εξετάσεις, καθώς και τη συμπλήρωση ενός ερωτηματολογίου. Ειδικότερα, προσδιορίσθηκαν τα ανθρωπομετρικά χαρακτηριστικά αθλητριών και πληθυσμού ελέγχου, οι αθλήτριες συμπλήρωσαν ένα ερωτηματολόγιο που αφορούσε σε στοιχεία γυναικολογικού και αθλητικού ιστορικού, ενώ συλλέχθηκαν δείγματα σιέλου από τις αθλήτριες και τις μάρτυρες. Οι ορμονικοί προσδιορισμοί στο σίελο αφορούσαν σε μετρήσεις επιπέδων ρεζιστίνης, βισφατίνης, αντιπονεκτίνης, κορτιζόλης και ινσουλίνης σε κατάσταση ηρεμίας και μετά από άσκηση. Αποτελέσματα Προσδιορισμός των συγκεντρώσεων λιποκινών στο σίελο Οι λιποκίνες ρεζιστίνη και αντιπονεκτίνη ανιχνεύονται στο σίελο του ανθρώπου και τα επίπεδά τους συσχετίζονται με τα αντίστοιχα επίπεδα στον ορρό (r=0.441, p=0.003 και r=0.347, p=0.019 αντίστοιχα). Αντίθετα, η λιποκίνη βισφατίνη ανιχνεύεται στο σίελο του ανθρώπου, όμως τα επίπεδά της δε συσχετίζονται με τα αντίστοιχα επίπεδα στον ορρό (r=-0.128, p=0.4431). Η επίδραση της άσκησης και του αρνητικού ενεργειακού ισοζυγίου στην ορμονική ομοιοστασία του λιπώδους ιστού Παρατηρήθηκαν μειωμένα επίπεδα αντιπονεκτίνης (p<0.001) και αυξημένα επίπεδα βισφατίνης (p<0.05) στο σάλιο RGs, ενώ δεν παρατηρήθηκαν διαφορές στα επίπεδα ινσουλίνης, κορτιζόλης και ρεζιστίνης στο σάλιο μεταξύ αθλητριών και πληθυσμού ελέγχου. Στις RGs κορυφαίου επιπέδου η επίδραση της μικρής διάρκειας, έντονης αναερόβιας άσκησης του επίσημου αγώνα οδήγησε σε αύξηση των επιπέδων ινσουλίνης (p<0.001), μείωση των επιπέδων αντιπονεκτίνης και βισφατίνης (p<0.001και p<0.05 αντίστοιχα) και καμιά μεταβολή στα επίπεδα κορτιζόλης και ρεζιστίνης στο σάλιο. Επίσης, καταγράφηκε απουσία διημερήσιας διακύμανσης της κορτιζόλης. Τέλος, τα επίπεδα αντιπονεκτίνης ηρεμίας παρουσίασαν σημαντική συσχέτιση με τις παραμέτρους έντασης της άσκησης. Συμπεράσματα Προσδιορισμός των συγκεντρώσεων λιποκινών στο σίελο Οι λιποκίνες ρεζιστίνη, αντιπονεκτίνη και βισφατίνη ανιχνεύονται στο σίελο του ανθρώπου. Όμως, τα ορμονικά επίπεδα στο σίελο συσχετίζονται με τα αντίστοιχα επίπεδα στον ορρό μόνο για τη ρεζιστίνη και την αντιπονεκτίνη. Η επίδραση της άσκησης και του αρνητικού ενεργειακού ισοζυγίου στην ορμονική ομοιοστασία του λιπώδους ιστού Σε RGs κορυφαίου επιπέδου η χρόνια εντατική άσκηση και το χρόνιο αρνητικό ενεργειακό ισοζύγιο αυξάνουν τα επίπεδα αντιπονεκτίνης και μειώνουν τα επίπεδα βισφατίνης στο σάλιο, ενώ η μικρής διάρκειας, έντονη αναερόβια άσκηση καταστέλλει τα επίπεδα τόσο της αντιπονεκτίνης, όσο και της βισφατίνης στο σάλιο. Επιπλέον, τα επίπεδα αντιπονεκτίνης στο σίελο συσχετίζονται με τη συνήθη ένταση της άσκησης, αντανακλώντας, μάλλον, την επιδείνωση του ενεργειακού ισοζυγίου παρά την κλιμάκωση της έντασης της άσκησης καθαυτής, υποδεικνύοντας έναν πιθανός ρόλο των συγκεντρώσεων αντιπονεκτίνης στο σάλιο στην εκτίμηση της επιδείνωσης του ενεργειακού ισοζυγίου, με ενδεχόμενη χρησιμότητα στην κλινική έρευνα και την ιατρική της άσκησης. / Exercise represents a physical stress challenging homeostasis, disturbing the energy balance and leading to adaptive changes in central and peripheral regulatory mechanisms. The aim of the present study was to evaluate the effect of negative energy balance, acute and chronic exercise on adiponectin, resistin and visfatin levels, their interaction with salivary cortisol and insulin levels and the relationship of the specific adipokines with training frequency and intensity. Elite Rhythmic Gymnasts (RGs) were used as a model of energy homeostasis and metabolism. Elite RGs begin exercise at an early age, undergo physical and psychological stress and adopt negative energy balance to retain a lean physique. The main problem in studying hormonal responses in elite athletes on the field of competition lies on the difficulty in obtaining blood samples. The determination of salivary hormone levels provides a convenient, non-invasive and stress-free alternative to blood analysis. As part of the study design, we invested interest in introducing the methodology for detecting and measuring adipokine levels in saliva and evaluating their association with relative serum levels. Materials and Methods Detection and measurement of adipokine levels in human saliva Resistin, visfatin and adiponectin levels were measured in serum and saliva of 50 healthy adult volunteers (17 male and 33 female) using commercial enzyme immunoassay kits for plasma with minor modifications. The influence of acute and chronic intensive physical training and negative energy balance on salivary adipokine levels in elite RGs The study included 51(fifty one) elite female athletes of RG from 8 European countries, participating in the top level tournament of “Kalamata 2010 Rhythmic Gymnastics World Cup” in Kalamata, Greece on April 2010. 24 (twenty four) healthy pubertal girls not engaged in strenuous sports activities were used as controls. The study protocol included noninvasive clinical and laboratory investigations as well as the completion of a questionnaire. The athletes completed a questionnaire including personal data (age of training onset, usual weekly training intensity and number of participations in international championships per year). Baseline and post exercise salivary cortisol, insulin, adiponectin, resistin and visfatin levels were measured. Results Detection and measurement of adipokine levels in human saliva The present study documented the determination of resistin and adiponectin levels in saliva and the significant correlation of salivary with their respective serum levels (r=0.441, p<0.01 and r=0.347, p<0.05, respectively). Moreover, the identification of visfatin in saliva was achieved, but no significant correlation with serum visfatin levels was observed. The influence of acute and chronic intensive physical training and negative energy balance on salivary adipokine levels in elite RGs At baseline, a significant inverse correlation was documented between salivary insulin and adiponectin levels (r=-0.316, p<0.05), after controlling for the effect of age and BMI. Salivary adiponectin levels were higher (p<0.001) and visfatin lower (p<0.05) in RG’s compared with controls, while no significant changes were observed regarding salivary cortisol, insulin and resistin levels. In elite RG’s short term intensive anaerobic exercise led to increased salivary insulin levels (p<0.001), reduced salivary adiponectin (p<0.001) and visfatin levels (p<0.05) and no changes in salivary resistin levels. Moreover, diurnal variation of salivary cortisol was lost. In addition, salivary adiponectin levels are associated with the intensity of training. Conclusions Detection and measurement of adipokine levels in human saliva The adipokines resistin, visfatin and adiponectin can be detected in human saliva. However, significant correlation between salivary and their relative serum levels are documented only for resistin and visfatin, but not for visfatin. The influence of acute and chronic intensive physical training and negative energy balance on salivary adipokine levels in elite RGs. Chronic intensive physical training and negative energy balance up-regulate salivary adiponectin and down-regulate salivary visfatin levels, while acute glucoregulatory response to short term intensive anaerobic exercise down-regulates salivary adiponectin and visfatin levels. Moreover, salivary adiponectin levels are associated with the intensity of training, reflecting the deterioration of energy balance rather than the training stress. Thus, salivary adiponectin could be introduced as a possible marker of significant energy deprivation, with potential usefulness in clinical and sports medicine.

Cardiovascular disease (CVD) and erectile dysfunction (ED) have common etiologies, such as increased adiposity and chronic diseases. Incident ED is known to be a sentinel of CVD, providing a unique opportunity for early lifestyle interventions to attenuate the progression of disease. The internal pudendal artery (IPA) plays an important role in controlling resistance to penile blood flow and thereby erections. Although morphological and functional disturbances in the IPA have been associated with ED, few studies have characterized changes in the IPA as it relates to increased adiposity and chronic diseases (e.g., chronic kidney disease [CKD]). Finally, although both vascular calcification and ED have been shown to be prevalent in patients with CKD, there has yet to be an assessment of associated mechanisms. The effect of lifestyle modifications on erectile function was evaluated in both experimental and clinical settings. Specifically, the studies assessed the effect of caloric restriction (CR) in rats and of chronic exercise in sedentary, overweight or obese male and female subjects. In rats, structural and functional changes of the IPA and erectile responses were characterized in relation to increasing adiposity and to CKD. Experimentally, the susceptibility of various vascular beds to calcification in CKD was determined. Clinically, erectile and female sexual function was assessed in patients with Stage 3 to 5 CKD, who had no history of CVD. In rats, CR blunted the accumulation of abdominal adiposity, and attenuated progression of both endothelial dysfunction and ED, independently of morphological changes in the IPA. Rats with CKD had an increased frequency of ED, greater endothelial dysfunction, and altered vascular morphology, yet vascular calcification per se did not account for ED. In the clinical study, sedentary and overweight or obese males with ED, but not females, had a significantly higher body mass index (BMI) and waist circumference. Chronic exercise significantly improved ED and female sexual dysfunction (FSD). Clinically, CKD was associated with ED and FSD as well as increased coronary artery calcification and endothelial dysfunction. These findings support the concept that early detection of cardiovascular abnormalities, using incident ED as a sentinel, should facilitate early interventions in otherwise asymptomatic populations. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2013-09-30 22:33:20.436

Multipotente mesenchymale Stromazellen (MSCs) stellen nicht nur beim humanen Patienten, sondern auch in der Veterinärmedizin einen vielversprechenden Therapieansatz in der Behandlung erkrankter muskuloskelettaler Gewebe dar. Ziel der Behandlung ist dabei die Regeneration der betroffenen Strukturen im Vergleich zur Reparation nach konservativer Therapie. Vor allem im Bereich von Sehnenerkrankungen können nach MSC-Applikation vielversprechende Ergebnisse im Hinblick auf niedrigere Rezidivraten beobachtet werden. Dennoch sind noch nicht alle Umstände einer optimalen MSC-Anwendung geklärt. Hierbei sind unter anderem Fragen bezüglich der Herkunft und Gewinnung von MSCs offen, da Unterschiede von MSCs aufgrund ihrer Gewebezugehörigkeit bereits nachgewiesen wurden. Grundlegende umfassende Arbeiten zum Vergleich von equinen MSCs aus verschiedenen Quellen sowie deren mögliche Beeinflussung durch die Isolierung aus dem Gewebe lagen bislang noch nicht vor. Ziel dieser Studie war es daher, equine MSCs aus verschiedenen Quellen zu gewinnen und mögliche Unterschiede in vitro aufzuzeigen. Weiterhin sollten Unterschiede zwischen den Zelleigenschaften nach Anwendung verschiedener Isolationsprotokolle untersucht werden. In der hier vorliegenden Studie wurden MSCs aus Fett- und Sehnengewebe, Knochenmark, Nabelschnurblut und Nabelschnurgewebe von Pferden isoliert und vergleichend charakterisiert. Dabei wurden für die soliden Körpergewebe zwei unterschiedliche Isolationsmethoden, die Digestion und die Explantation, angewendet, um mögliche Einflüsse auf die gewonnen Zellen zu ermitteln. Die untersuchten Kriterien beinhalteten Zellertrag, Proliferation, Differenzierungspotenz und das Migrationsverhalten von MSCs. Hinblickend auf eine Anwendung von MSCs bei Sehnenerkrankungen wurde auch die Expression von Sehnenmarkern verglichen. In der vorliegenden Studie konnte gezeigt werden, dass sich die MSCs aus verschiedenen Quellen hinsichtlich der Zellausbeute und ihres Wachstumspotentials unterschieden. Aus soliden Geweben konnten mittels Digestion im Vergleich zu Körperflüssigkeiten signifikant mehr MSCs isoliert werden (p < 0,001). Dabei erbrachte die Isolation von MSCs mittels Digestionsmethode einen deutlich höheren Zellertrag nach der Passage 0 im Vergleich zur Explantationsmethode (p < 0,05). Im weiteren Verlauf der Kultivierung zeigten MSCs aus Sehnengewebe und Fettgewebe ein signifikant besseres Proliferationsverhalten im Vergleich zu Knochenmark-MSCs und Nabelschnurblut-MSCs. Im Hinblick auf das Differenzierungspotential konnten signifikante Unterschiede zwischen den MSCs aus den verschiedenen Quellen beobachtet werden. MSCs aus Knochenmark zeigten eine sehr gute osteogene Differenzierungsfähigkeit im Vergleich zu MSCs aus den geburtsassoziierten Geweben (p < 0,05). Im Gegensatz dazu zeichneten sich diese MSCs durch eine deutlich bessere chondrogene Differenzierung im Vergleich zu Knochenmark-MSCs aus (p < 0,05). Im Hinblick auf die Isolationsmethode konnten keine Unterschiede im Differenzierungspotential beobachtet werden. Weitere Unterschiede aufgrund der Zellquelle lassen sich in der Genexpression der Sehnenmarker erkennen. MSCs aus Fettgewebe und Sehnengewebe exprimierten Kollagen 1A2 auf höchstem Niveau. Sklexaris hingegen wurde von MSCs aus Nabelschnurblut und Sehnengewebe am höchstem exprimiert. Dabei zeigten MSCs, die mittels Digestionsmethode isoliert worden waren, ein signifikant höheres Expressionslevel von Skleraxis im Vergleich zur Explantationsmethode (p < 0,05). Die Ergebnisse der vorliegenden Studie lassen einen Einfluss der Zellquelle auf die Zellcharakteristika erkennen. MSCs aus Fettgewebe stellen dabei eine vielversprechende Alternative zu Knochenmark-MSCs dar. Allerdings scheint für eine klinische Anwendung von MSCs eine selektive Auswahl der Zellquelle entsprechend der vorliegenden Erkrankung von Vorteil zu sein. Dabei ist eine Isolierung von MSCs aus soliden Geweben mittels Digestionsverfahren zu empfehlen, da hier deutlich höhere Zellzahlen gewonnen werden können. Eine negative Beeinflussung der Zelleigenschaften durch die enzymatische Digestion lässt sich nach den vorliegenden Ergebnissen nicht vermuten. Inwiefern die beobachteten Unterschiede bei in-vivo-Anwendungen von Bedeutung sind, muss jedoch noch umfassend untersucht werden. / Not only in humans but also in veterinary medicine, multipotent mesenchymal stromal cells (MSCs) are a promising treatment option in the therapy of injured musculoskeletal tissues. This is due to the improved tissue regeneration instead of the insufficient reparation following conventional therapies. With regard to an application of MSCs for treatment of tendinopathies in horses, lower rates of reinjury have been reported. However, further investigations to optimize the MSC treatment are still outstanding. Differences in MSCs from different origins have been already reported, but there are still remaining questions about the influence of origin and isolation procedures of MSCs. Fundamental research on equine MSCs derived from different sources and their potential impact due to the isolation process has not been published so far. The aim of this study was to isolate equine MSCs from different sources and to demonstrate potential differences in vitro. Furthermore, differences in cell features following different isolation methods were investigated. In the present study, MSCs from horses were isolated from adipose tissue, tendon tissue, bone marrow, umbilical cord blood and umbilical cord tissue and subsequently subjected to comparative characterization. In case of the solid tissues, two different isolation methods, digestion and explantation, were performed in order to analyze influences on obtained cells. Investigated cell features included cell yield, proliferation, differentiation and migration potential. Furthermore, expression of tendon markers was evaluated with regard to an application of MSCs in tendinopathies. In the present study it was shown that MSCs derived from different sources differ distinctly in cell yield and proliferation potential. In comparison to body fluids, significantly more MSCs could be isolated from solid tissues when using the digestion method (p < 0.001). Furthermore, the cell yield at first cell harvest was distinctly higher when performing the isolation by digestion in comparison to isolation by explantation (p < 0.05). With regard to further cultivation, MSCs derived from tendon tissue and adipose tissue displayed a significantly better proliferation potential compared to MSCs derived from other sources. Considering the differentiation potential, significant differences were obvious between the MSCs derived from different sources. Bone marrow-MSCs showed an excellent osteogenic differentiation capacity in comparison to MSCs derived from umbilical cord blood and tissue (p < 0.05). In contrast, the birth-associated MSCs displayed a distinctly better chondrogenic differentiation than MSCs derived from bone marrow (p < 0.05). No difference in the differentiation potential was noticeable following the different isolation procedures. Furthermore, differences in the gene expression of tendon markers were evident with regard to the cell source. MSCs derived from adipose tissue and tendon tissue expressed collagen 1A2 on the highest level. On the other hand, scleraxis was expressed highest in MSCs derived from umbilical cord blood and tendon tissue. In these cells, MSCs isolated by the digestion method showed a significantly higher expression level of scleraxis in comparison to MSCs isolated by explantation (p < 0.05). Based on the results obtained so far, a relevant impact of the source of MSCs on cell features was evident. MSCs derived from adipose tissue are a promising alternative to bone marrow-MSCs. However, with regard to a clinical application of MSCs, a selection of the MSC source depending on the respective intended use seems to be advantageous. For routine isolation of MSCs from solid tissues, the digestion method could be recommended due to the higher obtainable cell numbers. Furthermore, a negative influence of the enzymatic digestion on the cell features was not detectable. However, to what extent the observed differences in vitro are relevant for in-vivo-applications needs to be further investigated.

Résumé : L’obésité est un enjeu de société de première importance; elle est un facteur de risque de plusieurs maladies et engendre d’importantes dépenses en santé. Outre l’alimentation, la sédentarité et les prédispositions génétiques, il semble que l’environnement fœtal soit un facteur déterminant dans le développement de l’obésité. En effet, il a été démontré que les nouveau-nés exposés à un environnement intra-utérin défavorable ont un risque accru de développer, à l’adolescence et à l’âge adulte, l’obésité ainsi que les désordres métaboliques qui y sont associés. Le diabète gestationnel (DG) est l’une des complications de santé maternelle les plus fréquentes et est associé à un risque accru à long terme pour la santé métabolique de l’enfant. Malgré les nombreuses données probantes épidémiologiques concernant le phénomène de la programmation fœtale associée au DG, les mécanismes moléculaires impliqués ont été très peu étudiés. Il est cependant de plus en plus évident que l’épigénétique soit l’un de ces mécanismes. Cette thèse a pour objectif d’identifier les changements de méthylation de l’ADN, la modification épigénétique la plus stable et la plus connue, chez les nouveau-nés exposés in utero au DG. Dans un premier temps, la méthylation de l’ADN de 44 échantillons de placenta et de sang de cordon a été analysée à l’échelle du génome. Cette approche a permis de démontrer que les gènes épigénétiquement modifiés suite à une exposition au DG sont majoritairement retrouvés dans les voies biologiques associées aux maladies métaboliques. Des analyses dans une cohorte indépendante (n=80) ont confirmé l’effet de la glycémie maternelle sur la méthylation de l’ADN des gènes BRD2, LRP1B et CACNA1D impliqués dans la régulation du métabolisme des lipides et du glucose et du système rénine-angiotensine respectivement. Dans un second temps, l’approche par gènes candidats a démontré que l’exposition au DG est associée à la méthylation de l’ADN de gènes du métabolisme des lipides (LPL et ABCA1) du placenta. L’analyse de la méthylation de la LEP et de l’ADIPOQ dans le sang et les tissus adipeux de sujets sévèrement obèses a permis d’identifier des sites de méthylation pouvant potentiellement être utilisés dans le sang comme marqueur de susceptibilité à l’obésité. L’ensemble des résultats de cette thèse démontrent que le DG modifie le profil épigénétique de gènes impliqués dans les voies biologiques des maladies métaboliques (métabolisme énergétique et des lipides) et supportent l’importance de la méthylation de l’ADN dans la programmation de la santé métabolique du nouveau-né ayant été exposé in utero au DG. / Abstract : Obesity has reached epidemic proportions worldwide in both adult and childhood populations and is now recognized as a major public health issue. Obesity is associated with higher incidence of cardiometabolic complications including type 2 diabetes (T2D), dyslipidemia and hypertension as well as with increased health care costs. The fetal environment now appears, with genetics and the environment, as one cause of the obesity epidemic. Indeed, according to the fetal programming hypothesis, newborns exposed to a detrimental fetal environment are more susceptible to develop obesity, T2D and other related chronic disorders when they become teenagers or adults. Many studies have associated gestational diabetes mellitus (GDM) exposure with these long-term metabolic health risks for the newborn. Although, numerous studies show epidemiological evidence to support the fetal programming hypothesis, only a few studies have been undertaken to understand the underlying molecular mechanisms. However, several studies now suggest that epigenetics may be involved. The objective of this thesis is to study changes in DNA methylation, the more stable and studied epigenetic system, in newborns that have been exposed to GDM in utero. First, a genome-wide DNA methylation analysis (BeadChip) was performed in a sample set of 44 placenta and cord blood samples to identify genes and metabolic pathways dysregulated by GDM. This approach showed that genes epigenetically affected by GDM are predominantly involved in metabolic diseases. The associations between maternal glycemia and DNA methylation levels were confirmed, in an independent birth cohort, for BRD2, LRP1B and CACNA1D gene loci involved in the regulation of lipid and glucose metabolism and the renin-angiotensin system respectively. Then, using a candidate gene approach we reported that DNA methylation levels at gene loci involved in lipid metabolism (LPL and ABCA1) are modified in the placenta following exposure to GDM. Furthermore, analyses of LEP and ADIPOQ DNA methylation levels in blood and adipose tissues of severely obese men and women allowed the identification of CpG sites that might be used in blood as a marker of obesity susceptibility. Altogether the results of this thesis show that GDM affects the epigenetic signature of genes involved in metabolic disease pathways (energy and lipid metabolism) and support the role of DNA methylation in metabolic health programming of the newborn exposed to GDM.

Obesity is a worldwide problem affecting both children and adults. Genetic, physiological, environmental, psychological, social and economic factors interact in varying degrees, influencing body weight and fat distribution and the progress of obesity. Moreover, some anthropogenic chemicals have proven to be endocrine disrupting chemicals (EDCs) with the potential to interfere with different actions of hormones in the body. EDCs may thereby disrupt homeostasis, modifying developmental, behavioral and immune functions in humans and animals, and also promoting adiposity. Because hormones generally act at low concentrations, small changes in the endocrine system may lead to extensive effects. Based on data from experimental and epidemiological studies this thesis elucidates the relationship between a large number of environmental contaminants and obesity. The experimental studies demonstrated that fructose supplementation in the drinking water resulted in unfavorable metabolic alterations such as a higher liver somatic index (LSI), an increase in plasma triglycerides and increased plasma levels of apo A-I. Fructose in combination with exposure to bisphenol A (BPA) increased liver fat content and plasma levels of apo A-I in juvenile female Fischer 344 rats. The experimental studies also showed that the retro-peritoneal fat, which in rats is a distinct fat depot easy to distinguish and dissect, correlated well with the measurements of total fat mass analyzed with MRI, and could therefore be used as a substitute for total fat mass in rats. The epidemiological studies showed that circulating levels of persistent organic pollutants (POPs) were related to fat mass measured by DXA. OCDD, HCB, TNC, DDE and the less chlorinated PCBs were positively related to fat mass, while the more highly chlorinated PCBs showed a negative association. Further, circulating levels of BPA were positively associated with levels of the hormones adiponectin and leptin, but negatively related with ghrelin, hormones which are involved in the regulation of hunger and satiety. However, serum BPA levels were not related to measures of fat mass in the elderly individuals in the PIVUS cohort. This thesis concludes that environmental contaminants such as BPA and POPs most likely are contributors, along with genetic, social and behavioral factors, to the development of obesity.