A cirurgia bariátrica confere proteção cardiometabólica à indivíduos obesos, contribuindo para uma redução do risco de mortalidade. No entanto, a extensão do benefício metabólico pode estar sujeita a mudanças no estilo de vida do paciente após a intervenção cirúrgica. Embora o exercício físico pareça melhorar os efeitos da cirurgia na sensibilidade à insulina, o mecanismo de ação subjacente permanece em grande parte sem explicação. Especula-se que mudanças potenciais na matriz extracelular do músculo esquelético (ECM) poderiam estar associadas à melhora da sensibilidade à insulina induzida pelo exercício físico em pacientes pós-bariátricos. Além disso, não se sabe se os benefícios da cirurgia bariátrica sobre a função endotelial, importante marcador precoce de aterosclerose, são sustentáveis sem alterações no estilo de vida, como a inclusão de exercícios físicos. Dessa forma, foram objetivos do presente estudo, investigar os efeitos do exercício físico sobre a sinalização intracelular envolvida na sensibilidade à insulina e remodelamento da matriz extracelular do músculo esquelético (Estudo 1) e sobre a função endotelial da artéria braquial de pacientes submetidos à cirurgia bariátrica (Estudo 2). Sessenta e duas mulheres foram randomizados após a cirurgia bariátrica para um programa de exercícios físicos de 6 meses ou tratamento padrão. No início do estudo, 3 e 9 meses após a cirurgia, a sensibilidade à insulina foi avaliada pelo teste oral de tolerância à glicose (TOTG), análise da função endotelial e amostras de músculo esquelético foram obtidas a partir do vasto lateral. As amostras de músculo esquelético foram submetidas a análises abrangentes, incluindo expressão de genes e proteínas, fenótipo do músculo esquelético, transcriptoma e identificação de novas vias de sinalização celular. O treinamento físico após a cirurgia bariátrica melhorou a sensibilidade à insulina no músculo esquelético. Esta resposta foi mediada por alterações moleculares e fenotípicas na ECM. A cirurgia bariátrica per se foi incapaz de solucionar completamente a resistência à insulina e a expansão da ECM no músculo esquelético. Candidatos relevantes modulados pelo exercício emergiram como alvos terapêuticos para o tratamento da resistência à insulina do músculo esquelético, nomeadamente a via TGF \'beta\' 1 SMAD 2/3 e seu antagonista folistatina. Em resumo, empregamos uma abordagem \"top-down approach\" para fornecer evidências de que a ECM do músculo esquelético desempenha um papel fundamental nos efeitos sobrepostos da cirurgia bariátrica e do exercício físico sobre a sensibilidade à insulina em mulheres obesas. Além disso, este estudo demonstrou que o treinamento físico evitou a reversão da melhora da função endotelial por meio da melhora do padrão de fluxo sanguíneo e redução de marcadores inflamatórios. Em conclusão, ao revelar um novo mecanismo pelo qual o exercício pode contrabalançar a resistência à insulina em pacientes pós-bariátricos (isto é, atenuar a espessura da ECM) e preservar a função endotelial, este estudo endossa que o exercício físico deve ser adotado como relevante medida terapêutica a fim de garantir os melhores resultados cardiometabólicos em pacientes submetidos à cirurgia bariátrica / Bariatric surgery provides cardiometabolic protection to obese individuals, contributing to a reduction in mortality risk. However, the extent of metabolic benefit may be subject to changes in the patient\'s lifestyle after surgical intervention. Although exercise seems to improve the effects of surgery on insulin sensitivity, the underlying mechanism of action remains largely unexplained. It is speculated that potential changes in the skeletal muscle extracellular matrix (ECM) could be associated with improved insulin sensitivity induced by physical exercise in post-bariatric patients. In addition, it is not known whether the benefits of bariatric surgery on endothelial function, an important marker of early atherosclerosis, are sustainable without changes in lifestyle, such as the inclusion of physical exercise. Thus, the aims of the present study were to investigate the effects of exercise on intracellular signaling involved in insulin sensitivity and skeletal muscle ECM remodeling (Study 1) and the effects of exercise on the brachial artery vasodilator response of patients undergoing bariatric surgery (Study 2). Sixty-two women were randomized after bariatric surgery to a 6-month exercise program or standard of treatment. At the beginning of the study, 3 and 9 months after surgery, insulin sensitivity was assessed by the oral glucose tolerance test (OGTT), endothelial function analysis and skeletal muscle samples were obtained from the vastus lateralis. Skeletal muscle samples were subjected to comprehensive analysis, including gene and protein expression, skeletal muscle phenotype, transcriptome and identification of new cell signaling pathways. Exercise training after bariatric surgery improved insulin sensitivity in skeletal muscle. This response was mediated by molecular and phenotypic changes in ECM. Bariatric surgery per se was unable to completely resolve insulin resistance and skeletal muscle ECM expansion. Relevant exercise-modulated candidates emerged as therapeutic targets for the treatment of skeletal muscle insulin resistance, namely the TGFβ1/SMAD 2/3 pathway and its follistatin antagonist. In summary, we employed a \"top-down approach\" to provide evidence that skeletal muscle ECM plays a key role in the overlapping effects of bariatric surgery and exercise on insulin sensitivity in obese women. In addition, this study demonstrated that physical training avoided reversal of endothelial function improvement by improving blood flow pattern and reducing inflammatory markers. In conclusion, in revealing a new mechanism by which exercise can counterbalance insulin resistance in post-bariatric patients (i.e., attenuate ECM thickness) and preserve endothelial function, this study endorses that exercise should be adopted as a relevant therapeutic measure in order to guarantee the best cardiometabolic results in patients undergoing bariatric surgery.

Long interspersed nuclear element-1 (L1) is a genetic element that mobilizes throughout the mammalian genome via retrotransposition and damages host DNA via mutational insertions, chromosomal rearrangements, and reprogramming of gene expression. The cellular mechanisms responsible for aberrant L1 expression during cancer pathogenesis are unclear. Previously, we have shown that L1 reactivation in several human cell lines is dependent upon the activation of aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor member of the PAS superfamily of proteins. We also showed that ectopic expression of L1 reprograms the HepG2 genome leading to epithelial-to-mesenchymal transition (EMT). Here we present evidence that reactivation of L1 and modulation of EMT in HepG2 cells by the AhR ligand benzo(a)pyrene (BaP) is effected through the canonical TGF-β1 signaling pathway. BaP increased TGF-β1 mRNA, SMAD2 phosphorylation and decreased expression of E-Cadherin. The functional relevance of these interactions and the involvement of TGFBR1/ALK5 and SMAD2/3 were confirmed by siRNA interference. Furthermore, expression of L1-encoded ORF1p was positively correlated with the activation of TGF-β1 signaling in human hepatocarcinoma samples at various stages of malignant progression. These results indicate that ligand-mediated AhR activation regulates L1 via canonical TGF-β1 signaling and raise important questions about the molecular etiology of human hepatocarcinomas.

Articular cartilage functions to maintain joint mobility. The loss of healthy, functional articular cartilage due to osteoarthritis or injury can severely compromise quality of life. To address this issue, cartilage tissue engineering approaches are currently in development. Bone marrow-derived mesenchymal progenitor cells (MPCs) hold much promise as an alternative cell source for cartilage tissue engineering. While previous studies have established that MPCs from humans and multiple other species undergo in vitro chondrogenic differentiation, additional research is needed to define conditions that will enhance MPC differentiation, increase matrix production by differentiating cultures, and support development of functional tissue-engineered cartilage constructs. Mechanical loading may be an important factor regulating chondrogenic differentiation of MPCs and cartilage matrix formation by chondrogenic MPCs. This thesis work evaluated the influence of oscillatory unconfined compressive mechanical loading on in vitro MPC chondrogenic activity and biosynthesis within hydrogel suspension. Loading was conducted using MPCs cultured in media supplements supporting chondrogenic differentiation. Possible interactions between the number of days in chondrogenic media preceding loading initiation and the ability of the MPC culture to respond to mechanical stimulation were explored in two different loading studies. The first loading study investigated the effects of 3 hour periods of daily oscillatory mechanical stimulation on subsequent chondrogenic activity, where chondrogenic activity represented an assessment of cartilage matrix production by differentiating MPCs. This study found that oscillatory compression of MPCs initiated during the first seven days of culture did not enhance chondrogenic activity above the level supported by media supplements alone. The second loading study evaluated changes in biosynthesis during a single 20 hour period of oscillatory mechanical stimulation to assess mechanoresponsiveness of the MPC cultures. This study found that MPCs modulated proteoglycan and protein synthesis in a culture time-dependent and frequency-dependent manner upon application of oscillatory compression. Together the two loading studies provide an assessment of dynamic compressive mechanical loading influences on MPC cultures undergoing chondrogenic differentiation. The information gained through in vitro studies of differentiating MPC cultures will increase basic knowledge about progenitor cells and may also prove valuable in guiding the future development of cartilage tissue engineering approaches.

Thirty five million people are currently living with HIV-1 today with women accounting for half of infected individuals globally. Sexual transmission is the main route of HIV transmission with approximately 40% of HIV infections occurring when the mucosal lining of the female genital tract (FGT) is exposed to HIV in semen from an infected male partner. Seminal plasma (SP), the fluid portion of semen, is a complex fluid which plays an immunomodulatory role in the FGT for successful conception, largely due to its high concentrations of TGF-β1. Several factors in SP from HIV-uninfected men have been shown to either inhibit or enhance HIV infection in target cells, however it is not clear how SP from HIV infected men would modulate genital epithelial cells (GECs), the first cells that encounter HIV in the FGT. The overall goals of this thesis were to compare inflammatory and regulatory cytokine concentrations in SP from HIV-uninfected and infected men, and subsequently compare GEC cytokine responses following exposure to SP from HIV-uninfected and HIV-infected men. I also investigated how SP and TGF-β regulated cytokine production and barrier function in GECs in the presence of HIV. The results summarized in this thesis demonstrated that HIV infection leads to different cytokine profiles in SP, based on stage of HIV-1 infection. HIV-infected men in acute stage contained higher levels of proinflammatory cytokines in their SP compared to HIV-uninfected and chronically infected men (CI men) which subsequently lead to higher levels of proinflammatory cytokines from GECs compared to CI men. In the follow up to this study we found that active TGF-β, which was found in higher concentrations in SP from CI men and led to decreased inflammatory response from GECs, was compartmentalized between blood plasma and seminal plasma. Higher levels of active TGF-β in SP correlated with decreased semen viral load and the immune activation marker sCD14 leading us to believe that ART-naive CI men in our cohort were naturally controlling their immune activation status, as active TGF-β levels were lower in ART-treated men. Short-term exposure of GECs to SP from CI men or TGF-β at comparable concentrations to SP protected the GEC barrier against HIV by decreasing inflammatory cytokines and preventing tight junction breakage. However, long-term exposure to TGF-β in the presence of HIV further increased inflammation in GECs suggesting a biphasic role for TGF-β in the FGT. This body of work summarized in this thesis demonstrates for the first time how semen from HIV-infected men modulates FGT epithelial cell cytokine responses and barrier function, an important consideration in the design of local therapeutic strategies to protect the FGT against HIV infection. / Thesis / Doctor of Philosophy (Medical Science)

Wachstumsfaktoren sind wichtige Steuerelemente des Knochenzellmetabolismus. Im Verlauf der Frakturheilung kommt es zur Ausschüttung von zahlreichen Wachstumsfaktoren, Zytokinen und Botenstoffen im und um den Bereich des Frakturspalts, die systemisch oder lokal, endokrin, parakrin oder autokrin wirksam werden können. Für verschiedene Wachstumsfaktoren konnten in zahlreichen Studien osteoinduktive und die Frakturheilung positiv beeinflussende Wirkungen nachgewiesen werden. In vitro und in vivo Studien belegen, dass einige dieser Faktoren wie Insulin-like growth factor-I (IGF-I), Transforming growth factor-beta1 (TGF-beta1) und Bone morphogenetic protein-2 (BMP-2) einen stimulierenden Effekt auf osteo- und chondrogene Zellen aufweisen und somit die Knochenheilung stimulieren. Der genaue Wirkmechanismus dieses positiven Effektes der Wachstumsfaktoren und ihre Interaktion im Verlauf der Frakturheilung ist nicht bekannt. Die lokale Applikation der Faktoren für einen therapeutischen Einsatz bei der Frakturheilung stellte bisher jedoch ein Problem dar. Mit der entwickelten biodegradierbaren Poly(D,L-Laktid)-Beschichtung von Implantaten können eingearbeitete Wachstumsfaktoren kontrolliert und lokal direkt an der Fraktur freigesetzt werden. Das beschichtete Implantat dient dabei der Stabilisation der Fraktur und gleichzeitig als Wirkstoffträger. Die Beschichtung weist eine hohe mechanische Stabilität auf. Die eingearbeiteten Wachstumsfaktoren behalten ihre biologische Aktivität in der Beschichtung und werden kontrolliert lokal freigesetzt. Um den Effekt lokal applizierter Wachstumsfaktoren auf die Frakturheilung zu untersuchen, wurde ein standardisiertes geschlossenes Frakturmodell entwickelt, das der klinischen Situation möglichst nahe ist und reproduzierbar durchgeführt werden kann. Untersucht wurde der Effekt der Wachstumsfaktoren IGF-I, TGF-beta1 und BMP-2 und des Trägermaterials PDLLA sowie lokale und systemische unerwünschte Wirkungen. Die Ergebnisse zeigten einen signifikant grösseren stimulierenden Effekt von IGF-I auf die Frakturheilung im Vergleich zur TGF-beta1 Applikation. Die kombinierte Gabe beider Faktoren ergab einen signifikant grösseren Effekt auf die torsionale Stabilität und die Kallusreifung im Vergleich zur Einzelapplikation. Beide Faktoren scheinen einen synergistischen Effekt auf die Frakturheilung zu haben. Die lokale Applikation von BMP-2 beschleunigte ebenso, wie die lokale Freisetzung von IGF-I und TGF-beta1 die Frakturheilung signifikant. Deutliche Unterschiede zwischen IGF-I / TGF-beta1 und BMP-2 konnten nicht festgestellt werden.Allerdings zeigte sich bei der Verwendung von BMP-2 auch ausserhalb der Frakturzone eine grössere Mineralisation der Kortikalis, die bei IGF-I / TGF-beta1 nicht zu beobachten ist. Auch im Grosstiermodell bestätigte sich die Wirksamkeit dieser bioaktiven Oberflächen-beschichtung auf die Osteotomieheilung. Die PDLLA-Beschichtung alleine, ohne eingearbeitete Wachstumsfaktoren, zeigte bereits einen positiven Effekt auf die Frakturheilung. Die Untersuchungen belegen, dass die lokale Freisetzung von Wachstumsfaktoren aus einer biodegradierbaren PDLLA-Beschichtung von Implantaten die Frakturheilung signifikant beschleunigt, wobei keine unerwünschten lokalen oder systemischen Wirkungen beobachtet werden konnten. Bei dem Vergleich lokaler (durch Wachstumsfaktoren) mit systemischer Stimulationsmöglichkeit (durch Wachstumshormon) der Frakturheilung lässt sich zusammenfassend feststellen, dass die kombinierte Anwendung beider Stimulationsmöglichkeiten zu keiner weiteren Steigerung der Heilungsvorgänge führte. Weitere Untersuchungen wurden hinsichtlich der genauen Rolle und Interaktion der Wachstumsfaktoren durchgeführt. Vor allem die Frühphase scheint hierbei eine entscheidende Rolle bei der Frakturheilung einzunehmen. Es zeigte sich hierbei eine deutliche Stimulation der Osteoblastendifferenzierung mit einer Erhöhung der Kollagen-1 Produktion in vitro sowie eine Steigerung der Proliferationsrate und Angiogenese mit einem schnelleren Ablauf der Phasen der Frakturheilung in vivo durch lokal appliziertes IGF-I und TGF-beta1. Weitere Anwendungen der entwickelten Beschichtungstechnologie stellen die lokale Applikation von Wachstumsfaktoren von beschichteten PDLLA-Cages bei der intervertebralen Spondylodese sowie die lokale Applikation von Antibiotika aus einer PDLLA-Beschichtung von Implantaten zur Prophylaxe der Implantat-assoziierten Osteomyelitis dar.Basierend auf diesen Ergebnissen steht der Einsatz PDLLA-Gentamicin beschichteter intramedullärer Tibianägel kurz vor der klinischen Anwendung.Eine Zulassung durch die entsprechenden Behörden ist erfolgt.Die klinische Anwendung Wachstumsfaktoren-beschichteter Implantate ist bereits in der Vorbereitung. / Growth factors are important regulators of bone metabolism. During fracture healing many growth factors or cytokines were locally released at the facture site. In several studies, different growth factors demonstrated osteoinductive and fracture stimulating properties. In vitro and in vivo studies showed a stimulating effect of Insulin-like growth factor-I (IGF-I), Transforming growth factor-beta1 (TGF-beta1) and Bone morphogenetic protein-2 (BMP-2) on osteo- and chondrogenetic cells. The exact effectiveness and the interaction of these growth factors during fracture healing is not known so far. Further, the local application of these factors for therapeutically use in fracture treatment is still a problem. The developed biodegradable poly(D,L-lactide)-coating of implants allows the local and controlled release of incorporated growth factors directly at the fracture site. The coated implant serves on the one hand for fracture stabilization and on the other hand as a drug delivery system. The coating has a high mechanical stability. The incorporated growths factors remain biologically active in the coating and were released in a sustained and controlled manner. To investigate the effect of locally released growth factors IGF-I, TGF-beta1 and BMP-2 and the carrier PDLLA on fracture healing, standardised closed fracture models were developed with a close relationship to clinical situation. Further, possible local and systemic side effects were analysed. The results demonstrated a significantly higher stimulating effect of IGF-I on fracture healing compared to TGF-beta1. The combined application of both growth factors showed a synergistic effect on the mechanical stability and callus remodeling compared to single treatment. The local release of BMP-2 also enhanced fracture healing significantly - comparable to combination of IGF-I and TGF-beta1. However, a higher rate of mineralisation was measurable outside the fracture region using BMP-2 in a rat fracture model. Using a large animal model on pigs with a 1 mm osteotomy gap, the effectiveness of locally released growths factors could be confirmed. Further, the PDLLA-coating without any incorporated growth factors demonstrated a significantly effect on healing processes in both models. These investigations showed, that the local release of growth factors from PDLLA coated implants significantly stimulate fracture healing without any local or systemic side effects. Comparing systemic with local stimulation techniques, we found an improvement of fracture healing by systemic administration of growth hormone and local application of IGF-I and TGF-beta1. However, the combined use of both simulation techniques did not lead to a further increase of healing processes. Investigations on the effectiveness and the interaction of growth factors during fracture healing demonstrated an dramatic effect in the early phases of healing processes. The growth factors stimulate the differentiation of osteoblasts with a higher production of collagen I in vitro and increase osteogenesis and vascularisation of the fracture callus in vivo. Further applications of the coating technology are the use of PDLLA and growth factor coated cages for the stimulation of intervertebral fusion and the use of PDLLA and Gentamicin coated implants in order to prevent implant associated infections. The clinical use of antibiotic and growth factor coated implants are in preparation.

Myofibroblast expansion is a critical event in the pathogenesis of liver fibrosis. The activation of hepatic stellate cells (HSC) to myofibroblast (MFB) results in the enhanced production of extracellular matrix (ECM). We have studied the effect of fibroblast growth factor 1 (FGF-1) on liver MFB. In the second part we investigated effect of transforming growth factor β1 (TGF-β1) and FGF-1 on cell line HSC-T6. Cells were cultured on plastic dishes and in 3D collagen gel mimicking fibrotic tissue. MFB were isolated by repeated passaging of nonparenchymal liver cell fraction. The transfer of MFB from plastic dishes to collagen gel resulted in the change in their shape and phenotype. The expression of cytokine TGF-β1 and of MFB markers, α-smooth muscle actin (α-SMA) and cellular fibronectin (EDA-FN) on protein level was significantly decreased in collagen gel. The experiments with SB 431542, the inhibitor of TGF-β receptor type I, showed that EDA-FN and α-SMA are differently regulated. EDA-FN expression is dependent on TGF-β1, while the expression of α-SMA is primarily determined by the environment and modified by TGF-β1. EDA-FN is more sensitive to the U0126, the inhibitor of protein kinases MEK 1 and 2. Collagen gel does not change the expression of metalloproteinase MMP-2 but activates the proenzyme....