In this project I sought to analyse the effects of different free fatty acids (FFAs) on INS-1E β-cells. The saturated fatty acid palmitate is considered toxic whereas the monounsaturated fatty acid oleate is harmless. In my working hypothesis I assumed an additional protective effect of oleate when used in combination with palmitate. Furthermore I aimed to explore in detail the possible causes and signalling pathways responsible for apoptosis or sustained cell survival. I examined the Endoplasmic Reticulum (ER) stress response, called unfolded protein response (UPR), as one essential criterion deciding about cell death or life. Analysis of viability and apoptosis confirmed the deleterious effect of palmitate on INS-1E β-cells after 24h of incubation. Oleate proved not to be harmful and even reversed the toxicity of palmitate. When the main components of the UPR were assessed using Western blot analyses and quantitative PCR was performed I found positive proof that palmitate activated the UPR and ultimately led to apoptosis. By contrast, oleate completely prevented UPR signalling. I conclude that oleate rescues INS-1E β-cells by inhibiting ER stress and its signalling.

Type 2 diabetes is a disease characterized by the inability of pancreatic β-cells to secrete sufficient amounts of insulin to maintain normoglycemia. Increased levels of saturated fatty acids such as palmitate are believed to contribute to β-cell failure and the development of the disease. In the present thesis, mechanisms behind palmitate-induced β-cell apoptosis were explored. Palmitate augmented insulin secretion after short exposure to the fatty acid, but attenuated the secretory response after longer exposure. Elevated levels of palmitate increased endoplasmic reticulum (ER) stress and induced apoptosis. When insulin secretion was inhibited by diazoxide, palmitate-induced ER stress and apoptosis were reduced. In comparison to palmitate, the mono-unsaturated fatty acid oleate increased neither ER stress nor apoptosis. Furthermore, shuttling of fatty acids into triglycerides and β-oxidation was favored in cells exposed to oleate compared to palmitate. When the levels of stearoyl-CoA desaturase 1 (SCD1), the enzyme responsible for conversion of saturated to mono-unsaturated fatty acids, were reduced, up-regulation of ER chaperones and components of the proteasome was observed. Cells with reduced levels of SCD1 showed increased sensitivity to palmitate, as exposure to the fatty acid increased levels of ER stress and apoptosis. Palmitate-induced apoptosis of the β-cell has been linked to alterations in sphingolipid metabolism. In cells with reduced levels of sphingosine kinase (SphK) 2, palmitate failed to induce apoptosis, and ER stress was reduced. Furthermore, SphK2 was required for the palmitate-induced activation of c-Jun N-terminal kinase (JNK). In contrast, knockdown of SphK1 sensitized the cell to palmitate-induced apoptosis independently of ER stress. In summary, palmitate induces β-cell apoptosis, which is partly dependent on the induction of ER stress. The mechanisms investigated support the notion that increased protein load on the ER, low degree of triglyceride formation and β-oxidation, and perturbations in sphingolipid metabolism contribute to palmitate-induced apoptosis in insulin-producing β-cells.

Resumo: As hipóteses mais modernas baseiam o processo de envelhecimento cutâneo em duas teorias: o intrínseco (ou cronológico) e o extrínseco (ou fotoenvelhecimento). Tanto no envelhecimento intrínseco como no extrínseco, as principais alterações da pele são caracterizadas por formação de rugas, flacidez e perda da elasticidade. O palmitato de retinol (PR) é um análogo sintético da vitamina A indicado topicamente para prevenir ou retardar algumas mudanças estruturais e funcionais da pele associadas ao processo de envelhecimento cutâneo, porém é um princípio ativo que apresenta estabilidade química limitada frente à umidade, oxigênio, ácidos, metais e exposição à luz. Assim, o objetivo deste trabalho foi desenvolver um sistema líquido-cristalino à base de silicone para a liberação prolongada e maximização da estabilidade do palmitato de retinol. Utilizando-se o método de titulação da fase aquosa, foram construídos dois diagramas ternários de fases, ambos constituídos por Procetyl AWS® (tensoativo) e água e por silicone DC 5330® (fase oleosa no diagrama 1) ou silicone DC 5329® (fase oleosa no diagrama 2). Nos estudos de estabilidade preliminar, as formulações do diagrama 1 (A, B, C e D) e do diagrama 2 (E, F, G e H) foram avaliadas nos seguintes estudos: microscopia de luz polarizada, centrifugação, pH, condutividade eletrolítica, índice de refração e reologia. Dentre estas formulações estudas preliminarmente, a formulação GPR foi selecionada para ser conduzida aos estudos posteriores após a incorporação de 1% do princípio ativo, pois demonstrou a formação de fases líquido-cristalinas evidenciadas sob luz polarizada no microscópio óptico, apresentou-se compatível com o pH fisiológico da pele, apresentou pequeno coeficiente de variação das medidas realizadas referentes aos parâmetros físico- químicos e menor capacidade de desestruturar... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Current hypotheses based on the process of skin aging in two theories: the intrinsic (or chronological) and extrinsic (or photoaging). Both in the extrinsic as intrinsic aging, the main skin disorders are characterized by wrinkles, flaccidity and loss of elasticity. Retinyl palmitate (RP) is a synthetic analogue of vitamin A given topically to prevent or delay some structural and functional changes of the skin associated with aging, but is an active principle that has limited chemical stability against moisture, oxygen, acids , metals and exposure to light. Thus, the aim of this study was to develop liquid crystalline system silicon-based for extended release and maximizing the stability of retinyl palmitate in order to optimize the use of topically active principle in the prevention and / or mitigation of aging skin. Using the method of titration of the aqueous phase, was built two ternary phase diagrams, both consist of Procetyl AWS ® (surfactant) and water and silicone DC 5330 ® (oil phase diagram 1) or silicone DC 5329 ® (phase oil in diagram 2). In preliminary studies of stability, the formulations of the diagram 1 (A, B, C and D) and the formulations of the diagram 2 (E, F, G and H) were evaluated in the following studies: polarized light microscopy, centrifugation, pH, electrolitic conductivity, refraction index and rheology. Among these formulations studied preliminarily, the formulation G RP-loaded was selected to be conducted to further studies, since it showed the formation of liquid crystalline phases evidenced by polarized light in an optical microscope, presented consistent with the physiological pH of skin, had a small coefficient of variation of measurements related to the physicochemical and less ability to disrupt the lipid bilayer of the skin due to have a lower concentration of surfactant. The validated analytical methodology for chemical quantification of retinyl... (Complete abstract click electronic access below) / Orientador: Maria Virgínia Costa Scarpa / Coorientador: Marlus Chorilli / Banca: Eneida de Paula / Banca: Sandra Helena Pulcinelli / Mestre

Submitted by MARCIA ROVADOSCHI (marciar@unifra.br) on 2018-08-15T14:23:12Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_KellyCristineZatta.pdf: 2655855 bytes, checksum: 40a81657cb3b6d7945a2bc834a0dacb6 (MD5) / Made available in DSpace on 2018-08-15T14:23:12Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_KellyCristineZatta.pdf: 2655855 bytes, checksum: 40a81657cb3b6d7945a2bc834a0dacb6 (MD5) Previous issue date: 2011-03-21 / Keeping in mind the problematic that photoaging and hyperpigmentation represent, the development of a topical formulation containing vitamin C (ascorbyl palmitate) and açai oil associated to nanoparticules may represent a protection of the cellular membrane against oxidation, due to a better permeation and the synergic acting of both active. This study has as its objective the development and the characterization of cream-gel formulations containing ascorbyl palmitate associated to nanocarriers, in the presence or absence of the açai oil as the oily nucleus. Associated to that, the stability of the freeze-dried nanocoated active was tested after the incorporation in a silicon basis. Suspensions containing the AP associated t o the nanocapsules (NCAP), nanoemulsions (NEAP) and nanodispersions (NDAP) were developed, being the latter used as active protection comparative parameter without the active (NCBC). With the purpose of assessing the influence of the water on the stability of the active, AP was incorporated in a methanolic solution (SMAP), to which was put in an aging study during 30 days in environment temperature (22 ºC + 2 ºC), refrigeration (4 ºC + 2 ºC), greenhouse (40 ºC + 2 ºC) and chamber UVC (254 nm), along with the formulations cited previously. The sample characterizations was carried out soon after its obtaining and on days 7, 15, 21 and 30, as to the AP content quantification, average particle diameter, polydispersity index, zeta potential and pH. The NCAP, NEAP, NCBC suspensions were incorporated in semi-solid basis of Hostacerin SAF® cream-gel and later stored for the accelerated aging study (21 days) and alternated cycles of heating-cooling, which were analysed at the beginning and end of this period through the determination of the particle average diameter, polydispersity index, zeta potential, pH, active content quantification, organoleptic characteristic determination, spreadability determination and rheological behavior, and centrifugation test. NCAP and NCBC suspension samples were freeze-dried and incorporated to the silicon base (CSNCAPLIO and CSNBCLIO, respectively), and stored in heating-freezing cycles. In this same basis, the active in the free form (CSAP) was incorporated. Soon after its obtaining, the suspensions presented active content equal to 97,51%±0,93 to NCAP, 80,68% + 1,25 to NEAP and 83,10% + 3,10 to NDAP, and remaining desirable paramaters to nanometric systems. However, in the 30th day of storing, all the physical-chemical characteristics suffered significant alteration, being evident in the active loss, which was detected only in the NCAP and NEAP samples conditioned under refrigeration (24,42 % + 1,0 e 21,37 % + 1,27,, respectively). Contrasting with these results, the SMAP kept the active stable and in effective concentrations during 90 days of storing, revealing the fragility of the AP structure in the presence of an aqueous environment. As to the cream-gel formulations, all presented physical-chemical parameters appropriate for the nanoparticle formulations and slightly acid pH, satisfactory for the structure of the active in the non-dissociated form (smaller than the AP pKa value), homogeneous aspect of the sensorial optimum. For those, a proper spreadability was obtained, considering the function and action local, intended. The formulation rheogram demonstrated to have a non-Newtonian character and pseudoplastic flux, which is desired in pharmaceutical formulations, for the initial resistance for the semi-solid formulation to flow diminished, reflecting the application easiness. The greatest active concentration for the samples containing the nanocoated active was obtained, suggesting a greater initial protection of the active in the presence of polymer film and the açai oil. However, at the end of the 21-day period, it was verified the destabilization of the formulation containing the active by the total freeze-drying of those, not being possible to carry out the final tests. The cream in silicone basis samples were analyzed only according to the active content soon after the incorporation of the freeze-dried, which demonstrated an average initial loss of 30% under the previous to the freeze-drying quantification, while for the sample containing the free active, the initial content was superior. This result may be justified possibly by the prolonged exposition to humidity and light during the freeze-drying process. After the 7-day storing in ES, the essay was repeated, obtaining for the CSNAPLIO an average of 15 % + 2,03 of AP content, being that for the CSAP the quantification due to the active oxidation total was not possible. These data restate the destabilization of the active faced with the heat, even for the water free formulation. From the obtained results it was possible to verify the AP instability faced with the heat, light and water conditions. However, keeping in mind its therapy potentiality t is considered relevant to the continuity of this research with the purpose of searching for more ascorbyl palmitate stability keeping it viable for topical application. It is suggested the incorporation to the drying of the suspensions for nano-spry-drying and the incorporation of those in different semi-solid basis, with emulsions, silicones and non-ionic bases for the long-term stability study, searching for evidences which take to definite conclusions on the product stability. / Tendo-se em vista a problemática que o fotoenvelhecimento e a hiperpigmentação representam, o desenvolvimento de uma formulação tópica contendo vitamina C (palmitato de ascorbila) e óleo de açaí associada à nanopartículas pode representar a proteção das membranas celulares contra a oxidação, devido à melhor permeação e a atuação sinérgica de ambos os ativos. Este estudo teve por objetivo o desenvolvimento e caracterização de formulações de creme-gel contendo palmitato de ascorbila associado à nanocarreadores, na presença e ausência do óleo de açaí como núcleo oleoso. Associado a isso, foi testada a estabilidade de liofilizados do ativo nanoencapsulado após incorporação em base de silicone. Foram desenvolvidas suspensões contendo o AP associado à nanocapsulas (NCAP), nanoemulsões (NEAP) e nanodispersões (NDAP), tendo sido esta última utilizada como parâmetro comparativo de proteção do ativo pela total ausência de filme polimérico e núcleo oleoso. Como branco utilizou-se uma suspensão de nanocápsulas sem o ativo (NCBC). A fim de avaliar a influência da água sobre a estabilidade do ativo, incorporou-se AP em uma solução metanólica (SMAP), a qual foi colocada em estudo de envelhecimento durante 30 dias em temperatura ambiente (22 ºC + 2 ºC), refrigeração (4 ºC + 2 ºC), estufa (40 ºC + 2 ºC) e câmara climatizada UVC (254 nm), juntamente com as formulações citadas anteriormente. A caracterização das amostras foi realizada logo após sua obtenção e nos dias 7, 15, 21 e 30, quanto à quantificação do teor de AP, diâmetro médio de partícula, índice de polidispersão, potencial zeta e pH. As suspensões de NCAP, NEAP, NCBC foram incorporadas em bases semissólidas de creme-gel Hostacerin SAF® e posteriormente armazenadas para estudo de envelhecimento acelerado (21 dias) em ciclos alternados de aquecimento – resfriamento, as quais foram analisadas ao início e final deste período através das determinações de diâmetro médio de partícula, índice de polidispersão, potencial zeta, pH, quantificação do teor de ativo, determinação das características organolépticas, determinação da espalhabilidade e comportamento reológico, e teste de centrifugação. Amostras de suspensões de NCAP e NCBC foram liofilizadas e incorporadas em base de silicone (CSNCAPLIO e CSNCBCLIO, respectivamente), e armazenadas em ciclos de aquecimento-resfriamento. Nesta mesma base, foi incorporado o ativo na forma livre (CSAP). Logo após sua obtenção, as suspensões apresentaram teor de ativo iguais a 97,51% + 0,93 para NCAP, 80,68% + 1,25 para NEAP e 83,10% + 3,10 para NDAP, e demais parâmetros desejáveis para sistemas nanométricos. Contudo, no 30o dia de armazenamento, todas as características físico-químicas sofreram significativa alteração, sendo evidente a perda de ativo, o qual foi detectado somente apenas nas amostras de NCAP e NEAP acondicionadas sob refrigeração (24,42 % + 1,0 e 21,37 % + 1,27, respectivamente). Contrastando com estes resultados, a SMAP manteve o ativo estável e em concentrações efetivas durante 90 dias de armazenamento, revelando a fragilidade da estrutura do AP em presença de meio aquoso. Quanto às formulações de creme-gel, todas apresentaram parâmetros físico-químicos adequados para formulações de nanopartículas e pH levemente ácido, satisfatório para a estrutura do ativo na forma não-dissociada (menor que o valor de pKa do AP), aspecto homogêneo de ótimo sensorial. Para as mesmas obteve-se espalhabilidade adequada, considerando a função e local de ação, pretendidos. O reograma das formulações demonstrou haver caráter não-newtoniano e fluxo pseudoplástico, o qual é desejado em formulações farmacêuticas, pois a resistência inicial para a formulação semissólida fluir diminui, refletindo a facilidade de aplicação. Obteve-se a maior concentração de ativo para as amostras contendo o ativo nanoencapsulado, sugerindo a maior proteção inicial do ativo em presença do filme polimérico e do óleo de açaí. Contudo, ao final do período de 21 dias, verificou-se desestabilização das formulações contendo o ativo pela total liquefação das mesmas, não sendo possível a realização dos testes finais. As amostras de creme em base de silicone foram analisadas somente segundo o teor de ativo logo após a incorporação do liofilizado, o qual demonstrou uma perda inicial média de 30 % sob a quantificação anterior à liofilização, enquanto que para a amostra contendo o ativo livre, o teor inicial foi superior. Este resultado pode ser justificado possivelmente pela exposição prolongada à umidade e luz durante o processo de liofilização. Após os primeiros 7 dias de armazenamento em ES, o ensaio foi repetido, obtendo-se para o CSNCAPLIO uma média de 15 % + 2,03 de teor de AP, sendo que para o CSAP não foi possível a quantificação devido a total oxidação do ativo. Estes dados reafirmam a desestabilização do ativo frente ao calor, mesmo para formulação isenta de água. A partir dos resultados obtidos foi possível verificar a instabilidade do AP frente a condições de calor, luz e água. Contudo, tendo em vista sua potencialidade terapêutica considera-se relevante a continuidade desta pesquisa de forma a buscar maior estabilidade do palmitato de ascorbila mantendo-o viável para aplicação tópica. Sugere-se a incorporação a secagem das suspensões por nano-spray-drying e incorporação das mesmas em diferentes bases semissólidas, como emulsões, silicones e bases não-iônicas para estudo de estabilidade de longo prazo, buscando indícios que levam a conclusões definitivas sobre a estabilidade do produto.

Os retinóides têm sido amplamente utilizados na clínica dermatológica e nos produtos cosméticos com finalidades preventivas e reparadoras dos efeitos indesejáveis do envelhecimento cutâneo. Considerando que a concentração de retinóides, como por exemplo, o ácido retinóico ou o palmitato de retinila, pode influenciar na eficácia e segurança de uso dos mesmos em formulações tópicas, é de fundamental importância o desenvolvimento e avaliação dos efeitos benéficos e/ou colaterais de formulações dermocosméticas contendo diferentes concentrações de ácido retinóico (0,025%, 0,05% e 0,1%) e palmitato de retinila (0,25%, 0,5% e 1,0%), visando a obtenção de uma concentração que proporcione a máxima eficácia possível e risco mínimo à pele. Assim, o presente trabalho tem por objetivos avaliar a influência de diferentes concentrações de retinóides (ácido retinóico ou palmitato de retinila) em formulações dermocosméticas com finalidades antienvelhecimento, na pele de camundongos sem pêlo, por estudos histopatológicos, morfométricos, estereológicos e por Bioengenharia Cutânea. Para tal foram preparadas três formulações de géis creme à base de hidroxietilcelulose (HEC) e microemulsão de silicone e octanoato de octila (formulações de nos 1 e 3) e à base de complexo lipídico contendo álcool batílico e lecitina de soja, HEC e octanoato de octila (formulação de nº 2) as quais foram submetidas a testes preliminares de estabilidade. A formulação de nº 1 (F1) foi considerada a mais estável e, portanto, selecionada como veículo para a avaliação dos efeitos do ácido retinóico e do palmitato de retinila na pele de camundongos sem pêlo. Para a realização do ensaio biológico, amostras das formulações, acrescidas ou não (veículo) de 0,025; 0,05 ou 0,1% ácido retinóico ou 0,25; 0,5 ou 1,0% de palmitato de retinila foram aplicadas no dorso de camundongos sem pêlo. Após cinco dias da aplicação diária destas formulações, foram obtidas medidas de índice de eritema pelo equipamento Mexameter® MX16 e medidas do conteúdo aquoso do estrato córneo pelo equipamento Corneometer® CM825. Em seguida, os camundongos foram mortos e posteriormente foram colhidos fragmentos de pele das áreas que receberam aplicação das formulações, bem como da área que não foi aplicada nenhuma formulação (controle) e, a seguir, obtidos cortes histológicos para a realização dos estudos histopatológicos, morfométricos e estereológicos. De acordo com as metodologias empregadas, foi possível observar que, na avaliação do conteúdo aquoso do estrato córneo, somente as formulações que continham ácido retinóico em diferentes concentrações, provocaram mudanças significativas, reduzindo este conteúdo, ou seja, ocasionou um ressecamento na superfície da pele. Apenas as formulações contendo 0,025 e 0,1% de ácido retinóico e 1,0% de palmitato de retinila provocaram um aumento no índice de eritema. Além disso, tanto o ácido retinóico quanto o palmitato de retinila, atuaram na epiderme, porém de modo e intensidade diferentes, sendo que, o ácido retinóico teve um efeito mais pronunciado em relação às variáveis estudadas. As três diferentes concentrações de ácido retinóico e de palmitato de retinila ocasionaram aumento significante da espessura das camadas epiteliais, sem alteração da camada córnea. O ácido retinóico e o palmitato de retinila atuaram ainda aumentando os volumes nuclear, citoplasmático e celular, um dos fatores que ocasionou aumento da espessura do epitélio. Finalizando, ao analisar todas as variáveis histopatológicas, morfométricas e estereológicas, bem como o índice de eritema e o conteúdo aquoso do estrato córneo estudados, podemos sugerir que as formulações que continham as concentrações intermediárias, tanto do ácido retinóico (0,05%) quanto do palmitato de retinila (0,5 %), foram as que apresentaram melhores resultados, principalmente no que se refere a uma relação risco / benefício adequada e aceitável. / Retinoids has been widely used in dermatological clinic and in cosmetics products with preventive purposes as well as for the repairmen of the cutaneous aging undesirable effects. Considering that the concentration of retinoids, i.e., the retinoic acid or the retinyl palmitate, can influence their efficacy and safety in topical formulations, the development and evaluation of the beneficial and/or collateral effect of dermocosmetic formulations containing different concentrations of retinoic acid (0.025%, 0.05% e 0.1%) and retinyl palmitate (0.25%, 0.5% e 1.0%) is very important, aiming at the attainment of a concentration that provides to the maximum possible efficacy and minimum risk to the skin. Thus, the aim of this study was to evaluate the influence of different concentrations of retinoids (retinoic acid or retinyl palmitate) in dermocosmetics formulations with antiaging purposes, in hairless mice, using histopathological, morphometric and stereologic studies and Skin Bioengineering Techniques. For this purpose, three gel cream formulations were developed, containing hydroxyethylcellulose (HEC) and silicone microemulsion and octyl octanoate (formulations nº 1 and 3) and a complex lipidic based formulation containing batyl alcohol and lecithin, HEC and octyl octanoate (formulation nº 2), which were submitted to preliminary stability tests. The formulation nº1 (F1) was considered the most stable, therefore, it was selected as the vehicle for the evaluation of the effects of the retinoic acid and the retinyl palmitate in hairless mice skin. For the accomplishment of the biological assay, samples of the formulations, supplemented or not (vehicle) of 0.025, 0.05 or 0.1% retinoic acid or 0.25, 0.5 or 1.0% retinyl palmitate were applied in the dorsal skin of hairless mice. After five days of daily application of these formulations, the erythema index was measured by reflectance spectrophotometry using a Mexameter® MX16 as well as the water content of the stratum corneum using Corneometer® CM825. After that, the hairless mice were sacrificed and later skin fragments were obtained for each area that received application of the formulations, as well as of the area that was not applied any formulation (control) and, after that histologic sections were obtained and submitted to histopathological, morphometric and stereologic studies. In accordance with the used methodologies, it was possible to observe that in the evaluation of the water content of the stratum corneum, only the formulations that contained retinoic acid in different concentrations provided significant changes, enhancing skin surface dryness. Only the formulations containing 0.025 and 0.1% of retinoic acid and 1.0% of retinyl palmitate provided an increase in erythema index. Moreover, both retinoic acid and retinyl palmitate acted in the epidermis, however in different intensity and way, since retinoic acid had more pronounced effects in relation to the studied variables. The three different concentrations of retinoic acid and retinyl palmitate caused a significant increase of the epithelial layers thickness, without alteration of the horny layer. Retinoic acid and retinyl palmitate also increased the nucleus, cytoplasmic and cell volumes, which was one of the factors that influenced the increase of the epithelium thickness. Finishing, when analyzing all the histopathological, morphometric and stereologic variables, as well as the erythema index and the water content of the stratum corneum studied, we can suggest that the formulations that contained the intermediate concentrations (such as 0.05% of retinoic acid and 0.5% of retinyl palmitate) presented the best results, mainly when an adequate and acceptable risk/ benefit relationship is considered.

Adipöse Patienten und Typ-2-Diabetiker zeigen ein erhöhtes Risiko für Harnwegsinfekte. Die Ursache der höheren Prävalenz ist noch nicht nachhaltig geklärt. Bekannt ist, dass Typ-2-Diabetiker erhöhte Konzentrationen freier Fettsäuren im Blut aufweisen. Der veränderte Fettstoffwechsel könnte neben bakteriellen Ursachen ein möglicher Grund für abakterielle Entzündungsreaktionen der Harnblase sein. Zur Prüfung dieser Hypothese wurden zeit- und konzentrationsabhängig kultivierte humane Detrusormyozyten im Vergleich zur Lipopolysaccharid (LPS) induzierten Entzündungsreaktion mit Palmitat stimuliert. Es wurde geprüft, ob eine autokrine und/oder endokrine Regulation des IL-6-Signalwegs vorliegt. Im Fokus standen insbesondere die IL-6- und MCP-1-Expression und deren möglichen regulatorischen Proteine gp80, gp130, NF-κB, STAT3, SOCS3 und MEK1. Die Stimulationsversuche mit LPS und Palmitat zeigen einen differenten zeit- und konzentrationsabhängigen Effekt auf die IL-6- und MCP-1-Expression in den humanen Detrusormyozyten. LPS und Palmitat induzieren eine zeitabhängige autokrine Regulation der IL-6-Signalkaskade über phosphoryliertes STAT3 und Feedback-mechanismen via SOCS3. Sowohl LPS als auch Palmitat bewirken über 48h eine mögliche endokrine Regulation des IL-6-Signalwegs. Zusammenfassend zeigt die Palmitatstimulation zeit- und konzentrationsabhängig einen stärkeren Effekt auf die IL-6-Signalwirkung als die Stimulation mit LPS.:Abkürzungsverzeichnis 1. Hintergrund und Ziel der Arbeit 2. Methoden 3. Ergebnisse 3.1 Struktur und Funktion der suburothelialen Myofibroblasten 3.2 Entzündungsmechanismen der Harnblase 3.3 Einfluss von Palmitat auf die IL-6 and MCP-1 Expression humaner Detrusormyozyten - Link zwischen Stoffwechsel und immunologischer Reaktion der Harnblasenmuskulatur 3.4 IL-6 - JAK/STAT - Signalweg in den Detrusormyozyten 3.5 Schlussfolgerungen 4. Literaturverzeichnis 5. Publikationen 5.1 Struktur und Funktion der suburothelialen Myofibroblasten in der humanen Harnblase unter normalen und pathologischen Bedingungen 5.2 Regulation von Interleukin-6 durch Lipopolysaccharid-Stimulation in kultivierten Detrusormyozyten 5.3 Palmitate induced IL-6 and MCP-1 expression in human bladder smooth muscle cells provides a link between diabetes and urinary tract infections 6. Zusammenfassung der Arbeiten Anlagen / Background: Urinary tract infections (UTI) are more frequent in type-2 diabetes mellitus patients than in subjects with normal glucose metabolism. The mechanisms underlying this higher prevalence of UTI are unknown. However, cytokine levels are altered in diabetic patients and may thus contribute to the development of UTI. Increased levels of free fatty acids (FFA), as observed in obese patients, can induce IL-6 production in various cell types. Therefore we studied the effects of the free fatty acid palmitate and bacterial lipopolysaccharide (LPS) on interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) expression and secretion in cultured human bladder smooth muscle cells (hBSMC). Methodology/Principal Findings: Biopsies were taken from patients undergoing cystectomy due to bladder cancer. Palmitate or LPS stimulated hBSMC were analysed for the production and secretion of the IL-6, gp80, gp80soluble, gp130, MCP-1, pSTAT3, SOCS3, NF-kB and SHP2 by quantitative PCR, ELISA, Western blotting, and confocal immunofluorescence. In signal transduction inhibition experiments we evaluated the involvement of NF-kB and MEK1 in IL-6 and MCP-1 regulation. Palmitate upregulates IL-6 mRNA expression and secretion via NF-kB dependent pathways in a concentration- and timedependent manner. MCP-1 was moderately upregulated by palmitate but was strongly upregulated by LPS involving NF-kB and MEK1 dependent pathways. Soluble IL-6 receptor (gp80soluble) was downregulated by palmitate and LPS, while membrane-bound gp80 was moderately upregulated. LPS increased SOCS3 and SHP2, whereas palmitate only induced SOCS3. Secondary finding: most of the IL-6 is secreted. Conclusions/Significance: Bacterial infection (LPS) or metabolic alterations (palmitate) have distinct effects on IL-6 expression in hBSMC, (i) short term LPS induced autocrine JAK/STAT signaling and (ii) long-term endocrine regulation of IL-6 by palmitate. Induction of IL-6 in human bladder smooth muscle cells by fatty acids may represent a pathogenetic factor underlying the higher frequency and persistence of urinary tract infections in patients with metabolic diseases.:Abkürzungsverzeichnis 1. Hintergrund und Ziel der Arbeit 2. Methoden 3. Ergebnisse 3.1 Struktur und Funktion der suburothelialen Myofibroblasten 3.2 Entzündungsmechanismen der Harnblase 3.3 Einfluss von Palmitat auf die IL-6 and MCP-1 Expression humaner Detrusormyozyten - Link zwischen Stoffwechsel und immunologischer Reaktion der Harnblasenmuskulatur 3.4 IL-6 - JAK/STAT - Signalweg in den Detrusormyozyten 3.5 Schlussfolgerungen 4. Literaturverzeichnis 5. Publikationen 5.1 Struktur und Funktion der suburothelialen Myofibroblasten in der humanen Harnblase unter normalen und pathologischen Bedingungen 5.2 Regulation von Interleukin-6 durch Lipopolysaccharid-Stimulation in kultivierten Detrusormyozyten 5.3 Palmitate induced IL-6 and MCP-1 expression in human bladder smooth muscle cells provides a link between diabetes and urinary tract infections 6. Zusammenfassung der Arbeiten Anlagen

Le syndrome de Leigh, type canadien français (LSFC) est une maladie infantile orpheline causée par une mutation du gène lrpprc. Elle se caractérise par une déficience tissu spécifique de cytochrome c oxydase (COX), une dysfonction mitochondriale et la survenue de crises d’acidose lactique fatales dans plus de 80% de cas. Selon les familles des patients, ces crises apparaissent lors d’une demande excessive d’énergie. Malheureusement, les mécanismes sous-jacents à l’apparition des crises et notamment la physiopathologie du LSFC demeurent inconnus. Afin de mieux comprendre les mécanismes de régulation du métabolisme énergétique chez les patients LSFC, nous avons examiné la régulation de la protéine kinase activée par l’AMP (AMPK), une enzyme clé de l'homéostasie énergétique, de même que certaines de ses voies cibles (SIRT1/PGC1α et Akt/mTOR) dans les fibroblastes de patients LSFC et de témoins en conditions basales et conditions de stress. En conditions basales, l’activité de l’AMPK était similaire dans les cellules LSFC et les témoins. Par contre, les cellules LSFC montraient une surexpression significative des voies Akt/mTOR et SIRT1/PGC1α comparativement aux cellules témoins. Nous avons aussi examiné ces voies de signalisation suite à une incubation de 4h avec 10 mM de lactate et 1 mM de palmitate (LP), nous permettant de mimer les conditions de « crise ». Nos résultats ont démontré que le LP augmentait les niveaux de phosphorylation de l’AMPK de 90% (p<0,01) dans les cellules témoins mais pas dans les cellules LSFC. Pourtant, l’AMPK est activée dans les cellules LSFC en réponse à une hypoxie chimique induite par le 2,4 dinitrophénol. Dans les cellules témoins, le LP augmentait aussi les niveaux d’expression de SIRT1 (57%, p<0,05), de LRPPRC (23%, p=0,045) et de COXIV (19%, p<0,05). Un prétraitement de 48h au ZMP, un activateur pharmacologique de l’AMPK, a eu un effet additif avec le LP et des augmentations de SIRT1 phosphorylée (120%, p<0,05), de SIRT1 total (75%, p<0,01), de LRPPRC (63%, p<0,001) et de COXIV (38%, p<0,001) ont été observées. Tous ces effets étaient aussi abolis dans les cellules LSFC. En conclusion, nos résultats ont démontré des altérations importantes de la régulation du métabolisme énergétique dans les fibroblastes de patients LSFC. / Leigh syndrome French Canadian type (LSFC) is an orphan infantile disease caused by mutations in the LRPPRC gene. It is characterized by a tissue-specific cytochrome c oxidase deficiency (COX), mitochondrial dysfunction and fatal lactic acidosis crises which occur in more than 80% of cases. According to parents, these crises occur during stressful situations. The pathophysiology underlying this disease and the factors that precipitate these crises remain unknown. To better understand the regulation of energy metabolism in LSFC patients, we examined the activity of AMP activated protein kinase (AMPK), a key regulator of energy balance, and its downstream targets (SIRT1/PGC1α and Akt/mTOR) in LSFC and control fibroblasts under basal and stress conditions. Our results showed that AMPK activity was similar in LSFC and control cells under basal conditions. On the other hand, Akt/mTOR and SIRT1/PGC1α pathways were up regulated in LSFC cells compared to controls. We next examined AMPK activity in cells treated with 10 mM lactate and 1mM palmitate (LP) for 4h, thus mimicking the conditions of “crisis”. Following this treatment, AMPK phosphorylation levels increased significantly (90%, p<0.01) in control cells but not in LSFC cells. Nevertheless, AMPK seems functional in LSFC cells because the enzyme was activated in response to chemical hypoxia induced by 2,4 dinitrophenol. LP also increased the expression of SIRT1 (57%, p<0.05), LRPPRC (23%, p=0.045) and COXIV (19%, p<0.05), in controls cells. Furthermore, pretreatment with ZMP, a pharmacological activator of AMPK, had an additive effect with LP leading to a further increase in the activity of SIRT1 (120%, p<0.05), as well as the expression levels of SIRT1 (75%, p<0.01), LRPPRC (63%, p<0.001) and COXIV (38%, p<0.001). All these effects were abolished in LSFC cells and thus, our data highlight alterations in the regulation of key enzymes of energy metabolism, including the activation of AMPK, in LFSC fibroblasts.

Obesity-associated metabolic disorders are amongst the most prevalent causes of death worldwide. Understanding how obesity leads to the development of the Metabolic Syndrome (MetS) and cardiovascular disease (CVD) will enable the development of novel therapies that dissociate obesity from its cardiometabolic complications. Our laboratory views the functional capacity of white adipose tissue (WAT), the organ designed for safe lipid storage, as a key factor in the development of MetS and CVD. At a genetically-defined stage of the aberrant WAT expansion that occurs during obesity, adipocytes undergo a functional failure, resulting in an impaired control of serum free fatty acid (FFA) concentration. In such setting, FFAs and their metabolic derivatives accumulate in other organs, where they cause lipotoxicity, leading to the development of insulin resistance and CVD. We therefore aim to understand the pathophysiological mechanisms that induce adipocyte dysfunction. The past two decades of research have established the immune system as an important regulator of WAT function. The number of adipose tissue macrophages (ATMs), the most abundant immune cell type in WAT, increases during obesity, resulting in WAT inflammation. Multiple genetic and pharmacological intervention studies of murine models of obesity have assigned a causal link between ATM pro-inflammatory activation and WAT dysfunction. However, while the propagation of inflammation in ATMs during obesity has been extensively studied, factors triggering ATM inflammatory activation are less clear. Recently, our lab has observed lipid accumulation in the ATMs isolated from obese mice. Lipid-laden ATMs were pro-inflammatory, leading us to hypothesise that aberrant lipid build-up in macrophages triggers WAT inflammation during obesity. This thesis expands on the initial findings from our lab and describes two novel mechanisms that potentially contribute to lipid-induced inflammatory activation of ATMs. In chapter 3, the role of de novo phosphatidylcholine (PC) synthesis pathway during lipotoxicity in macrophages is addressed. The first part of the chapter demonstrates that lipotoxic environment increased de novo PC synthesis rate in bone marrow-derived macrophages (BMDMs) and ATMs, and that loss of rate-limiting enzyme in de novo PC synthesis pathway, CTP:phosphocholine cytidylyltransferase a (CCTa) diminished saturated FFA-induced inflammation in BMDMs. In the second part, I show that macrophage-specific CCTa deletion did not impact on the development of WAT inflammation or systemic insulin resistance, but had a minor benefitial effect on hepatic gene transcription during obesity. Chapter 4 develops on recent observations of interactions between sympathetic nerves and macrophages in WAT. In the first part of the chapter, I demonstrate that stimulating B2-adrenergic receptor (B2AR), the main receptor for sympathetic neurotransmitter norepinephrine in macrophages, enhanced intracellular triglyceride storage by up-regulating diacylglycerol O-acyltransferase 1 (Dgat1) gene expression in BMDMs. The second part of the chapter shows that macrophage-specific B2AR deletion did not modulate systemic glucose and lipid metabolism during obesity, but mice lacking B2ARs in macrophages demonstrated augmented hepatic glucose production on a chow diet. Furthermore, systemic B2AR blockade or macrophage-specific B2AR deletion in mice did not affect the thermogenic response to cold exposure. Chapter 5 includes the characterisation of B2AR stimulation-induced changes to the global cellular proteome of BMDMs, and a subsequent validation of the role of candidate transcription factors in regulating B2AR agonism-induced gene expression in BMDMs.

Neste projeto, demonstramos a modulação da enzima NAD(P)H oxidase pela glicose, palmitato e interleucina - 1? através da análise da expressão protéica do componente p47PHOX e pela atividade dessa enzima via produção de superóxido e peróxido de hidrogênio. Demonstramos também a participação da enzima NAD(P)H oxidase no processo de secreção de insulina induzido pela glicose pois a inibição da enzima pelo DPI e oligonucleotídeo anti p47PHOX promoveu uma diminuição da secreção do hormônio. A partir desse dado passamos a avaliar o mecanismo de ação da enzima no processo secretório e demonstramos que a inibição dessa enzima promove uma inibição de genes essenciais no processo de secreção de insulina como GLUT-2 e glicocinase.Assim podemos concluir que a enzima NAD(P)H oxidase é modulada pela glicose, palmitato e interleucina 1? e que essa enzima participa do processo de secreção insulina modulando genes essenciais para o processo secretório como GLUT-2 e glicocinase. / The expression and activity of the componenents of NAD(P)H oxidase in pancreatic islets were described for the first time in our laboratory (OLIVEIRA, HR et ai, 2003). It was shown the gene and protein expression of the components of this enzyme in Seta cells and that enzyme activation is mediated by glucose. Glucose induced insulin secretion was followed by increase in EROS generation and this increase was in part mediated by NAD(P)H oxidase activation (the same mechanism observed in phagocytes). In this study, the modulation of NAD(P)H oxidase activity by glucose, palmitate and interleukin 1ß as investigated through protein expression of p47phox vity of this enzyme through superoxide and hydrogen peroxide production. To determinate the role of NAD(P)H oxidase in the process of glucoseinduced insulin secretion the enzyme was inhibited by DPI and oligonucleotide anti p47phox, in the both cases the enzyme inhibition produced a decrease on insulin secretion. In order to investigated NAD(P)H oxidase mechanism of action in insulin secretion, we shown that the inhibition enzyme by DPI reduced the GLUT-2 and glucokinase gene expression. We can concluded hat NAD(P)H oxidase was modulated by glucose, palmitate and interleukin 1ß and that enzyme participed in process of glucoseinduced insulin secretion through modulation of GLUT-2 and glucokinase gene expression.

Insulin and glucagon are released in pulses from pancreatic β- and α-cells, respectively. Both cell types are electrically excitable, and elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i) due to depolarization with voltage-dependent entry of the cation is the main trigger of hormone secretion. Store-operated Ca2+ entry  (SOCE) also contributes to the [Ca2+]i elevation and this process has been suggested to be particularly important for glucagon secretion. cAMP is another important messenger that amplifies Ca2+-triggered secretion of both hormones, but little is known about cAMP dynamics in islet cells. In type-2 diabetes, there is deteriorated β-cell function associated with elevated concentrations of fatty acids, but the underlying mechanisms are largely unknown. To clarify the processes that regulate insulin and glucagon secretion, cAMP signalling and the store-operated pathway were investigated in β- and α-cells, primarily within their natural environment in intact mouse and human islets of Langerhans. Fluorescent biosensors and total internal reflection microscopy were used to investigate signalling specifically at the plasma membrane (PM). Adrenaline increased and decreased the sub-PM cAMP concentration ([cAMP]pm) in immuno-identified α-cells and β-cells, respectively, which facilitated cell identification. Glucagon elicited [cAMP]pm oscillations in α- and β-cells, demonstrating both auto- and paracrine effects of the hormone. Whereas glucagon-like peptide 1 (GLP-1) consistently elevated [cAMP]pm in β-cells, only few α-cells responded, indicating that GLP-1 regulates glucagon secretion without changes of α-cell [cAMP]pm. Both α- and β-cells responded to glucose with pronounced oscillations of [cAMP]pm that were partially Ca2+-dependent and synchronized among islet β-cells. The glucose-induced cAMP formation was mediated by plasma membrane-bound adenylyl cyclases. Several phosphodiesterases (PDEs), including the PDE1, -3, -4, and -8 families, were required for shaping the [cAMP]pm signals and pulsatile insulin secretion. Prolonged exposure of islets to the fatty acid palmitate deteriorated glucose-stimulated insulin secretion with loss of pulsatility. This defect was associated with impaired cAMP generation, while [Ca2+]i signalling was essentially unaffected. Stromal interacting molecule 1 (STIM1) is critical for activation of SOCE by sensing the Ca2+ concentration in the endoplasmic reticulum (ER). ER Ca2+ depletion caused STIM1 aggregation, co-clustering with the PM Ca2+ channel protein Orai1 and SOCE activation. Glucose, which inhibits SOCE by filling the ER with Ca2+, reversed the PM association of STIM1. Consistent with a role of the store-operated pathway in glucagon secretion, this effect was maximal at the low glucose concentrations that inhibit glucagon release, whereas considerably higher concentrations were required in β-cells. Adrenaline induced STIM1 translocation to the PM in α-cells and the reverse process in β-cells, partially reflecting the opposite effects of adrenaline on cAMP in the two cell types. However, cAMP-induced STIM1 aggregates did not co-cluster with Orai1 or activate SOCE, indicating that STIM1 translocation can occur independently of Orai1 clustering and SOCE.