Orientador: Hélio Amante Miot / Resumo: A patogênese do melasma, especialmente o papel dos queratinócitos e fibroblastos no desenvolvimento e manutenção da doença não é bem compreendida. Alterações dérmicas como dano estrutural à zona de membrana basal, melanócitos em pêndulo, elastose solar, celularidade, proliferação vascular, além da expressão de mediadores inflamatórios, fatores de crescimento, expressão epitelial de melanocortina e receptores dos hormônios sexuais; sugerem interação entre a unidade epidermo-melânica e a derme na fisiopatologia do melasma. A pigmentação melânica da pele pode ser estimulada por diferentes vias de sinalização, sendo a radiação ultravioleta, citocinas dérmicas e inflamação epidérmica, os modelos mais usuais. Neste estudo, objetivamos comparar a morfologia nuclear e a textura da cromatina entre queratinócitos basais no melasma facial e pele adjacente, investigar a ativação das diferentes vias de estímulo à pigmentação, além do envolvimento da derme, com foco na zona de membrana basal e colágeno, no melasma facial. Para a sua execução, foram coletados pares de biópsias faciais (2mm) de mulheres adultas com melasma e de pele adjacente (<2 cm). Processaram-se para coloração de PAS e picrosirius red; imunofluorescência para p53, p38, IL- 1α, αMSH, MC1R e COX2; imunoistoquímica para Melan-Acontracorada com PAS; Microscopia Eletrônica de Transmissão, além de cultura primária de fibroblastos para real-timePCR array e marcação para SA-β-gal. Foram avaliados: núcleos de quer... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The melasma pathogenesis, specially the role of keratinocytes and fibroblast in the disease development and maintenance are not completely understood. Dermal alterations such as basal membrane structural damage, pendulous melanocytes, solar elastosis, cellularity, vascular proliferation, in addition to the expression of inflammatory mediators, grow factors, epithelial melanocortin and sexual hormones receptors, suggest there is an interaction between the epidermal melanin unit and dermis in melasma physiopathology. The melanin skin pigmentation can be stimulated by different signaling pathways. UVR, dermal cytokines and epidermal inflammation are the most common models. These study aims to compare the nuclear morphology and chromatin texture in basal keratinocytes between melasma and adjacent skin, to investigate the activation of different pigmentation signaling pathways, and the dermal involvement, focusing on basal membrane zone and collagen. Therefore, facial skin biopsies (2 mm) from women were taken from melasma and normal skin (<2 cm apart) and processed for PAS and picrosirius red; immunofluorescence for p53, p38, IL-1α, αMSH, MC1R and COX2, immunohistochemistry for Melan-A counterstaining with PAS, and transmission electronic microscopy. Furthermore, primary fibroblast culture for real-timePCRarray and SA-β-gal staining. The nuclei of basal keratinocytes were evaluated as nuclear morphometry and chromatin texture; the fluorescence intensity was quantified in the epid... (Complete abstract click electronic access below) / Doutor

Submitted by GABRIELLI BRIANEZI null (gabrielli.brianezi@hotmail.com) on 2016-02-26T15:36:13Z No. of bitstreams: 1 20160224 Tese Gabrielli Brianezi.pdf: 3073309 bytes, checksum: 8fd0793265926aa1a58aea12aad689a6 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-02-26T20:04:11Z (GMT) No. of bitstreams: 1 brianezi_g_dr_bot.pdf: 3073309 bytes, checksum: 8fd0793265926aa1a58aea12aad689a6 (MD5) / Made available in DSpace on 2016-02-26T20:04:11Z (GMT). No. of bitstreams: 1 brianezi_g_dr_bot.pdf: 3073309 bytes, checksum: 8fd0793265926aa1a58aea12aad689a6 (MD5) Previous issue date: 2016-01-26 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A patogênese do melasma, especialmente o papel dos queratinócitos e fibroblastos no desenvolvimento e manutenção da doença não é bem compreendida. Alterações dérmicas como dano estrutural à zona de membrana basal, melanócitos em pêndulo, elastose solar, celularidade, proliferação vascular, além da expressão de mediadores inflamatórios, fatores de crescimento, expressão epitelial de melanocortina e receptores dos hormônios sexuais; sugerem interação entre a unidade epidermo-melânica e a derme na fisiopatologia do melasma. A pigmentação melânica da pele pode ser estimulada por diferentes vias de sinalização, sendo a radiação ultravioleta, citocinas dérmicas e inflamação epidérmica, os modelos mais usuais. Neste estudo, objetivamos comparar a morfologia nuclear e a textura da cromatina entre queratinócitos basais no melasma facial e pele adjacente, investigar a ativação das diferentes vias de estímulo à pigmentação, além do envolvimento da derme, com foco na zona de membrana basal e colágeno, no melasma facial. Para a sua execução, foram coletados pares de biópsias faciais (2mm) de mulheres adultas com melasma e de pele adjacente (<2 cm). Processaram-se para coloração de PAS e picrosirius red; imunofluorescência para p53, p38, IL- 1α, αMSH, MC1R e COX2; imunoistoquímica para Melan-Acontracorada com PAS; Microscopia Eletrônica de Transmissão, além de cultura primária de fibroblastos para real-timePCR array e marcação para SA-β-gal. Foram avaliados: núcleos de queratinócitos da camada basal quanto à morfometria nuclear e textura da cromatina; quantificada a intensidade de fluorescência nos compartimentos da epiderme e derme; avaliadas organelas citoplasmáticas e zona de membrana basal; além do colágeno dérmico e densidade de melanócitos: totais e em pêndulo. Em relação à pele adjacente sem melasma: núcleos de queratinócitos basais da epiderme com melasma facial apresentaram índices morfométricos e textura da cromatina alterados; houve aumento da expressão epitelial de αMSH e MC1R, sem diferença quanto ao p53, p38, IL1α e COX2; houve maior número de organelas citoplasmáticas e melanossomas em estágios de maturação maiores nos queratinócitos e melanócitos; áreas danificadas na zona de membrana basal com presença de microvesículas adjacentes à membrana basal; maior número de melanócitos em pêndulo; colágeno dérmico desestruturado. Entre os fibroblastos, houve maior marcação para SA-β-gal e expressão alterada dos genes COL4A1, IL6, ESR2, DKK3, CCL2, WIF1, WNT3A, HGF, IL1B, MMPs 1-7-9 no melasma. As alterações encontradas nos queratinócitos, na derme e zona de membrana basal, sugerem que o fenótipo do melasma pode resultar de alterações em toda unidade epidermo-melânica, não somente de uma hipertrofia dos melanócitos, as vias inflamatórias da epiderme e dependentes de p53 não se mostraram proeminentes, além de ser identificado um possível papel do processo de reparo/dano da derme e da senescência de fibroblastos na patogênese da doença. / The melasma pathogenesis, specially the role of keratinocytes and fibroblast in the disease development and maintenance are not completely understood. Dermal alterations such as basal membrane structural damage, pendulous melanocytes, solar elastosis, cellularity, vascular proliferation, in addition to the expression of inflammatory mediators, grow factors, epithelial melanocortin and sexual hormones receptors, suggest there is an interaction between the epidermal melanin unit and dermis in melasma physiopathology. The melanin skin pigmentation can be stimulated by different signaling pathways. UVR, dermal cytokines and epidermal inflammation are the most common models. These study aims to compare the nuclear morphology and chromatin texture in basal keratinocytes between melasma and adjacent skin, to investigate the activation of different pigmentation signaling pathways, and the dermal involvement, focusing on basal membrane zone and collagen. Therefore, facial skin biopsies (2 mm) from women were taken from melasma and normal skin (<2 cm apart) and processed for PAS and picrosirius red; immunofluorescence for p53, p38, IL-1α, αMSH, MC1R and COX2, immunohistochemistry for Melan-A counterstaining with PAS, and transmission electronic microscopy. Furthermore, primary fibroblast culture for real-timePCRarray and SA-β-gal staining. The nuclei of basal keratinocytes were evaluated as nuclear morphometry and chromatin texture; the fluorescence intensity was quantified in the epidermis and dermis; cytoplasm organelles and basal membrane zone were evaluated; in addition to dermal collagen and melanocytes density: total and pendulous. Regarding adjacent healthy skin, the melasma skin showed alterations in morphometric index and chromatin texture; there was greater epithelial expression of αMSH and MC1R, but without difference for p53, p38, IL1α and COX2; cytoplasm organelles and melanossomas in higher maturity were in greater number in keratinocytes and melanocytes; there were commoner damaged areas in basal membrane zone, presence of microvesicules adjacent to basal membrane; and higher number of pendulous melanocyte; dermal collagen was less structured. There were greater SA-β-gal staining and altered expression of COL4A1, IL6, ESR2, DKK3, CCL2, WIF1, WNT3A, HGF, IL1B, MMPs 1-7-9 genes in melasma fibroblasts.The alterations presented in keratinocytes, dermis and basal membrane zone suggest the melasma phenotype can result from alteration in entire epidermal melanin unit, not just hypertrophic melanocytes, the epidermal inflammatory pathways and p53 dependents do not show prominence, in addition to the possible role of the repair/ damage process identified at dermis and fibroblast senescence in the melasma pathogenesis. / FAPESP: 12/09233-5 / FAPESP: 12/05004-1

Background: Muscular strengthening activity (MSA) has been shown to be inversely associated with insulin resistance (IR). The associations between quartiles of the homeostatic model assessment of insulin resistance (HOMA-IR) and self-reported MSA in a nationally representative sample of euglycemic U.S. adults were examined. Methods: Sample included adult participants (≥20 years of age [n=2,543]) from the 1999-2004 National Health and Nutrition Examination Survey (NHANES). HOMA-IR was categorized into quartiles based on every 25th percentile. No MSA was the dependent variable. Results: Following adjustment for covariates, those with HOMA-IR values in third (p Conclusions: Having a higher HOMA-IR value is associated with greater odds of reporting no MSA in euglycemic U.S. adults.

ABSTRACT Purpose: The purpose of this study was to examine the relationship between self-reported sedentary time (ST) and the cumulative risk of preserved ejection fraction heart failure (HFpEF) and reduced ejection fraction heart failure (HFrEF) using a diverse cohort of U.S. adults 45-84 years of age. Methods: Using data from the Multi-Ethnic Study of Atherosclerosis (MESA), we identified 6,814 subjects (52.9% female). All were free of baseline cardiovascular disease. Cox regression was used to calculate the hazard ratios (HR) associated with baseline ST and risk of overall heart failure (HF), HFpEF, and HFrEF. Weekly self-reported ST was dichotomized based on the 75th percentile (1,890 min/wk). Results: During an average of 11.2 years of follow-up there were 178 first incident HF diagnoses; 74 HFpEF, 69 HFrEF and 35 with unknown EF. Baseline ST >1,890 min/wk was significantly associated with an increased risk of HFpEF (HR [95% CI]; 1.87 [1.13 – 3.09], p= 0.01), but not HFrEF (HR [95% CI]; 1.30 [0.78 – 2.15], p= 0.32). The relationship with HFpEF remained significant in separate fully adjusted models including either waist circumference (HR [95% CI]; 2.16 [1.23 – 3.78], p < 0.01) or body mass index (HR [95% CI]; 2.17 [1.24 – 3.80], p < 0.01). Additionally, every 60 minute increase in weekly ST was associated with a significant 3% increased risk of HFpEF (HR [95% CI]; 1.03 [1.01 – 1.05], p < 0.01). Conclusions: Sedentary time > 1,890 min/wk (~4.5 h/d) is a significant independent predictor of HFpEF, but not HFrEF.

Objectives: We sought to examine the gender differences between C - reactive protein (CRP) and muscle strengthening activity (MSA) in U.S. adults (≥20 years of age) Background: Elevated levels of CRP have been shown to be associated with an increase in risk of cardiovascular disease (CVD). Studies analyzing the relationship between physical activity (PA) and CRP by gender have produced mixed results. Methods: The sample (n=9,135) included participants in the 1999-2004 National Health and Nutrition Examination Survey (NHANES). Three categories of reported MSA participation were created: no MSA (referent group), some MSA (≥1 to/wk), and meeting the 2008 Department of Health and Human Services (DHHS) recommendation (>2 d/wk). The dependent variable was elevated CRP (>3 to 10 mg/L). Results: Gender stratified analysis revealed significantly lower odds of having elevated CRP for women reporting some MSA (OR 0.61; 95% CI 0.45-0.83, P=0.0023), or volumes of MSA meeting the DHHS recommendation (OR 0.66; 95% CI 0.54-0.82, P=0.0004). Significantly lower odds of men having elevated CRP was observed in those reporting MSA volumes meeting the recommendation (OR 0.73; 95% CI 0.61-0.88, P=0.0011). Following adjustment for waist circumference (WC) these odds remained significant in men but not women. Conclusions: Women reporting any MSA were found to have lower odds of having elevated CRP when compared to those reporting no MSA prior to adjustment for WC. Significantly lower odds in men were only observed in those meeting the recommendation. These results suggest that WC may mediate the associations between MSA and CRP and this relationship may be stronger in women.

Indiana University-Purdue University Indianapolis (IUPUI) / Immunotherapy for cancer has held much promise as a potent modality of cancer treatment. The ability to selectively destroy diseased cells and leave healthy cells unharmed has been the goal of cancer immunotherapy for the past thirty years. However, the full capabilities of cancer immunotherapies have been elusive. Cancer immunotherapies have been consistently hampered by limited immune reactivity, a diminishing immune response over time, and a failure to overcome self-tolerance. Many of these deficiencies have been borne-out by immunotherapies that have focused on the adoptive transfer of activated or genetically modified mature CD8+ T cells. The limitations inherent in therapies involving terminally differentiated mature lymphocytes include limited duration, lack of involvement of other components of the immune system, and limited clinical efficacy. We sought to overcome these limitations by altering and enhancing long-term host immunity by genetically modifying then transplanting HSCs. To study these questions and test the efficiency of gene transfer, we cloned a tumor reactive HLA-DR4-restricted CD4+ TCR specific for the melanocyte differentiation antigen TRP-1, then constructed both a high expression lentiviral delivery system and a TCR Tg expressing the same TCR genes. We demonstrate with both mouse and human HSCs durable, high-efficiency TCR gene transfer, following long-term transplantation. We demonstrate the induction of spontaneous autoimmune vitiligo and a TCR-specific TH1 polarized memory effector CD4+ T cell population. Most importantly, we demonstrate the destruction of subcutaneous melanoma without the aid of vaccination, immune modulation, or cytokine administration. Overall, these results demonstrate the creation of a novel translational model of durable lentiviral gene transfer, the induction of spontaneous CD4+ T cell immunity, the breaking of self-tolerance, and the induction of anti-tumor immunity.