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Agradecimentos: The equipment used for the development of this work was purchased with the help of Fapesp (the São Paulo Research Foundation; 08/57906-3) and CNPq (the National Council for Scientific and Technological Development; 573913/2008-0). We reviewed the content of the manuscript, followed by Ms Diane Ellis, B.A. in education. Biostatistician Cleide Aparecida Moreira Silva, Research Committee, Biostatistics Division, Medical Sciences School, Unicamp, provided statistical consultation Abstract: Solid tumors typically contain high levels of fibrillar collagen. The increased stromal collagen deposition usually promotes cancer progression since biochemical and biophysical cues from tumor-associated collagen fibers stimulate neoplastic cells. Few studies have investigated the relationship between Merkel cell carcinoma (MCC) and the extracellular matrix (ECM), but there are no works evaluating collagen. This is an observational, analytical, retrospective study including 11 patients with MCC. Primary tumor-stained sections were evaluated by second harmonic generation microscopy and texture analysis. Peritumoral texture features (area fraction, mean gray value, entropy, and contrast) showed much lower values than normal skin (P < .0001) revealing extensively altered structure of peritumoral collagen fibers. These differences were not significant between tumors with unfavorable and favorable known prognostic factors. Profound changes in collagen fibers present in the stroma accompanying primary MCC may contribute to the aggressive behavior of this tumor. Our results indicate that whatever MCC histological subtype, size or anatomical location, MCC promotes the same type of ECM for its development. As an outlook, therapies using ECM macromolecules or fibroblasts (the architects of ECM remodeling) as target could be useful in the treatment of MCC FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ Aberto

Orientadores: José Vassallo, Carmen Silvia Passos Lima Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: O câncer de mama é a neoplasia não-cutânea mais frequente em mulheres acima dos 50 anos de idade. Apesar dos esforços em se diagnosticar a doença precocemente, ainda há atraso considerável no diagnóstico, causando prejuízo as pacientes. Os marcadores prognósticos podem auxiliar na estratificação de risco para eventos danosos e clarear a fisiopatologia da doença. Em paralelo, sabe-se que o colágeno possui importante papel na carcinogênese e progressão da neoplasia, porém seu valor prognóstico continua subestimado. Notavelmente, a microscopia não-linear, mais especificamente, a microscopia por geração de segundo-harmônico (SHG), permite a identificação e a avaliação quantitativa não-destrutiva do colágeno depositado nos tecidos. Nesse sentido, os objetivos desse trabalho foram avaliar os parâmetros do colágeno baseado nas imagens geradas a partir da microscopia por SHG para: (1) descrever as imagens nos diversos subtipos histológicos de câncer de mama e (2) avaliar seu papel prognóstico. Para o primeiro objetivo, as imagens produzidas a partir da SHG dos cortes histológicos de tecidos fixados e parafinados foram avaliados de acordo com sua classificação: subtipo histológico especial de mama (n= 99) e carcinoma ductal invasivo de mama (n= 21). O microarray tecidual dessas amostras foi realizado para categorizar o subtipo molecular, baseado na avaliação imunoistoquímica. Para o segundo objetivo, propusemos a mesma avaliação, partindo, entretanto, do estroma tumoral selecionado de 60 pacientes com diagnóstico de carcinoma ductal invasivo de mama. Para a avaliação das imagens utilizamos o software ImageJ, em associação com o plug-in OrientationJ. As análises estatísticas foram elaboradas no software R, sendo significativos valores de p< 0.05. Nossos resultados permitiram agrupar os diversos subtipos histológicos de câncer de mama em 3 grandes grupos baseados nos parâmetros do colágeno obtidos das imagens. Ademais, identificamos que a maior deposição de colágeno, bem como a deposição de fibras mais espessas são fatores associados a pior prognóstico. Em conclusão, nossos dados permitem inferir que pode haver padrões estereotipados de deposição de colágeno dentre os diversos subtipos histológicos de câncer de mama e este pode refletir no prognóstico da paciente Abstract: Breast cancer (BC) is the most common non-cutaneous neoplasm in women over 50 years of age. Despite efforts for early diagnosis, there is still considerable delay, causing poor prognosis to patients. Prognostic markers can aid in the stratification of risk for harmful events and clarify the pathophysiology of the disease. In parallel, it is known that collagen plays an important role in carcinogenesis and in tumor progression, but its prognostic value remains undervalued. Notably, nonlinear microscopy, more specifically, second-harmonic generation microscopy (SHG), allows the identification and non-destructive quantitative evaluation of the collagen deposited in tissues. In this sense, the objectives of the present study were to evaluate collagen parameters based on images generated by SHG to: (1) describe the images in the various histological subtypes of BC and (2) asses the prognostic value of collagen features. For the first objective, images obtained from the SHG of histological sections of fixed and paraffin-embedded tissues were evaluated according to their classification: special histological subtype of BC (n = 99) and invasive ductal carcinoma of the breast (n= 21). Tissue microarray of these samples was performed to categorize the molecular subtype, based on immunohistochemical evaluation. For the second objective, we proposed the same evaluation, starting, however, from selected stromal tumor areas of 60 patients with the diagnosis of invasive ductal carcinoma of the breast. For image evaluation we used the ImageJ software in association with the OrientationJ plug-in. Statistical analyzes were performed in R software, with significance levels at p< 0.05. Our results allowed grouping the various histological subtypes of breast cancer in three large groups based on collagen parameters obtained from the images. In addition, we have identified that the greater deposition of collagen, as well as the deposition of thicker fibers, were associated to unfavorable prognosis. In conclusion, our data support the existence of stereotyped patterns of collagen deposition across the various histological subtypes of breast cancer. Such patterns might be related to patients¿ outcomes Doutorado Clínica Médica Doutor em Ciências CNPQ 573913/2008-0, 312049/2014-5 FAPESP 08/57906-3, 11/51959-0

Agradecimentos: Contract grant sponsor: FAPESP; Contract grant number: 2009/16150-6 (to H.F.C.); Contract grant sponsor: FAPESP; Contract grant number: 2011/51591-3; Contract grant sponsors: CEPOF (Optics and Photonics Research Center, FAPESP), INFABIC (National Institute of Photonics Applied to Cell Biology, FAPESP and CNPq), PICTO UNER-INTA-CAFEGS; Contract grant number: 2009-209; Contract grant sponsor: PIO CONICET-UNER Res: 4337/15; Contract grant number: No 14620140100004 CO) Abstract: A plethora of optical techniques is currently available to obtain non-destructive, contactless, real time information with subcellular spatial resolution to observe cell processes. Each technique has its own unique features for imaging and for obtaining certain biological information. However none of the available techniques can be of universal use. For a comprehensive investigation of biological specimens and events, one needs to use a combination of bioimaging methods, often at the same time. Some modern confocal/multiphoton microscopes provide simultaneous fluorescence, fluorescence lifetime imaging, and four-dimensional imaging. Some of them can also easily be adapted for harmonic generation imaging, and to permit cell manipulation technique. In this work we present a multimodal optical workstation that extends a commercially available confocal microscope to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools. The nonlinear microscopy capabilities were added to the commercial confocal microscope by exploiting all the flexibility offered by the manufacturer. The various capabilities of this workstation as applied directly to reproductive biology are discussed FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ Fechado

Agradecimentos: This study was supported by the Fundação do Amparo à Pesquisa do Estado de São Paulo - FAPESP (www.fapesp.br). FAPESP Process No 2010/12565-4. FAPESP-INCT-INFABIC Process No 2008/57906-3. The funding institution had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript Abstract: Nonlinear optical (NLO) microscopy techniques have potential to improve the early detection of epithelial ovarian cancer. In this study we showed that multimodal NLO microscopies, including two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG), third-harmonic generation (THG) and fluorescence lifetime imaging microscopy (FLIM) can detect morphological and metabolic changes associated with ovarian cancer progression. We obtained strong TPEF + SHG + THG signals from fixed samples stained with Hematoxylin & Eosin (H&E) and robust FLIM signal from fixed unstained samples. Particularly, we imaged 34 ovarian biopsies from different patients (median age, 49 years) including 5 normal ovarian tissue, 18 serous tumors and 11 mucinous tumors with the multimodal NLO platform developed in our laboratory. We have been able to distinguish adenomas, borderline, and adenocarcinomas specimens. Using a complete set of scoring methods we found significant differences in the content, distribution and organization of collagen fibrils in the stroma as well as in the morphology and fluorescence lifetime from epithelial ovarian cells. NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for serous and mucinous ovarian tumors. The results provide a basis to interpret future NLO images of ovarian tissue and lay the foundation for future in vivo optical evaluation of premature ovarian lesions FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP Aberto

Agradecimentos: The authors are grateful to J.R. Castro for technical assistance and to the funding support of CNPq 301.282/2009-9 Abstract: Apolar fibers wired into a mesh-like microstructure forming a coating with a contact angle larger than 160 degrees and fabricated by polycyanoacrylate polymerization are described. Interconnected fibers with diameters measuring approximately 5 mu m are formed by texturized linear or folded nanowires. The structure forming the deposited film occupies similar to 1.5% of the coating's top geometric area. This value agrees with the water/coating contact area given by the Cassie-Baxter contact-angle model (similar to 1.5%). The spatial distribution of the surface in contact with water was determined by Raman spectral imaging (similar to 1.5%) using the polycyanoacrylate lines and by scanning electron microscopy (similar to 2.0%) CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ Fechado

Agradecimentos: The authors are grateful to FAPESP. This work is also linked with the CEPOF (Optics and Photonics Research Center, FAPESP) and National Institute of Photonics Applied to Cell Biology (INFABIC) Abstract: We show that combined multimodal nonlinear optical (NLO) microscopies, including two-photon excitation fluorescence, second-harmonic generation (SHG), third harmonic generation, and fluorescence lifetime imaging microscopy (FLIM) can be used to detect morphological and metabolic changes associated with stroma and epithelial transformation during the progression of cancer and osteogenesis imperfecta (01) disease. NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for different types of human breast cancer, mucinous ovarian tumors, and skin dermis of patients with OI. Using a set of scoring methods (anisotropy, correlation, uniformity, entropy, and lifetime components), we found significant differences in the content, distribution and organization of collagen fibrils in the stroma of breast and ovary as well as in the dermis of skin. We suggest that our results provide a framework for using NLO techniques as a clinical diagnostic tool for human cancer and OI. We further suggest that the SHG and FLIM metrics described could be applied to other connective or epithelial tissue disorders that are characterized by abnormal cells proliferation and collagen assembly FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP Aberto