Proteomic techniques were used to analyze the protein expression profile of the early-stage differentiation of pig embryonic stem cells (ES cells). The pig ES cells were induced to develop to neuronal cells by all-trans retinoic acid (ATRA) in vitro by Tainan Livestock Research Institute. The ES cells were cultured with ATRA and collected at time intervals of 0, 1, 2, 4, 8 and 10 days. The cell lysates were analyzed by two-dimensional electrophoresis, and the differentially expressed proteins are identified by MALDI-TOF. Our data shows that the expression profile of pig ES cells is similar to other mammalian models but with some differences. Preliminary pig ES cells 2D database was set up. Six spots each with up or down-regulation in neurogenesis were identified by MS. These proteins may become the good markers of pig ES cells into neural cells by RA. Among those proteins, vimentin, prohibitin and annexin A10 were up-regulated, zinc finger protein 482 (ZNF482), fyn-related kinase (FRK) and annexin A1 were down-regulated during differentiation of pig ES cells to neural cells. Addtionally, we ultilized RT-PCR technique to investigate mRNA expression during neurogenesis, vimentin and prohibitin was up-regulated, anxa1(annexin A1) was slightly down-regulated, neuroD1 and neurogenin 2 were high expression on day 10, beta-catenin was high expression on day 8 to 10.

Introdução: Os marcadores moleculares têm sido utilizados para identificar subgrupos de tumores com comportamento clínico distinto, inclusive quanto ao padrão de recidiva. Objetivo: Caracterizar a expressão imunoistoquímica de vimentina (VIM) e E-caderina (CDH1) em carcinomas ductais invasivos (CDI) de mama e sua associação com a expressão de citoqueratina 5 (CK5), e características clínico-patológicas. Métodos: Microarranjos teciduais (TMA) foram construídos a partir de 82 amostras de CDI de mama. Imunoistoquímica (IHQ) foi realizada para determinar os receptores hormonais (RH; receptores de estrógeno e progesterona) e receptor do fator de crescimento epidérmico humano 2 (HER2), VIM, CDH1, CK5 e Ki-67. Os tumores foram classificados como luminal A (RH+, HER2-), luminal B (RH+, HER2+ ou Ki-67 alto), HER2 hiperexpresso (RH-, HER2+) e triplo negativo (TN; RH-, HER2-). Resultados: O fenótipo VIM+/CDH1-/low não foi observado nos tumores luminal A, B e HER2 hiperexpresso, enquanto que este fenótipo estava presente em 61,9% dos TN (p= 0,0001). A mediana de Ki-67 em tumores VIM+/CDH1-/low foi 13,6 (variação, 17,8-45,4), em comparação com 9,8 (variação de 4,1-38,1) nos não- VIM+/CDH1-/low (p= 0,0007). O acometimento linfonodal foi menos freqüente em pacientes com VIM+/CDH1-/low do que nos não- VIM+/CDH1-/low (23% X 61%, teste de 2, p = 0,01). A sobrevida livre de doença em 5 anos foi de 61,5% e 83,7% (teste de log-rank, p= 0,02) e a sobrevida global em 5 anos foi de 51,2% e 83,5% (teste de log-rank, p= 0,03) em pacientes com tumores com fenótipo VIM+/CDH1-/low e não VIM+/CDH1-/low, respectivamente. Conclusão: A expressão de VIM e CDH1 identificaram um subconjunto de CDI de mama com fenótipo mesenquimal com alto grau histológico e alto índice mitótico. / Introduction: Molecular markers have been used to identify subgroups of tumors with distinct clinical behavior, including recurrence pattern. Objective: To characterize the immunohistochemical expression of vimentin (VIM) and E-cadherin (CDH1) in invasive ductal carcinoma (IDC) of the breast and its association with cytokeratin 5 (CK5) expression and clinicopathological features. Methods: A tissue microarray was constructed from 82 IDC breast cancer specimens. Immunohistochemistry (IHC) was used to determine hormone receptor (HR; estrogen and progesterone receptors) status and human epidermal growth factor receptor 2 (HER2), VIM, CDH1, CK5, and Ki-67 expression. Tumors were classified as luminal A (HR+, HER2-), luminal B (HR+, HER2+ or high Ki-67), HER2 enriched (HR-, HER2+), and triple negative (TNBC; HR-, HER2-). Results: The VIM+/CDH1-/low phenotype was not observed in luminal A, luminal B and HER2 enriched tumors, whereas this phenotype was present in 61.9% of triple negative (TNBC) tumors (p = 0.0001). The median Ki-67 index in VIM+/CDH1-/low tumors was 13.6 (range, 17.8-45.4) compared with 9.8 (range, 4.1-38.1) in non-VIM+/CDH1-/low tumors (p= 0.0007). The presence of lymph node metastasis was less frequent in patients with VIM+/CDH1-/low tumors than in those with non-VIM+/CDH1-/low tumors (23% vs. 61%; 2 test, p= 0.01). The 5-years disease-free survival was 61.5% and 83.7% (log-rank test; p= 0.02) and the 5-year overall survival was 51.2% and 83.5% (log-rank test; p= 0.03) in patients with VIM+/CDH1-/low phenotype and non-VIM+/CDH1-/low phenotype tumors, respectively. Conclusion: The expression of VIM and CDH1 identified a subset of IDC breast cancer of the mesenchymal phenotype with high histological grade and high mitotic index.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-10-03T15:00:55Z No. of bitstreams: 1 fernandaoliveiradepaula.pdf: 152498 bytes, checksum: 3eda55fca7b7172518cf488b112dbc87 (MD5) / Approved for entry into archive by Diamantino Mayra (mayra.diamantino@ufjf.edu.br) on 2016-10-03T15:17:08Z (GMT) No. of bitstreams: 1 fernandaoliveiradepaula.pdf: 152498 bytes, checksum: 3eda55fca7b7172518cf488b112dbc87 (MD5) / Made available in DSpace on 2016-10-03T15:17:08Z (GMT). No. of bitstreams: 1 fernandaoliveiradepaula.pdf: 152498 bytes, checksum: 3eda55fca7b7172518cf488b112dbc87 (MD5) Previous issue date: 2010-08-13 / O objetivo deste estudo foi avaliar, pelo método de imunoistoquímica, a expressão de vimentina e osteopontina durante as fases iniciais de reparo de defeitos ósseos criados em fêmures de ratos Wistar albinus tratados com enxerto ósseo bovino orgânico Gen-ox® em associação com terapia laser de baixa intensidade. Foram selecionadas de forma aleatória 24 amostras emblocadas de arquivo provenientes de trabalho experimental desenvolvido anteriormente, no qual foram efetuadas análises histológicas e histomorfométricas de fêmures de cinquenta ratos. Obtiveram-se lâminas histológicas dos blocos de diferentes animais, os quais estavam previamente divididos, de acordo com o tratamento realizado, da seguinte forma: grupo I (controle), grupo II (Gen-Ox®) e grupo III (LLLT e Gen-Ox®). Foram elaboradas quatro lâminas para cada um dos tempos experimentais de 3, 5, 7 e 15 dias para cada grupo. Duas lâminas foram utilizadas para analisar a expressão da osteopontina e duas para vimentina, totalizando 48 lâminas. Em cada lâmina considerou-se dois campos para análise, sendo um campo na região da interface osso-defeito e o outro próximo ao periósteo, em um total de 96 campos. Para realização da reação imunoistoquímica anti-vimentina e anti-osteopontina utilizou-se o método clássico do complexo avidina-biotina peroxidase anti-peroxidase. A marcação positiva foi determinada pela identificação de coloração castanha intracitoplasmática nas reações com ambos os anticorpos. Os cortes foram analisados em microscópio Zeiss em aumentos de 200x, 400x e 1000x, em toda extensão, por dois diferentes observadores. Determinou-se, por método de contagem semiquantitativo, a média de intensidade de células positivas marcadas nos campos dos tratamentos propostos em cada período, o qual foi classificado pelo sistema de escore de acordo com os seguintes parâmetros: 0 = ausência de marcação; + = marcação leve (até 1/3 de células positivas); ++ = marcação moderada (até 2/3 de células positivas); e +++ = marcação intensa (acima de 2/3 de células marcadas). Os resultados mostraram que todos os grupos apresentaram marcação para vimentina e para osteopontina em todos os períodos do experimento. Observou-se marcação celular mais acentuada para vimentina no período cicatricial inicial no grupo III. Não foram verificadas diferenças na marcação para osteopontina nos animais submetidos à terapia laser de baixa intensidade associada ao enxerto quando comparado aos outros grupos. / The aim of this study was to evaluate, by immunohistochemistry, the expression of vimentin and osteopontin during the early stages of repair of bone defects created in femurs of Wistar albinus rats treated with organic bovine bone graft Gen-ox® and associated with low level laser therapy. Twenty four embedded samples were randomly selected from the file of a previous experimental work, in which histological analysis and histomorphometry of the femurs of fifty rats was performed. Histological slides were obtained from blocks of different animals which were divided in accordance to previous treatment as follow: group I (control), group II (Gen-Ox ® ) and group III (LLLT and Gen-Ox ®). Four slides were prepared for each of the experimental time of 3, 5, 7 and 15 days for each group. Of the four slides, two were assessed for the expression of osteopontin and two of vimentin, in a total of 48 slides. On each slide two fields were considered for analysis: one in the bone-defect interface and the other near the periosteum, in a total of 96 fields. To perform the immunohistochemistry anti-vimentin and anti-osteopontin, we used the classical avidin-biotin peroxidase anti-peroxidase method. The positive staining was determined by identification of intracytoplasmic brown color in the reactions with both antibodies. The sections were analyzed in Zeiss microscope at a magnification of 200x, 400x and 1000x by two different observers. The average intensity of positive cells stained in the fields in each period was determined by a semiquantitative counting method which was classified by a scoring system as follow: 0 = no marking; + = mild labeling (up to one third of positive cells); + + = moderate labeling (up to two thirds of positive cells) and + + + = intense labeling (over two thirds of labeled cells). The results showed that all groups had marked for vimentin and osteopontin in all periods of the experiment. We observed a stronger cell labeling for vimentin in the initial healing period in group III. There were no differences in cell labeling for osteopontin in animals subjected to low level laser therapy associated with graft as compared to other groups.

Chlamydia is the most reported sexually transmitted infection in the US and is caused by the obligate intracellular bacterium Chlamydia trachomatis. Typically, this presents as a lower genital tract infection (cervicitis or urethritis), but can ascend to the upper genital tract, causing pelvic inflammatory disease, tubal infertility, epididymitis, or ectopic pregnancy. While chlamydia infections can be cured with a single-dose oral antibiotic, repeat infections are common and having multiple chlamydial infections increases a woman’s risk of developing serious chronic conditions. Previous research has shown that estrogen has a positive effect on C. trachomatis infections—an important finding, connecting fluctuating estrogen levels in females to variance in pathogenesis.The mechanism behind this hormonal influence remains unknown; however, previous work in our laboratory indicates that estrogen-stimulated stromal cell effectors play a role in enhancing C. trachomatis infections in a polarized endometrial epithelial Ishikawa (IK)/stromal (SHT-290) cell co-culture model. Specifically, our data indicate that estrogen exposure stimulates osteopontin and vimentin release from stromal cells in co-culture with endometrial epithelial cells. Furthermore, we noted that Chlamydia-infected, polarized Ishikawa cells exposed to a combination of recombinant osteopontin and estrogen released significantly more infectious chlamydia than cultures exposed to estrogen alone. Most tissue culture models being used today employee non-polarized cells. Given the fact that epithelial cell polarization is known to impact C. trachomatis serovar E development, in the current study we sought to determine if the estrogen-induced stromal cell effectors, osteopontin and vimentin, affect C. trachomatis viability and infectivity in non-polarized Ishikawa cells. Non-polarized Ishikawa cells were exposed to osteopontin or vimentin in the presence or absence of estrogen, infected with C. trachomatis serovar E, and collected for examination of chlamydial infectivity and progeny production. Our initial data show that osteopontin and vimentin impact chlamydial progeny production in a concentration dependent fashion, with higher concentrations of recombinant effectors +/- estrogen significantly decreasing progeny production. These data suggest that polarization of host cells influences the way hormone-stimulated effectors interact with the cell to impact on chlamydial infection. Future research goals are to explore other stromal effectors such as fibronectin with estrogen and to study the cell signaling mechanism osteopontin and vimentin use to affect chlamydial infections in polarized epithelial cell cultures.

Huntington's disease (HD) is an inherited autosomal dominant neurodegenerative disorder caused by an unstable expansion of the CAG repeat sequence within the huntingtin gene. Huntingtin associates with ubiquitin-proteasome system that ensures degradation of particular proteins including β-catenin which is an important molecule whose equilibrated degradation is necessary for the proper functioning of the Wnt signaling pathway. The binding of β-catenin to the destruction complex is altered in HD, leading to the toxic stabilization of β-catenin. The main goal of my thesis was to determine whether the accumulation of β-catenin due to the presence of mutant huntingtin is also characteristic of Liběchov minipigs, a large animal model of Huntington's disease stably expressing N-truncated human mutant huntingtin. Using immunoblot and specific antibodies, we have revealed age-dependent accumulation of mutant huntingtin in transgenic minipigs. Unlike endogenous huntingtin, no decrease of the level of mutant huntingtin was observed in the striatum of transgenic animals. Surprisingly, this was followed by a decrease of phosphorylated β-catenin. Nevertheless, our results demostrate the accumulation of β-catenin in mesenchymal stem cells isolated from the oldest boars during ontogenesis. Furthermore, we have revealed a...

Modificações nas histonas são conhecidas por regular a estrutura conformacional da cromatina e a expressão gênica em células adultas e células-tronco pluripotentes. Tem sido postulado que a acetilação e deacetilação das histonas podem influenciar a expressão de genes envolvidos na iniciação, progressão e metástase tumoral, além de contribuir para o desenvolvimento de resistência à quimioterapia. Assim, buscou-se avaliar a influência das modificações nas histonas sobre a biologia do carcinoma epidermoide de cabeça e pescoço (CECP) e sua respectiva subpopulação de células semelhantes às células-tronco (CSC). Inicialmente, foi checado os níveis de acetilação da histona H3 (membro das histonas nucleares associado à compactação da cromatina) em um painel representativo de linhagens celulares de CECP. Posteriormente, para estudar a influência do estroma tumoral no padrão de acetilação da histona H3, o microambiente do tumor foi mimetizado através da utilização de meio condicionado derivado do cultivo de fibroblastos e cultura primária de células endoteliais humanas. Além disso, validamos esses resultados in vitro por meio de amostras humanas de CECP. Finalmente, a acetilação e deacetilação da cromatina foi induzida, respectivamente, pela administração dos inibidores das enzimas histona deacetilase tricostatina A (TSA) e histona acetiltransferase curcumina, em linhagens celulares de CECP. Foi feita a análise da formação de esferas (ensaio funcional de células-tronco), juntamente com a verificação dos níveis de ALDH, marcador de células-tronco (citometria de fluxo - FACS), além da determinação do índice de proliferação tumoral (Ki-67) e realização dos ensaios de invasão e migração celular. Linhagens celulares de CECP apresentaram níveis baixos de acetilação da histona H3 e demonstraram capacidade de retenção de uma subpopulação de CSC. Apenas o meio condicionado de células endoteliais humanas foi capaz de alterar a conformação da cromatina, uma vez que induziu o aumento da acetilação da histona H3. Interessantemente, foi também notado um concomitante aumento da agressividade de linhagens celulares de CECP (aumento dos níveis de BMI-1 e vimentina). Esses resultados foram confirmados em amostras humanas de CECP que mostraram, apenas no fronte de invasão, células com cromatina acetilada. Curiosamente, essas mesmas células também expressaram vimentina. Os tratamentos com TSA e curcumina resultaram na diminuição significativa da subpopulação de CSC, interrompendo a formação de esferas e reduzindo os níveis de ALDH. Além disso, o tratamento com curcumina mostrou resultados muito interessantes, uma vez que gerou uma redução evidente da invasão celular e impactou por completo o potencial de migração tumoral, sendo nesse sentido mais eficiente que a cisplatina, droga antineoplásica bem estabelecida. Por outro lado, o tratamento com TSA induziu a transição epitélio-mesenquimal nas linhagens celulares de CECP, detectada pelo aumento da expressão de vimentina e indução de um fenótipo fusiforme, juntamente com o aumento da invasão tumoral e os níveis de BMI-1. Portanto, a organização da cromatina está envolvida na modulação da presença de CSC e os altos níveis de acetilação das histonas intensificam o comportamento agressivo de células de CECP. / Histone modifications are known to regulate chromatin conformation structure and gene expression in adult cells and pluripotent stem cells. It has been postulated that histone acetylation and deacetylation could influence the expression of genes involved in cancer initiation, progression, metastasis, and development of resistance to chemotherapies. Here, we sought to evaluate the influence of histone modifications over the biology of head and neck squamous cell carcinoma (HNSCC) and its stem cell-like subpopulation (CSC). Initially, we checked the status of histone H3 acetylation (a member of the core histones associated to chromatin compaction) in a representative set of HNSCC cell lines. Subsequently, to analyze the influence of tumor stroma over the histone H3 acetylation, we mimicked the tumor microenvironment by using conditioned medium from fibroblasts and primary human endothelial cells. Further we validated these in vitro findings through human samples of HNSCC. Finally, we induced chromatin acetylation and deacetylation by the administration of the histone deacetylase inhibitor trichostatin A (TSA) and histone acetyltransferase inhibitor curcumin, respectively, in HNSCC cell lines. The analysis of spheres formation (stem cell functional assay), along with the levels of stem cells marker ALDH (showed by flow cytometry - FACS), tumor proliferation index (Ki-67), invasion and migration cellular potencial were verified. HNSCC cell lines showed lower levels of histone H3 acetylation and ability to retain a subpopulation of CSC. Only conditioned media from human endothelial cells was able to alter the conformation of chromatin, since it induced the increase of histone H3 acetylation. Interestingly, it was also noted a concomitant augment of HNSCC cell lines aggressiveness (enhanced BMI-1 and vimentin levels). These findings were confirmed in human samples of HNSCC that showed, only at the invasive front, cells with acetylated chromatin. Curiously, these same cells also expressed vimentin. TSA and curcumin treatments resulted in significant decrease of the CSC subpopulation by disrupting the spheres and reducing the levels of ALDH. Also, curcumin treatment showed exciting results since it caused an evident reduction of cellular invasion and it impacted the tumoral migration potential, being more efficient than cisplantin, a well-established antineoplastic drug. However, TSA induced epithelial to mesenchymal transition in HNSCC cell lines detected by the upregulation of vimentin and the induction of a fusiform phenotype along with augmented tumor invasion and the levels of BMI-1. Chromatin organization is involved in the modulation of CSC where high levels of histone acetylation intensify the aggressive behavior of HNSCC cells.

Modificações nas histonas são conhecidas por regular a estrutura conformacional da cromatina e a expressão gênica em células adultas e células-tronco pluripotentes. Tem sido postulado que a acetilação e deacetilação das histonas podem influenciar a expressão de genes envolvidos na iniciação, progressão e metástase tumoral, além de contribuir para o desenvolvimento de resistência à quimioterapia. Assim, buscou-se avaliar a influência das modificações nas histonas sobre a biologia do carcinoma epidermoide de cabeça e pescoço (CECP) e sua respectiva subpopulação de células semelhantes às células-tronco (CSC). Inicialmente, foi checado os níveis de acetilação da histona H3 (membro das histonas nucleares associado à compactação da cromatina) em um painel representativo de linhagens celulares de CECP. Posteriormente, para estudar a influência do estroma tumoral no padrão de acetilação da histona H3, o microambiente do tumor foi mimetizado através da utilização de meio condicionado derivado do cultivo de fibroblastos e cultura primária de células endoteliais humanas. Além disso, validamos esses resultados in vitro por meio de amostras humanas de CECP. Finalmente, a acetilação e deacetilação da cromatina foi induzida, respectivamente, pela administração dos inibidores das enzimas histona deacetilase tricostatina A (TSA) e histona acetiltransferase curcumina, em linhagens celulares de CECP. Foi feita a análise da formação de esferas (ensaio funcional de células-tronco), juntamente com a verificação dos níveis de ALDH, marcador de células-tronco (citometria de fluxo - FACS), além da determinação do índice de proliferação tumoral (Ki-67) e realização dos ensaios de invasão e migração celular. Linhagens celulares de CECP apresentaram níveis baixos de acetilação da histona H3 e demonstraram capacidade de retenção de uma subpopulação de CSC. Apenas o meio condicionado de células endoteliais humanas foi capaz de alterar a conformação da cromatina, uma vez que induziu o aumento da acetilação da histona H3. Interessantemente, foi também notado um concomitante aumento da agressividade de linhagens celulares de CECP (aumento dos níveis de BMI-1 e vimentina). Esses resultados foram confirmados em amostras humanas de CECP que mostraram, apenas no fronte de invasão, células com cromatina acetilada. Curiosamente, essas mesmas células também expressaram vimentina. Os tratamentos com TSA e curcumina resultaram na diminuição significativa da subpopulação de CSC, interrompendo a formação de esferas e reduzindo os níveis de ALDH. Além disso, o tratamento com curcumina mostrou resultados muito interessantes, uma vez que gerou uma redução evidente da invasão celular e impactou por completo o potencial de migração tumoral, sendo nesse sentido mais eficiente que a cisplatina, droga antineoplásica bem estabelecida. Por outro lado, o tratamento com TSA induziu a transição epitélio-mesenquimal nas linhagens celulares de CECP, detectada pelo aumento da expressão de vimentina e indução de um fenótipo fusiforme, juntamente com o aumento da invasão tumoral e os níveis de BMI-1. Portanto, a organização da cromatina está envolvida na modulação da presença de CSC e os altos níveis de acetilação das histonas intensificam o comportamento agressivo de células de CECP. / Histone modifications are known to regulate chromatin conformation structure and gene expression in adult cells and pluripotent stem cells. It has been postulated that histone acetylation and deacetylation could influence the expression of genes involved in cancer initiation, progression, metastasis, and development of resistance to chemotherapies. Here, we sought to evaluate the influence of histone modifications over the biology of head and neck squamous cell carcinoma (HNSCC) and its stem cell-like subpopulation (CSC). Initially, we checked the status of histone H3 acetylation (a member of the core histones associated to chromatin compaction) in a representative set of HNSCC cell lines. Subsequently, to analyze the influence of tumor stroma over the histone H3 acetylation, we mimicked the tumor microenvironment by using conditioned medium from fibroblasts and primary human endothelial cells. Further we validated these in vitro findings through human samples of HNSCC. Finally, we induced chromatin acetylation and deacetylation by the administration of the histone deacetylase inhibitor trichostatin A (TSA) and histone acetyltransferase inhibitor curcumin, respectively, in HNSCC cell lines. The analysis of spheres formation (stem cell functional assay), along with the levels of stem cells marker ALDH (showed by flow cytometry - FACS), tumor proliferation index (Ki-67), invasion and migration cellular potencial were verified. HNSCC cell lines showed lower levels of histone H3 acetylation and ability to retain a subpopulation of CSC. Only conditioned media from human endothelial cells was able to alter the conformation of chromatin, since it induced the increase of histone H3 acetylation. Interestingly, it was also noted a concomitant augment of HNSCC cell lines aggressiveness (enhanced BMI-1 and vimentin levels). These findings were confirmed in human samples of HNSCC that showed, only at the invasive front, cells with acetylated chromatin. Curiously, these same cells also expressed vimentin. TSA and curcumin treatments resulted in significant decrease of the CSC subpopulation by disrupting the spheres and reducing the levels of ALDH. Also, curcumin treatment showed exciting results since it caused an evident reduction of cellular invasion and it impacted the tumoral migration potential, being more efficient than cisplantin, a well-established antineoplastic drug. However, TSA induced epithelial to mesenchymal transition in HNSCC cell lines detected by the upregulation of vimentin and the induction of a fusiform phenotype along with augmented tumor invasion and the levels of BMI-1. Chromatin organization is involved in the modulation of CSC where high levels of histone acetylation intensify the aggressive behavior of HNSCC cells.

The lens of the eye is a vital tissue in the visual system, responsible for the collection and focusing of light on to the retina. Comprised of epithelial cells at differing stages of differentiation, the transparency of the lens is dependent on the highly ordered crystalline structure of lens proteins. The lens consists of several proteins including crystallins (α, β, γ) that make up 90% of the soluble protein, and the lens cytoskeletal proteins. Cytoskeletal proteins contribute only a fraction of the total lens protein, but are thought to play an important role in the establishment and maintenance of transparency. Calpain-induced degradation of these proteins may be involved in the development of cataracts. This has been an area of research at Lincoln University where a flock of sheep genetically predisposed to cataract maintained as a cataract development model. The aim of this research was to investigate the distribution of cytoskeletal proteins in the lens, and to examine the effects of calpain proteolysis on these proteins, with the goal of establishing the role of the lens cytoskeletal proteins in the ovine cataract model. A combination of techniques was used including immunohistochemistry, which required the development of a specific protocol for ovine lenses. Cytoskeletal proteins were identified using immunohistochemistry in lens tissue sections and exhibited characteristic distributions. Actin displayed preferential distribution in the short sides of the fibre cells in the cortex of the lens but was absent in the lens nucleus, while spectrin in the cortex and nucleus was associated with the fibre cell membrane. Filensin was observed in the outer cortex of lens sections associated with the fibre cell membrane and cytoplasm, although the pattern of localisation was indistinct due to the abundance of filensin breakdown products. Vimentin displayed membrane and cytoplasmic association in the outer cortex that diminished toward the lens nucleus, with membrane associated vimentin only persisting in the deeper regions of the cortex and nucleus. Additionally, the effect of novel calpain inhibitors (Cat0059 and Cat811) in preventing proteolysis of lens cytoskeletal protein was investigated and compared with calpain inhibitors developed elsewhere (SJA6017). The inhibitors were tested at between 10 and 0.1 μM (100 nM). All inhibitors were effective at 10 μM. SJA6017 provided significant protection to vimentin at 1 μM. Cat0059 was found to protect spectrin and filensin at 1 μM, but not vimentin, while inhibitor Cat811 was found to protect spectrin only. SJA6017 added to assays at 100 nM offered significant protection to spectrin, and Cat0059 was found to protect filensin and spectrin to a significant degree at 100 nM, indicating the novel inhibitors were comparable to those developed elsewhere in terms of their effectiveness. Taken together, the evidence presented in this thesis shows the cytoskeletal proteins as crucial elements in the lens. Their pervasive presence coupled with evidence that lens cytoskeletal proteins are sensitive to calpain-induced proteolysis that is inhibited with novel calpain inhibitors suggests that the lens cytoskeletal proteins may be useful targets in cataract prevention for future research.

No description available.

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-08-03T15:13:23Z No. of bitstreams: 1 mariacristinacardosodemendonca.pdf: 5593456 bytes, checksum: 3765ccabf0d5e517d8bc0b46070c90b6 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-08-28T13:46:27Z (GMT) No. of bitstreams: 1 mariacristinacardosodemendonca.pdf: 5593456 bytes, checksum: 3765ccabf0d5e517d8bc0b46070c90b6 (MD5) / Made available in DSpace on 2018-08-28T13:46:27Z (GMT). No. of bitstreams: 1 mariacristinacardosodemendonca.pdf: 5593456 bytes, checksum: 3765ccabf0d5e517d8bc0b46070c90b6 (MD5) Previous issue date: 2018-07-12 / Introdução: A aparência física da população, que vive cada dia mais, vem assumindo um papel significativo quando nos referimos à saúde e bem-estar geral. Portanto as questões dermatológicas assumem, a cada dia, uma grande importância médica. Os peelings químicos são um importante arsenal terapêutico, sendo o peeling de fenol classicamente recomendado para tratamento de clareamento de pele, atenuação das rugas estáticas e flacidez cutânea da face, exigindo sedação em ambiente hospitalar. Na técnica pontuada, sua execução pode ser feita em ambiente ambulatorial, com segurança. Objetivo: investigar os mecanismos pelos quais a técnica se mostra eficaz quando aplicado de forma pontuada, avaliando as alterações da matriz extracelular colagenosa, das fibras elásticas e as fibras colágenas Tipo I e Tipo III, por morfometria automática, e o aumento de células de origem mesenquimal da derme através da imuno-histoquímica. Métodos: Foram utilizados blocos de biópsias de pele da face de pacientes do sexo feminino (n = 17) realizadas antes e depois do tratamento que foram atendidas no Serviço de Dermatologia do Hospital Universitário da Universidade Federal de Juiz de Fora, submetidas ao protocolo de cinco sessões de peeling de fenol pontuado. Os cortes histológicos foram submetidos à análise histopatológica de rotina (HE), análise histoquímica para fibras elásticas (Verhöeff) e para fibras colágenas Tipo I e Tipo III (picrosirius red) e à análise imuno-histoquímica para células com expressão vimentina positiva na derme. Três pacientes foram excluídas, por material insuficiente nos blocos para todas essas colorações. Os dados foram expressos em média aritmética simples ± desvio padrão, mediana e expostos em boxplot. Resultados: Ao compararmos as concentrações de matriz extracelular colagenosa pelo HE e de fibras elásticas (Verhöeff) houve uma relação inversa na histomorfometria, onde das oito pacientes que apresentavam as menores concentrações de fibras colágenas do grupo, seis possuíam as maiores concentrações de fibras elásticas. Das seis pacientes que apresentavam as maiores concentrações de fibras colágenas do grupo, todas apresentavam concentração abaixo da média do grupo para fibras elásticas. Quanto a análise da relação entre fibras colágenas Tipo III e Tipo I, houve uma inversão de valores por maior ganho de fibras colágenas Tipo I em relação as fibras colágenas Tipo III. Na imuno-histoquímica, 64,28% das pacientes (n = 9) apresentou aumento da celularidade mesenquimal. Conclusão: os resultados sugerem que a melhora clínica no tratamento do fotoenvelhecimento facial com a técnica proposta pode estar correlacionada com o maior equilíbrio entre material colágeno e elástico, com maior produção de fibras colágenas Tipo I em relação as fibras colágenas Tipo III e com o aumento do número de células mesenquimais nas amostras pós-tratamento. Diante dos resultados sugerimos que novos estudos sejam realizados, tanto clínicos quanto experimentais, para melhor elucidar os mecanismos de ação da técnica. / Introduction: The physical appearance of the population has been increasingly taking on a significant role when we speak of general health and well-being. Thus, dermatological questions take on an ever greater medical significance. Chemical peels are an important therapeutic arsenal, with phenol peeling being classically recommended for treatment of skin lightening, attenuation of static wrinkles and facial skin flaccidity, requiring sedation in a hospital environment. In the punctuated technique, treatment can safely be done in an outpatient setting. Objective: investigating the mechanisms by which the technique proves effective when applied in a punctuated form and evaluating the changes in the collagenous extracellular matrix, elastic fibers and Type I and Type III collagen fibers, by automatic morphometry, and the increase of dermal mesenchymal cells, through immunohistochemistry. Methods: Facial skin biopsy blocks from female patients (n = 17), taken before and after treatment at the Dermatology Service of the Universidade Federal de Juiz de Fora University Hospital, were used. Patients were submitted to the protocol of five punctuated phenol peeling sessions. The histological sections were submitted to routine histopathological analysis (HE), histochemical analysis for elastic fibers (Verhöeff) and for Type I and Type III collagen fibers (picrosirius red), and immunohistochemical analysis for cells with positive vimentin expression in the dermis. Three patients were excluded because of insufficient material in the blocks for all of these stains. The data were expressed as simple arithmetic mean ± standard deviation, median, and shown in boxplot. Results: Upon comparing the collagenous extracellular matrix concentrations by HE and for elastic fibers (Verhöeff), there was an inverse relation in the histomorphometry, where of the eight patients with the lowest concentrations of collagen fibers in the group, six had the highest concentrations of elastic fibers. Of the six patients who had the highest concentrations of collagen fibers in the group, all of them had a concentration for elastic fibers below the group mean. Regarding the analysis of the relationship between Type III and Type I collagen fibers, there was an inversion of values due to the higher gain of Type I collagen fibers in relation to Type III collagen fibers. In the immunohistochemistry, 64. 28% of the patients (n = 9) presented increased mesenchymal cellularity. Conclusion: the results suggest that the clinical improvement in the treatment of facial photoaging with the proposed technique may be correlated with the greater balance between collagen and elastic material, with a higher production of Type I collagen fibers in relation to Type III collagen fibers, and with the increase of the number of mesenchymal cells in the post-treatment samples. In view of the results, we suggest that new studies be conducted, both clinical and experimental, to better elucidate the action mechanisms of the technique.