The nBmp2 protein was first identified in a DNA affinity chromatography/mass spectrometry screen designed to detect proteins that interact with a cartilage-specific enhancer element (called D/E) from the type XI collagen gene Col11a2. The transcription factor SOX9, a protein from the Sox (SRY-related HMG box) family, binds to and activates gene expression from this enhancer. nBmp2 has no transcriptional activity of its own on this enhancer, but when co-transfected with SOX9 it increases SOX9's activation of D/E nearly 2-fold. SOX9 also activates cartilage-specific enhancer elements from the Col2a1, Col27a1, and Col9a1 genes. The purpose of this project was to determine 1) whether nBmp2 similarly effects SOX9-dependent expression from these enhancers, and 2) whether it does so by binding (either directly or indirectly) to the Col2a1, Col27a1, and Col9a1 enhancers. The work described in this thesis has shown that nBmp2 increases luciferase levels produced from three enhancer/reporter plasmids, but it does so without binding directly to the enhancers. This work has opened up a new area of exploration into the function of the novel protein nBmp2 to examine its potential effects on a variety of different gene regulatory processes.

Orientadores: Ângela Cristina Malheiros Luzo, Sara Teresinha Olalla Saad / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T14:04:31Z (GMT). No. of bitstreams: 1 AndreoliRisso_MarilisaFerruda_M.pdf: 14754073 bytes, checksum: 6e3b6aa9d09a03a1f2873ad414c83bdf (MD5) Previous issue date: 2012 / Resumo: Lesões cartilaginosas raramente curam-se espontaneamente. As atuais opções terapêuticas cirúrgicas e não são limitadas ao alívio dos sintomas, mando a necessidade de futura substituição total de joelho. Terapia baseada em células tronco adultas representa uma alternativa promissora para os procedimentos existentes. As células-tronco mesenquimais (MSC) apresentam alta plasticidade celular e podem ser obtidas de diversas fontes teciduais, opção que exige grande aporte celular expondo a necessidade de criopreservação, processo vantajoso. Contudo, protocolos convencionais estão diretamente associados a possíveis danos e mortalidade celular, sendo desencadeados por formação de cristais de gelo intracelular, toxicidade do crioprotetor adotado e desidratação celular. Este projeto tem como objetivo investigar a interferência da criopreservação com dimetilsufóxido (Me2SO-) (DMSO) na capacidade de MSCs em diferenciação em linhagens mesodermais e arranjo de fibras de colágeno produzidas na diferenciação condrogência. MSCs foram obtidas de tecido adiposo (ADSC). Em quarta passagem foram caracterizadas por citometria de fluxo e diferenciadas em linhagem mesodermais. Diferenciação comprovada por análise morfológica e expressão gênica por de RT-PCR. Genes escolhidos ADIPOQ, FABP4 e PPARG linhagem adipogênica, AGCAN, SOX9, COL1A1 e COL2A1 linhagem condrogênica e OC, OPN e COL1A1 linhagem osteogênica. Diferenciação condrogênica realizada pela técnica de micromassa. Arquitetura das fibras colágenas observada por microscopia de Geração de Segundo Harmônico (SHG) de MSCs e images analisadas pelos algoritmos Fast Fourier Transform (FFT) e Gray Level Co-occurrence Matrix (GLCM). ADSCs criopreservadas em taxa controlada de congelamente com solução criprotetora de soro fetal bovino e 10% de DMSO, mesmos ensaios realizados após descongelamento. Células descongeladas submetidas aos mesmos protocolos de caracterização. Características morfológicas obtidas por SHG expressão gênica das células frescas e criopreservadas analisadas estatisticamente. Amostras de ADSC, fresca e criopreservadas, foram capazes de se diferenciar em linhagens mesodermais. Perfil de expressão gênica da linhagem adipogênica foi semelhante ao esperado para tal processo de diferenciação, em ambas amostras, criopreservadas e frescas, estas últimas com maior expressão (p=ns). Na diferenciação para linhagem osteogênica também houve o perfil de expressão esperado para os genes estudas, sendo mais expresso no grupo criopreservado (p=ns). Apesar do fato da expressão do gene COL2A1 de linhagem condrogênica ter sido maior no grupo criopreservado, quando o perfil de expressão gênica do grupo fresco foi analisado, este mostrou-se mais consistente com o perfil esperado normal. Análise das imagens de SHG demonstraram que a arquitetura das fibras colágenas foi mais organizada (p=ns) e uniforme (p>0,0001) nas amostras frescas. O grupo criopreservado demonstrou maior entropia (p=ns) e contraste (p=0,0167), demonstrando haver maior tendência direcional das fibras de colágeno nas amostras frescas. Os resultados de expressão gênica sugerem que a criopreservação pode interferir de forma positiva na diferenciação osteogênica e negativamente nas linhagens adipogênica e condrogênica. A arquitetura da rede tridimensional de colágeno foi modulada negativamente pelo processo de criopreservação, dados esses confirmados por análise das imagens obtidas por SHG, o que poderia interferir com as propriedades físico/mecânicas características do tecido colagenoso, importante na manutenção da cartilagem articular baseada em terapia celular. O uso das imagens de SHG tornou-se uma importante ferramenta na melhor avaliação das fibras colágenas / Abstract: Cartilage defects rarely heal spontaneously. Current surgical and non-surgical therapeutic interventions are limited to symptom relief and future total knee replacement continues to be necessary. Adult stem cell based therapy could represent a promising alternative to this procedure. Mesenchymal Stem Cells (MSCs) have great capacity of differentiation and can easily be obtained from various sources. Cryopreservation offers many advantages for practitioners engaged in cell-based therapies. However, conventional slow freezing, despite using cryoprotectant solution, has always been associated with damage and mortality due to intracellular ice formation, cryoprotectant toxicity, and dehydration. The aim of this work is to investigate whether the Dimethyl Sulfoxide (Me2SO-) (DMSO) cryopreservation process interferes with the MSCs capacity of differentiation to mesodermal lineages and/or with collagen fiber network architecture, produced by chondrogenic cells, comparing fresh and thawed MSCs. MSCs were obtained from adipocyte tissue (ADSCs). At the fourth passage cells were characterized as ADSCs by flow cytometry analysis and differentiation to mesodermic lineages was confirmed by morphology (light optical microscopy) and gene expression analysis (RT-PCR). The following genes were chosen ADIPOQ, FABP4 and PPARG for adipogenic lineage, AGCAN, SOX9, COL2A1 and COL1A1 for chondrogenic and OC, OPN and COL1A1 for osteogenic lineage. Chondrogenic differentiation was carried out by micromass technique. Collagen fibers architecture was observed by Second Harmonic Generation (SHG) microscopy. Images were analyzed by Fast Fourier Transform (FFT) and Gray Level Cooccurrence Matrix (GLCM) algorithms. ADSCs were cryopreserved in a controlledrate freezing device with bovine serum fetal and 10% DMSO as cryoprotectant solution. Thawed cells were submitted to the same characterization protocols. SGH morphologic features and gene expression results from thawed and fresh cells were statistically analyzed. Both, thawed and fresh ADSCs were able to differentiate into mesodermal lineages. Gene expression profile of adipogenic lineage was similar to that expected for the differentiation process in both, thawed and fresh cells, with greater expression in fresh ones (p=ns). Osteogenic lineage also demonstrated the expected gene expression profile, being more expressed in the thawed group (p=ns). Despite the fact that COL2A1 gene expression in chondrogenic lineage was greater in the thawed group, when the gene expression profile was analyzed the fresh group was more consistent with the expected normal profile. SHG images results demonstrated that collagen fibers architecture was more organized (p=ns) and uniform (p<0, 0001) in fresh samples. The thawed group showed more entropy (p=ns) and contrast (p=0, 0167) demonstrating that a directional trend in the collagen fibers was only observed in the fresh group. Gene expression results suggested that cryopreservation could interfere in a positive manner with osteogenic differentiation, and negatively on adipogenic and chondrogenic lineages. Collagen network tridimensional architecture was negatively modulated by cryopreservation, confirmed by SHG analysis, which could interfere with the desirable collagen mechanical properties, important for the maintenance of articular cartilage in cell based therapy. SHG analysis has become an important tool for better evaluate collagen fibers / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestre em Fisiopatologia Médica

Osteoarthritis (OA) is a degenerative joint disease that affects more than 60% of Americans 65 and older. Because human subjects and samples are not readily available for research, animal models are an invaluable resource for the study of OA. Disproportionate micromelia (Dmm) is one such model that develops OA early in life due to a deletion in the c-propeptide of the Col2a1 gene. Light microscope analysis of the articular cartilage in Dmm has been completed, but is insufficient to show the cellular effects of the deletion mutation in Dmm in adequate detail. The present study explores the changes that occur in the rough endoplasmic reticulum (ER) of chondrocytes in the articular cartilage of Dmm heterozygous mutants (D/+). Immunohistochemical analysis in Dmm has shown that type II collagen is absent extracellularly in articular cartilage of Dmm homozygous mutants and reduced in the heterozygotes. Because preprocollagens are processed through the endoplasmic reticulum (ER), it has been hypothesized that due to improper folding this mutation prevents newly synthesized collagen from leaving the ER, as a result large dilations are seen in the ER of Dmm mice. Furthermore, matrix area fractions should be reduced in the D/+ group if indeed type II collagen is not secreted. Data collected indicated that at 4 months and older, large distensions in the ER disappear. At age 0 months, there is significant dilation in the ER of the D/+ (p=.0013), and at .75 months significant dilation is also observed (p=.0063). In pooled age groups, the D/+ has a 1.77% greater ER fraction than the +/+ (p=.0022). The matrix area fraction was also significantly lower in the D/+ compared to the +/+ (p=.0037). Apoptosis was prominent in older ages, but did not appear to be different between +/+ and D/+ mice. Because decreased matrix and dilation of ER have been documented in OA, Dmm is a good model of OA that can be further used to study the molecular changes and deficiencies that occur in the pathogenesis of OA.

Deafblindness is a combined impairment of vision and hearing with an incidence of about 1: 8000 children and 1: 5500 adults. The most common genetic causes are the Stickler (STL) and Usher (USH) syndromes. The main goal of this work is to provide an up-to-date overview of STL and USH in the Czech and Slovak Republic (CR and SR), to determine the correlations between the genotype and phenotype in our population and the associated diagnostic criteria. Using sequencing and MLPA we examined 45 patients from 28 families for suspected STL. We found potentially causal variants of STL genes in 39 patients from 22 families. Fifteen different COL2A1 variants (8 being novel) were found in 28 patients from 18 families and 4 novel COL11A1 variants were found in 11 patients from 4 families. We identified the cause of the disease in 79 % of the families. The USH study involved 30 patients from 27 families. The most frequent cause was USH2A pathogenic variants, i.e. 19 variants in 14 families, 9 being novel. Less common were pathogenic variants in MYO7A (6 variants in 3 families, 5 being novel), USH1C and CDH23 (3 variants, 2 being novel, in 2 families both) genes. In 2 families, compound heterozygosity was found for variants in two different USH genes. The deafblindness etiology was clarified for 24 patients from...