O zebrafish (Danio rerio) tem se destacado na pesquisa biomédica por sua homologia fisiológica e genética aos humanos. No entanto, há poucos relatos sobre a criopreservação ovariana desta espécie. Assim, pesquisamos a utilização de um recipiente de metal na vitrificação de tecido ovariano de zebrafish. O objetivo foi avaliar a sobrevivência e o desenvolvimento in vitro de folículos de zebrafish após a vitrificação de fragmentos ou ovários inteiros usando a cápsula de metal. Primeiro, testamos quatro soluções de vitrificação (VS1 – 1,5 M metanol + 4,5 M propilenoglicol; VS2 – 1,5 M metanol + 5,5 M Me2SO; VS3 – 1,5 M metanol + 4,5 M propilenoglicol + 0,5 M sacarose; VS4 – 1,5 M metanol + 5,5 M Me2SO + 0,5 M sacarose) e cinco estágios de desenvolvimento folicular utilizando o teste de coloração supravital iodeto de propídio combinado com diacetato de fluoresceína. Estes resultados mostraram que os folículos em estágio I, imaturos, apresentaram as maiores taxas de sobrevivência celular e que VS1 foi a melhor solução em termos de viabiidade. No Experimento 2, utilizou-se VS1 para vitrificar o tecido ovariano em diferentes dimensões (fragmentos ou ovários inteiros) e em dois diferentes recipientes (palheta de plástico ou cápsula de metal). Para avaliar a sobrevivência e o crescimento folicular dos folículos em estádio I, o diâmetro dos folículos foi mensurado antes e depois de cultivo in vitro por 24 horas. A morfologia folicular foi analisada por microscopia de luz após vitrificação utilizando a cápsula de metal. Os dados mostraram que a morfologia de folículos imaturos foi bem preservada após a criopreservação. A taxa de sobrevivência folicular foi maior (P <0,05) em fragmentos vitrificados, quando comparados com a vitrificação de ovários inteiros. Não houve diferenças significativas na sobrevivência e crescimento folicular entre os dois recipientes de vitrificação, palheta de plástico ou cápsula de metal. No entanto, a cápsula de metal diminui os riscos de contaminação, pois é hermeticamente fechada evitando contato com nitrogênio líquido e poder ser esterilizada, em vista que é manufaturada em aço inoxidável. Por essas razões, acreditamos que a cápsula de metal tem um uso potencial em reprodução humana para a vitrificação em grau clínico de tecido ovariano. / Zebrafish (Danio rerio) has excelled in biomedical research for its physiological and genetic homology to humans. However, there are few reports on ovarian cryopreservation of this specie. Thus, we studied the use of a metal capsule to vitrify zebrafish ovarian tissue. The aim of this study was to assess the survival and in vitro development of zebrafish follicles after vitrification of fragmented or whole ovaries using the metal capsule. First, we tested four vitrification solutions (VS1 - 1.5 M methanol + 4.5 M propylene glycol; VS2 - 1.5 M methanol + 5.5 M Me2SO; VS3 - 1.5 M methanol + 4.5 M propylene glycol + 0.5 M sucrose; VS4 - 1.5 M methanol + 5.5 M Me2SO + 0.5 M sucrose) and five follicular developmental stages using fluorescein diacetate and propidium iodide supravital staining test. These results showed that immature follicles, stage one, presented the highest survival rates and VS1 the best vitrification solution in terms of viability. In Experiment 2, we used VS1 to vitrify ovarian tissue in different dimensions (fragments or whole ovaries) and tested two different carriers (plastic straw or metal capsule). To evaluate follicular survival and growth of stage I, we measured follicle diameter before and after twenty-four-hour in vitro culture. The follicular morphology was analyzed by light microscopy after vitrification using the metal capsule. Data showed that the immature follicles morphology was well preserved after cryopreservation. Follicular survival rate was higher (P<0.05) on vitrified fragments, when compared to whole ovaries. There were no significant differences on follicular survival and growth between the two vitrification devices, plastic straw or metal capsule. However, the metal capsule being tightly sealed and manufactured in stainless steel avoids contact with liquid nitrogen and can be sterilized reducing contamination risk. These reasons lead us to believe that the metal capsule has a potential use in human reproduction for the clinical grade vitrification of ovarian tissue.

O zebrafish (Danio rerio) tem se destacado na pesquisa biomédica por sua homologia fisiológica e genética aos humanos. No entanto, há poucos relatos sobre a criopreservação ovariana desta espécie. Assim, pesquisamos a utilização de um recipiente de metal na vitrificação de tecido ovariano de zebrafish. O objetivo foi avaliar a sobrevivência e o desenvolvimento in vitro de folículos de zebrafish após a vitrificação de fragmentos ou ovários inteiros usando a cápsula de metal. Primeiro, testamos quatro soluções de vitrificação (VS1 – 1,5 M metanol + 4,5 M propilenoglicol; VS2 – 1,5 M metanol + 5,5 M Me2SO; VS3 – 1,5 M metanol + 4,5 M propilenoglicol + 0,5 M sacarose; VS4 – 1,5 M metanol + 5,5 M Me2SO + 0,5 M sacarose) e cinco estágios de desenvolvimento folicular utilizando o teste de coloração supravital iodeto de propídio combinado com diacetato de fluoresceína. Estes resultados mostraram que os folículos em estágio I, imaturos, apresentaram as maiores taxas de sobrevivência celular e que VS1 foi a melhor solução em termos de viabiidade. No Experimento 2, utilizou-se VS1 para vitrificar o tecido ovariano em diferentes dimensões (fragmentos ou ovários inteiros) e em dois diferentes recipientes (palheta de plástico ou cápsula de metal). Para avaliar a sobrevivência e o crescimento folicular dos folículos em estádio I, o diâmetro dos folículos foi mensurado antes e depois de cultivo in vitro por 24 horas. A morfologia folicular foi analisada por microscopia de luz após vitrificação utilizando a cápsula de metal. Os dados mostraram que a morfologia de folículos imaturos foi bem preservada após a criopreservação. A taxa de sobrevivência folicular foi maior (P <0,05) em fragmentos vitrificados, quando comparados com a vitrificação de ovários inteiros. Não houve diferenças significativas na sobrevivência e crescimento folicular entre os dois recipientes de vitrificação, palheta de plástico ou cápsula de metal. No entanto, a cápsula de metal diminui os riscos de contaminação, pois é hermeticamente fechada evitando contato com nitrogênio líquido e poder ser esterilizada, em vista que é manufaturada em aço inoxidável. Por essas razões, acreditamos que a cápsula de metal tem um uso potencial em reprodução humana para a vitrificação em grau clínico de tecido ovariano. / Zebrafish (Danio rerio) has excelled in biomedical research for its physiological and genetic homology to humans. However, there are few reports on ovarian cryopreservation of this specie. Thus, we studied the use of a metal capsule to vitrify zebrafish ovarian tissue. The aim of this study was to assess the survival and in vitro development of zebrafish follicles after vitrification of fragmented or whole ovaries using the metal capsule. First, we tested four vitrification solutions (VS1 - 1.5 M methanol + 4.5 M propylene glycol; VS2 - 1.5 M methanol + 5.5 M Me2SO; VS3 - 1.5 M methanol + 4.5 M propylene glycol + 0.5 M sucrose; VS4 - 1.5 M methanol + 5.5 M Me2SO + 0.5 M sucrose) and five follicular developmental stages using fluorescein diacetate and propidium iodide supravital staining test. These results showed that immature follicles, stage one, presented the highest survival rates and VS1 the best vitrification solution in terms of viability. In Experiment 2, we used VS1 to vitrify ovarian tissue in different dimensions (fragments or whole ovaries) and tested two different carriers (plastic straw or metal capsule). To evaluate follicular survival and growth of stage I, we measured follicle diameter before and after twenty-four-hour in vitro culture. The follicular morphology was analyzed by light microscopy after vitrification using the metal capsule. Data showed that the immature follicles morphology was well preserved after cryopreservation. Follicular survival rate was higher (P<0.05) on vitrified fragments, when compared to whole ovaries. There were no significant differences on follicular survival and growth between the two vitrification devices, plastic straw or metal capsule. However, the metal capsule being tightly sealed and manufactured in stainless steel avoids contact with liquid nitrogen and can be sterilized reducing contamination risk. These reasons lead us to believe that the metal capsule has a potential use in human reproduction for the clinical grade vitrification of ovarian tissue.

Orientador: Frederico Andrade e Silva / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-08T12:40:24Z (GMT). No. of bitstreams: 1 Ramos_GuilhermedaGama_D.pdf: 1761630 bytes, checksum: 72bd7cf357aeaf44614f32847aebecdf (MD5) Previous issue date: 2007 / Resumo: Este estudo teve como objetivo comparar, por meio de radiografias transcranianas (técnica Accurad), duas técnicas descritas na literatura para a obtenção da relação centrica e posição das cabeças da mandibula nas fossas mandibulares. Uma delas, a manipulação bilateral descrita por DAWSON em 1974. A outra, um traçado gráfico dos movimentos mandibulares no plano horizontal, denominado por GYSI, em 1910, de arco gotico, obtido por meio de um dispositivo, chamado de registro intra-oral. Foi utilizada uma amostra composta de vinte voluntarios (10 homens e 10 mulheres). Os resultados foram submetidos a uma analise estatística pelo teste não paramétrico de Wilkocson com significância de 5%. O alto valor do coeficiente de variação mostrou que a técnica que utiliza a manipulação bilateral, apresentou maiores variações quando comparada à técnica que utiliza o registro intra-oral para a obtenção do arco gótico de Gysi. A técnica que utiliza o registro intra-oral do arco gótico de Gysi posicionou as cabeças da mandíbula aproximadamente 1,5mm para tres em relação a posição das cabeças da mandíbula, quando as mesmas estavam em oclusão centrica. A tecnica da manipulação não posicionou as cabeças da mandibula na região mais superior e anterior como o preconizado pela técnica / Abstract: The purpose of this study was a radiographs investigate variations in condilar positions using two different methods of determining centric relation; bilateral mandibular manipulation advocated by DAWSON since 1974 and the arch tracing for the mandible movements in horizontal plane. This arch was denominate arch gothic in 1910 by Gysi. The sample consisted of 20 adults, 10 females and 10 males. On the basis of the analysis of the Wilkocson test (sampling error of 5%), it can be concluded that high coefficient of variation showed the most variation of bilateral mandibular manipulation method when compareted with Gysi gothic arch method. Both techniques showed condyles positioned more posteriorly and inferiorly than the mandibular position of maximum intercuspation. The distance existent between centric oclusion and the vertex of the gothic arc was approximately 1,5 mm / Doutorado / Protese Dental / Doutor em Clínica Odontológica

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O interesse de pesquisadores pelo estudo do sistema proprioceptivo vem crescendo nas Ãltimas dÃcadas. Isto à decorrente da importÃncia da integridade deste sistema no tratamento das enfermidades ortopÃdicas. Os mecanorreceptores e as terminaÃÃes nervosas livres sÃo as unidades microscÃpicas fundamentais da propriocepÃÃo e do sistema nervoso aferente. Eles transmitem ao sistema nervoso central informaÃÃes fisiolÃgicas em forma de potencial de aÃÃo, quando as estruturas articulares, cÃpsula, ligamentos e mÃsculos, sÃo submetidas ao estresse do movimento. Foram avaliadas a presenÃa e a densidade dos mecanorreceptores na cÃpsula, ligamento da cabeÃa femoral e labrum acetabular de 45 quadris masculinos. Destes, 30 foram obtidos de pacientes do sexo masculino com artrose secundÃria (grupo I) durante artroplastia e 15 de cadÃveres frescos com articulaÃÃo do quadril normal (grupo II). A idade mÃdia do grupo I foi de 56,5 e do grupo II foi de 35,6 anos. Os fragmentos obtidos foram mensurados em 2mm e corados com cloreto de ouro a 1%. ApÃs fixaÃÃo os fragmentos foram seccionados com criostato, em sÃrie de 6 micrÃmetros de espessura e submetidos à microscopia de luz. Foram identificados trÃs tipos de mecanorreceptores: Ruffini, Pacini e Golgi, alÃm das terminaÃÃes nervosas livres de acordo com a classificaÃÃo de Freeman e Wike (1967). Em cada grupo foi determinado o nÃmero e a densidade de mecanorreceptores e em seguida foram comparados os resultados. Concluiu-se que o predomÃnio das terminaÃÃes tipo Pacini no gurpo controle foi significante quando comparado com os corpÃsculos tipo Rufini (p < 0,01) e Golgi (p < 0,001).Enquanto que no grupo artrose a densidade do tipo Golgi foi menor que o tipo Pacini(p < 0,001) e terminaÃÃes nervosas livres( p < 0,01).Por outro lado, quando comparados a densidade total das terminaÃÃes nervosas nos dois grupos observou-se uma reduÃÃo significante nos quadris artrÃsicos (p = 0.008). Isto sugere fortemente que a integridade do sistema proprioceptivo parece sofrer modificaÃÃes em quadris artrÃsicos, como conseqÃÃncia da reduÃÃo do nÃmero de terminaÃÃes nervosas. Estudos eletrofisiolÃgicos futuros serÃo necessÃrios para definir o papel das terminaÃÃes nervosas e o padrÃo proprioceptivo do quadril normal do quadril artrÃsico. / Proprioceptive studies have reported growing interest in investigators in the last few decades. This is result of relevant integrity of this system in the treatment of orthopedic diseases. Mechanoreceptors and free nerve endings are the microscopy basic units from proprioception and peripheral nervous system. They transmit to the central nervous system physiological information with detection threshold when the joint are submitted to mechanical stress. The presence and density of mechanoreceptors were investigated in the capsule, teres ligament and acetabulum labrum from 45 hips joint. Of these 30 were obtained from male patients (Group I) with secondary arthrosis at open arthoplasty and 15 of fresh cadaver with normal hip joint (Group II). The mean age of group I was 56,6 and the group II was 36,5 years. The fragments obtained were measured up to 2mm and stained with gold chloride 1%. After fixation the fragments were sectioned with cryostat at serial sections of 6Âm thickness and examined using light microscopy. We identified 3 types of mechanoreceptors: Ruffini, Pacini and Golgi corpuscles, as well free nerve endings according of Freeman and Wike classified 1967. Each group was determined the number and density of mechanoreceptors and then it was compared. We conclude that the number of Pacini type was significative when it was compared with Rufini ( p < 0,01 ) and Golgi types (p< 0,001), in the normal group. However, the denstity of the Golgi type was minor compared to the Pacini ( p < 0,001) and free nerve ending ( p < 0,01 ) in the arthrosis group. Therefore, when the density total of nerve endings in normal hips were compared with arthosis hips we founded that the number decreased (p = 0.008). This is suggests strongly that the integrity of proprioceptive system seems to be modified by arthosis in consequence of nerve endings numbers. In the future, electrophysiological studies will be necessary to determine the rule of nerve endings and proprioceptive system in the normal and arthosis hip.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Ce mémoire est consacré à la synthèse et à l'étude de coordinats phosphorés originaux construits sur des résorcinarènes rigidifiés. Trois types de ligands ont été élaborés : 1) des phosphines tertiaires obtenues par ancrage de groupes –PPh2 sur des cycles aromatiques d'un résorcinarène générique. Celles-ci réagissent avec des espèces cationiques en formant des complexes chélate P,O résultant du clivage d'une liaison C–O localisée en ortho du cycle phosphoré. Ce type de cassure, inédit, permet d'accéder à des catalyseurs d'oligomérisation d'éthylène pour la production de chaînes courtes; 2) des diiminophosphoranes obtenus par fonctionnalisation distale d'un résorcinarène-cavitand. Un ligand de cette famille donne lieu à des sélectivités de forme en hydrogénation (Rh) de mélanges d'oléfines; 3) des phosphino-cavitands doubles permettant d’engendrer, par chélation, des métallo-capsules. L'une d'entre elles (Pt) conduit à des sélectivités remarquables en hydroformylation du styrène. / This thesis describes the synthesis of three types of phosphorus-containing resorcinarene cavitand: a) compounds with diphenylphosphino groups grafted to the wider rim of a generic cavity. These phosphines undergo facile, directed C–O bond breaking upon reaction with transition metal ions in the presence of exogenous or endogenous nucleophiles. A nickel complex of this type was shown suitable for the low pressure production of short α-olefins starting from ethylene; b) cavitands distally-substituted by two iminophosphoranyl groups (–Ph2P=NAr). One of them (Ar = p-anisyl), when combined with Rh(I), resulted in a shape-selective olefin hydrogenation catalyst; 3) double phosphino-cavitands, which upon chelation gave metallo-capsular complexes in which the metal centre is either fully or partially embedded in the resulting cavity. A platinum complex of this type resulted in the selective hydroformylation of styrene into the expected branched aldehyde.

Abstract This study was undertaken to identify mechanisms of adhesion of intraocular lenses (IOLs) to the capsular bag after cataract surgery and IOL implantation. It was also done to challenge the sandwich theory presented for posterior capsular opacification (PCO): If the IOL is made of a bioactive material it would allow a single lens epithelial cell layer to bond both to the IOL and the posterior capsule at the same time. This would produce a sandwich pattern including the IOL, the cell monolayer and the posterior capsule. The sealed sandwich structure would prevent further epithelial ingrowth. The degree of bioactivity of the IOL could explain the basic difference in the incidence of PCO and capsulotomy rates with different IOL materials. The sandwich theory was put forward on the basis of a search for a keratoprosthesis material, which would allow maximal adhesion of the prosthesis to corneal tissue. Titanium and glass-ceramic coated titanium were found to develop better adhesion than poly (methyl methacrylate) (PMMA). The adhesion of PMMA to the corneal stromal tissue was loose, and down growth of corneal epithelial cells was seen around the prosthesis. The differences between various IOL materials were first tested with rabbit corneal tissue cultures. There was better adhesion of corneal tissue to soft, hydrophobic acrylate than to PMMA, heparin surface modified (HSM)-PMMA, silicone or hydrogel IOLs. To assess differences in protein adhesion to IOL surfaces, different IOLs were incubated for 24 hours with radioactive iodine labeled fibronectin. Soft hydrophobic acrylate (AcrySof®) showed the highest binding of fibronectin, and the differences relative to all the other materials were significant (p &lt; 0.01-0.001), except to PMMA (p = 0.31). The sandwich theory and the results with rabbit corneal tissue cultures and the protein adhesion study in vitro were evaluated against the results found in pseudophakic autopsy eyes. Altogether, 70 autopsy eyes were analyzed. From 38 autopsy eyes containing PMMA, silicone, soft hydrophobic acrylate or hydrogel IOLs histological sections were prepared from the capsular bag and immunohistochemical analyses were performed for fibronectin, vitronectin, laminin and collagen type IV. A total of 152 specimens were analyzed. From 32 autopsy eyes containing IOLs made of PMMA, silicone, acrylate or hydrogel, IOLs were explanted from the capsular bag and immunohistochemical analysis was done on both sides of the IOLs for fibronectin, vitronectin, laminin or collagen type IV. Soft hydrophobic acrylate IOLs had significantly more adhesion of fibronectin to their surfaces than PMMA or silicone IOLs. Also, more vitronectin was attached to acrylate IOLs than to the other IOL materials. Silicone IOLs had more collagen type IV adhesion in comparison to the other IOL materials studied. In histologic sections a sandwich-like structure (anterior or posterior capsule-fibronectin-one cell layer-fibronectin-IOL surface) was seen significantly more often in eyes with acrylate IOLs than in PMMA, silicone or hydrogel IOL eyes. These studies support the sandwich theory for posterior capsule opacification after cataract surgery with IOLs. The results suggest that fibronectin may be the major extracellular protein responsible for the attachment of acrylate IOLs to the capsular bag. This may represent a true bioactive bond between the IOL and the lens epithelial cells, and between the IOL and the capsular bag. This may explain the reason for clinical observations of less posterior capsular opacification and lower capsulotomy rates with the soft hydrophobic acrylate material of AcrySof® IOLs compared to the other IOL materials studied.

Encapsulated Escherichia coli are responsible for a number of life threatening infections of man. These range from urinary tract infections to septicemia and neonatal meningitis. A common property of these E. coli strains is the expression of a polysaccharide capsule or K antigen. The expression of a capsule is an essential virulence factor protecting the bacterium from host defenses. Like many virulence factors capsule gene expression is regulated by temperature, such that at 37 0C inside the host the capsule is expressed whereas at 20 0C it is not. The project used the K5 capsule gene cluster as a model system to study in detail the regulation of capsule gene expression. Expression of E. coli K5 gene cluster is regulated at the transcriptional level by two convergent promoters PR1 and PR3. The temperature regulation-dependent expression is in part controlled at the level of transcription by complex regulatory network involving the regulators SlyA, H-NS and IHF acting at PR1 and PR3. A large 5’ untranslated region (5’ UTR) is involved in transcriptional regulation by interacting with global regulator proteins. In this study, a combination of lacZ reporter gene fusions, 5’ RACE analysis and site-direct mutagenesis at promoter functional elements were used to investigate the promoter. These studies identified that the PR1 promoter was more complex than initially thought and contains, in addition to previously characterized PR1-1 promoter at +1, three additional tandem promoters PR1-2, PR1-3 and PR1-4 transcribing in the same direction from the site +133, +142 and +182, respectively. In order to analyse the contribution for the transcription from PR1 among these multiple promoters, these multiple tandem promoters’ activities were measured by β-galactosidase assay and Real-time quantitative reverse PCR assay. We determined that PR1-2 and PR1-3 are two cryptic promoters with very low transcription activity while PR1-1 and PR1-4 are the major promoters that contributed evenly to the total transcripts into kps operon in the mid-exponential phase. Furthermore, we demonstrated that the promoter PR1-1 and PR1-4 are tightly coupled and the activity of PR1-4 can be co-ordinately reduced by disrupted PR1-1.Different minimal PR1-lacZ promoter fusions were also transformed into strains with mutations in the genes that encode these regulatory proteins (IHF, SlyA and H-NS) and the transcription activity was examined by β-galactosidase assay at both 37 0C and 20 0C. IHF is required indirectly for maximum transcription at PR1-1 promoter but directly represses transcription from PR1-4 due to binding at +160 region at 37 0C. Global regulator H-NS represses the transcription at both 37 0C and 20 0C at PR1 and plays an important role for transcriptional temperature regulation at PR1 region. The anti-repressor SlyA activates transcription at PR1-1 at 37 0C. This study identified for the first time growth phase dependent expression from the PR1 promoter. Also, this study discovered different temporal patterns of promoter PR1-1 and PR1-4 transcription was coordinated with bacterial growth cycle. Overall this study will be helpful to decipher the complex regulation of capsule gene expression in E. coli.

Esta dissertação relata e traduz o percurso de estudos, reflexões, pesquisas e procedimentos, dedicados à elaboração de um conjunto de trabalhos realizados com a terra, com a cerâmica, sobre a temática dos ninhos e casulos a partir das vivências que meu corpo atual e consciente, tem com a natureza que me cerca. As reflexões desencadeadas por essas vivências fazem uma relação análoga dos ninhos e casulos com a casa e com o corpo enquanto ninho primordial. / This paper reports and translates the way studies, reflections, researches and procedures, dedicated to a work set elaboration that was done with earth, about the nestles thematic, started to the experiences that my conscious body has with that nature that surround me. The reflections unchained by those experiences do a nestles analog relation with the house and the body as a prime nestle.

Thesis provides an overview and discussion of current findings in the field of biometrics. In particular, it focuses on facial recognition subject. Special attention is payed to convolutional neural networks and capsule networks. Thesis then lists current approaches and state-of-the-art implementations. Based on these findings it provides insight into engineering a very own solution based of CapsNet architecture. Moreover, thesis discussed advantages and capabilitied of capsule neural networks for identification of a person by its face.