Nitric oxide (NO) is an important mediator of angiogenesis and is primarily produced endogenously through the action of nitric oxide synthase (NOS). An alternate pathway for NO production is the conversion of nitrite to NO, which depends on the presence of hemoglobin (Hb) and hypoxic conditions. The angiogenic effects of topically applied sodium nitrite on two vascular beds in the ex ovo chicken embryonic model of angiogenesis were assessed. Gas barrier films were used to modulate local oxygen levels in the chorioallantoic membrane (CAM), a respiratory vascular network, and the area vasculosa (AV) on the yolk sac, a typical peripheral vascular network. The low-permeable film polyvinylidene chloride (PVDC) and highly permeable regenerated cellulose (RC) were applied to the surface of the vasculature to alter oxygen diffusion and transport and produce a local environment of low or high oxygen, respectively. Phosphorescence Quenching Microscopy (PQM) was used to verify the oxygen levels in the vascular membranes underneath the films. Following 48 hours of continuous application of sodium nitrite (330 μg/kg/day), saline, or sodium nitrite + cPTIO (a NO scavenger) (1mg/kg/day), the angiogenic response was quantified by measuring vascular density and network complexity. The PVDC film reduced CAM PO2 to 17.9±5.5 mmHg and AV PO2 to 29.5±3.6 while the RC film maintained a PO2 of 115 mmHg. At the edge of PVDC film, there was found to be a small area of transition between the nearby low and high PO2 regions. After nitrite application, significant increases in vascularity were observed in the AV under hypoxic conditions, but not normoxic conditions (p<0.03). cPTIO inhibited nitrite-induced angiogenesis and returned vascularity to levels observed with saline application. No significant changes were observed in the CAM, but a trend of reduced angiogenesis after nitrite application was observed compared to saline and saline+cPTIO. These results indicate that two highly diffusible gases, NO and O2, play important roles in the growth of new blood vessels, but in a way that appears to depend on the gas exchange function of the vascular network.

O cristalino se origina de um epitélio simples e cuboidal que recobre a vesícula óptica. Neste estádio, os filamentos de actina são distribuídos ao longo do eixo apicobasal. As células do ectoderma pré- placodal, em contato com a vesícula óptica, formam um epitélio pseudoestratificado, chamado de placóide do cristalino, com acúmulo de actina no domínio apical. Nós propusemos estudar o papel da proteína PAR3 e sua fosforilação no estabelecimento de actina apical. A superexpressão de PAR3 no placóide forma pontos ectópicos de PAR3 na membrana baso-lateral e induz o recrutamento de actina ectópica para esses pontos. O recrutamento de actina e aPKC ectópicos é independente do estado de fosforilação da treonina 833, resíduo localizado no domínio de ligação do PAR3 ao aPKC. Além disso, no ectoderma peri-placoidal, onde a actina localiza-se baso-lateralmente, PAR3 induz o recrutamento ectópico de actina apical e esse recrutamento é independente da fosforilação da treonina 833. Esses dados nos sugerem que PAR3 é suficiente para recrutar actina no placóide do cristalino. / The lens originates from a simple cuboidal epithelium that overlies the optic vesicle. At this stage, the actin filaments are distributed along its apical-basal sides. The pre-placodal ectoderm, in contact with the optic vesicle, forms a pseudostratified tissue, the lens placode, with accumulation of actin network at the apical domain. Here, we focused on the role of the polarity protein PAR3 and its phosphorylation in the establishment of this apical actin network. Overexpression of PAR3 in the lens placode, induced formation of ectopic actin clusters in the basolateral membrane of the lens placode. The formation of these actin clusters, as well as recruitment of aPKC was independent of Threonine 833 phosphorylation at the PAR3 aPKC-binding site. In addition, PAR3 induced ectopic actin networks in the apical membrane of the periplacodal ectoderm independent of the Threonine 833 phosphorylation. Taken together, these data suggest that PAR3 is sufficient for actin recruitment in the lens placode.

SCRATCH2 é um fator de transcrição envolvido no desenvolvimento neural expresso em células pós-mitóticas. Identificamos que a retenção nuclear de SCRATCH2 é dada pelo domínio zinc-finger. A atividade repressora é modulada pelo domínio SCRATCH e não depende do domínio SNAG. O alinhamento de ortólogos de SCRATCH2 identificou uma sequência conservada na região N-terminal contendo os resíduos de fosforiláveis Y77 e S78. Mutação em Y77 ou S78 reduz a capacidade repressora de SCRATCH2, enquanto mutações em ambos resíduos resgatam sua função. Nossos dados sugerem que o domínio zinc-finger é responsável pela localização nuclear enquanto a atividade repressora é mediada pelo domínio SCRATCH. Já a identidade de Y77 e S78 é importante para a conformação correta proteína. O nocaute de SCRATCH2 aumenta a expressão de marcadores de progenitores intermediários (IP) e reduz o a expressão de marcadores de neurônios pós-mitóticos durante a neurodiferenciação de células-tronco. Esses dados sugerem que SCRATCH2 atua na manutenção de IP, e participa do início da diferenciação neural. / SCRATCH2 is a transcription factor involved in neural development expressed in postmitotic neural cells. Here, we identify that the nuclear retention of SCRATCH2 is controlled by the zinc-finger domain. The repressor activity is modulated by the SCRATCH domain and is independent of the SNAG domain. An analysis of SCRATCH2 through homology comparison identified a N-terminal conserved sequence containing two phosphorylatable residues, Y77 and S78. Single mutation in Y77 or S78 reduces SCRATCH2 repression ability while concomitant mutation in both rescue SCRATCH2 function. Our data suggest that the zinc-finger domain is responsible for nuclear retention of SCRATCH2 while residues Y77 and S78 are relevant for the protein correct conformation. In mouse embryonic stem cells neural induced towards corticogenesis, SCRATCH2 KO increases the levels of intermediate progenitors (IP) markers and reduces the level of early born neurons markers. This data suggests that SCRATCH2 plays a role in the maintenance of IP pool, thereby regulating the onset of neural differentiation.

<p>Developmental biology is a field in which explorations are made to answer how an organism transforms from a single cell to a complex system made up of trillions of highly organized and highly specified cells. This field, however, is not just for discovery, it is crucial for unlocking factors that lead to diseases, defects, or malformations. The one key ingredient that contributes to the success of studies in developmental biology is the technology that is available for use. Optical coherence tomography (OCT) is one such technology. OCT fills a niche between the high resolution of confocal microscopy and deep imaging penetration of ultrasound. Developmental studies of the chicken embryo heart are of great interest. Studies in mature hearts, zebrafish animal models, and to a more limited degree chicken embryos, indicate a relationship between blood flow and development. It is believed that at the earliest stages, when the heart is still a tube, the purpose of blood flow is not for convective transport of oxygen, nutrients and waster, bur rather to induce shear-related gene expressions to induce further development. Yet, to this date, the simple question of "what makes blood flow?" has not been answered. This is mainly due limited availability to adequate imaging and blood flow measurement tools. Earlier work has demonstrated the potential of OCT for use in studying chicken embryo heart development, however quantitative measurement techniques still needed to be developed. In this dissertation I present technological developments I have made towards building an OCT system to study chick embryo heart development. I will describe: 1) a swept-source OCT with extended imaging depth; 2) a spectral domain OCT system for non-invasive small animal imaging; 3) Doppler flow imaging and techniques for quantitative blood flow measurement in living chicken embryos; and 4) application of the OCT system that was developed in the Specific Aims 2-5 to test hypotheses generated by a finite element model which treats the embryonic chick heart tube as a modified peristaltic pump.</p> / Dissertation

O cristalino se origina de um epitélio simples e cuboidal que recobre a vesícula óptica. Neste estádio, os filamentos de actina são distribuídos ao longo do eixo apicobasal. As células do ectoderma pré- placodal, em contato com a vesícula óptica, formam um epitélio pseudoestratificado, chamado de placóide do cristalino, com acúmulo de actina no domínio apical. Nós propusemos estudar o papel da proteína PAR3 e sua fosforilação no estabelecimento de actina apical. A superexpressão de PAR3 no placóide forma pontos ectópicos de PAR3 na membrana baso-lateral e induz o recrutamento de actina ectópica para esses pontos. O recrutamento de actina e aPKC ectópicos é independente do estado de fosforilação da treonina 833, resíduo localizado no domínio de ligação do PAR3 ao aPKC. Além disso, no ectoderma peri-placoidal, onde a actina localiza-se baso-lateralmente, PAR3 induz o recrutamento ectópico de actina apical e esse recrutamento é independente da fosforilação da treonina 833. Esses dados nos sugerem que PAR3 é suficiente para recrutar actina no placóide do cristalino. / The lens originates from a simple cuboidal epithelium that overlies the optic vesicle. At this stage, the actin filaments are distributed along its apical-basal sides. The pre-placodal ectoderm, in contact with the optic vesicle, forms a pseudostratified tissue, the lens placode, with accumulation of actin network at the apical domain. Here, we focused on the role of the polarity protein PAR3 and its phosphorylation in the establishment of this apical actin network. Overexpression of PAR3 in the lens placode, induced formation of ectopic actin clusters in the basolateral membrane of the lens placode. The formation of these actin clusters, as well as recruitment of aPKC was independent of Threonine 833 phosphorylation at the PAR3 aPKC-binding site. In addition, PAR3 induced ectopic actin networks in the apical membrane of the periplacodal ectoderm independent of the Threonine 833 phosphorylation. Taken together, these data suggest that PAR3 is sufficient for actin recruitment in the lens placode.

One of the first embryonic structures generated during early human development is the neural tube. The embryonic process of neurulation, including neural tube closure, is necessary for proper brain and spinal cord development, whereas improper closure leads to neural tube defects including anencephaly, spina bifida, and craniorachischisis. The mechanism by which these defects occur is unknown, but some evidence suggest that redox disruption may play a role. Cellular redox state is important in regulating key processes during neural tube closure, including differentiation, proliferation, gene expression, and apoptosis. This study aims to determine whether redox potential shifts and these key processes are affected similarly or differentially after treatment with three neural tube defect-inducing developmental toxicants: ceramide (C2), valproic acid (VPA), and fumonisin (FB1). Using the P19 cell model of neurogenesis, in both undifferentiated and terminally differentiated cells, we analyzed glutathione (GSH) redox (Eh) potential to evaluate the effect of each toxicant over time. We show that in C2 and VPA treated cultures an oxidizing shift occurs, but interestingly, FB1 treatment results in a reducing shift in embryonic GSH Eh as compared to untreated cultures. Using the chick embryo model, comparable redox shifts were observed as were seen in P19 cells, supporting similarity between the models. To better understand how differential shifts in the redox state can result in similar defects, we then examined potential variances in neuronal differentiation and cellular proliferation, survival, metabolism, adhesion, and gene expression under each treatment. We report changes to cellular and embryonic endpoints that support dysmorphogenesis, likely the result of oxidizing or reducing stress that altered redox state. These results support the need for broad comparative analyses such as this to determine whether toxicants that cause the same types of defects, whether NTDs or others, act through similar or different mechanisms. This can better inform preventative measures used to reduce the risk and occurrence of birth defects.

No description available.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Acredita-se que os primeiros progenitores da hematopoese definitiva surjam da diferenciação do endotélio da aorta dorsal, na altura da região da Aorta-Gônada-Mesonefros (AGM). Com o intuito de estudar esta região e o fenótipo das células do endotélio da aorta dorsal nesta posição topográfica, ovos galados de Gallus gallus domesticus L. foram incubados em chocadeira, classificados em estádios de E16 a E25 e processados histotecnologicamente para obtenção de secções seriadas na altura da região AGM. Estas passaram por coloração por Hematoxilina-Eosina, histoquímica para PAS, PAS-diastase e Alcian Blue pH 1.0 e pH 2.5, histoquímica por lectinas fluoresceinadas e imunofluorescência para moléculas de superfície, citoesqueleto e matriz extracelular. Foi observada hipertrofia endotelial no assoalho da aorta nos estádios observados, o qual se apresentava positivo ao PAS, com ocorrência frequente de vacuolizações basais PAS negativas, e o surgimento ocasional de grupamentos celulares intravasculares. Nestes, as células que se destacavam da membrana basal do endotélio expressavam progressivamente mais material PAS positivo, o qual, no entanto, em nenhum momento pareceu se tratar de glicogênio. Em relação às glicosaminoglicanas, notamos a presença predominante de ácido hialurônico por todo o mesênquima da região e em outras estruturas como periferia da notocorda, tubo neural e mesoderma lateral. Ocorreu co-expressão de fibronectina e &#945;-actina de músculo liso em células circunjacentes à aorta, na face ventral do vaso. GFAP e BMP-4 são expressas entre as células do tubo neural e em sua periferia, assim como na notocorda do embrião. As lectinas Abrus precatorius, Lens culinarise Ricinus communis mostraram-se positivas principalmente na região subedotelial do assoalho da aorta nos estádios observados neste trabalho. Bandeiraea simplicifolia exibiu pouca marcação na aorta dorsal e a Arachis hypogeae foi negativa. Outras estruturas da região AGM também expressaram resíduos de açúcares revelados por estas lectinas, tais como: notocorda, tubo neural, mesênquima, intestino primitivo e saco vitelínico. Estes resultados acrescentam elementos morfológicos e bioquímicos ao conhecimento sobre a região AGM de embriões de galinha e sobre o endotélio, possivelmente hemogênico, da aorta dorsal. / Nowadays it is known that firsts definitive hematopoietic progenitors arise from endothelium differentiation of dorsal aorta, at Aorta-Gonad-Mesonephros (AGM) site. In order to study those events and cells phenotype of dorsal aorta endothelium in this topographical site, fertilized eggs of Gallus gallus domesticus L. were incubated, from E16 to E25 and to be processed by histotechonology to obtain serial sections of the AGM site. After this, they were stained with Hematoxilin-Eosin, histochemistry to PAS, diastase PAS and Alcian Blue pH 1.0 and pH 2.5, to obtain a better overview and characterization from cells, also, histochemistry to fluorescein lectins and immunofluorescense to surface molecules, cytoeskeleton andextracellular matrix were performed. Our results showed endothelial hypertrophy of the aorta floor in the stages analyzed, which shown positive for PAS, with frequent appearance of PAS negatives basal vacuolizations and an occasional intravascular cell clusters arise of. It was also observed that cells which were separated from endothelium basal membrane shown progressively were more PAS positive which however in any time seems to be glycogen. Regard of glycosaminoglycans we noted the main presence of hialuronic acid for all the mesenchymes site and in others structures like notochord periphery, neural tube and lateral mesoderm. It was observed fibronectin and smooth muscle &#945;-actin co-expression on aorta surrounding walls, at vessels ventral face. GFAP and BMP-4 are express between the cells of neural tube and surrounding it, as well as at embryo notochord. The lectins Abrus precatorius, Lens culinaris and Ricinus communis showed mostly positive expression on the sub endothelium site of the dorsal aorta floor at the stages analyzed in this work. Bandeiraea simplicifolia showed low expression on dorsal aorta and in Arachis hypogeae it was negative. Other structures of AGMs site also expressed sugar residues revealed by these lectins like: notochord, neural tube, mesenchyme, primitive gut and yolk sac.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Acredita-se que os primeiros progenitores da hematopoese definitiva surjam da diferenciação do endotélio da aorta dorsal, na altura da região da Aorta-Gônada-Mesonefros (AGM). Com o intuito de estudar esta região e o fenótipo das células do endotélio da aorta dorsal nesta posição topográfica, ovos galados de Gallus gallus domesticus L. foram incubados em chocadeira, classificados em estádios de E16 a E25 e processados histotecnologicamente para obtenção de secções seriadas na altura da região AGM. Estas passaram por coloração por Hematoxilina-Eosina, histoquímica para PAS, PAS-diastase e Alcian Blue pH 1.0 e pH 2.5, histoquímica por lectinas fluoresceinadas e imunofluorescência para moléculas de superfície, citoesqueleto e matriz extracelular. Foi observada hipertrofia endotelial no assoalho da aorta nos estádios observados, o qual se apresentava positivo ao PAS, com ocorrência frequente de vacuolizações basais PAS negativas, e o surgimento ocasional de grupamentos celulares intravasculares. Nestes, as células que se destacavam da membrana basal do endotélio expressavam progressivamente mais material PAS positivo, o qual, no entanto, em nenhum momento pareceu se tratar de glicogênio. Em relação às glicosaminoglicanas, notamos a presença predominante de ácido hialurônico por todo o mesênquima da região e em outras estruturas como periferia da notocorda, tubo neural e mesoderma lateral. Ocorreu co-expressão de fibronectina e &#945;-actina de músculo liso em células circunjacentes à aorta, na face ventral do vaso. GFAP e BMP-4 são expressas entre as células do tubo neural e em sua periferia, assim como na notocorda do embrião. As lectinas Abrus precatorius, Lens culinarise Ricinus communis mostraram-se positivas principalmente na região subedotelial do assoalho da aorta nos estádios observados neste trabalho. Bandeiraea simplicifolia exibiu pouca marcação na aorta dorsal e a Arachis hypogeae foi negativa. Outras estruturas da região AGM também expressaram resíduos de açúcares revelados por estas lectinas, tais como: notocorda, tubo neural, mesênquima, intestino primitivo e saco vitelínico. Estes resultados acrescentam elementos morfológicos e bioquímicos ao conhecimento sobre a região AGM de embriões de galinha e sobre o endotélio, possivelmente hemogênico, da aorta dorsal. / Nowadays it is known that firsts definitive hematopoietic progenitors arise from endothelium differentiation of dorsal aorta, at Aorta-Gonad-Mesonephros (AGM) site. In order to study those events and cells phenotype of dorsal aorta endothelium in this topographical site, fertilized eggs of Gallus gallus domesticus L. were incubated, from E16 to E25 and to be processed by histotechonology to obtain serial sections of the AGM site. After this, they were stained with Hematoxilin-Eosin, histochemistry to PAS, diastase PAS and Alcian Blue pH 1.0 and pH 2.5, to obtain a better overview and characterization from cells, also, histochemistry to fluorescein lectins and immunofluorescense to surface molecules, cytoeskeleton andextracellular matrix were performed. Our results showed endothelial hypertrophy of the aorta floor in the stages analyzed, which shown positive for PAS, with frequent appearance of PAS negatives basal vacuolizations and an occasional intravascular cell clusters arise of. It was also observed that cells which were separated from endothelium basal membrane shown progressively were more PAS positive which however in any time seems to be glycogen. Regard of glycosaminoglycans we noted the main presence of hialuronic acid for all the mesenchymes site and in others structures like notochord periphery, neural tube and lateral mesoderm. It was observed fibronectin and smooth muscle &#945;-actin co-expression on aorta surrounding walls, at vessels ventral face. GFAP and BMP-4 are express between the cells of neural tube and surrounding it, as well as at embryo notochord. The lectins Abrus precatorius, Lens culinaris and Ricinus communis showed mostly positive expression on the sub endothelium site of the dorsal aorta floor at the stages analyzed in this work. Bandeiraea simplicifolia showed low expression on dorsal aorta and in Arachis hypogeae it was negative. Other structures of AGMs site also expressed sugar residues revealed by these lectins like: notochord, neural tube, mesenchyme, primitive gut and yolk sac.

This thesis deals with optimization of MRI measuring of phantoms containing the chicken embryo. The theoretical part of thesis is dedicated to the MRI fundamentals and description of pulse sequences. In the next part the options of keeping and monitoring the temperature are being discussed. The thesis also deals with design and realization of phantoms which are used for measuring. A browser of images captured by MRI was programmed in Matlab environment. The program allows users to look through the images with different settings of pulses sequences or kind of phantom. The final part of thesis describes the anatomical structures of chicken embryo and discussion of collected results.