Em vista das limitações éticas em torno da obtenção de células-tronco de fetos humanos, o cão é uma alternativa para estes estudos. Além disso, a terapia celular proporciona novas expectativas para o tratamento na espécie. Realizamos o estudo comparativo das células isoladas de saco vitelino, fígado e medula óssea de fetos caninos. As células foram analisadas microscopicamente e ultra estruturalmente. O imunofenótipo das células foi avaliado através de marcadores. Caracterizamos a plasticidade, o cariótipo e o potencial teratogênico destas células. Após expansão as células progenitoras formaram colônias com morfologia fibroblastóide. As células progenitoras do saco vitelino e medula óssea são compostas por: células com alta proporção núcleo-citoplasma e células com citoplasma rico em organelas, enquanto que as células progenitoras do fígado eram semelhantes à célula epitelial e células ricas em organelas. As células-progenitoras dos três tecidos fetais foram positivas para os anticorpos nestina e vimentina, mas negativas para CD45 e CD13. Células progenitoras de medula óssea foram positivas para o marcador CD44. Células progenitoras do fígado e medula óssea expressaram a proteína citoqueratina-18, enquanto as do saco vitelino expressaram ve-caderina. Células positivas para Oct3/4 foram detectadas em todas as células progenitoras. As células-progenitoras do saco vitelino e medula óssea diferenciaram-se em tecidos ósseo, cartilaginoso e muscular; já as do fígado para tecido ósseo e muscular. Nenhum tipo celular diferenciou-se em adipócitos. As células progenitoras da medula óssea diferenciaram em células semelhantes a neurônios. Sugere-se a presença de progenitores semelhantes a células mesenquimais e epiteliais. Todas as células mantiveram o cariótipo estável e não formaram tumores. Células progenitoras de medula óssea apresentaram maior capacidade de proliferação e diversidade de diferenciação. Sugere-se que estas células são possíveis candidatas para a terapia celular. / The use the human fetuses for stem cells isolation have ethical limitations. In this context the dog is an excellent candidate to fetal stem cells. Furthermore, these cells can be used in cell therapy of canine diseases We aimed at isolation and comparative characterization of progenitor cells from yolk sac, liver and bone marrow of canine fetuses. Cells were characterized using stem cells antibodies. Differentiation assays as well as karyotype analysis were performed. Teratogenic properties this cells were evaluated. After establishment of primary culture, best proliferation potential was observed in bone marrow progenitor cells. Bone marrow and liver progenitor cells were more efficient in CFU-F assay, then yolk sac progenitor cells. Evidenced by TEM cells with a high nuclear-to-cytoplasmic ratio and cells with cytoplasm rich in organelles. Cells isolated from liver showed epithelial-like morphology and cytoplasm rich in organelles. The yolk sac, liver bone marrow cells reacted positively with nestin and vimentin, being negative to CD45 and CD13 antibodies. Additionally bone marrow progenitor cells were positive to CD44. Bone marrow and liver progenitor cells reacted positively with cytokeratin 18. Yolk sac progenitor cells were positive to ve-cadherin. A few Oct3/4 positive cells were found in yolk sac, liver and bone marrow. Yolk sac and bone marrow progenitor cells showed successful osteogenic, chondrogenic, myogenic differentiation. Differentiation liver progenitor cells were able to bone and muscle cells. The bone marrow progenitor cells were able to produce neuron-like cells. None of progenitor cells showed adipogenic differentiation. The study suggests the presence of mesenchymal-like and epithelial-like progenitor cells. All the karyotype remained and failed to induce the formation of tumors. Stem cells from bone marrow showed high diversity of differentiation than other cell types. It is suggested that these cells are possible candidates for cell therapy.

Orientador: Carlos Ducatti / Resumo: Estudos avícolas usando isótopos estáveis como traçadores em suas dietas fornecem estimativas de quão rapidamente os isótopos estáveis dos tecidos são substituídos por isótopos derivados da dieta. Estabeleceu-se taxas de turnover, meia-vida e substituição total do 13C no sangue, ovo (albúmen + gema), albúmen e gema de aves de postura, pela troca de dietas compostas de plantas dos ciclos fotossintéticos C3 e C4. E avaliou o efeito das dietas quanto ao fracionamento isotópico dieta-tecido e bromatologia do albúmen e da gema. O turnover é influenciado pela qualidade da dieta oferecida a ave. A comparação entre as meias-vidas do sangue, ovo, gema e albúmen, respectivamente, mostra que a dieta RC4 apresentou valores de meia-vida maiores que os outros tratamentos (RC4 = 13,3; 4,8; 5,5 e 5,2d; RC3 = 5,3; 3,8; 4,5 e 2,5d; RMC4 = 10,5; 3,8; 4,8 e 3,2d e RMC3 = 9,2; 3,5; 4,6 e 1,5d). Os tecidos estudados, pelo fato de apresentarem baixo fracionamento isotópico (?dieta-tecido = 0,1 l 0,5%), refletem bem a dieta consumida. Quanto a bromatologia da gema e albúmen não encontrou diferença significativa. / Abstract: Poultry studies using stable isotopes as tracers in their diets supply information of how fast the stable isotopes from tissues are replaced by those diet-derivated. Established turnover rates, half-life and 13C total replacement in blood, egg (albumen + yolk), albumen and egg yolk from lay hens, through exchanging diets composed of C3 and C4 photosynthetic cycle plants. It also evaluated the effect of diets with regard to diet-tissue isotopic fractionation and bromatology of albumen and egg yolk. Turnover is influenced by quality of the diet used to feed the hens. The comparison among the half-lives of the blood, egg, yolk and albumen, respectively, shows that the diet RC4 presented half-life values higher than the other treatments (RC4 = 13,3; 4,8; 5,5 and 5,2days; RC3 = 5,3; 3,8; 4,5 and 2,5days; RMC4 = 10,5; 3,8; 4,8 and 3,2days and RMC3 = 9,2; 3,5; 4,6 and 1,5days). The studied tissues, due to the fact of presenting low isotopic fractionation (?diet-tissue = 0,1 l 0,5%), reflect well the diet intook. With relation to the albumen and yolk bromatology, none significant difference was found. / Mestre

Submitted by Jaqueline Silva (jtas29@gmail.com) on 2014-10-06T19:08:19Z No. of bitstreams: 2 Tese - Jussara de Souza Carneiro - 2013.pdf: 1644698 bytes, checksum: 3faeafe59bfc34f56dc772cbdb1cf640 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-10-06T19:15:39Z (GMT) No. of bitstreams: 2 Tese - Jussara de Souza Carneiro - 2013.pdf: 1644698 bytes, checksum: 3faeafe59bfc34f56dc772cbdb1cf640 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-10-06T19:15:39Z (GMT). No. of bitstreams: 2 Tese - Jussara de Souza Carneiro - 2013.pdf: 1644698 bytes, checksum: 3faeafe59bfc34f56dc772cbdb1cf640 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-08-01 / Aimed at feeding laying hen, corn has been substituted by other cheaper food. However, since these foods have little capacity to stain egg yolk, pigments are usually added to the animal’s feed. Synthetic carotenoids, such as canthaxanthin, and the natural pigments present in bran annatto are among the most used food coloring. However, little is known about the impact of those pigments to the animal’s health, and it is important to point out that the choice of the dye and its dose are based solely on the eggs color. In the present study we aimed at evaluating if the pigments have some beneficial or toxic effects on the laying hen health, and test a new simple, fast and non subjective computerized method to analyze the egg color. Thus, laying hens of the ISA Brown lineage were fed with three different sorghum meal: without dye; with bran annatto (3%); or with cathaxanthin (8 ppm) + apocaroten (25 ppm). Animal performance, eggs quality, the liver and kidney functions, as well as the oxidative stress inferred from the levels of malondialdehyde in red blood cells were determined. None of the treatments altered the laying hen performance, the liver and kidney integrity and functions, and the oxidative stress. However, all the dyes tested increased yolks egg weight and color. Finally, the results clearly suggest that yolks color intensity was more accurately determined by the computerized method when compared to the traditional colorimetric fan assay. / O milho vem sendo substituído por outros alimentos na alimentação de galinhas poedeiras. Contudo, como tais alimentos apresentam baixa capacidade de corar as gemas dos ovos, pigmentantes são comumente adicionados à ração dos animais. Carotenóides sintéticos como cantaxantina, bem como pigmentos naturais encontrados no farelo de urucum estão entre os mais utilizados. Contudo, sabe-se muito pouco sobre o impacto da utilização de tais pigmentantes na saúde das galinhas, além da determinação da dose ser baseada somente na coloração das gemas dos ovos. No presente estudo avaliou-se se os pigmentantes apresentam algum efeito sobre a saúde das galinhas, bem como testou-se um novo método computadorizado, simples, rápido e não subjetivo, para a avaliação da coloração das gemas. Assim, galinhas poedeiras vermelhas foram alimentadas com três diferentes rações a base de sorgo: sem pigmentante; com 3% de farelo de urucum ou com cantaxantina (8 ppm) + apocaroteno (25 ppm). Foram avaliados o desempenho das galinhas, a qualidade dos ovos, as funções renais e hepáticas, e o estresse oxidativo (inferido a partir dos níveis de malondealdeído de eritrócitos). Nenhum dos tratamentos alterou o desempenho das galinhas, a qualidade dos ovos e as funções hepáticas e renais. Contudo, todos os corantes aumentaram a coloração e o peso das gemas dos ovos. Por fim, os resultados sugerem claramente que a intensidade da coloração das gemas foi determinada com maior precisão pelo método computadorizado quando comparado ao ensaio colorimétrico tradicional.

Os roedores murídeos são melhor conhecidos em relação à placentação, porém descrições para outros grupos como os cricetídeos incluindo os \"camundongos do Novo Mundo\" ainda são escassos. Recentemente à placenta e as membranas fetais tem sido considerados fontes promissoras para a obtenção de células-tronco. Em particular, o saco vitelino é estruturalmente diverso e desempenha várias e importantes funções. Por essa razão, o objetivo deste trabalho foi descrever a placentação corioalantóidea e vitelina em Necromys lasiurus. Além de avaliar o potencial do saco vitelino como uma fonte de células-tronco mesenquimais. Para tanto, um total de 10 placentas variando de início ao final de gestação foram analisadas através de técnicas de histologia, imunohistoquímica e microscopia eletrônica, bem como através do cultivo e diferenciação celular, citometria de fluxo e imunocitoquímica. A placenta corioalantóidea discoidal era organizada em uma zona labiríntica, zona juncional e decidua. O labirinto era a região mais importante para as trocas materno-fetal. Próximo ao final da gestação ele apresentou uma barreira hemotricorial com espessura média de 2,41 µm. A zona juncional era composta por sincício e citotrofoblasto. Células trofoblásticas gigantes localizavamse entre a zona juncional e a decidua, assim como nas margens laterais da placenta. A morfologia do saco vitelino visceral invertido variou de acordo com a sua localização e relação com a placenta e o útero. Quando cultivadas, as células aderentes do saco vitelino formaram colônias fibroblastóide (92,13%) e expressaram marcadores de células-tronco mesenquimais e alguns para células precursoras de células-hematopoiéticas. As células apresentaram sucesso quanto às diferenciações osteogênica, adipogênica e condrogênica e não desenvolveram formação tumoral quando injetadas em camundongo nude. Com isso, essas células-tronco despontam como uma fonte terapêutica promissora para a terapia celular. / Murine rodents are well investigated in regard to placentation, but data for other groups such as cricetids including New World mice or Sigmodontinae are sparse. Recently the placenta and fetal membranes are regarded to be promising sources for obtaining stem cells. In particular, the yolk sac is structurally diverse and shows important functions throughout gestation. For this reason, this study aims to describe the chorioallantoic and yolk sac placentation in Necromys lasiurus. In addition, the potential of the yolk sac as a source for mesenchymal stem cells that are not well studied so far will be evaluated. In total, 10 individuals from early gestation to near term were investigated by means of histology, immunohistochemistry and electron microscopy as well as cell culture and differentiation, flow citrometry and immunocytochemistry. The discoidal chorioallantoic placenta was organized in a labyrinth zone, junctional zone, and decidua. The labyrinth was most import for maternal-fetal exchange processes. It possessed a hemotrichorial barrier of about 2.41 µm thickness near term. The junctional zone included syncytial areas and cytotrophoblasts. Trophoblast giant cells were located between the junctional zone and decidua as well as in the lateral margins of the placenta. The morphology of the inverted visceral yolk sac varies according to its location and relationship with the placenta and uterus. When cultured, the adherent cells of the yolk sac formed fibroblastoid colonies (92.13%) and expressed mesenchymal stem cells markers, and some hematopoietic precursor cells markers. They showed successful osteogenic, adipogenic, and chondrogenic differentiation and did not develop tumors when transferred to nude mice. Thus, these cells resulted as stem cells with promising therapeutic values for cell therapy.

O saco vitelino é o único anexo embrionário presente em todas as espécies animais cuja função é manter o desenvolvimento primário do feto até que a placenta assuma esta função. Além disso, ele desempenha funções importantes como: nutrição do embrião, síntese proteica, atividade fagocitária, transferência de materiais e hematopoiese. A utilização de células-tronco embrionárias gera muitas discussões devido às questões éticas associadas à obtenção destas células, além de questões religiosas e também as relações de baixa plasticidade das células-tronco adultas motivam os pesquisadores ao crescente interesse em outras fontes de célulastronco. Com isso, células provenientes de tecidos como as dos anexos embrionários chamam cada vez mais atenção devido à sua facilidade de acesso, crescimento rápido e boa plasticidade. Nosso grupo tem demonstrado que as células do saco vitelino têm boa capacidade de proliferação e são multipotentes. Vários pesquisadores vêm demonstrando que o período no qual as células são obtidas pode ser de fundamental importância para a diferenciação. Sendo assim, neste projeto tivemos como objetivo estabelecer e caracterizar células-tronco de saco vitelino de cães em dois estágios gestacionais, visando analisar de forma comparativa o perfil de expressão de marcadores mesenquimais, hematopoiéticos e pluripotência de forma a entender o comportamento das mesmas. Neste trabalho observamos que as células de saco vitelino obtidas de gestações com 40 e 50 dias aderiram ao plástico, tiveram morfologia fibroblastóide, boa proliferação celular, se diferenciaram em adipócitos, condrócitos e ostéocitos e expressaram alguns marcadores mesenquimais, sugerindo assim ser uma célula-tronco mesenquimal. Além disso, não levaram à formação tumoral quando injetadas em camundongos imunossuprimidos, nudes. / The yolk sac is an embryonic attachment present in all animal species, which function is to keep the primary development of the fetus until the placenta assumes this function. Moreover, it plays important roles as: nutrition of the embryo, protein synthesis, phagocytic activity, transfer of materials and hematopoiesis. The use of embryonic stem cells generates many discussions because of the ethical and religious issues associated with the obtaintion of these cells. Also the relationship of low plasticity of adult stem cells motivates researchers to explore other sources of stem cells. Thus, cells from tissues such as the embryonic anexes call more attention, due to its ease of access, rapid growth and good plasticity. Our group has shown that cells of the yolk sac has good ability to proliferate and are multipotent. Several researchers have shown that the period in which the cells are obtained can be critical for differentiation. Therefore, in this project we aimed to establish and characterize stem cells from dog yolk sac in two stages of pregnancy, in order to analyze comparatively the expression profile of mesenchymal, hematopoietic and plurypotency markers, and study their behavior and plasticity potencial in vitro. In this study we observed that yolk sac cells obtained from pregnancies at 40 and 50 days adhered to plastic, had fibroblast like-morphology, good cell proliferation and differentiated into adipocytes and osteocytes and expressed some mesenchymal markers, therefore suggesting to be a mesenchymal stem cell. Also, they did not lead to tumor formation when injected into immunosuppressed mice, nude.

Calbindin-D28K is a cytosolic calcium binding protein found in a variety of cells that transport calcium. The chorioallantoic membrane and yolk sac of oviparous squamate reptiles (lizards and snakes) transport calcium from the eggshell and yolk to the developing embryo. I used immunohistochemistry to localize calbindin-D28K expression in the chorioallantoic membrane and yolk sac of two species of oviparous scincid lizards, Plestiodon fasciatus and Saproscincus mustelinus. Calbindin-D28K was detected in the chorioallantoic membrane and yolk sac of both lizard species by a polyclonal anti-snake calbindin antibody and a monoclonal anti-cow calbindin antibody. Calbindin-D28K was localized in the chorionic epithelium and allantoic epithelium of the chorioallantoic membrane and in endodermal cells scattered throughout the yolk mass of both species. This is the first demonstration of calbindin-D28K in allantoic epithelium and in endodermal cells of the yolk sac of lizards.

Elevated yolk progesterone has been shown to impair prenatal, but facilitate postnatal auditory learning in bobwhite quail chicks. Elevated yolk testosterone has facilitated prenatal learning, but its effects on postnatal auditory learning in quail are unknown. Either testosterone or an oil vehicle was injected into bobwhite quail eggs prior to incubation. Control eggs were unmanipulated. Following hatching, chicks were exposed to a conspecific maternal call (A or B) for 240 min. At 48 hr, chicks were tested for their preference for the familiarized vs. novel call. All groups demonstrated a preference for the familiar call (p < .05), but minimal between group difference were found. Contrary to previous research, elevated yolk testosterone neither facilitated nor impaired postnatal auditory learning in bobwhite quail chicks. Further research will examine underlying mechanisms responsible for differential effects and explore if similar systems are involved in other species-typical processes such as social motivation. / Includes bibliography. / Thesis (M.A.)--Florida Atlantic University, 2017. / FAU Electronic Theses and Dissertations Collection

The extraembryonic endoderm (XEN) defines the yolk sac, a set of membranes that provide essential support for mammalian embryos. Recently, the committed XENprecursor was identified in the embryonic Inner Cell Mass (ICM) as a group of cells that intermingles with the closely related, anatomically indistinguishable epiblast (EPI)- precursor that gives rise to the fetus. In vitro, the EPI-precursor is represented by the well-known embryonic stem (ES) cell lines, but cell lines representing the XENprecursor are not known. Furthermore, since the XEN-precursor cells were discovered only very recently, the unexpected fact that they express the key pluripotency marker Oct4 has not been explored. Recently, however, our laboratory has isolated rat XEN cell lines that express Oct4, leading to the following two questions: (i) Do these new XEN cell lines represent XEN-precursor cells? (ii) Is their Oct4 expression regulated similarly as previously known from ES cells? These two questions are addressed here by lineage marker and reporter gene analyses. Whole culture analyses showed that rat XEN cell lines expressed markers of all XEN stages including XEN-precursor, primitive endoderm (PrE) and/or visceral endoderm (VE), and parietal endoderm (PE) but trophoectoderm and EPI-precursor markers were missing. In line with this, immunocytochemistry demonstrated heterogeneity and directly visualized the XEN-precursor, PrE/VE, and PE subpopulations. Low-density plating and time-dependent immunocytochemistry on resulting colonies strongly suggested that XEN-precursor cells generate the other XEN stages. Moreover, by analyzing single-cell derived clones, it was shown that culture heterogeneity results from the self-renewal and differentiation of a single cell. Reporter gene analyses using the 5’ regulatory region of the mouse Oct4 gene revealed that a DNA fragment containing the previously described distal enhancer drove reporter gene expression only in ES cells whereas inclusion of an upstream fragment led to high expression in both mouse ES and rat XEN cells. In conclusion, our rat XEN cell lines contain XEN-precursor cells that differentiate extensively, providing for the first time an in vitro model that mimics the natural process of early XEN differentiation. In addition, they regulate Oct4 gene transcription differently than ES cells suggesting heterogeneous Oct4 regulation within the mammalian ICM.

Pre-mature hatching of fertilized eggs of cuttlefishes and squids, which are Taiwan¡¦s major fishing species, exists in the late embryonic development before yolk sacs are fully absorbed. It is so far unknown whether there is any difference in survival rate between pre-maturely developed juveniles and the fully developed ones. Hence, by laboratory incubation, this study aimed to discuss the relationship between the yolk size in the embryonic development process and embryo, as well as the difference in survival rate of juveniles developed at different developmental stages. The impact of the incubation time on the survival rate is explored in case of changing physical and chemical environmental factors (temperature, salinity, ammonia concentration, vibration etc.) The embryonic development of Sepia pharaonis can be divided into 40 stages according to the external shape and quality of the embryo. The embryo mantle length and the yolk diameter vary by time, while the increasing rate of the mantle length does not(F = 1.88, p = 0.06), increasing or decreasing in a linear relationship respectively. However, under the same environmental conditions, the consumption rate and the mantle size may vary in different batches(yolk diameter: F = 8.77, p < 0.01. mantle length: F = 92.14, p < 0.01). There is no difference in the surviving time of juveniles artificially and naturally incubated at the same developmental stage, and the surviving time will be longer if the artificially incubated juveniles are at later embryonic developmental stages(F = 34.66¡Ap < 0.01). With regard to the feeding ratio of juveniles pre-maturely hatched at different stages, the feeding ratio of the juveniles incubated after the 36th stage will increase with the developmental stages(F = 93.10¡Ap < 0.01). In this study, the temperature limit of the embryonic development of Sepia pharaonis should never be lower than 10¢J or higher than 35¢J, and the most suitable temperate range is between 17-28¢J. In case of sudden change in temperature, temperature increase can more effectively affect the pre-mature hatching than temperature decrease. In case of either the 36th or 39th stage embryos, if the temperature rises or drops by more than 10 degrees, pre-mature hatching can exist in some of the embryos. Some embryos may die if salinity is lowered suddenly below 20 psu. Meanwhile, pre-mature hatching may occur within one hour if it is suddenly lowered below 10 psu, and the unhatched embryos may die. When increasing the ammonia concentration suddenly to 1 and 5 ppm, the embryo incubation time may be lengthened compared with the group without the addition: some embryos may die if it is increased to 5 ppm. Meanwhile, most embryos are hatched pre-maturely within 17 minutes when it is increased to 1000 ppm, while those unhatched ones may die. In case of various environmental stimuli of the experiments, a higher percentage of embryos at the 39th stage got away from the hostile environment by pre-mature hatching, while a higher percentage of embryos at 36th stage continued the development until natural incubation or died. The vibration experiment is to produce vibration by a vertical vibration instrument. In case of 30-minute vibration at frequency of 350 times/minute and maximum amplitude at 2 cm, there is no effect on incubation time and mode of embryos at both stages. This factor is still open and subject to further discussion.

During gastrulation, a set of highly coordinated morphogenetic movements creates the shape and internal organization of the embryo. In teleostean fishes, these morphogenetic movements involve not only the embryonic progenitor cells (deep cells) but also two extra-embryonic tissues: an outer sheet of epithelial cells (EVL) and a yolk syncytial layer (YSL). Epiboly is characterized by the spreading of the blastoderm (deep cells and EVL) to cover the large yolk cell, whereas convergence and extension leads, respectively, to mediolateral narrowing and anteroposterior elongation of the embryo. Recent studies have shown that the nuclei of the YSL undergo epiboly and convergence and extension movements similarly to the overlying deep cells, suggesting that these tissues interact during gastrulation. However, it is so far not clear whether and how the movements of YSL nuclei and deep cells influence each other. In the first part of this thesis, the convergence and extension movement of YSL nuclei was quantitatively compared to the movement of the overlying mesendodermal progenitor (or “hypoblast)” cells. This revealed that, besides the similarity in the overall direction of movement, YSL nuclei and hypoblast cell movements display differences in speed and directionality. Next, the interaction between YSL and hypoblast was addressed. The movement of the blastoderm was analyzed when YSL nuclei movement was impaired by interfering with the YSL microtubule cytoskeleton. We found that YSL and blastoderm epiboly were strongly reduced, while convergence and extension were only mildly affected, suggesting that YSL microtubules and YSL nuclei movement are required for epiboly, but not essential for convergence and extension of the blastoderm. We also addressed whether blastodermal cells can influence YSL nuclei movement. In maternal-zygotic one-eyed pinhead (MZoep) mutant embryos, which lack hypoblast cells, YSL nuclei do not undergo proper convergence movement. Moreover, transplantation of wild type hypoblast cells into these mutants locally rescued the YSL nuclei convergence phenotype, indicating that hypoblast cells can control the movement of YSL nuclei. Finally, we propose that the hypoblast influences YSL nuclei movement as a result of shape changes caused by the collective movement of cells, and that this process requires the adhesion molecule E-cadherin.