Submitted by Silvana Teresinha Dornelles Studzinski (sstudzinski) on 2015-11-30T11:09:24Z No. of bitstreams: 1 Jaqueline Brummelhaus_.pdf: 909672 bytes, checksum: a67cf8e4fb697cea521fe93f204b4187 (MD5) / Made available in DSpace on 2015-11-30T11:09:24Z (GMT). No. of bitstreams: 1 Jaqueline Brummelhaus_.pdf: 909672 bytes, checksum: a67cf8e4fb697cea521fe93f204b4187 (MD5) Previous issue date: 2013-11-21 / UNISINOS - Universidade do Vale do Rio dos Sinos / O pinguim-antártico (Pygoscelis antarcticus) tem suas populações distribuídas principalmente nas Ilhas Sandwich do Sul, Georgia do Sul e Shetlands do Sul e na região da Península Antártica. Algumas de suas características são o baixo dimorfismo sexual, a monogamia e o comportamento filopátrico. Esta tese tem como objetivos: 1) quantificar o dimorfismo sexual através de medidas morfológicas, testar uma função discriminante e avaliar o dimorfismo sexual entre duas áreas de reprodução distantes (Ilhas Rei George e Elefante, Shetlands do Sul); 2) caracterizar a distribuição espacial da variabilidade genética populacional entre colônias de reprodução nas Ilhas Rei George e Elefante, através de marcador mitocondrial. Na avaliação do dimorfismo sexual de tamanho, através de caracteres morfológicos, foi encontrado que machos são 6 a 9,4% maiores que as fêmeas e a equação discriminante formulada classifica corretamente 80,6% das aves. Não foi encontrada diferença no dimorfismo sexual entre as colônias de reprodução das Ilhas Rei George e Elefante. Mesmo sendo uma alternativa na determinação sexual, as equações discriminantes devem ser usadas com cautela em locais diferentes do que foram produzidas por causa dos erros de classificação. Quando equações discriminantes das Ilhas Deception e Rei George foram testadas para os dados da Ilha Elefante, obteve-se apenas 67,7% e 71% de acerto. Desta forma, a abordagem molecular é uma opção eficiente na resolução de dúvidas relacionadas à sexagem. Quanto à variabilidade genética com uso de marcador mitocondrial, foram encontrados 38 haplótipos para 61 indivíduos analisados, sendo apenas dois compartilhados nas colônias e todos os demais são exclusivos. Os valores de FST e da AMOVA revelam que a divergência entre as populações é baixa e que a maioria da variação genética (98,3%) ocorreu dentro das populações. Isso poderia ser justificado por um alto fluxo gênico entre as populações, mas não corrobora com o comportamento filopátrico da espécie. Os testes de neutralidade e de expansão demográfica apontam para uma evolução neutra e possibilidade de expansão, que ocorreu há mais de dois milhões de anos atrás e no último um milhão de anos o tamanho efetivo populacional manteve-se constante. Os resultados ressaltam a ocorrência de uma expansão populacional a partir de uma população geneticamente homogênea e a manutenção do tamanho efetivo em longa escala de tempo pode ter amplamente contribuído para a falta de estruturação genética entre as colônias recentes de pinguim-antártico. / Chinstrap penguin (Pygoscelis antarcticus) has their populations distributed mainly in South Sandwich, South Georgia and South Shetlands Islands, and the Antarctic Peninsula. This specie presents low sexual dimorphism, monogamy and philopatric behavior. This thesis aims to: 1) to evaluate sexual dimorphism among males and females and among two breeding areas (King George and Elephant Islands) using morphological characters and to obtain a discriminant function based on the characters that best identify the sex of Chinstrap penguins; 2) to determine the spatial structuring of population genetic variation among breeding colonies at King George and Elephant Islands, using mitochondrial control region. In the assessment of sexual dimorphism using morphological characters, were found that males were 6 to 9.4% larger than females and discriminant equation formulated correctly classifies 80.6% of the birds. There was no difference in sexual dimorphism between the breeding colonies of King George and Elephant Islands. However, the discriminant function should be used with caution in different locations than are produced because penguins may be misclassified. When discriminant equations from Deception and King George Islands were tested for Elephant Island data, we obtained only 67.7% and 71% accuracy. Where there is doubt in the field, it would be interesting to apply molecular sexing technique. For genetic variability using mitochondrial control region, were found 38 haplotypes for 61 individuals analyzed, only two were shared in the colonies and all others are exclusive. FST and AMOVA values revealed that the divergence between populations is low and that most of genetic variation (98.3%) occurred within populations. This could be explained by a high gene flow among populations, but does not corroborate with the philopatric behavior of this specie. The neutrality tests and Mismatch distribution point to a neutral evolution and possibility of expansion, which occurred more 2 Mya and the last 1 Mya, the effective population size remained constant. The results show the occurrence of a population expansion from a genetically homogeneous population and maintenance of effective size in long time scale can have widely contributed to the lack of genetic structuring among the current colonies of Chinstrap penguin.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-10-17T16:03:22Z No. of bitstreams: 1 2015 - Paula Cardoso de Almeida Silva.pdf: 2121155 bytes, checksum: b827c0e7a20bdec7ae36fca85e2c8455 (MD5) / Made available in DSpace on 2016-10-17T16:03:22Z (GMT). No. of bitstreams: 1 2015 - Paula Cardoso de Almeida Silva.pdf: 2121155 bytes, checksum: b827c0e7a20bdec7ae36fca85e2c8455 (MD5) Previous issue date: 2015-11-06 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Embryo transfer and other biotechnologies are intensivelyused for equine reproduction and other species as well. Even though there is an expansion of the mule market and an increase in the number of animals, researches working with reproduction of these animals are still scarce. The aim of this study was to evaluate aspects of embryo transfer, embryo morphology and gender identification of hybrid (horses x donkeys) embryos, checking either their similarity or divergence with the characteristics already known of equine embryos. Attempts of embryo collection on Day 6 -9 after OV were performed in mares previously bred with a P?ga donkey. The embryo recovery rates, the characteristics related to age, morphology and embryonic diameter were evaluated. After these assessments, a group of embryos was cut using an adapted technique, where, the cutting process was carried out with the aid of an ophthalmic scalpel blade. The resulting biggest cut portion was used for embryo transfer, and the smallest parts and the others whole embryos to determination of the embryo gender. Data were analyzed using Fisher's exact test, with 5% significance, except the daily growth of embryos, which was analyzed by linear regression. The overall embryo recovery rate was 55,9% (71/127), and for each group D6, D7, D8 and D9, was 57,1% (28/49), 51% (25/49), 63% (15/24) 60% (3/5), respectively. The developmental stage of the collected embryos were, morula: 18.3% (13/71); early blastocysts: 26,8% (19/71); blastocyst: 29,6% (21/71); and expanded blastocysts: 25,3% (18/71). The measured diameter revealed that the size of the embryos ranged from 147 to 1688?m, the mean diameter of all collected embryos was 438,04?m, and according to different groups, the average size (smallest and largest diameter) of embryos were D6 (n = 29) - 183,4?m (147 - 253?m), D7 (n = 24) - 463,2?m (168 - 886?m), D8 (n = 15) - 727,2?m (422 - 1224?m ) and D9 (n = 3) - 1350,6?m (844 - 1688?m), and daily growth rate was 312,7?m. After the section, the 23 embryos were transfered, only one recipient mare was diagnosed as pregnant at 15 days after ovulation, however after 30 days the embryo was lost. The efficiency of the sex identification by PCR using the primers SRY and ZFX / ZFY was 85,9% (61/71), being 55,7% (34/61) determined as female: and 39,3% (27/61) as male. Embryo transfer has shown to be favorable to aid mule reproduction, whereas the recovery rate and the characteristics of the embryos are similar to equine embryos. Altough, the methodology used to section of embryos had unable the gestational development, the most part of the biopsy derived cutting allowed sexing of the embryos / A transfer?ncia de embri?es e outras biotecnologias reprodutivas s?o cada vez mais utilizadas para a produ??o de equinos e de outros animais, entretanto, mesmo com a expans?o do mercado de muares e com crescimento do n?mero de animais, as pesquisas relacionadas com a produ??o desses animais ainda s?o raras. O presente estudo avaliou as caracter?sticas relacionadas ? transfer?ncia de embri?es, a morfologia e a identifica??o do sexo de embri?es h?bridos (muares), verificando sua semelhan?a ou diverg?ncia com as caracter?sticas j? conhecidas em equinos. Foram realizadas colheitas de embri?es provenientes do cruzamento de ?guas com um jumento P?ga, nos dias 6, 7, 8 e 9 ap?s a ovula??o, a taxa de recupera??o embrion?ria, e as caracter?sticas relacionadas com a idade, morfologia e di?metro embrion?rio foram avaliadas para os diferentes dias. Ap?s essas avalia??es, uma parte dos embri?es coletados foi seccionada, com uma t?cnica adaptada onde o corte foi realizado com o aux?lio de uma l?mina de bisturi oftalmol?gico. A maior por??o resultante do corte foi destinada para transfer?ncia de embri?es e a menor parte, juntamente com os embri?es inteiros, foram utilizados para verificar a efici?ncia dos primers SRY e ZFX/ZFY, na an?lise molecular para a determina??o do sexo dos embri?es. Os dados foram analisados pelo teste Exato de Fisher, com 5% de signific?ncia, exceto o crescimento di?rio dos embri?es que foi analisado atrav?s da regress?o linear. A taxa de recupera??o embrion?ria total foi de 55,9% (71/127), e para os diferentes dias de colheita D6, D7, D8 e D9, foi 57,1% (28/49), 51% (25/49), 63% (15/24), 60% (3/5), respectivamente. Os embri?es coletados apresentavam-se nos seguintes est?gios de desenvolvimento, m?rulas: 18,3% (13/71); blastocistos iniciais: 26,8% (19/71); blastocistos: 29,6% (21/71); e blastocistos expandidos: 25,3% (18/71). O di?metro mensurado revelou que o tamanho dos embri?es variou entre 147 - 1688?m, a m?dia do di?metro de todos os embri?es recolhidos foi de 438,04?m, e de acordo com os diferentes dias de colheita, o tamanho m?dio (maior e menor di?metro) dos embri?es foi de: D6 (n = 29) ? 183,4?m (147 - 253?m), D7 (n = 24) ? 463,2?m (168 - 886?m), D8 (n = 15) ? 727,2?m (422 - 1224?m) e D9 (n = 3) ? 1350,6?m (844 ? 1688?m), e a taxa de crescimento di?ria foi de 312,7?m. Ap?s a sec??o e transfer?ncia de 23 embri?es, apenas uma receptora foi diagnosticada como gestante aos 15 dias, mas aos 30 dias, o embri?o tinha sido absorvido. A identifica??o do sexo atrav?s da t?cnica de PCR, utilizando os primers SRY e ZFx/ZFy foi de 85,9% (61/71), sendo determinado f?meas: 55,7% (34/61) e machos: 39,3% (27/61). A transfer?ncia de embri?es ? favor?vel para auxiliar na produ??o de muares, a taxa de recupera??o e as caracter?sticas dos embri?es s?o semelhantes aos embri?es equinos. A metodologia de sec??o dos embri?es, inviabilizou o desenvolvimento gestacional, entretanto a biopsia oriunda do corte permitiu a sexagem da maioria dos embri?es coletados.

Μελέτες στη Μεσογειακή μύγα έχουν οδηγήσει στον χαρακτηρισμό πέντε άρρενο-ειδικών πρωτεϊνών (MSSPs) οι οποίες είναι ομο- και ετερο- διμερή δύο συγγενών, τύπου α και β, πολυπεπτιδίων (MSSP-α και –β). Τα πολυπεπτίδια αυτά κωδικοποιούνται από τουλάχιστον 7 γονίδια τα οποία με βάση την ομοιότητα των αλληλουχιών τους κατατάσσονται σε 3 ομάδες: msspα (α1 και α2), msspβ (β1, β2 και β3) και msspγ (γ1 και γ2). Λειτουργική ανάλυση των υποκινητών των γονιδίων msspα2 και msspβ2 έδειξε ότι το γονίδιο msspα2 εκφράζεται σε υψηλά επίπεδα στο λιπώδη ιστό των ενήλικων αρσενικών ατόμων ενώ το γονίδιο msspβ2 εκφράζεται σε χαμηλά επίπεδα στο έντερο και των δύο φύλων. Με στόχο την αρρενο- ειδική υπερέκφραση τής αλκοολικής αφυδρογονάσης τής D. melanogaster σε άτομα C. Capitata για την κατασκευή ενός Στελέχους Διαλογής Φύλου στη Μεσογεική μύγα στο οποίο παρουσία υψηλών επιπέδων αλκοόλης θα πεθαίνουν τα θηλυκά άτομα, ενώ τα αρσενικά θα επιβιώνουν, κατασκευάστηκαν διαγονιδιακά στελέχη τα οποία έφεραν το γονίδιο DmadhFAST υπό τον έλεγχο της περιοχής -522/+37 (α2PL) του γονιδίου msspα2. Η κατασκευή των διαγονιδιακών στελεχών έγινε με γενετικό μετασχηματισμό χρησιμοποιώντας το σύστημα μετασχηματισμού Minos. Από την ανάλυση 9 μετασχηματισμένων σειρών σε επίπεδο RNA (Northern ανάλυση και RT-PCR) και σε επίπεδο πρωτεΐνης (Western ανάλυση και Adh assay) προέκυψαν τα εξής συμπεράσματα: α) Ο υποκινητής α2PL είναι κατάλληλος για υψηλή φύλο-ειδική έκφραση διαγονιδίων στα ενήλικα αρσενικά άτομα της Μεσογειακής μύγας και β) Το γονίδιο της ADH της D. melanogaster πιθανόν δεν είναι το κατάλληλο γονίδιο επιλογής για τη δημιουργία στελεχών γενετικού διαχωρισμού του φύλου στη Μεσογειακή μύγα. Επιπλέον, στην παρούσα διατριβή επιλέχθηκε να μελετηθεί ο τρόπος έκφρασης του γονιδίου msspβ1. Όπως και με το msspα2 γονίδιο μελετήθηκε η λειτουργία του msspβ1 γονιδίου, in vivo, σε μετασχηματισμένες σειρές χρησιμοποιώντας το τμήμα -485/+35 (β1PL) του υποκινητή και το γονίδιο lacZ ως γονίδιο αναφοράς. Οι αναλύσεις έκφρασης του διαγονιδίου lacZ, τόσο στο επίπεδο του RNA (RT-PCR) όσο και στο επίπεδο της πρωτεΐνης (β-gal assay), σε 7 διαφορετικές σειρές έδειξαν ότι το τμήμα β1PL είναι αρκετό για την ορθή χρονο- ειδική έκφραση του διαγονιδίου σύμφωνα με το φυσιολογικό πρότυπο έκφρασης των mssp-α και -β γονιδίων, εντούτοις, δεν παρουσιάζει αυστηρά άρρενο-ειδικό πρότυπο έκφρασης, αν και η έκφραση στα αρσενικά άτομα ήταν εντονότερη από ότι στα θηλυκά. Από τη σύγκριση των επιπέδων μεταγραφής του διαγονιδίου lacZ υπό τον έλεγχο του υποκινητή β1PL με εκείνα του διαγονιδίου DmadhFAST υπό τον έλεγχο του υποκινητή α2PL, φαίνεται ότι η ισχύς του υποκινητή του γονιδίου msspβ1 είναι εκατοντάδες φορές μικρότερη από εκείνη του msspα2. Τα αποτελέσματα αυτά σε συνδυασμό με προηγούμενη μελέτη υποδηλώνουν ότι το δεύτερο γονίδιο mssp που εκφράζεται σε υψηλά επίπεδα και με αρρενο-ειδικό τρόπο θα πρέπει να είναι το msspβ3 γονίδιο ή κάποιο άλλο mssp γονίδιο του οποίου το προϊόν είναι ένα MSSP πολυπεπτίδιο τύπου β. / Studies in the medfly have led to the characterization of five male-specific serum proteins (MSSPs) that are homo- and hetero- dimmers of two major polypeptide types, MSSP-α and –β. These polypeptides are coded by at least 7 distinct genes which, based on their homology, are classified in three subgroups, MSSP-α (α1 and α2), MSSP-β (β1, β2 and β3) and MSSP-γ (γ1 and γ2). Functional analysis of the msspa2 and msspβ2 promoters showed that msspa2 is expressed in high levels in the fat body of adult male individuals, whereas msspβ2 is expressed in low levels in the midgut of both sexes. In order to overexpress in a male-specific manner the D. melanogaster alcohol dehydrogenase (ADH) in C. capitata for the construction of a Genetic Sexing Strain (GSS) in the medfly, which in the presence of high alcohol concentration will lead to the death of female and the survival of the male individuals, we constructed transgenic medfly strains using DmadhFAST under the control of the -522/+37 fragment (α2PL) of the msspa2 promoter, via Minos-mediated germline transformation. The RNA analysis (Northern and RT-PCR) and the protein analysis (Western and Adh assay) of 9 transgenic strains showed that: a) The α2PL fragment is sufficient for high levels of male-specific expression of transgenes in the medfly and b) The D. melanogaster ADH gene may not be the appropriate gene for the construction of GSS strains in the medfly. In addition in the present study we performed functional analysis of the msspβ1promoter in vivo in medfly transgenic adults generated by Minos-mediated germ line transformation. For this analysis we used the -485/+37 fragment of the msspβ1 promoter and the lacZ reporter gene. The RNA analysis (RT-PCR) and the protein analysis (b-gal assay) performed on 7 transgenic strains showed that the β1PL fragment is sufficient for the proper time-specific expression of the transgene in accordance to the expression pattern of mssp-a and –β genes, not in male-specific manner, although the level of expression in males was higher than in females. The comparison of the expression levels of the lacZ and the DmadhFAST transgenes showed that the α2PL promoter fragment is a hundred time stronger than the β1PL. These results indicate that msspβ3 or another β type mssp gene may be expressed in high levels in the male adults of the medfly.

A forma e o tamanho de um determinado organismo devem caracterizar aspectos ecológicos, uma vez que a morfometria é resultado da evolução. Diferenças nos caracteres morfológicos podem ter sido causadas por isolamento geográfico, mesmo em períodos de tempo relativamente curtos. O estudo da morfologia ecológica é uma tentativa de compreender a relação funcional entre variação morfológica e a ecologia dos animais. A variação nos atributos morfométricos de tamanho corpóreo entre os sexos pode ser um resultado da ação da seleção sexual. O presente estudo aborda uma comparação intrasexual e entre área continental e insular da morfologia de Conopophaga melanops (Vieillot, 1818), tendo sido realizado em uma área na Ilha Grande e em outra área na Reserva Ecológica Rio das Pedras (ReRP), RJ. A espécie, endêmica de Mata Atlântica e estritamente florestal, apresenta dimorfismo sexual, contudo indivíduos jovens possuem plumagem similar a de fêmeas. As aves foram capturadas com redes neblina, e doze medidas morfométricas foram obtidas de 51 indivíduos. A confirmação do sexo foi realizada por métodos moleculares baseados no DNA em 69 amostras. O percentual de erro na identificação do sexo em campo, pela plumagem, foi de 9,7%. A confirmação molecular do sexo é uma importante ferramenta que têm potencial de revelar padrões demográficos em estudos comportamentais e reprodutivos desta espécie. Na ReRP o comprimento da asa e a variável distância da cabeça até a ponta do bico apresentaram uma diferença significativa, sendo maior para machos do que para fêmeas. Já na Ilha Grande, as únicas variáveis que apresentaram diferença significativa foram comprimento da cauda (maior em machos) e altura do bico na base (maior em fêmeas). As diferenças de tamanho da asa entre os sexos corroboram com padrões de diversas outras espécies Neotropicais. A diferença morfométrica do bico pode estar associada à ecologia alimentar desta espécie. Tanto fêmeas quanto machos foram maiores na ilha do que no continente com relação ao comprimento total e comprimento da asa, além de comprimento da cauda maior para os machos. / Size and shape of a certain organism must characterize ecological aspects, once morphometrics is a result of evolution. Differences on the morphological traits could have been caused by geographical isolation even within short periods of time. Ecological morphology survey is an attempt to understand the relationship between morphological variation and ecology of the animals. Variation in morphometric traits of body size between sexes can be a result of sexual selection. The present study approach intrasexual and mainland-island environment in the morphology of Conopophaga melanops (Vieillot, 1818), been accomplished in an area of Ilha Grande and another in the Rio das Pedras Ecological Reserve (ReRP), RJ State. This species is endemic to the Atlantic Forest and depends to the forest. It has sexual dimorphism, but young individuals have female like plumage. Birds were captured with mist nets and 12 morphometrics traits were measured for 51 individuals. DNA molecular sexing was conducted in 69 blood samples. The percentage of error in field identification of sex by plumage was 9.7%. The molecular sexing is an important tool with potential to reveal demographic patterns in behavior and in reproductive studies of this species. In ReRP Reserve wing length and head and bill variable showed significant difference, being larger in males than in females. Yet in Ilha Grande, the unique variables which showed significant difference were tail length (larger in males) and bill depth at the base (larger in females). The differences in wing length between sexes corroborate with patterns of others Neotropical species. The difference associated to the bill could be related to the feeding ecology of this species. Both females and males were larger on island than in mainland in relation to total body and wing length, the same as tail length in males.

A forma e o tamanho de um determinado organismo devem caracterizar aspectos ecológicos, uma vez que a morfometria é resultado da evolução. Diferenças nos caracteres morfológicos podem ter sido causadas por isolamento geográfico, mesmo em períodos de tempo relativamente curtos. O estudo da morfologia ecológica é uma tentativa de compreender a relação funcional entre variação morfológica e a ecologia dos animais. A variação nos atributos morfométricos de tamanho corpóreo entre os sexos pode ser um resultado da ação da seleção sexual. O presente estudo aborda uma comparação intrasexual e entre área continental e insular da morfologia de Conopophaga melanops (Vieillot, 1818), tendo sido realizado em uma área na Ilha Grande e em outra área na Reserva Ecológica Rio das Pedras (ReRP), RJ. A espécie, endêmica de Mata Atlântica e estritamente florestal, apresenta dimorfismo sexual, contudo indivíduos jovens possuem plumagem similar a de fêmeas. As aves foram capturadas com redes neblina, e doze medidas morfométricas foram obtidas de 51 indivíduos. A confirmação do sexo foi realizada por métodos moleculares baseados no DNA em 69 amostras. O percentual de erro na identificação do sexo em campo, pela plumagem, foi de 9,7%. A confirmação molecular do sexo é uma importante ferramenta que têm potencial de revelar padrões demográficos em estudos comportamentais e reprodutivos desta espécie. Na ReRP o comprimento da asa e a variável distância da cabeça até a ponta do bico apresentaram uma diferença significativa, sendo maior para machos do que para fêmeas. Já na Ilha Grande, as únicas variáveis que apresentaram diferença significativa foram comprimento da cauda (maior em machos) e altura do bico na base (maior em fêmeas). As diferenças de tamanho da asa entre os sexos corroboram com padrões de diversas outras espécies Neotropicais. A diferença morfométrica do bico pode estar associada à ecologia alimentar desta espécie. Tanto fêmeas quanto machos foram maiores na ilha do que no continente com relação ao comprimento total e comprimento da asa, além de comprimento da cauda maior para os machos. / Size and shape of a certain organism must characterize ecological aspects, once morphometrics is a result of evolution. Differences on the morphological traits could have been caused by geographical isolation even within short periods of time. Ecological morphology survey is an attempt to understand the relationship between morphological variation and ecology of the animals. Variation in morphometric traits of body size between sexes can be a result of sexual selection. The present study approach intrasexual and mainland-island environment in the morphology of Conopophaga melanops (Vieillot, 1818), been accomplished in an area of Ilha Grande and another in the Rio das Pedras Ecological Reserve (ReRP), RJ State. This species is endemic to the Atlantic Forest and depends to the forest. It has sexual dimorphism, but young individuals have female like plumage. Birds were captured with mist nets and 12 morphometrics traits were measured for 51 individuals. DNA molecular sexing was conducted in 69 blood samples. The percentage of error in field identification of sex by plumage was 9.7%. The molecular sexing is an important tool with potential to reveal demographic patterns in behavior and in reproductive studies of this species. In ReRP Reserve wing length and head and bill variable showed significant difference, being larger in males than in females. Yet in Ilha Grande, the unique variables which showed significant difference were tail length (larger in males) and bill depth at the base (larger in females). The differences in wing length between sexes corroborate with patterns of others Neotropical species. The difference associated to the bill could be related to the feeding ecology of this species. Both females and males were larger on island than in mainland in relation to total body and wing length, the same as tail length in males.

Made available in DSpace on 2016-08-17T18:39:41Z (GMT). No. of bitstreams: 1 4098.pdf: 4555828 bytes, checksum: eeec91702a4d283144c291e30559f09c (MD5) Previous issue date: 2011-02-18 / In this paper we proposed and analyzed methodologies using the technology of Cell-Free Fetal Nucleic Acid-Free (cffDNA = Cell Free Fetal DNA) in noninvasive prenatal diagnosis (NIPD). Due to the modern technologies employed and their repercussion among the involved families, we sought to discuss some ethical, social and legal implications. Contrary to the popular belief that the placenta forms an impermeable barrier between mother and child, there is bidirectional traffic between the fetus and mother during pregnancy. Several studies have shown that not only intact cells but also fetal cell-free fetal nucleic acids (cffNA, ie, DNA and RNA) cross the placenta and travels in the mother's bloodstream. Four different applications of analysis technology were identified: cffDNA: a) Prenatal Sex Determination, used in pregnancies under the risk of sexual transmitted diseases and performed through the detection of the Y chromosome b) The diagnosis of certain diseases of a single gene through the detection of paternally inherited mutation c) Fetal Aneuploidy, such as Down syndrome, where chromosomal abnormalities may occur d) identifying the type of fetal blood in pregnancies under the risk of incompatibility, especially RhD. To carry out this work it was used the pre-natal determination of sex in 53 pregnant women at different gestational periods. For that it was proposed and tested methods of DNA extraction, amplification of DNA obtained by PCR (Polymerase Chain Reaction) and a new methodology called LAMP (Loop Mediated Isothermal Amplification) and the analysis of final results. Furthermore, the efficiency of LAMP and PCR was compared by amplifying different segments of the Y chromosome: DSY14 and TSPY. In three cases the samples were discarded because there was no fetal sex confirmation after molecular tests due to loss of contact with pregnant women. In two other cases the results pointed to male fetuses whilst the ultrasound confirmed these fetuses as females due to contamination. Finally it was obtained 28 male samples (58.33%) with amplification of the sequences of the Y chromosome and 20 female samples (41.67%) that did not amplify the sequence of the Y chromosome but only for the control. These results showed that the amplification lamp is more efficient than PCR, in the analysis of DSY14, the limit of detection is 10 pg and 0.1 pg for LAMP and PCR respectively. It was concluded that the amplification using the LAMP method is faster (60 min) and has a high sensitivity and specificity and does not require sophisticated equipment for reaction if compared to the PCR method. These characteristics make this methodology feaseable in laboratories with limited resources. / Neste trabalho propusemos e analisamos metodologias empregadas no uso da tecnologia dos Ácidos Nucléicos Livres de Células Fetais (cffDNA = Cell Free Fetal DNA) no diagnóstico pré-natal não invasivo (DPIN = Non-invasive Prenatal Diagnosis). Dada a modernidade das tecnologias empregadas e a sua repercussão nas famílias envolvidas, procuramos discutir algumas implicações éticas, sociais e legais. Ao contrário da crença popular de que a placenta constitui uma barreira impermeável entre mãe e filho, há tráfego bidirecional entre o feto e a mãe durante a gravidez. Vários estudos têm demonstrado que tanto células fetais intactas e ácidos nucléicos livres de células fetais (cffNA, ou seja, DNA e RNA) atravessam a placenta e circulam na corrente sanguínea materna. Identificamos quatro diferentes aplicações da tecnologia de análise do cffDNA: a) Determinação pré-natal do sexo, utilizada em gestações com risco de uma doença ligada ao sexo e realizada através da detecção do cromossomo masculino Y; b) O diagnóstico de certas doenças de gene único, normalmente através da detecção de mutação herdada paternalmente; c) Aneuploidia fetal, tal como a Síndrome de Down, onde há alterações cromossômicas; d) Detecção do tipo de sangue fetal em gestações com risco de incompatibilidade, sobretudo RhD. Para realizar este trabalho utilizamos a determinação pré-natal do sexo em 53 gestantes em diferentes períodos gestacionais. Para tal, testamos e propusemos metodologias de extração do DNA; amplificação do DNA extraído através da técnica da PCR (Polymerase Chain Reaction) e de uma nova metodologia denominada LAMP (Loop Mediated Isothermal Amplification); e a análise final dos resultados. Além disso, comparamos a eficiência da PCR e LAMP amplificando diferentes segmentos do cromossomo Y: DSY14 e TSPY. Em três casos as amostras foram descartadas porque não houve a confirmação do sexo fetal após os testes moleculares devido à perda de contato com as gestantes. Em outros dois casos os resultados apontaram para fetos masculinos, entretanto, no ultra-som eles foram confirmados como femininos, provavelmente devido a uma contaminção. No final obtivemos 28 amostras (58,33%) masculinas, com a amplificação das sequências do cromossomo Y e 20 amostras (41,67%) femininas que não amplificaram a sequência do cromossomo Y e apenas para o controle. Nossos resultados demonstraram que a amplificação por LAMP é mais eficiente que a PCR na análise de DSY14, sendo que o limite de detecção é de 10 pg e 0,1 pg, para a PCR e o LAMP, respectivamente. Concluímos que a amplificação utilizando a metodologia de LAMP é mais rápida (60 minutos) e apresenta uma alta especificidade, sensibilidade e não requer equipamentos sofisticados para reação, comparada com a técnica da PCR. Tais características viabilizam esta metodologia em laboratórios com poucos recursos.

The in ovo sexing of chicken eggs is a current task and a prerequisite to overcome the mass killing of male day-old chicks from laying lines. Although various methods have been developed and tested in recent years, practicable methods for sex determination are still missing which can be applicated in poultry hatcheries before the chicken embryo is capable of nociception and pain sensation. Optical spectroscopic methods enable an early determination of the sex. In this study, a novel method based on two-wavelength in ovo fluorescence excitation is described. More than 1600 eggs were examined. In ovo fluorescence was sequentially excited at 532 nm and 785 nm. The fluorescence intensities of the spectral regions behave inversely with respect to sex. It is shown that the observed sex-related differences in the fluorescence intensities are based on the embryonic hemoglobin synthesis. The accuracy of sex determination is 96% for both sexes. The hatching rate is not reduced compared to an equivalent reference group.

Bislang wurden Bullen in der Milchrinderzucht anhand des Töchterleistungsvergleiches bewertet, was zur Folge hatte, dass die Zuchtwertschätzung erst nach fünf Jahren vorlag (REENTS UND REINHARDT, 2007). Durch die 2010 eingeführte genomische Zuchtwertschätzung werden Bullenkälber bereits in den ersten Lebensmonaten geprüft und die erfolgreichen Bullen ab einem Alter von 12 Monaten zur Spermaproduktion von den Besamungsstationen eingesetzt. Zu bedenken ist dabei, dass Jungbullen unter 2 Jahren lediglich ein durchschnittliches Ejakulatvolumen von 2-4 ml aufweisen und die Spermienkonzentration deutlich unter der von Altbullen liegt. Es müssen daher entweder mehr Jungbullen zum Einsatz kommen oder die Besamungsportionen müssen in ihrer Spermienkon-zentration reduziert werden. Eine am Institut für Nutztiergenetik Mariensee ent-wickelte Methode, der Sperm-Intra-Fallopian-Transfer (SIFT®), ermöglicht es, minimal dosierte Spermaportionen unchirurgisch in den Eileiter zu übertragen (GROSSFELD ET AL., 2011B). Ein wesentlicher Vorteil dieser Methode ist die Re-duzierung des Besamungsvolumens unter Beibehaltung des Volu-men/Mengenverhältnisses. Zielsetzung dieser Arbeit war es, eine in Volumen und Spermienzahl reduzierte Spermaportion neu zu konfektionieren und ein angepasstes Kühl- sowie Gefrierprotokoll zu entwickeln. Hierbei sollten vergleichbare Auftauqualitäten wie in einer üblichen Besamungsportion erreicht werden. Die Qualität des Spermas wurde dabei sowohl unter Laborbedingungen als auch in Testbesamungen an zufällig ausgewählten Färsen und Kühen evaluiert. In ersten Screeningversuchen wurde zunächst ein Tiefgefrierverfahren für gering dosierte Verpackungssysteme (Nanostraw) mit einem Volumen bis 50 µl entwickelt. Dabei sollten vergleichbare Auftauqualitäten üblicher Besamungs-portionen erreicht werden. Es wurden sechs verschiedene Kühlkurven (A-D, D+ und E) entwickelt. Dabei zeigte sich, dass aufgrund der Stickstoff-volumeneinströme ein Haltepunkt bei -8°C in keiner der Einfrierkurven für kleine Volumina zu etablieren war. Dies ist u.a. auf die bauartbedingten Vorgaben des verwendeten Einfrierautomaten zurückzuführen. Ebenso wurde der Zusatz von 7,5% Glyzerin im Verdünner bei dem Gefrierprotokoll D+ aufgrund der Datenlage des Vorversuchs für den Hauptversuch verworfen. Die Proben wurden bullenindividuell erhoben, zur besseren statistischen Absicherung jedoch für die Auswertung zusammengefasst. Aus den Screeningversuchen erschien das Kryokonservierungsprotokoll D+ für den Nanostraw unter Laborbedingungen am geeignetsten und wurde im Thermoresistenztest, der den Bedingungen im Hinblick auf die Temperatur im Uterus bzw. Eileiter ähnlich ist, getestet. Weiterhin fand ein Vergleich der spermatologischen Qualitätsparameter nach dem Auftauen zwischen der Kontroll- und der Nanostrawgruppe mit der Kühlkurve D+ im Thermoresistenztest statt. Im praktischen Teil der Arbeit wurde die Methode SIFT® zunächst anhand eines Versuchs mit geschlechtsdifferenziertem Frischsamen praktiziert. Dabei wurden 20 Färsen mittelseiner Prid®α 1, 55g Progesteron-Spirale und zehn Kühe mit einem Ovsynch Programm synchronisiert. In einem weiteren Besamungsversuch konnte das Sperma aus den Nanostraws verwendet werden, das vorher kryokonserviert worden ist. Es wurden nur spontan brünstige Färsen und Kühe in den Eileiter besamt. Die Untersuchungen führten zu folgenden Ergebnissen: 1) In den Screeningversuchen zeigte sich, dass der Anteil motiler Spermien signifikant (p≤0,05) abhängig von der Gefriergeschwindigkeit der Kühlkurve war. Der höchste Anteil motiler Spermien aus dem Nanostraw war in Kühlkurve D+ mit einem Anteil von 52,9% zu verzeichnen. Eine signifikante Zunahme des Anteils membranintakter Spermien von Kurve A zu B und D zu D+ war zu beobachten. Die Hälfte der Nanostrawspermien zeigten in der Kühlkurve D+ eine intakte Membranintegrität. Die Kontrollspermien lagen bei 56,9%. In Kurve D+ zeigten 66,6% der Spermien aus dem Nanostraw keine morphologischen Veränderungen, in der Kontrollgruppe waren dies 75,4%. Die Änderung der Glyzerinkonzentration im Verdünnungsmedium zeigte keine signifikante Verbesserung der Spermaqualitätsparameter nach dem Auftauen. 2) Im Thermoresistenztest erreichten die Spermien aus dem Nanostraw in allen gemessenen Parametern die Mindestanforderungen an die Spermaqualität nach Rath et al. (2009). So belief sich der Anteil membranintakter Spermien aus dem Nanostraw nach dreistündiger Inkubation bei 37°C auf 56,4%, die Kontrollstrawspermien lagen bei einem Anteil von 67,2%. Dabei zeigte der Faktor Bulle einen signifikanten Effekt (p≤0,01) auf den Anteil motiler und progressiver Spermien sowie den Anteil PI-negativer Spermien. Mögliche Effekte des Bullen auf die Qualität und Befruchtungsfähigkeit der Spermien nach dem Auftauen zeigten andere Versuchsanstellungen und Forschungshypothesen (JANUSKAUSKAS ET AL., 1996, KATHIRAVAN ET AL., 2011). 3) In dem ersten Besamungsversuch wurden 20 Färsen und zehn Kühen geschlechtsdifferenziertes Sperma mit der Methode SIFT® in den Eileiter über-tragen. 37,5% der Färsen und 33% der Kühe wurden tragend. Dabei konnte ein bullenindividueller dosisabhängiger Effekt festgestellt werden. 50% der Kühe, die mit 500.000 Samenzellen inseminiert wurden und 16% der Kühe, die mit 250.000 Samenzellen besamt wurden, wurden tragend. 4) Im Besamungsversuch mit dem kryokonservierten Sperma aus dem Na-nostraw wurden sechs spontan brünstige Rinder mittels SIFT®-Spermien in den Eileiter übertragen. Alle Tiere wurden als Kälber für die Follikelpunktion in einem anderen Versuch verwendet. Fünf Tiere wurden mit dem Bullen A besamt. 40% der Tiere wurden tragend. Lediglich ein Tier wurde mit dem Bullen B besamt. Das Trächtigkeitsergebnis war negativ. Die Kühe wurden mit dem Bullen A besamt, dieser erzielte eine Trächtigkeitsrate von 33%.

Made available in DSpace on 2017-07-10T18:13:29Z (GMT). No. of bitstreams: 1 Daniele M Albuquerque.pdf: 425418 bytes, checksum: 257e51c8fd4dccafbaa92e976f8b7c0e (MD5) Previous issue date: 2011-02-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objective was to evaluate the water quality, performance, phenotypic sexing, corporal indexes, macro and micro composition, proximate composition, partial operating cost of Nile tilapia, GIFT strain, fed with diets formulated with probiotic Bacillus cereus var. Toyoi and Bacillus subtilis C-3102, during 113 days of cultivation in the sex reversal and nursery. Had been used 1200 post-larvae with an average initial weight of 24.7±0.50 mg. Fish were randomly distributed to a system of 24 aquaria of 0.03 m³ in a completely randomized design consisting of three treatments and eight replications, consisting of two probiotics and without probiotic. Initially during the sex reversal was considered an experimental unit, a aquarium containing 50 fish with a volume of 0.03 m³ and during the nursery an aquarium with 30 fingerlings. The data of performance, body índices, indexes, macro and micro composition, partial operation costs were subjected to ANOVA with 5% significance level, and sex ratio data were subjected to chi-square (&#967;2). There were differences (p<0.05) in the parameters of final weight, weight gain, average daily weight gain, specific growth rate, final biomass, biomass gain during sex reversal and nursery. Except for the specific growth, the fishes of nursery showed values of performance higher than sexual reversion. There was no association between the proportion of males and females (&#967;2=1.42, p=0.4926) and inclusion of the probiotics. There were no differences (p<0.05) in the indexes, proximate composition of the fishes fed with probiotics or absent in the rations. The inclusion of any of probiotics was influenced (p=0.00458) on calcium levels under carcass of the juveniles in relation to fish absent of probiotics. The COP was affected significantly (p=0.02159) for the treatment with Bacillus subtilis in its formulation in comparison to fish that were not fed probiotics. There was an increased colonization by bacteria and total coliforms during the nursery in relation to sex reversal (p<0.05). The utilization of the Bacillus subtilis and Bacillus cereus improves the performance and partial operating cost, and doesn t negatively affect the corporal indexes, intestinal microbiology, proximate composition, macro and micronutrients in the cultive fingerlings of Nile tilapia, GIFT strain. / Objetivou-se avaliar qualidade de água, desempenho produtivo, sexagem fenotípica, índices corpóreos, composição centesimal, macro micronutrientes, custos operacionais parciais (COP) e microbiologia intestinal de tilápia do Nilo, linhagem GIFT, alimentadas com ração formulada com probióticos Bacillus cereus var. Toyoi e Bacillus subtilis C-3102, durante 113 dias de cultivo nas fase de reversão sexual e alevinagem. Foram utilizados 1200 pós-larvas com peso médio inicial de 24,7±0,50 mg. Os peixes foram distribuídos aleatoriamente em um sistema de 24 aquários de 0,03 m³ num delineamento inteiramente casualizado composto por três tratamentos e oito repetições, constituído por dois probióticos e, tratamento isento de probiótico. Inicialmente na fase de reversão sexual considerou-se uma unidade experimental um aquário contendo 50 peixes com volume útil de 0,03 m³ e, na fase de alevinagem um aquário contendo 30 peixes. Os dados de desempenho produtivo, índices corporais, composição centesimal, macro e micronutrientes, COP e microbiologia intestinal foram submetidos à ANOVA a 5% de significância e, sexo fenotípico foi submetido ao Qui-Quadrado (&#967;2). Houve diferenças (p<0,05) entre as fases de reversão sexual e alevinagem nos parâmetros peso final, ganho em peso médio diário, crescimento específico, ganho em biomassa. Exceto o crescimento específico, os peixes da fase de alevinagem apresentaram valores de desempenho superiores aos da fase de reversão sexual. Não houve associação entre a proporção de machos e fêmeas (&#967;2 =1,42; p=0,4926) e a inclusão de probióticos. Não houve diferenças (p>0,05) nos índices corporais, composição centesimal dos peixes alimentados com probióticos ou ausentes nas rações. A inclusão de quaisquer dos probióticos foi influenciada (p=0,00458) sobre valores de cálcio na carcaça dos alevinos em relação aos peixes ausentes de probióticos. O COP foi afetado significantemente (p=0,02159) para o tratamento que continha na sua formulação Bacillus subtilis em comparação aos peixes que não foram alimentados de probióticos. Houve uma maior colonização por bactérias totais e coliformes totais na fase de alevinagem em relação à fase de reversão sexual (p<0,05). A utilização de Bacillus subtilis ou Bacillus cereus melhora o desempenho produtivo e custos operacionais parciais e, não afetam negativamente os indices córporeos, microbiologia intestinal, composição centesimal, macro e micronutrientes no cultivo de alevinos de tilápia do Nilo, linhagem GIFT.

Nous définissons l’hybridation littéraire comme la combinaison d’éléments a priori disparates aboutissant à la création d’un ensemble à la fois un et multiple, qui garde les traces de ses parties constitutives tout en étant autre, différent, nouveau. L’hybride englobe les sphères de représentation de ses éléments-parents tout en les dépassant. Par conséquent, l’hybridation tend vers l’extension de toutes les frontières, littéraires et culturelles, dans le but de nous offrir une vision du monde et du sujet toujours plus complète. Dans Oranges are not the Only Fruit, Sexing the Cherry, Written on the Body et The PowerBook de Jeanette Winterson, l’hybridation se manifeste à tous les niveaux du texte. L’hybridité physique et sexuelle des narrateurs est ainsi une des manifestations de leurs identités et vies plurielles et paradoxales. Ces hybrides incarnés (monstres, travestis ou androgynes) se construisant au fil de leurs histoires, qui s’inspirent toujours de récits antérieurs, narrateurs et narrations se démultiplient conjointement, s’entremêlent et se redéfinissent sans cesse. Le texte fluctue au gré de l’hybridation générique et de l’intertextualité. De l’entrecroisement de genres réalistes – tels que l’autobiographie, le récit historique ou le discours scientifique – et fictionnels – tels que le conte ou la romance – naît un hybride générique à résonances parodiques représentant la nature du sujet, de sa vie, de la réalité et de la vérité. Enfin, en hybridant des textes préexistants à des motifs personnels, l’auteur élabore une narration originale qui réécrit les schémas sexistes relayés par ses ancêtres et retranscrit sa vision de l’individu, du monde et de l’art. / We understand literary hybridization as the combination of seemingly different elements resulting in the creation of an entity that is simultaneously single and multiple. Indeed, while it is utterly other and new, the hybrid still shows the marks of its constituents. The hybrid incorporates its ‘parents’’ initial fields of representation while reaching beyond them. Consequently, hybridization is a process that pushes all boundaries, be they literary or cultural, to offer an ever more complete vision of the subject and his/her life. In Jeanette Winterson’s Oranges are not the Only Fruit, Sexing the Cherry, Written on the Body and The PowerBook, hybridization permeates every level of the text. Thus, the narrators’ physical and sexual hybridity is a manifestation of their plural and paradoxical identities. These hybrid creatures (monsters, transvestites or androgynous beings) build their identities through the stories they tell and that are always inspired by existing narratives. Therefore, the narrators and their narratives proliferate conjointly, intermix and redefine each other constantly. The shape of the text fluctuates with generic hybridization and intertextuality. Realistic narratives – such as autobiographical, historical or scientific discourses – and fictional ones – such as fairy tales or romances – interact to produce generic hybrids with parodic undertones that represent the nature of the subject, his/her life, reality and truth. Finally, by hybridizing existing texts and personal literary devices, the author elaborates original narratives that rewrite her ancestors’ sexist discourses and reflect how she perceives the individual, the world and art.