Members of the class Aves possess integumentary structures which distinguish them from other vertebrate lineages. The characteristic integumentary structure that defines the Aves from other vertebrates are the feathers, whose functions include insulation, camouflage, visual display, gliding, and powered flight. The recent discoveries of theropod dinosaur fossils displaying feather-like structures have led to interest in the morphological innovations of the feathers, which are associated with the evolution of flight in Aves. Most modern birds, display a highly ordered, hexagonal arrangement of feather follicles, which aids in the streamlining of the body to increase aerodynamic efficiency. Using the chicken embryo as a developmental model, I address the cellular and molecular processes involved in the initiation and formation of a high fidelity periodic pattern of feather primordia. From my studies, I propose a model in which the induction of individual feather primordia begins with the activation of FGF20 expression. This gene encodes a protein that serves as a chemoattractant. Aggregation of cells towards sources of FGF20 stimulates and reinforces FGF20 expression and also induces the expression of BMP4. Via a reaction-diffusion-like mechanism, BMP4 acts to limit the growth of the cell aggregate and promotes lateral inhibition to prevent fusions between neighbouring feather primordia through transcriptional regulation of FGF20. In order to achieve a high fidelity periodic pattern of feather primordia, three components are required; 1) a competent epidermis displaying β-Catenin and EDAR expression, 2) wave-like propagation of EDA expression, which acts synergistically with β-Catenin expression to activate FGF20 expression at the β-Catenin/EDA junction, 3) and a dermis of sufficient cell density. The spatiotemporal wave-like propagation of EDA expression, specifically, promotes the sequential induction of new feather primordium rows and is associated with the formation of a high fidelity periodic pattern. The importance of these three components appears to be evolutionarily conserved among the Aves and differences in the periodic pattern of feather primordia between species can be explained by how the three components are expressed or regulated in individual species. Independent losses of flight in ratites, such as ostriches and emus, are associated with the loss of feather pattern fidelity. In emus, this loss of pattern fidelity results from the delayed formation of a dermis of sufficient cell density, which prevents the induction of feather primordium formation within the dorsal tract, despite the presence of a fully primed and competent epidermis. These studies demonstrate how the precise feather pattern arises during embryonic development in birds, and how feather patterns can be modified through differential regulation of the molecular and cellular toolkit involved in feather primordium induction in different bird species.

Correct number and pattern of digits is determined in a time and concentration-dependent manner by a gradient of Sonic Hedgehog (SHH) across the anterior-posterior axis of the embryonic limb bud. Owing to the potent morphogenic/mitogenic capabilities of SHH, transcription of the SHH gene in the limb is tightly regulated by feedback loops with other signalling pathways and by the Zone of Polarising Activity regulatory sequence (ZRS). The ZRS is a long-range, cis-regulatory limb-specific enhancer of SHH, and is essential for correct limb SHH expression. The Silkie, a polydactylous breed of chicken, possesses a C > A mutation in the ZRS, resulting in ectopic SHH expression in the anterior limb and hindlimb-specific polydactyly. We employ the Silkie mutant to investigate how SHH is regulated by the ZRS, and how Hedgehog signalling can modulate SHH expression in an autoregulatory manner. We further characterise the effects that the Silkie mutation has on subsequent limb development; investigating the dependence of increased posterior SHH, increased Hedgehog-dependent growth and necessary genotype in both the posterior and anterior limb bud. Several fundamental questions regarding SHH during limb development have yet to be fully addressed: how much SHH protein is present, and does it form a gradient as hypothesised by Wolpert’s Morphogen Gradient Model? By developing a standard curve-based method to assess absolute quantities of processed SHH protein, N-SHH, we find that the quantity of N-SHH protein increases through limb development, and does indeed form a quantifiable gradient across the posterior limb. By comparing quantity of N-SHH protein in equivalently staged mouse, rat, emu and chicken limbs, we find that there is no significant link between N-SHH protein quantity and digit number between mammalian and avian species, and investigate how digit number is modulated in the late limb. A number of species exhibit reduced numbers of digits, including the wings of the emu, cassowary and kiwi. Unlike in mammalian examples of digit loss (i.e. cow, pig) the emu wing has delayed and significantly reduced SHH expression. Through sequencing and functional in vivo testing of ZRS sequences of ratite bird species, we investigate whether the ZRS has a role in evolutionary digit loss. We also demonstrate the aspects of digit loss and Hh signalling are shared with examples of mammalian digit loss. This thesis presents novel research into multiple aspects of genetic regulation, limb development, and evolutionary developmental biology; elucidating both long held dogmas and upcoming areas of limb development.

Made available in DSpace on 2014-06-11T19:32:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-07-19Bitstream added on 2014-06-13T21:03:40Z : No. of bitstreams: 1 ramos_sb_me_jabo.pdf: 520535 bytes, checksum: 6d50ca4d7611871079ff37f8e15c9d68 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Medidas periódicas de peso corporal de avestruzes (Struthio camelus) foram utilizadas para avaliar o desenvolvimento corporal por meio de modelos não lineares e para obter estimativas de herdabilidade para pesos ao nascer (PN), aos 90 (P90) e aos 180 dias de idade (P180). As funções estudadas foram: Brody, Richards, von Bertalanffy, Gompertz e Logístico, pelo método de Gauss Newton, por meio de regressão não-linear das características em função da idade (dias). As estimativas de herdabilidade foram obtidas pelo método de máxima verossimilhança restrita. Todos os modelos não-lineares ajustaram parâmetros para o crescimento corporal, exceto Richards e Brody. O modelo Gompertz foi o mais adequado para representar o crescimento, conforme a interpretação biológica dos parâmetros e a qualidade do ajuste. As estimativas de herdabilidade foram de 0,42±0,05, 0,16±0,04 e 0,24±0,08 para PN, P90 e P180, respectivamente. Todas as características poderão responder à seleção / Periodic measurements of body weight of ostriches (Struthio camelus) was used to evaluate the body development by nonlinear models and to fit heritability estimates of hatched chick weight (CHWT) and live weights recorded at 90 (LW90) and 180 (LW180) days of age. The studied models were: Brody, Richards, von Bertalanffy, Gompertz and Logistic, by Gauss-Newton method, on nonlinear regression of the traits in function of the age (days). The heritability estimates were obtained by restricted maximum likelihood method. All the nonlinear models fitted parameters for body growth, except Richards and Brody. The Gompertz model was the more appropriate to represent the growth, considering the biological interpretation of the parameters and the quality of the fitting. The heritability estimates were 0,42±0,05, 0,16±0,04 and 0,24±0,08 for CHWT, LW90 and LW180 respectively. All the traits can respond to selection

Orientador: Danísio Prado Munari / Banca: Maria Estela Gaglianone Moro / Banca:João Ademir de Oliveira / Resumo: Medidas periódicas de peso corporal de avestruzes (Struthio camelus) foram utilizadas para avaliar o desenvolvimento corporal por meio de modelos não lineares e para obter estimativas de herdabilidade para pesos ao nascer (PN), aos 90 (P90) e aos 180 dias de idade (P180). As funções estudadas foram: Brody, Richards, von Bertalanffy, Gompertz e Logístico, pelo método de Gauss Newton, por meio de regressão não-linear das características em função da idade (dias). As estimativas de herdabilidade foram obtidas pelo método de máxima verossimilhança restrita. Todos os modelos não-lineares ajustaram parâmetros para o crescimento corporal, exceto Richards e Brody. O modelo Gompertz foi o mais adequado para representar o crescimento, conforme a interpretação biológica dos parâmetros e a qualidade do ajuste. As estimativas de herdabilidade foram de 0,42±0,05, 0,16±0,04 e 0,24±0,08 para PN, P90 e P180, respectivamente. Todas as características poderão responder à seleção / Abstract: Periodic measurements of body weight of ostriches (Struthio camelus) was used to evaluate the body development by nonlinear models and to fit heritability estimates of hatched chick weight (CHWT) and live weights recorded at 90 (LW90) and 180 (LW180) days of age. The studied models were: Brody, Richards, von Bertalanffy, Gompertz and Logistic, by Gauss-Newton method, on nonlinear regression of the traits in function of the age (days). The heritability estimates were obtained by restricted maximum likelihood method. All the nonlinear models fitted parameters for body growth, except Richards and Brody. The Gompertz model was the more appropriate to represent the growth, considering the biological interpretation of the parameters and the quality of the fitting. The heritability estimates were 0,42±0,05, 0,16±0,04 and 0,24±0,08 for CHWT, LW90 and LW180 respectively. All the traits can respond to selection / Mestre

Submitted by Erika Demachki (erikademachki@gmail.com) on 2015-03-09T10:52:16Z No. of bitstreams: 2 Dissertação - Renato Clini Cervi - 2014.pdf: 5637849 bytes, checksum: 220f849214b2dacc6a1ac6cba29e1ce1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Rejected by Erika Demachki (erikademachki@gmail.com), reason: on 2015-03-09T10:52:35Z (GMT) / Submitted by Erika Demachki (erikademachki@gmail.com) on 2015-03-09T10:53:43Z No. of bitstreams: 2 Dissertação - Renato Clini Cervi - 2014.pdf: 5637849 bytes, checksum: 220f849214b2dacc6a1ac6cba29e1ce1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2015-03-09T10:54:03Z (GMT) No. of bitstreams: 2 Dissertação - Renato Clini Cervi - 2014.pdf: 5637849 bytes, checksum: 220f849214b2dacc6a1ac6cba29e1ce1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-03-09T10:54:03Z (GMT). No. of bitstreams: 2 Dissertação - Renato Clini Cervi - 2014.pdf: 5637849 bytes, checksum: 220f849214b2dacc6a1ac6cba29e1ce1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-02-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The egg is an animal product with excellent nutritional value used in human food. The Ema (Rhea americana) is an animal of the Brazilian wildlife with potential to produce eggs that differ from other species in its microbiological resistance and percentages of nutrients. In order to obtain information about the chemical composition, resistance to Salmonella Enteritidis, and appropriate forms of storage for preservation of quality, were evaluated five lots of eggs from emus to assess quality in periods of seven , 14 , 21, 28 and 35 days of storage at refrigeration temperature , and three lots on 10 , 20 and 30 days to contamination of Salmonella Enteritidis. According to the results, it can be concluded that the internal quality changes with storage time, remaining viable up to 21 days of storage. The fractions of the egg change with protein degradation, and significant reduction in quality values, expressed in Haugh Unit. The eggs had high resistance to contamination by salmonella Enteritidis in all storage periods. Protein fractions of white and yolk and rheological parameters show differences from chicken egg, providing information to appropriate use of these products by the industry. / O ovo é um produto de origem animal de excelente valor nutricional utilizado na alimentação humana. A Ema (Rhea americana) é um animal da fauna silvestre brasileira com potencial de produção de ovos que diferem de outras espécies em sua resistência microbiológica e porcentagens de nutrientes. Com o objetivo de obter informações sobre a composição centesimal, resistência à Salmonella Enteritidis, e formas de armazenamento adequadas para preservação de qualidade para o consumo humano, foram analisados cinco lotes de ovos de emas para avaliação da qualidade, em períodos de sete, 14, 21, 28 e 35 dias de armazenamento a temperatura refrigerada, e três lotes em períodos de 10, 20 e 30 dias para contaminação de Salmonella Enteritidis De acordo com os resultados obtidos pode-se concluir que a qualidade interna se altera com o tempo de armazenamento, permanecendo viável até 21 dias de armazenamento. As frações do ovo sofreram alterações com a degradação protéica, e redução significativa dos valores de qualidade expressos em unidade Haugh. Os ovos apresentam alta resistência à contaminação por Salmonella em todos os períodos de armazenamento. As frações protéicas de albúmen e gema e os parâmetros reológicos apresentaram diferenças em relação ao ovo da galinha, fornecendo informações para adequação da utilização destes produtos pela indústria.

Unambiguous gender identification (ID) is needed to assess parameters in studies of population dynamics, behavior, and evolutionary biology of Caribbean Flamingo (Phoenicopterus ruber ruber) and other birds. Due to its importance for management and conservation, molecular (DNA-based) avian gender ID assays targeting intron-size differences of the Chromosome Helicase ATPase DNA Binding (CHD) gene of males (CHD-Z) and females (CHD-W) have been developed. Male (ZZ) and female (WZ) genotypes are usually scored as size polymorphisms through agarose or acrylamide gels. For certain species, W-specific restriction sites or multiplex polymerase chain-reaction (PCR) involving CHD-W specific primers are needed. These approaches involve a minimum of three steps following DNA isolation: PCR, gel electrophoresis, and photo-documentation, which limit high throughput scoring and automation potential. In here, a short amplicon (SA) High-resolution Melting Analysis (HRMA) assay for avian gender ID is developed. SA-HRMA of an 81-Base Pair (bp) segment differentiates heteroduplex female (WZ) from homoduplex male (ZZ) genotypes by targeting Single-nucleotide Polymorphisms (SNPs) instead of intron-size differences between CHD-Z and CHD-W genes. To demonstrate the utility of the approach, the gender of Caribbean Flamingo (P. ruber ruber) (17 captive from the Dallas Zoo and 359 wild from Ria Lagartos, Yucatan, Mexico) was determined. The assay was also tested on specimens of Lesser Flamingo (P. minor), Chilean Flamingo (P. chilensis), Saddle-billed Stork (Ephippiorhynchus senegalensis), Scarlet Ibis (Eudocimus ruber), White-bellied Stork (Ciconia abdimii), Roseate Spoonbill (Platalea ajaja), Marabou Stork (Leptoptilos crumeniferus), Greater Roadrunner (Geococcyx californianus), and Attwater's Prairie Chicken (Tympanuchus cupido attwateri). Although the orthologous 81 bp segments of Z and W are highly conserved, sequence alignments with 50 avian species across 15 families revealed mismatches affecting one or more nucleotides within the SA-HRMA forward or reverse primers. Most mismatches were located along the CHD-Z gene that may generate heteroduplex curves and thus gender ID errors. For such cases, taxon and species-specific primer sets were designed. The SA-HRMA gender ID assay can be used in studies of avian ecology and behavior, to assess sex-associated demographics and migratory patterns, and as a proxy to determine the health of the flock and the degree by which conservation and captive breeding programs are functioning.