A produção de frangos de corte é um dos maiores segmentos em crescimento no mundo. É um importante fornecedor de proteínas para consumo humano, devido a sua facilidade e rapidez de produção. Devido a essa demanda, surgiram alguns problemas de manejo, que causam o aparecimento de problemas fisiológicos. Os problemas mais relevantes são relacionados com estresse, tanto físico como psicológico. No capítulo 1 estão descritas as alterações nas estruturas das miofibras e a distribuição de água na musculatura de frangos causada pelo estresse térmico em câmara climática. O estresse térmico pode causar alterações na fisiologia das aves, que levam a mudanças na cor, capacidade de retenção de água e maciez dos produtos cárneos, aspectos que influenciam diretamente na aquisição do produto pelo consumidor. Foram avaliados os seguintes parâmetros: hematócrito, creatina quinase plasmática, peso e rendimento de carcaça, vísceras, peito, pernas, asas e percentagem de água livre e ligada no peito. Conclui-se que a redução do hematócrito pode ser uma medida para prevenir uma possível hipovolemia devido à perda de água, principalmente pela hiperventilação. Embora exista efeito na estrutura da miofibra devido ao estresse térmico agudo, a drenagem de água de tecidos e órgãos nesta condição parece não envolver os músculos da ave, especialmente a musculatura do peito. No capítulo 2, estão descritas as modificações estruturais ao nível das proteínas musculares, que compõem a fibra muscular. O estresse térmico agudo causa alterações nas propriedades das miofibrilas, que afetam as características funcionais da carne, principalmente a capacidade de retenção de água. O presente experimento teve como objetivo identificar mudanças na proteólise miofibrilar e migração entre as frações miofibrilar e sarcoplasmática, decorrentes do estresse térmico pré-abate, através do índice de fragmentação miofibrilar (MFI), SDS-PAGE das frações miofibrilar e sarcoplasmática e imunodetecção de vinculina. Concluiu-se que a taxa de fragmentação miofibrilar pode ser prejudicada pelo estresse térmico agudo e que modificações na fração sarcoplasmática são observados em carne pálida, independente da condição ambiental pré-abate. / The production of broiler chickens is one of the biggest segments in growth in the world. It is an important protein supplier for human consumption, due to its easiness and velocity of production. Owed to this demand, some handling problems occurred and cause the appearance of physiological problems. The most important problems are related with stress, in such a way physicist as psychological. In chapter 1 the alterations in the structures of myofibril and the water distribution in the muscle of broilers caused by acute heat stress in climatic chamber are described. Acute heat stress can change birds physiology and leads to alterations in the colour, water holding capacity and tenderness of meat products, factors that affect consumers purchase decision. The following parameters had been evaluated: hematocrit, plasmatic creatine kinase, weight and yield of carcass, visceras, breast, legs, wings, back and percentage of free water in the breast. It is concluded that the hematocrit and creatine kinase can change due to acute heat stress. The weight of carcass and its parts is not altered. The percentage of free and bound water can change due to acute heat stress.In chapter 2, the structural modifications of the muscular proteins are described. The proteins are responsible for the structure of myofibrils and are related with the physical and biochemists processes which determine the characteristics of the meat product. Acute heat stress (AHS) causes alterations in the properties of myofibrils affecting functional characteristics of the meat, mainly the water holding capacity. The present experiment aimed to identify changes in myofibrillar proteolysis and migration between the myofibrillar and sarcoplasmatic fractions due to pre-slaughter heat stress. The myofibrillar fragmentation index (MFI), SDS-PAGE of the muscle protein fractions and western blot of vinculin were used. Its concluded that that AHS can be harmful in the process myofibrillar proteolysis. In the SDS-PAGE we observed modifications in sarcoplasmatic fraction, in pale meat, independent of the environmental condition.

Os grandes avanços obtidos em quantidade de massa muscular nos animais domésticos foram alcançados por programas de melhoramento genético baseados na seleção de características fenotípicas observados na fase adulta do animal. No entanto, o número de precursores miogênicos e conseqüentemente, de fibras musculares, é determinado nos estádios embrionários do desenvolvimento. Este fato direcionou o programa miogênico como alvo de busca de potenciais transcritos candidatos que possam causar impacto sobre a deposição muscular em animais domésticos. Este trabalho objetivou identificar transcritos candidatos originais associados ao desenvolvimento e crescimento do tecido muscular esquelético em frangos (Gallus gallus). Um total de 4.534 transcritos (ESTs) identificados em trabalhos anteriores foram imobilizados em membrana de náilon (microarranjo) para permitir a investigação das diferenças no padrão de expressão entre genótipos e tecidos. O perfil transcricional do tecido muscular peitoral entre linhagens contrastantes para potencial de crescimento e deposição muscular da Embrapa Suíno Aves permitiu identificar 98 transcritos diferencialmente expressos, que foram validados por amplificação quantitativa de transcritos reversos (RT-PCRq), e localizados em regiões coincidentes às anteriormente mapeadas por QTLs. Estes transcritos são agora os candidatos preferenciais para a identificação de mutações e desenvolvimento de marcadores específicos que possam ser empregados em programas de melhoramento e seleção precoce ou indireta de aves. O mesmo microarranjo foi utilizado para caracterizar os transcritos desconhecidos pelo padrão de expressão entre cinco tecidos (cérebro, coração, fígado, músculo e pele). Esta abordagem permitiu identificar transcritos com padrão de expressão tecido-específico e tecido-preferencialmente expressos e caracterizar 24 transcritos desconhecidos como co-expressos aos transcritos músculo esquelético-específicos. Por fim, o orientador de axônio RGM-A (Repulsive guidance molecule A), identificado como músculo esquelético-específico, foi selecionado para ser funcionalmente analisado in vivo, para investigar uma possível função deste transcrito atípico no tecido muscular e comprovar a eficiência dos microarranjos em orientar a seleção de transcritos candidatos. O padrão de expressão de RGM-A em embriões de frango e os efeitos da super-expressão sobre Pax3 indicaram uma possível associação deste orientador de axônio com a formação da musculatura esquelética dos vertebrados superiores. Os transcritos candidatos indicados por análises de microarranjo deste trabalho serão úteis não apenas para que os programas de melhoramento sejam capazes de manter ou aumentar os índices de crescimento das aves, que atualmente buscam por informações adicionais para superar os limites impostos pelo próprio perfil genético alcançado em anos de seleção. Estes resultados também abriram novas perspectivas para a caracterização funcional in vivo de transcritos desconhecidos, tendo embriões de frango como modelo animal. / by animal breeding programs along the years based on the observation of adult phenotypic characters. But, the number of myogenic precursor cells and, consequently, the number of muscle fibers is determined during the embryonic stages of development. Therefore, the embryonic myogenic program is an ideal target for searching potential candidate-transcripts, that can influence skeletal muscle deposition in domestic animals. This work aimed to identify original candidate-transcripts associated with skeletal muscle development and growth in chicken embryos (Gallus gallus). A total of previously identified 4,534 transcripts (Expressed Sequence Tags) were transferred to a nylon membrane (microarray) to investigate differences in expression patterns among genotypes and tissues. The pectoral skeletal muscle transcriptional pattern between broiler and layer chicken lines allowed the identification of 98 differentially expressed transcripts, whose expression were validated by quantitative RT-PCR (Reverse-transcription Polymerase Chain Reaction), and were co-localized in coincident regions with previously mapped QTLs (Quantitative Trait Loci). These transcripts are now preferential candidates to further identify mutations, and/or to develop specific markers to be used in breeding programs for early or indirect selection of superior animals. The same microarray was used to characterize unknown transcripts by expression patterns among five tissues (brain, heart, liver, muscle and skin). This approach allowed to identify tissue-specific and/or preferentially-expressed transcripts, and to characterize 24 unknown transcripts as co-expressed with skeletal musclespecific ones. Finally, the axon guidance molecule RGM-A, identified as skeletal muscle-specific transcript by microarrays, was selected to be functionally analyzed in vivo, to investigate the function of this atypical transcript in the skeletal muscle and to demonstrate the approach efficiency to guide selection of candidate transcripts. The RGM-A expression pattern and the Pax3 phenotype after over-expression in chicken embryos indicated a possible association of this axon guidance with the skeletal muscle formation. The candidate transcripts identified by mycroarray analyses may be useful in animal breeding programs to increase the present gains in chicken growth rate, since additional information is urgently required to surpass current limits of selection gains. The results have also opened a new range of possibilities to promote the functional characterization in vivo of unknown transcripts, having chicken embryos as models.

<p>Heart rate variability (<b>HRV</b>), or the variation in time between adjacent heart beats over time, is a non-invasive proxy measure of autonomic nervous system (<b>ANS</b>) function that has been used regularly in studies focused on evaluating livestock stress and welfare. The autonomic nervous system regulates involuntary physiological processes (<i>e.g.</i> respiration and heart rate) and consists of two main components, the parasympathetic (<b>PNS</b>), and sympathetic (<b>SNS</b>) branches, which act to maintain bodily homeostasis (PNS) or stimulate the “fight-or-flight” response after exposure to a stressor (SNS). Traditional linear HRV measures provide an estimation of overall autonomic activity or changes to the balance between the PNS and SNS branches by evaluating changes to the mean, variance, or frequency spectra of the R-R intervals. </p><p>To interpret HRV data obtained via linear HRV measures, particularly spectral HRV analysis, a linear assumption has to be assumed where SNS and PNS activity act in a purely antagonistic manner. However, this assumption is not always met. In many cases, ANS activity is altered in a nonlinear manner, which is reflected to some degree in the variability of heart rate output. Therefore, HRV measures that evaluate nonlinear changes to organizational or structural aspects of the R-R interval variability may be a useful compliment to traditional linear HRV measures for distinguishing between stressed and non-stressed states. The purpose of this dissertation was to evaluate the use of nonlinear HRV measures for evaluating dairy calf disbudding pain, piglet castration pain, growing pig heat stress, and as potential indicators of the subsequent immune response to a lipopolysaccharide (<b>LPS</b>) challenge in growing pigs.</p><p>Chapter 1 provides a knowledge base for understanding HRV and its use as a measure of autonomic stress in studies with livestock species. A brief explanation of animal welfare science, measures used to evaluate an animal’s welfare, and a demonstration of need for non-invasive physiological measures is provided before discussing the physiological basis of HRV. Relevant linear and nonlinear HRV measures are explained and examples of their use in livestock stress research are provided. Finally, a rationale for the studies conducted in this dissertation is presented.</p><p>Chapter 2 evaluates the use of HRV as an indicator of castration pain in 9-d-old piglets over a 3-d experimental period. Compared to sham castrated piglets, surgically castrated piglets exhibited greater low frequency to high frequency ratios (<b>LF/HF</b>), reduced sample entropy (<b>SampEn</b>), and greater percent determinism (<b>ÞT</b>) during the post-castration period. However, postural behavior was not different between treatments and serum cortisol concentrations only tended to differ between treatments at 1 and 24 h post-castration treatment, with surgically castrated pigs having numerically greater serum cortisol concentrations at both timepoints. These results demonstrate the ability of nonlinear HRV measures (SampEn and ÞT) to complement the physiological interpretation of linear HRV measures (LF/HF) in response to castration. Specifically, pigs who were surgically castrated exhibited more regularity (SampEn) and periodicity (ÞT) in their HRV data, and potentially more sympathetic activity (LF/HF) compared to sham castrated piglets, indicating greater pain-related stress. Additionally, HRV was a more sensitive measure of the stress response to castration than readily identifiable behaviors such as posture and the serum cortisol response.</p><p>Chapter 3 evaluates the use of HRV as an indicator of disbudding pain in dairy heifer calves (4 to 7-wk of age) over a 5-d experimental period. Calves who were given lidocaine and meloxicam prior to disbudding exhibited lower mean R-R interval (<b>RR</b>) values and a greater short-term detrended fluctuation analysis scaling exponent (<b>DFAα₁</b>) than sham disbudded calves. Together, these results indicate that calves who received pain mitigation exhibited greater pain-related stress (RR) and reduced physiological complexity in their heart rate signal (DFAα₁). Calves who were disbudded without pain mitigation had an intermediate response compared to sham disbudded calves and calves provided lidocaine and meloxicam. However, their numerical values closely followed those of calves provided lidocaine and meloxicam. These results demonstrate the usefulness of nonlinear HRV measures (DFAα₁) for evaluating nonlinear and correlational aspects of physiological complexity in response to disbudding. Additionally, the HRV results suggest that the provision of meloxicam does not reduce the amount of pain-related stress experienced by calves following disbudding.</p><p>Chapter 4 evaluates the use of HRV as an indicator of heat stress in growing pigs exposed to an acute heat episode. Heat stressed pigs exhibited greater body temperatures and spent less time in an active position compared to thermoneutral control pigs. Additionally, heat stressed pigs displayed an altered nonlinear HRV response to the acute heat phase compared to non-heat stressed control pigs. Specifically, heat stressed pigs exhibited lower SampEn and tended to exhibit greater ÞT, but no alterations to linear measures were observed in response to the acute heat episode. The low frequency to high frequency ratio was higher in heat stressed pigs during the period following the acute heat phase. Therefore, nonlinear HRV measures (particularly SampEn) may be more sensitive to the immediate physiological stress response to increased environmental temperature than traditional linear HRV measures.</p><p>Chapter 5 evaluates the use of baseline HRV as a potential indicator of the subsequent cortisol and pro-inflammatory cytokine response to an LPS challenge in growing pigs. The time for a pig to approach a human (<b>approach time)</b> prior to LPS administration was inversely related to baseline standard deviation of the R-R intervals (<b>SDNN</b>), and directly related to RR and the mean length of diagonal lines in a recurrence plot (<b>Lmean</b>). This result may have implications for the use of HRV as a measure of temperament in livestock species, since pigs with lower baseline SDNN (<i>i.e.</i> greater stress) and greater baseline Lmean (<i>i.e.</i> increased periodicity length in HRV data; greater stress) values took longer to approach a human observer before LPS administration (which occurred 1 d after HRV measurement). Area under the curve values for approach time following LPS administration were inversely related to high frequency spectral power (<b>HF</b>) and directly related to body weight, where pigs with low baseline HF values (<i>i.e. </i>lower parasympathetic activity) and higher body weights were slower to approach a human observer following LPS administration. Additionally, pigs with greater Lmean values had a greater change in body temperature following LPS administration. In conclusion, while baseline HRV measures were not directly representative of the cortisol or cytokine response following an LPS challenge, HF and Lmean may be useful indicators for evaluating certain aspects (sickness behavior and fever) of the innate immune response to an LPS challenge. <b></b></p><p> In conclusion, these studies demonstrate the usefulness of nonlinear HRV measures for evaluating livestock stress. Measures such as sample entropy and those derived from recurrence quantification analysis (ÞT, Lmean) seem to be particularly useful for complementing traditional linear HRV measures and, in some cases, are more sensitive measures of the physiological stress response (see chapter 4). Therefore, their inclusion in future studies on livestock HRV is warranted. However, further work is needed to fully elucidate the physiological significance of nonlinear HRV measures and their response to stress.</p>

Os grandes avanços obtidos em quantidade de massa muscular nos animais domésticos foram alcançados por programas de melhoramento genético baseados na seleção de características fenotípicas observados na fase adulta do animal. No entanto, o número de precursores miogênicos e conseqüentemente, de fibras musculares, é determinado nos estádios embrionários do desenvolvimento. Este fato direcionou o programa miogênico como alvo de busca de potenciais transcritos candidatos que possam causar impacto sobre a deposição muscular em animais domésticos. Este trabalho objetivou identificar transcritos candidatos originais associados ao desenvolvimento e crescimento do tecido muscular esquelético em frangos (Gallus gallus). Um total de 4.534 transcritos (ESTs) identificados em trabalhos anteriores foram imobilizados em membrana de náilon (microarranjo) para permitir a investigação das diferenças no padrão de expressão entre genótipos e tecidos. O perfil transcricional do tecido muscular peitoral entre linhagens contrastantes para potencial de crescimento e deposição muscular da Embrapa Suíno Aves permitiu identificar 98 transcritos diferencialmente expressos, que foram validados por amplificação quantitativa de transcritos reversos (RT-PCRq), e localizados em regiões coincidentes às anteriormente mapeadas por QTLs. Estes transcritos são agora os candidatos preferenciais para a identificação de mutações e desenvolvimento de marcadores específicos que possam ser empregados em programas de melhoramento e seleção precoce ou indireta de aves. O mesmo microarranjo foi utilizado para caracterizar os transcritos desconhecidos pelo padrão de expressão entre cinco tecidos (cérebro, coração, fígado, músculo e pele). Esta abordagem permitiu identificar transcritos com padrão de expressão tecido-específico e tecido-preferencialmente expressos e caracterizar 24 transcritos desconhecidos como co-expressos aos transcritos músculo esquelético-específicos. Por fim, o orientador de axônio RGM-A (Repulsive guidance molecule A), identificado como músculo esquelético-específico, foi selecionado para ser funcionalmente analisado in vivo, para investigar uma possível função deste transcrito atípico no tecido muscular e comprovar a eficiência dos microarranjos em orientar a seleção de transcritos candidatos. O padrão de expressão de RGM-A em embriões de frango e os efeitos da super-expressão sobre Pax3 indicaram uma possível associação deste orientador de axônio com a formação da musculatura esquelética dos vertebrados superiores. Os transcritos candidatos indicados por análises de microarranjo deste trabalho serão úteis não apenas para que os programas de melhoramento sejam capazes de manter ou aumentar os índices de crescimento das aves, que atualmente buscam por informações adicionais para superar os limites impostos pelo próprio perfil genético alcançado em anos de seleção. Estes resultados também abriram novas perspectivas para a caracterização funcional in vivo de transcritos desconhecidos, tendo embriões de frango como modelo animal. / by animal breeding programs along the years based on the observation of adult phenotypic characters. But, the number of myogenic precursor cells and, consequently, the number of muscle fibers is determined during the embryonic stages of development. Therefore, the embryonic myogenic program is an ideal target for searching potential candidate-transcripts, that can influence skeletal muscle deposition in domestic animals. This work aimed to identify original candidate-transcripts associated with skeletal muscle development and growth in chicken embryos (Gallus gallus). A total of previously identified 4,534 transcripts (Expressed Sequence Tags) were transferred to a nylon membrane (microarray) to investigate differences in expression patterns among genotypes and tissues. The pectoral skeletal muscle transcriptional pattern between broiler and layer chicken lines allowed the identification of 98 differentially expressed transcripts, whose expression were validated by quantitative RT-PCR (Reverse-transcription Polymerase Chain Reaction), and were co-localized in coincident regions with previously mapped QTLs (Quantitative Trait Loci). These transcripts are now preferential candidates to further identify mutations, and/or to develop specific markers to be used in breeding programs for early or indirect selection of superior animals. The same microarray was used to characterize unknown transcripts by expression patterns among five tissues (brain, heart, liver, muscle and skin). This approach allowed to identify tissue-specific and/or preferentially-expressed transcripts, and to characterize 24 unknown transcripts as co-expressed with skeletal musclespecific ones. Finally, the axon guidance molecule RGM-A, identified as skeletal muscle-specific transcript by microarrays, was selected to be functionally analyzed in vivo, to investigate the function of this atypical transcript in the skeletal muscle and to demonstrate the approach efficiency to guide selection of candidate transcripts. The RGM-A expression pattern and the Pax3 phenotype after over-expression in chicken embryos indicated a possible association of this axon guidance with the skeletal muscle formation. The candidate transcripts identified by mycroarray analyses may be useful in animal breeding programs to increase the present gains in chicken growth rate, since additional information is urgently required to surpass current limits of selection gains. The results have also opened a new range of possibilities to promote the functional characterization in vivo of unknown transcripts, having chicken embryos as models.

Os eritrócitos dos mamíferos são anucleados e desprovidos de organelas citoplasmáticas, contando somente com o ciclo da glicólise, o ciclo das pentoses e algumas enzimas anexas, o que garante o fornecimento de energia calórica sob a forma de adenosina-5\'-trifosfato (ATP) e de energia redutora sob a forma de nicotinamida adenosina dinucleotídeo reduzida (NADH), nicotinamida adenosina dinucleotídeo fosfato reduzida (NADPH) e glutationa reduzida (GSH). A via glicolítica possui o desvio denominado de ciclo de Rapaport-Luebering, onde há a síntese de 2,3- difosfoglicerato (2,3-DPG), que é um importante metabólito e atua como modulador da afinidade da hemoglobina ao O2. Havendo poucos estudos comparativos sobre o metabolismo eritrócitário dos mamíferos propôs-se investigar as atividades das enzimas glicolíticas, anexas (2,3-difosfogliceratomutase, glicose-6-fosfato desidrogenase e 6-fosfogliconato desidrogenase) e a concentração dos compostos intermediários adenosina-5\' -trifosfato e 2,3-difosfoglicerato. Mamíferos das ordens Primates, Rodentia, Camivora, Lagomorpha, Artiodactyla, Didelphimorphia e Xenarthra oriundos da Fundação Parque Zoológico de São Paulo e Centro de Bioterismo da Faculdade de Medicina da USP foram investigados. O sangue foi colhido em ACD, os eritrócitos foram lavados em solução fisiológica a 4°C e hemolisados em solução hemolisante 1:20 por congelamento e descongelamento e as atividades das seguintes enzimas foram determinadas de acordo com Beutler (Red Cell Metabolism, a Manual of Biochemical Methods, Ed. Grune & Stratton, 3rd ed, 1984): hexoquinase, glicose fosfato isomerase , fosfofrutoquinase, aldolase, triose fosfato isomerase, gliceraldeído 3-fosfato desidrogenase, fosfoglicerato quinase, monofosfogliceromutase, enolase, piruvato quinase, lactato desidrogenase, bem como a 2,3-difosfoglicerato mutase, glicose-6-fosfato desidrogenase, 6-fosfogluconato desidrogenase, os metabólitos intermediários 2,3-difosfoglicerato e adenosina-5\'-trifosfato. As enzimas e os compostos intermediários estudados apresentaram grande variabilidade entre as espécies de mamíferos estudadas. Foi observada correlação positiva entre a atividade da triose fosfato isomerase e a 2,3-difosfoglicerato mutase e os teores de adenosina-5\'-trifosfato das espécies, bem como correlação positiva entre a 2,3-difosfoglicerato mutase em relação ao 2,3-difosfoglicerato. Os teores de adenosina-5\'-trifosfato mantiveram-se dentro de um patamar estável, ao redor de 4.000 a 6.000 nmoles / gHb, com as exceções das espécies das ordens Carnivora (Panthera leo, Leopardus pardalis, Canis lupus and Chrysocyon brachyurus) e Artiodactyla (Cervus elaphus), que exibiram teores ao redor de 2.000 a 3.000 nmoles / g Hb. Já os valores da concentração de 2,3-difosfoglicerato apresentaram variação considerável entre as espécies e ordens estudadas. / Mammalia red cells are non-nucleated and do not have cytoplasm organeles as well, and present the glycolytc pathway, the pentose shunt to attend the requirements in caloric energy as adenosine-5-triphosphate (ATP) and reducing power as reduced nicotinamide adenosine dinucleotide (NADH) and reduced nicotinamide adenosine dinucleotide phosphate (NADPH) and reduced glutathione (GSH). The glycolytic pathway exhibit besides the Luebering-Rappaport shunt, in which the 2,3-diphosphoglycerate is formed, which regulates the hemoglobin affinity to the molecular oxygen. As there are not so many studies on comparative about mammalian red cell metabolism, it was decided to study the glycolytic enzyme activities, the glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, 2,3-diphosphoglycerate mutase and the metabolites adenosine-5-triphosphate and 2,3-diphosphoglycerate (2,3-DPG). Mammalia representatives from Primates, Rodentia, Carnívora, Lagomorpha, Artyodactyla, Didelphimorphia and Xenarthra orders, obtained from Fundação Parque Zoológico de São Paulo and Centro de Bioterismo da Faculdade de Medicina da USP, were studied. The blood was collected in ACD, the red cells were washed in saline at 4° C, lysed 1:20 in hemolysing solution by freeze-and-thaw, and the following enzymes were assayed according to Beutler (Red Cell Metabolism, a Manual of Biochemical Methods, Ed. Grune & Stratton, 3rd ed, 1984): hexokinase, glucose-6-phosphate isomerase, phosphofructo kinase, aldolase, triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, monophosphoglycerate mutase, enolase, pyruvate kinase, lactate dehydrogenase activities, as well as 2,3-diphosphoglycerate mutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase activities, and adenosine-5-triphosphate, 2,3-diphophoglycerate concentrations. A remarkable variation among the studied species was observed. However, it was detected a significant positive correlation between the adenosine-5-triphosphate concentrations and triosephosphate isomerase and 2,3-diphosphoglycerate mutase activities, as well as significant positive correlation between 2,3-diphosphoglycerate concentration and 2,3-diphosphoglycerate mutase activity in all studied species as a whole. Most of studied species exhibited a steady ATP concentration range between 4,000 and 6,000 nmoles.g Hb -1 but the Artiodactyla (Cervus elaphus) and Carnivora (Panthera leo, Leopardus pardalis, Canis Lupus and Chrysocyon brachyurus,) which presented values between 2,000 and 3,000 nmoles Hb - 1. However, the 2,3-DPG concentration showed remarkable variation among the studied species and orders.

The effect of regular exercise on blood rheology, in both healthy and unhealthy subjects, was examined. To facilitate viscosimetry, a new hematocrit-independent viscosity parameter, named viscous resistance (VR) was defined and methodology developed that abrogates the necessity for hematocrit normalization or manipulation. In addition, in order to evaluate possible mechanisms whereby exercise may influence effective blood ($\rm\mu\sb{eff}$) viscosity, the effects of plasma viscosity (PV), cell free margin (CFM), fibrinogen and RBC aggregation on blood viscosity were examined PV correlates positively with high flow $\rm\mu\sb{eff}$ and VR (20 nl/sec), but not low flow $\rm\mu\sb{eff}$ or VR (0.5 nl/sec). Fibrinogen is an important determinant of PV. CFM positively correlates with $\rm\mu\sb{eff}$ and VR, at both low and high flow rates. RBC aggregation and fibrinogen concentration are important contributors to CFM at low flow rates, but not high flow rates A cross sectional study found regular runners to have lower $\rm\mu\sb{eff}$ and VR, and elevated RBC conductance (C$\sb{\rm rbc}$) compared to non-exercisers. In addition, a prospective rheology study found marathon training to reduce $\rm\mu\sb{eff}$, VR, and PV, and to increase C$\rm\sb{rbc}$. The benefits of running to blood rheology are further supported by the finding that an individual's change in VR and C$\sb{\rm rbc}$, is inversely related to the initial degree of fitness. The change in VR and C$\rm\sb{rbc}$ positively correlates with changes in human performance testing (VO$\sb{\rm 2max}$), which demonstrates the importance of blood viscosity in oxygen delivery and aerobic performance Subjects with coronary heart disease (CHD) were found to have significantly increased $\mu\sb{\rm eff}$ and VR, and decreased C$\sb{\rm rbc}$, compared to age matched healthy controls. A supervised, twelve week, cardiac rehabilitation program reduced the CHD groups' VR, PV, and RBC aggregation index, and increased C$\sb{\rm rbc}$. The rheological improvements are inversely related to the subjects' initial values. The worse a subject's initial rheological profile, the greater the potential benefit from cardiac rehabilitation Elevations in blood viscosity, RBC aggregation, and most importantly, plasma viscosity have all been identified as major risk factors for CHD. The decrease in all three of these parameters with cardiac rehabilitation may at least partially explain the reduction in mortality and morbidity associated with cardiac rehabilitation / acase@tulane.edu

Leptin, the product of the obese (ob) gene, is a potential regulator of development in primate gestation. During pregnancy, maternal serum leptin levels increase prior to changes in body mass and decline rapidly post-delivery, indicating that factors other than adiposity regulate leptin biosynthesis. Therefore, it is hypothesized that maternal hyperleptinemia may be due to enhanced leptin production by adipose tissue under the influence of elevated estrogen levels inherent to primate pregnancy We utilized the baboon (Papio anubis/cynocephalus ), an established model for the study of human pregnancy, to examine the effects of estrogen on leptin biosynthesis during primate pregnancy. Maternal serum leptin levels were highly elevated during pregnancy (P < 0.01) and increased with advancing gestation (P < 0.005). Ob mRNA transcripts and leptin were expressed in placenta and two types of maternal adipose tissue, and both were modulated by estrogen. Thus, leptin production was higher in pregnancy (P < 0.02) and increased with advancing gestation (P < 0.04) in adipose tissue. Conversely, placental ob mRNA transcripts declined from early to late gestation (P < 0.02). Effects were reversed by estrogen withdrawal, as ob mRNA and leptin were higher in placental villous tissue and lower (P < 0.05) in adipose tissue in fetectomized baboons. However, maternal leptin levels were unchanged in fetectomized baboons, suggesting that non-estrogen dependent mechanisms have the potential for maintaining serum levels To examine the molecular mechanism of estrogen action on leptin biosynthesis, in vitro reporter gene assays were employed. MCF-7 breast cancer or JEG-3 choriocarcinoma cells were transfected with a leptin-luciferase construct and treated with estradiol. Although estradiol did not stimulate activity in MCF-7 cells, leptin-luciferase was stimulated by estradiol (P < 0.05) in JEG-3 cells co-transfected with estrogen receptor alpha. Intriguingly, anti-estrogens stimulated leptin-luciferase activity (P < 0.05) in JEG-3 cells co-transfected with estrogen receptor beta, implying that ob promoter activation may be cell specific and dependent upon estrogen receptor expression Results suggest that estrogen modulates leptin biosynthesis in a tissue-specific manner via transcriptional and post-transcriptional mechanisms. Furthermore, results indicate that activation of the ob promoter is cell-specific and dependent upon estrogen receptor expression. Collectively, results suggest that estrogen is a permissive regulator of leptin biosynthesis and that non-estrogen dependent mechanisms and the interplay between leptin and other gestational hormones may be imperative to the regulation of leptin biosynthesis during primate pregnancy / acase@tulane.edu

1,25-Dihydroxyvitamin D$\sb3$ (1,25(OH)$\sb2$D$\sb3$) acts through specific nuclear receptors (VDR) to bring about its biological effects. The following studies were performed to test the hypothesis that 1,25(OH)$\sb2$D$\sb3$ receptors in rat testis are different from those in kidney. Scatchard analysis revealed that VDR sites in testis have a 10-fold higher binding affinity than do the principal VDR sites in kidney. This analysis also demonstrated heterogeneity of VDR sites in kidney, with the lower affinity site being the most abundant Administration of 1,25(OH)$\sb2$D$\sb3$ upregulated VDR in kidney, but not in testis, heart, or lung. Scatchard analysis revealed that 1,25(OH)$\sb2$D$\sb3$ treatment upregulated the low affinity VDR site in kidney, while the high affinity site was no longer detectable. Intestine displayed upregulation of the single, low affinity VDR site, while testis showed no regulation of the single, high affinity site. Dexamethasone administration affected VDR levels as follows: increased in intestine, decreased in kidney, and no change in testis, heart, or lung. Scatchard analysis revealed that dexamethasone treatment upregulated the single, low affinity VDR site in intestine, but did not regulate the single, high affinity site in testis. In kidney, dexamethasone treatment completely downregulated the low affinity binding component, leaving only the high affinity site, whose binding affinity was similar to that of testis Competition for VDR revealed that synthetic analogs Ro24-2287 and KH-1060 display higher affinity for the principal kidney VDR form than for testis VDR, while analog EB-1089 showed the opposite pattern. Although the major VDR forms in kidney and testis showed no size differences based on immunoblot analysis, comparing the ability of VDR sites from kidney and testis to bind to DNA-cellulose revealed differences in the KCl concentration at which peak elution occurred Overall, these studies provide evidence in support of the hypothesis that the principal 1,25(OH)$\sb2$D$\sb3$ receptors in kidney and testis are different. In addition, the kidney appears to contain a second, high affinity binding site, which may be the same as the high affinity site in testis. These data thus provide evidence in support of the novel concept that there are multiple forms of the vitamin D receptor / acase@tulane.edu

Glaucoma is one of the three leading causes of blindness in the U.S., and significantly impairs visual function in 2% of Caucasian and 6% of black Americans over the age of 40. Elevated intraocular pressure (IOP) has long been assumed to play a causative role in glaucomatous damage to the optic nerve head because this neuropathy is most commonly seen in eyes with elevated IOP. We believe IOP is a principal contributing risk factor in glaucoma and susceptibility is determined by anatomic and physiologic factors which govern the biomechanical response of an individual eye to IOP The eye is a pressure vessel, subject to the effects of IOP-related stress at all levels of intraocular pressure. As such, one can model the biomechanics of the eye, employing the finite element method to determine the IOP-related stresses and strains present in the load-bearing tissues of the posterior pole. Finite element modeling requires a three-dimensional geometry of the load-bearing tissues, material properties for those tissues, and loading and boundary conditions To begin this investigation, we have determined the thickness and surface geometry of the sclera in the posterior pole of the perfusion-fixed monkey eye. We have shown that posterior monkey sclera thins in response to chronic exposure to moderately elevated IOP. We have determined the viscoelastic material properties of sclera from rabbit eyes and normal and glaucomatous monkey eyes. We have shown that the material properties of monkey sclera are altered by chronic exposure to elevated IOP. Through finite element modeling of a scleral tensile specimen, we have shown that the finite element method is valid for modeling scleral tissues under load and have validated our experimental measures of scleral material properties. Finally, we have shown that short-term exposure to moderate IOP elevations causes thinning in the posterior scleral shell and alters the viscoelastic material properties of the sclera This work establishes the foundation upon which future finite element models of the posterior pole will be built / acase@tulane.edu

Corneal transparency, on which our vision depends, is a function of the complex physiological, structural, and biochemical interaction of the limiting membranes with the highly organized stromal tissue. Membrane transport of solutes and water provides necessary nutrients, removes waste, and maintains intrastromal pressure The primary goal of this investigation was to characterize the response of the corneal transport system to NaCl osmotic perturbations. A secondary goal was the characterization of the serendipitous discovery of an anomalous corneal swelling response following mechanical epithelial removal To retrieve corneal membrane transport properties from osmotically induced thickness changes, one-dimensional differential and integral models of the corneal transport system, including unstirred water layers were developed. Previous epithelial and newly generated endothelial osmotic perturbation experiments were matched with the computer models. Software was designed to track intrastromal reflective structures and to quantify the anomalous swelling of the anterior cornea following mechanical debridement (under silicone oil) of the epithelium It was shown that unstirred layers did not interfere with membrane parameter extraction, that small ($\pm$15 mOsm) NaCl perturbations elicited a volume regulatory response from the corneal endothelium, and that removal of the epithelium resulted in an anomalous anterior fluid shift Confidence in corneal membrane transport parameters derived by numerical fitting of the thickness response to osmotic shocks has been improved by the characterization of unstirred layer effects. The active response of the endothelial membrane to small perturbations of NaCl demonstrates the existence of a new control mechanism. The anterior fluid shift secondary to mechanical debridement suggests a new etiology for the acute edema following injury to tissue. It is postulated that mechanical damage to the epithelium releases proteases that lyse stromal ground substance subsequently increasing local osmotic pressure / acase@tulane.edu