Dissertations / Theses on the topic 'Bovine retina'

Background: Diabetic vascular complications affect both the macro- and microvasculature. Microvascular pathology in diabetes may be mediated by biochemical factors that precipitate cellular changes at both the gene and protein levels. In the diabetic retina, vascular pathology is found mainly in microvessels, including the retinal precapillary arterioles, capillaries and venules. Macular oedema secondary to breakdown of the inner blood-retinal barrier is the most common cause of vision impairment in diabetic retinopathy. Müller cells play a critical role in the trophic support of retinal neurons and blood vessels. In chronic diabetes, Müller cells are increasingly unable to maintain their supportive functions and may themselves undergo changes that exacerbate the retinal pathology. The consequences of early diabetic changes in retinal cells are primarily considered in this thesis. Aims: This thesis aims to investigate the effect of perivascular cells (Müller cells, RPE, pericytes) on retinal endothelial cell permeability using an established in vitro model. Methods: Immunohistochemistry, cell morphology and cell growth patterns were used to characterise primary bovine retinal cells (Müller cells, RPE, pericytes and endothelial cells). An in vitro model of the blood-retinal barrier was refined by coculturing retinal endothelial cells with perivascular cells (Müller cells or pericytes) on opposite sides of a permeable Transwell filter. The integrity of the barrier formed by endothelial cells was assessed by transendothelial electrical resistance (TEER) measurements. Functional characteristics of endothelial cells were compared with ultrastructural morphology to determine if different cell types have barrier-enhancing effects on endothelial cell cultures. Once the co-culture model was established, retinal endothelial cells and Müller cells were exposed to different environmental conditions (20% oxygen, normoxia; 1% oxygen, hypoxia) to examine the effect of perivascular cells on endothelial cell permeability under reduced oxygen conditions. Barrier integrity was assessed by TEER measurements and permeability was measured by passive diffusion of radiolabelled tracers from the luminal to the abluminal side of the endothelial cell barrier. A further study investigated the mechanism of laser therapy on re-establishment of retinal endothelial cell barrier integrity. Müller cells and RPE, that comprise the scar formed after laser photocoagulation, and control cells (Müller cells and pericytes, RPE cells and ECV304, an epithelial cell line) were grown in long-term culture and treated with blue-green argon laser. Lasered cells were placed underneath confluent retinal endothelial cells growing on a permeable filter, providing conditioned medium to the basal surface of endothelial cells. The effect of conditioned medium on endothelial cell permeability was determined, as above. Results: Co-cultures of retinal endothelial cells and Müller cells on opposite sides of a permeable filter showed that Müller cells can enhance the integrity of the endothelial cell barrier, most likely through soluble factors. Low basal resistances generated by endothelial cells from different retinal isolations may be the result of erratic growth characteristics (determined by ultrastructural studies) or the selection of vessel fragments without true â barrier characteristicsâ in the isolation step. When Müller cells were co-cultured in close apposition to endothelial cells under normoxic conditions, the barrier integrity was enhanced and permeability was reduced. Under hypoxic conditions, Müller cells had a detrimental effect on the integrity of the endothelial cell barrier and permeability was increased in closely apposed cells. Conditioned medium from long-term cultured Müller cells and RPE that typically comprise the scar formed after lasering, enhanced TEER and reduced permeability of cultured endothelial cells. Conclusions: These studies confirm that bovine tissues can be used as a suitable model to investigate the role of perivascular cells on the permeability of retinal endothelial cells. The dual effect of Müller cells on the retinal endothelial cell barrier under different environmental conditions, underscores the critical role of Müller cells in regulating the blood-retinal barrier in health and disease. These studies also raise the possibility that soluble factor(s) secreted by Müller cells and RPE subsequent to laser treatment reduce the permeability of retinal vascular endothelium. Future studies to identify these factor(s) may have implications for the clinical treatment of macular oedema secondary to diseases including diabetic retinopathy.

Doctor of Philosophy (Medicine)
Background: Diabetic vascular complications affect both the macro- and microvasculature. Microvascular pathology in diabetes may be mediated by biochemical factors that precipitate cellular changes at both the gene and protein levels. In the diabetic retina, vascular pathology is found mainly in microvessels, including the retinal precapillary arterioles, capillaries and venules. Macular oedema secondary to breakdown of the inner blood-retinal barrier is the most common cause of vision impairment in diabetic retinopathy. Müller cells play a critical role in the trophic support of retinal neurons and blood vessels. In chronic diabetes, Müller cells are increasingly unable to maintain their supportive functions and may themselves undergo changes that exacerbate the retinal pathology. The consequences of early diabetic changes in retinal cells are primarily considered in this thesis. Aims: This thesis aims to investigate the effect of perivascular cells (Müller cells, RPE, pericytes) on retinal endothelial cell permeability using an established in vitro model. Methods: Immunohistochemistry, cell morphology and cell growth patterns were used to characterise primary bovine retinal cells (Müller cells, RPE, pericytes and endothelial cells). An in vitro model of the blood-retinal barrier was refined by coculturing retinal endothelial cells with perivascular cells (Müller cells or pericytes) on opposite sides of a permeable Transwell filter. The integrity of the barrier formed by endothelial cells was assessed by transendothelial electrical resistance (TEER) measurements. Functional characteristics of endothelial cells were compared with ultrastructural morphology to determine if different cell types have barrier-enhancing effects on endothelial cell cultures. Once the co-culture model was established, retinal endothelial cells and Müller cells were exposed to different environmental conditions (20% oxygen, normoxia; 1% oxygen, hypoxia) to examine the effect of perivascular cells on endothelial cell permeability under reduced oxygen conditions. Barrier integrity was assessed by TEER measurements and permeability was measured by passive diffusion of radiolabelled tracers from the luminal to the abluminal side of the endothelial cell barrier. A further study investigated the mechanism of laser therapy on re-establishment of retinal endothelial cell barrier integrity. Müller cells and RPE, that comprise the scar formed after laser photocoagulation, and control cells (Müller cells and pericytes, RPE cells and ECV304, an epithelial cell line) were grown in long-term culture and treated with blue-green argon laser. Lasered cells were placed underneath confluent retinal endothelial cells growing on a permeable filter, providing conditioned medium to the basal surface of endothelial cells. The effect of conditioned medium on endothelial cell permeability was determined, as above. Results: Co-cultures of retinal endothelial cells and Müller cells on opposite sides of a permeable filter showed that Müller cells can enhance the integrity of the endothelial cell barrier, most likely through soluble factors. Low basal resistances generated by endothelial cells from different retinal isolations may be the result of erratic growth characteristics (determined by ultrastructural studies) or the selection of vessel fragments without true ‘barrier characteristics’ in the isolation step. When Müller cells were co-cultured in close apposition to endothelial cells under normoxic conditions, the barrier integrity was enhanced and permeability was reduced. Under hypoxic conditions, Müller cells had a detrimental effect on the integrity of the endothelial cell barrier and permeability was increased in closely apposed cells. Conditioned medium from long-term cultured Müller cells and RPE that typically comprise the scar formed after lasering, enhanced TEER and reduced permeability of cultured endothelial cells. Conclusions: These studies confirm that bovine tissues can be used as a suitable model to investigate the role of perivascular cells on the permeability of retinal endothelial cells. The dual effect of Müller cells on the retinal endothelial cell barrier under different environmental conditions, underscores the critical role of Müller cells in regulating the blood-retinal barrier in health and disease. These studies also raise the possibility that soluble factor(s) secreted by Müller cells and RPE subsequent to laser treatment reduce the permeability of retinal vascular endothelium. Future studies to identify these factor(s) may have implications for the clinical treatment of macular oedema secondary to diseases including diabetic retinopathy.

Mice immunised with partially purified surface molecules of photoreceptor outer segments or with a synthetic peptide corresponding to the N-terminus of a cone-specific rhodopsin-like protein, gave rise to the production of monoclonal antibodies with only general immunoreactivity against the retina. Immunisation with interphotoreceptor matrix or crude photoreceptor outer segment preparations resulted in the production of hybridomas which secreted monoclonal antibodies 1001.A1 and 1001.A3. 1001.A1 binds to the interstitial retinol-binding protein (IRBP), associated with the rod photoreceptor cells and the IPM, as demonstrated by immunohisto chemistry and by Western blotting and dot blotting of IPM and purified IRBP. 1001.A3 binds a soluble high molecular weight chondroitin sulphase proteoglycan. Immunohistochemistry indicated the antigen to be present in the form of distinct sheath-like structures surrounding the photoreceptor cells. Gel filtration chromatography both in native conditions and in the presence of guanidinium chloride showed the antigen to have an apparent molecular mass of greater than 2000kDaltons and indicated the antigen was not a loosely associated aggregate of smaller components. Binding of 1001.A3 to fixed tissue sections of the bovine retina was completely abolished by their prior treatment with either chondroitinase ABC, chondroitinase AC, hyaluronidase (testicular) or trypsin. Prior treatment of tissue sections with either heparinase or neuraminidase had no effect on binding. Treatment of tissue sections with hyaluronidase (Streptomyces) had no effect on the ability of 1001.A3 to bind, but the structure of the antigen was altered. The sheath-like structure surrounding the photoreceptors was broken down and immunoreactivity was seen in the same area of the IPM, adjacent to the photoreceptors but with no defined structure. In conclusion, the antigen is a chondroitin sulphate molecule which is associated with hyaluronic acid molecules and which together form a defined sheath-like structure surrounding the photoreceptor cells.

La purification des facteurs de croissance du fibroblaste (fgf) acide et basique chez le poulet au cours du developpement embryonnaire a permis de confirmer la grande conservation des fgf au cours de l'evolution. Le photorecepteur est la cellule qui contient la plus grande activite mitogenique specifique en fgf

Biochemical and immunological studies indicate that rod outer segments (ROS) of bovine photoreceptor cells contain a Mr 240,000 polypeptide related to the ∝-subunit of red blood cell (RBC) spectrin. With the use of sodium dodecyl sulfate gel electrophoresis in conjunction with the immunoblotting technique, monoclonal antibody 4B2 was found to bind to a Mr 240,000 polypeptide in ROS that is distinct from the prominent Mr 220,000 concanavalin A binding glycoprotein. The Mr 240,000 polypeptide is highly susceptible to degradation by endogenous proteases. It does not appear to be an integral membrane protein but is tightly membrane associated since it can be partially extracted from ROS membranes with urea in the absence of detergent. The 4B2 antibody cross-reacted with RBC ghost membranes and bovine brain microsomal membranes. Radioimmune assays and immunoblotting analysis of purified bovine RBC spectrin further revealed that the 4B2 antibody predominantly labelled the ∝-chain of RBC spectrin having an apparent Mr of 240,000. Monoclonal antibody 3A6 was found to bind to a polypeptide with a slightly lower Mr than the 4B2-specific polypeptide. It is also highly susceptible to degradation by endogenous proteases, but unlike the 4B2 antibody, it predominantly labelled the β-chain of RBC spectrin having an apparent M of 220,000. Polyclonal anti-spectrin antibodies that bound to both the ∝ - and β-chain of RBC spectrin predominantly labelled a Mr 240,000 polypeptide of ROS membranes. Two faintly labelled bands in the Mr range of 210,000-220,000 were also observed. These components may represent variants of the β -chain of spectrin that are weakly cross-reacting or present in smaller quantities than the ∝-chain. Immunocytochemical labelling studies using the 4B2 antibody and immunogold-dextran markers indicated that the ROS spectrin-like protein is preferentially localized in the region where the discs come in close contact to the plasma membrane of ROS. Immunoblotting analysis indicated that rhodopsin and peripherin which constitute over 90% of total disc membrane proteins were selectively solubilized in Triton X-100, whereas a set of polypeptides including the 4B2-specific polypeptide and the Mr 220,000 concanavalin A-binding glycoprotein was only partially soluble. Electron microscopy of a negatively stained Triton-extracted ROS pellet revealed a filamentous network. These studies indicate that ROS contain a protein related to RBC spectrin, which may constitute a major component of a filamentous network lining the inner surface of the ROS plasma membrane as previously seen by electron microscopy. This membrane skeletal system may serve to stabilize the ordered ROS structure and maintain a constant distance between the rim region of the discs and the plasma membrane.
Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate

Rhodopsin, der Rezeptor der visuellen Kaskade, gehört zu größten Klasse A der G-Protein-koppelnden Rezeptoren (GPCRs) und gilt als Modell-Rezeptor in der GPCR-Forschung. Über 3 % des humanen Genoms kodieren für GPCRs, doch trotz der physiologischen Bedeutung dieser Proteinfamilie sind die fundamentalen Mechanismen, mit denen diese Rezeptoren extrazelluläre Signale in das Zellinnere weiterleiten noch nicht verstanden. In der vorliegenden Dissertation werden Aspekte des Aktivierungsmechanismus von Rhodopsin sowie der Kopplung und Aktivierung des G-Proteins Transduzin untersucht. Die Arbeit ist in drei Schwerpunkte unterteilt: I. Es wurde ein in GPCR’s hochkonserviertes NPxxYx(5,6)F Motiv (Aminosäuresequenz Asn-Pro-x-x-Tyr-x(5,6)-Phe) in der siebten und achten Helix charakterisiert. In diesem konservierten Motiv sind mehrere für die Ausbildung der aktiven Rezeptorkonformation wichtige Funktionen vereint: Verknüpfung zu einem Wasserstoffbrückennetzwerk, Helixflexibilität sowie die exakte Positionierung der achten Helix. Letzteres hat nicht nur bei der Rezeptoraktivierung sondern auch bei der nachfolgenden Interaktion mit dem G-Protein eine Bedeutung. II. Anhand von chimären Rezeptoren, bei denen Teile der achten Helix durch homologe Sequenzen des beta2-adrenergen Rezeptors ausgetauscht wurden, wurde die Rolle der achten Helix bei der Rezeptor-Aktivierung und Bindung des G-Proteins untersucht. Auch bei dieser Studie wurde gezeigt, dass die exakte Positionierung der achten Helix essentiell für die Interaktion mit dem G-Protein ist. Zudem wurde ein bezüglich der G-Protein-Aktivierung funktionsfähiger chimärer Rezeptor gefunden, was auf einen übergeordneten Mechanismus bei der Aktivierung von G-Proteinen durch GPCRs hindeutet. III. Die Funktion des ß-Ionon-Rings des Retinals beim Aktivierungsmechanismus von Rhodopsin wurde an einem Retinal studiert, bei welchem Teile des Retinal-Rings fehlten (azyklisches Retinal). Auch diesem azyklischen Retinal können Eigenschaften eines partiellen Agonisten zugeschrieben werden. Beim Vergleich zu Pigmenten mit dem nativen 11-cis-Retinal wurden starke Analogien bei der initialen Energieaufnahme durch die Retinal-Isomerisierung sowie bei der Weiterleitung der Lichtenergie ins Protein gefunden. Allerdings wird die Energie schlechter auf das Protein übertragen, wodurch wesentlich weniger der aktiven G-Protein bindenden Rezeptorkonformation gebildet wird. Als wichtigste Funktion des Retinal-Rings wurde die Aufrechterhaltung der aktiven Meta-II-Konformation identifiziert.
Rhodopsin, the receptor of the visual cascade, belongs to the largest group A of G-protein coupled receptors (GPCRs) and can be seen as a model receptor in GPCR research. More than 3 % of the human genome code for GPCRs. But despite their physiological relevance, the detailed mechanism of signal transduction from extra cellular signal to different cellular pathways remains to be fully understood. Different aspects of receptor activation and the coupling and activation of the G-protein transducin are investigated in this dissertation. The thesis focuses on the following three subjects: I. A NPxxYx(5,6)F motif (amino acid sequence Asn-Pro-x-x-Tyr-x(5,6)-Phe) has been characterized for rhodopsin. It is localized in helix VII and VIII and is highly conserved throughout the GPCR family. Various roles for rhodopsin activation are combined in this motif: linkage to a hydrogen-bond network, helix flexibility and the exact positioning of helix VIII. The latter is not only relevant for the activation of the receptor but also for interaction with its G-protein. II. The role of helix VIII for receptor activation and G-protein coupling was studied on chimeric receptors, in which parts of helix VIII were exchanged against homologous sequences of the beta2 adrenergic receptor. This study confirmed the importance of helix VIII’s position for G-protein coupling. Furthermore, a chimeric receptor was found, which was fully functional concerning G-protein activation. This indicates that GPCRs might use a single, generic mechanism for G-protein activation. III. The role of the ß-ionone-ring for the activation mechanism of rhodopsin was studied by means of an acyclic retinal, which lacks four carbon atoms of the ß-ionone-ring. This modified retinal could be classified as a partial agonist for rhodopsin. Energy input by retinal isomerization and formation of the G-protein binding Meta-II conformation were found to be very similar to rhodopsin when bound to its native 11-cis-retinal. However, the lack of the ring structure resulted in a lower amount of Meta-II and a fast decay of activity. It was concluded that the main role of the ring structure is to maintain the active state of rhodopsin.

The ageing tips of vertebrate rod photoreceptors, (known as rod outer segments, or ROS), are phagocytosed by the retinal pigment epithelial (RPE) cells, which lie in close apposition to them. It has been shown by several workers, using in vitro RPE cell cultures, that ROS bind to the RPE cells with a higher affinity than other particles. On the basis of this and other evidence in the literature, it has been proposed that there is a 'ROS receptor' on the RPE plasma membrane, and a corresponding ligand on the ROS plasma membrane. In this project, an attempt has been made to identify and characterise the ROS plasma membrane ligand. At an early stage in this project, another research group published an abstract in which they proposed that a high molecular weight ROS integral plasma membrane protein, now known to be a Na+/Ca2+ exchanger, was the ligand referred to above. In this project, the proteins of bovine ROS membranes were analysed by SDS-PAGE. It appeared that one particular Coomassie blue staining band, by virtue of its molecular weight, might have been the Na+/Ca2+exchanger and putative ligand. Moreover, the results of a vectorial ROS membrane protein radio-iodination study were apparently consistent with the band representing a plasma membrane spanning protein, rather than a protein located in the much more abundant ROS disc membranes, which are enclosed by the plasma membrane. However, neither an extension of the vectorial radio-iodination study, which involved the chemical cleavage of the radiolabelled Coomassie staining band into peptides, nor a Ficoll Flotation study, in which an attempt was made to fractionate ROS membranes into disc and plasma membrane components, provided evidence that the Coomassie staining band referred to above represented a plasma membrane spanning protein.

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The vitamin A is essential to animals because of its participation in a great number of biological functions. The investigation of this vitamin s concentrations is important to serve as reference to normality parameters. This study had as aim to analyse the serics and hepatics concentrations of vitamin A in two groups of bovines and to compare the hepatics concentrations to the present requeriments of vitamin A for pregnant women. It was also appraised the consume habit of bovine liver by pregnant women through of the alimentary frequency quest. Two groups of bovine were studied and the first was formad by Nelore bovine breed and the second by bovine without defined breed (WDB). It was analysed 120 samples: 60 of liver and 60 of serum. The method used to dose retinol was High Performance Liquid Cromatography (HPLC). The average (+ sd) of retinol concentrations in Nelore breed bovine and WDB liver were 16947,8 + 6866,9 and 5213,1 + 2517,2 ?g of retinol/100g and at serum 39,6 + 17,9 e 28,6 + 9,4 ?g of retinol/dL, respectively. No statistically significant correlation was found between hepatic and the serum retinol. The bovines in this study had adequate vitamin A levels. Independently of animal breed, the daily ingestion of bovine liver is not advised for pregnant women who show adequate support of vitamin A. The consume of bovine liver by pregnant women consulted on school maternity hospital Janu?rio Cicco, UFRN, Natal RN, was considered high
A vitamina A ? essencial aos animais devido sua participa??o em uma s?rie de fun??es biol?gicas. A investiga??o das concentra??es desta vitamina ? importante para servir como refer?ncia para par?metros de normalidade. O objetivo do presente estudo foi analisar as concentra??es s?ricas e hep?ticas de vitamina A em dois grupos de bovinos e comparar as concentra??es hep?ticas com os atuais requerimentos desta vitamina para gestantes. Avaliou-se tamb?m o h?bito de consumo de f?gado bovino por mulheres gestantes atrav?s do question?rio de freq??ncia alimentar. Dois grupos de bovinos foram estudados sendo o primeiro formado por bovinos da ra?a Nelore e o segundo por bovinos sem ra?a definida (SRD). Analisou-se 120 amostras: 60 de f?gado e 60 de soro. O m?todo utilizado para dosar retinol foi a Cromatografia L?quida de Alta Efici?ncia (CLAE). As m?dias (+ dp) das concentra??es de retinol no f?gado dos bovinos da ra?a Nelore e SRD foram 16947,8 + 6866,9 e 5213,1 + 2517,2 ?g de retinol/100g e no soro 39,6 + 17,9 e 28,6 + 9,4 ?g de retinol/dL, respectivamente. N?o foi encontrada correla??o estatisticamente significativa entre o retinol hep?tico e o s?rico. Os bovinos do presente estudo se encontravam com aportes adequados de vitamina A. Independentemente da ra?a do animal, a ingest?o di?ria de f?gado bovino ? desaconselhada para mulheres gr?vidas que apresentam aportes adequados de vitamina A. O consumo de f?gado bovino por mulheres gestantes atendidas na Maternidade Escola Janu?rio Cicco, UFRN, Natal RN, foi considerado alto

L'objet de ce travail a consiste en une etude biochimique du canal cationique active par la gmpc des batonnets retiniens de bovins. (1) des reactifs bloqueurs des fonctions thiol ou capables de former des ponts disulfure entre deux cysteines proches ont un double effet sur l'activite des canaux: a faible concentration ils provoquent une augmentation de l'affinite apparente du canal pour le nucleotide cyclique (activation) et une perte de la cooperativite de liaison du nucleotide. A de plus fortes concentrations, ils entrainent une reduction de l'amplitude et de la vitesse initiale maximales des efflux calciques induits par le nucleotide cyclique. Les resultats suggerent que le fait de bloquer au moins une des 3 cysteines cytoplasmiques, situees au voisinage ou dans le site de liaison du nucleotide cyclique, ou la formation d'un pont disulfure entre deux sous-unites adjacentes du canal entrainent l'activation. L'inhibition resulterait du blocage d'au moins une des deux autres cysteines (par les bloqueurs des -sh) ou des deux autres (formation d'un pont disulfure intramonomerique). La liaison du nucleotide cyclique engendrerait un changement conformationnel au niveau des sites nucleotidiques du canal. (2) le phenomene dit d'inactivation, qui se traduit par une liberation partielle du contenu ionique des vesicules membranaires a des concentrations non saturantes en nucleotide cyclique, ne peut s'expliquer par une desesibilisation du canal, ni par une inactivation consecutive a l'apparition d'un potentiel membranaire apres addition du nucleotide cyclique, ni par un blocage du canal par les ions ca#2#+ extracellulaires. Ce phenomene pourrait s'expliquer par la variation de la porportion de vesicules capables de decharger leur contenu ionique. (3) la sous-unite beta du canal peut etre phosphorylee dans les membranes natives

Folha de aprova??o n?o presente na vers?o entregue.
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O estudo foi conduzido com o objetivo de determinar os par?metros cin?ticos de degradabilidade in situ da mat?ria seca (MS) e da fibra em detergente neutro (FDN) e de passagem de materiais oriundos da silagem do res?duo da cultura do abacaxi (partes a?reas). Foram utilizados quatro tratamentos: 1- silagem do res?duo da cultura do abacaxi sob compacta??o de 600 Kg/m?, 2- silagem do res?duo da cultura do abacaxi sob compacta??o de 700 Kg/m?, 3- silagem do res?duo da cultura do abacaxi sob compacta??o de 900 Kg/m?, 4- silagem do res?duo da cultura do abacaxi sob compacta??o de 1000 Kg/m?. Ap?s a ensilagem do material oriundo da planta??o, avaliaram-se a cin?tica de tr?nsito de part?culas e cin?tica de degrada??o ruminal. Para a cin?tica de tr?nsito de part?culas foi utilizado como indicador o cromo para marcar a fibra. As taxas de passagem foram determinadas por meio da recupera??o dos indicadores nas fezes do animal. No ensaio de degrada??o foram incubadas as amostras em bolsas de n?ilon nos tempos 0, 6, 18, 48 e 96 horas. O comportamento apresentado nas curvas de regress?o das vari?veis analisadas descreve uma alta correla??o entre elas, ou seja, o tempo em que a silagem fica retida no r?men exerce influ?ncia na sua digestibilidade e na sua taxa de degrada??o. De fato, quanto maior o tempo em que um alimento estiver no r?men, mais a??es digestivas ele sofrer? e por consequ?ncia vai ser mais digerido. Embora a silagem sob press?o de compacta??o de 900Kg/m? apresente uma maior fra??o potencialmente digest?vel, recomenda-se o uso da silagem sob press?o de compacta??o de aproximadamente 750Kg/m?, devido ao menor custo e por apresentar menor tempo m?dio de reten??o r?men ret?culo e reple??o ruminal, aumentando assim a din?mica ruminal de degrada??o e passagem.
Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Zootecnia, Universidade Federal dos Vales do Jequitinhonha e Mucuri, [2011].
ABSTRACT The study was conducted to determine the kinetic parameters of in situ dry matter (DM) and neutral detergent fiber (NDF) and the movement of materials from the waste silage of pineapple crop (aerial parts). It was used four treatments: 1- crop residue silage pineapple under compression of 600 kg/m?, 2- silage crop residue pineapple under compression of 700 kg/m?, 3- silage crop residue pineapple under compression 900 kg/m?, 4- silage crop residue pineapple under compression of 1000 kg/m?. After ensiling the material from the plantation, it was evaluated the kinetics of passage of particles and kinetics of ruminal degradation. For the kinetics of passage of particles was used as an indicator to mark the chromium fiber. Leakage rates were determined by means of the indicators of recovery in the feces of the animal. In the degradation assay samples were incubated in nylon bags at 0, 6, 18, 48 and 96 hours. The behavior presented in the regression curves of the variables describing a high correlation between them, in other words, the time when the silage is retained in the rumen influence on its digestibility and its rate of degradation. In fact, the longer that a food is in the rumen, he will suffer more digestive actions and therefore will be more digestible. Although silage compression pressure 900kg/m? present a higher potentially digestible fraction, we recommend the use of silage compaction pressure of about 750kg/m?, due to lower cost and have lower average retention rumen and reticulum ruminal fill, thus increasing the dynamic ruminal degradation and passage.

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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES)
Funda??o de Amparo ? Pesquisa do Estado de Minas Gerais (FAPEMIG)
Caracter?sticas da carne de bovinos cruzados (Wagyu ? Red Angus) e matura??o da carne de Nelore. Orientador: Orientador: Orientador: Orientador: Orientador: Cleube Andrade BoariCleube Andrade Boari Cleube Andrade BoariCleube Andrade Boari Cleube Andrade BoariCleube Andrade BoariCleube Andrade BoariCleube Andrade BoariCleube Andrade BoariCleube Andrade BoariCleube Andrade BoariCleube Andrade Boari . Disserta??o (Mestrado em Zootecnia). Esta disserta??o foi elaborada com o resultado de duas pesquisas. A primeira foi conduzida com o objetivo de avaliar as caracter?sticas de qualidade da carne de bovinos cruzados Wagyu ? Angus (Red). A segunda foi conduzida com o objetivo de verificar os efeitos da matura??o de maminha de alcatra de Nelore em diferentes temperaturas (0?C e 4?C) e tempos (0 = carne fresca com 24 horas, 7, 14, 21 e 28 dias). Na primeira pesquisa foi analisada a carne dos m?sculos Longissimus thoracis, Semitendinosus e Triceps brachii obtidas do abate de machos castrados e de f?meas, aos 24 meses de idade. Na desossa foram mensurados no m?sculo Longissimus thoracis a espessura de gordura subcut?nea, cor da gordura (L*a*b*, C, H?), a ?rea de olho de lombo e escore para o grau de marmoriza??o. Foram analisados o pH final, a capacidade de reten??o de ?gua, a perda de peso por cozimento, a for?a de cisalhamento, cor (L*a*b*, C, H?) e os teores de umidade, massa seca, res?duo mineral fixo, prote?na e gordura. Por??es de carne do m?sculo Longissimus thoracis foram coletadas avaliar o efeito dos tempos de sete e 14 dias de matura??o no pH final, na capacidade de reten??o de ?gua, na perda de peso por cozimento, na for?a de cisalhamento, na cor da carne (L*a*b*, C, H?) e na cor da gordura (L*a*b*, C, H?). Para a carne de todos os m?sculos n?o houve efeito de sexo no pH final, na capacidade de reten??o de ?gua, na perda de peso por cozimento e no teor de prote?na. O Longissimus thoracis de f?meas recebeu os melhores escore para marmoreio, apresentou maior teor de gordura e menor for?a de cisalhamento e no Longissimus thoracis de machos apresentou maior ?rea de olho de lombo. A matura??o da carne do Longissimus thoracis proporcionou redu??o na for?a de cisalhamento e aumento do pH final. Na segunda pesquisa, por??es de carne (? 400 gramas) foram embaladas, a v?cuo, e estocadas em c?mara frigor?fica nas temperaturas (0?C e 4?C). Foram mantidas nestas temperaturas por 7, 14, 21 e 28 dias. Foram analisados o pH, capacidade de reten??o de ?gua, perda de peso por cozimento, for?a de cisalhamento, cor (L*, a*, b*, C, H?). N?o houve varia??o no pH em fun??o da temperatura utilizada. Entretanto, o pH aumentou com o decorrer do tempo de matura??o. Foi observada varia??o na luminosidade da carne em fun??o da temperatura, sendo menor a luminosidade na carne maturada a 0?C. O teor de vermelho desta carne aumentou linearmente em fun??o do tempo de matura??o e tamb?m foi influenciada pelas temperaturas de matura??o, sendo maior quando maturada a 0?C. A perda de peso por cozimento tamb?m apresentou aumento linear ao longo do tempo de matura??o. A capacidade de reten??o de ?gua e a for?a de cisalhamento apresentaram redu??o linear ao longo do tempo de matura??o. A matura??o por 28 dias ocasionou redu??o na for?a de cisalhamento da maminha de alcatra. Recomenda-se maturar a maminha de alcatra por 28 dias em temperatura de 4?C.
Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Zootecnia, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2015.
Characteristics of meat from crossbred cattle (Wagyu ? Red Angus) and maturation Nelore meat. Adviser: Cleube Andrade Boari. Adviser: Cleube Andrade Boari. Adviser: Cleube Andrade Boari. Adviser: Cleube Andrade Boari.Adviser: Cleube Andrade Boari. Adviser: Cleube Andrade Boari.Adviser: Cleube Andrade Boari. Adviser: Cleube Andrade Boari.Adviser: Cleube Andrade Boari.Adviser: Cleube Andrade Boari.Adviser: Cleube Andrade Boari.Adviser: Cleube Andrade Boari.Adviser: Cleube Andrade Boari. Adviser: Cleube Andrade Boari. Dissertation Dissertation (Master?s degree in Animal Science (Master?s degree in Animal Science (Master?s degree in Animal Science (Master?s degree in Animal Science (Master?s degree in Animal Science (Master?s degree in Animal Science (Master?s degree in Animal Science (Master?s degree in Animal Science (Master?s degree in Animal Science (Master?s degree in Animal Science (Master?s degree in Animal Science (Master?s degree in Animal Science (Master?s degree in Animal Science (Master?s degree in Animal Science (Master?s degree in Animal Science ). This paper was drawn up on the results of two surveys. The first was conducted in order to evaluate the quality characteristics of meat from crossbred cattle Wagyu ? Angus (Red). The second was conducted in order to verify the effects of maturation titty rump Nellore at different temperatures (0 ? C and 4 ? C) and time (0 = fresh meat with 24 hours, 7, 14, 21 and 28 days ). In the first study we analyzed the meat of Longissimus thoracis, Semitendinosus and Triceps brachii obtained from the slaughter of castrated males and females, at 24 months old. The bones were measured in the Longissimus thoracis muscle to fat thickness, color of fat (L * a * b *, C, H?), rib eye area and score to the degree of marbling. The final pH were analyzed, water retention capacity, weight loss by cooking, shear strength, color (L * a * b *, C, H?), and the moisture content, dry matter, mineral residue fixed, protein and fat. Meat portions of Longissimus thoracis muscle were collected to evaluate the effect of seven days and 14 days of aging at the end pH, water retention capacity, weight loss by cooking, the shear force on the meat color (L * a * b *, C, H?) and fat color (L * a * b *, C, H?). For the meat of all muscles of no gender effect on the ultimate pH, water retention capacity, by cooking weight loss and protein content. The Longissimus thoracis females received the best score for marbling, showed higher fat content and lower shear strength and Longissimus thoracis males showed greater loin eye area. The maturation of the meat of Longissimus thoracis provided a reduction in shear force and increase in final pH. In the second study, cam portions (400 ? grams) were packed, vacuum, and stored in the cold room temperatures (0 ?C and 4 ?C). They were maintained at these temperatures for 7, 14, 21 and 28 days. They analyzed the pH, water holding capacity, cooking weight loss, shearing force, color (L *, a *, b *, C, M ?). There was no variation in pH as a function of temperature used. However, the pH increase during maturation time. It was observed variation in brightness of the flesh a function of temperature being lower in brightness aged meat at 0 ?C. The content of this red meat increased linearly as a function of aging time and was also influenced by the temperature of ripeness, being higher when aged at 0 ?C. The cooking weight loss also showed a linear increase over time of aging. The water retention capacity and shear strength showed a linear decrease over time of aging. Maturation for 28 days decreased the shear force of titty rump. It is recommended to mature titty rump for 28 days in temperature

An analytical method was developed for the determination of the â-agonists clenbuterol, cimaterol and salbutamol in bovine retina. The method involved extraction into a pH 8.5 tris-HCl buffer, followed by protease enzyme digestion and immunoaffinity column cleanup before analysis by liquid chromatography with tandem mass spectrometry detection LC/MS/MS. The LOD for clenbuterol, salbutamol and cimaterol were 0.64, 1.20 and 1.92 ng/g respectively. The identities of the analytes were able to be confirmed to an acceptable standard. An analytical method was also developed for the analysis of the â-agonists clenbuterol, salbutamol, cimaterol, ractopamine, and mabuterol in bovine urine and emu muscle. The urine and muscle samples were digested with â-glcuronidase enzyme and cleaned up using a Bond Elute Certify SPE. The extracts were analysed by LC/MS/MS. Deuterated internal standards were used for quantitation. The LOD for urine [less than] 1ng g and for emu muscle it was [less than] 0.3 ng/g. The last part of the work describes the simultaneous gas chromatography-mass spectrometric analysis of diethylstilbestrol DES, hexestrol HEX, dienestrol DIEN, zeranol ZER, taleranol TAL and zeralenone ZON in fresh full cream and fresh skim milk. The analytes were analysed as their trimethyl silane (TMS) derivatives. A three phase solvent system was used for extraction and the extract was cleaned up using a combination of the anion exchange and hydrophobic properties of an anion exchange SPE. The detection limits for DES, DIEN, HEX, ZER, TAL and ZON were 9.6, 9.6, 16.8 , 7.2 , 13.5 and 34.8 ng/L respectively.
Doctor of Philosophy(PhD)

An analytical method was developed for the determination of the â-agonists clenbuterol, cimaterol and salbutamol in bovine retina. The method involved extraction into a pH 8.5 tris-HCl buffer, followed by protease enzyme digestion and immunoaffinity column cleanup before analysis by liquid chromatography with tandem mass spectrometry detection LC/MS/MS. The LOD for clenbuterol, salbutamol and cimaterol were 0.64, 1.20 and 1.92 ng/g respectively. The identities of the analytes were able to be confirmed to an acceptable standard. An analytical method was also developed for the analysis of the â-agonists clenbuterol, salbutamol, cimaterol, ractopamine, and mabuterol in bovine urine and emu muscle. The urine and muscle samples were digested with â-glcuronidase enzyme and cleaned up using a Bond Elute Certify SPE. The extracts were analysed by LC/MS/MS. Deuterated internal standards were used for quantitation. The LOD for urine [less than] 1ng g and for emu muscle it was [less than] 0.3 ng/g. The last part of the work describes the simultaneous gas chromatography-mass spectrometric analysis of diethylstilbestrol DES, hexestrol HEX, dienestrol DIEN, zeranol ZER, taleranol TAL and zeralenone ZON in fresh full cream and fresh skim milk. The analytes were analysed as their trimethyl silane (TMS) derivatives. A three phase solvent system was used for extraction and the extract was cleaned up using a combination of the anion exchange and hydrophobic properties of an anion exchange SPE. The detection limits for DES, DIEN, HEX, ZER, TAL and ZON were 9.6, 9.6, 16.8 , 7.2 , 13.5 and 34.8 ng/L respectively.

Dissertação de mestrado em Bioquímica, apresentada ao Departamento Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.
À perturbação metabólica provocada pela incapacidade ou pela diminuição da capacidade de produção de insulina no pâncreas, designamos comummente por Diabetes. Esta patologia de aparente simples definição, é de facto um processo bastante complexo e de difícil controlo, será talvez por tal facto que actualmente a apelidamos de “Epidemia do Mundo Moderno”. Não obstante à medida que esta patologia progride e se adensa a severidade da sintomatologia associada aumenta proporcionalmente, estando inerente um maior risco de complicações quer microvasculares, tais como retinopatia, nefropatia, neuropatia como macrovasculares. Consequentemente, as diabetes induzem alterações fisiológicas e metabólicas na retina nas quais a inflamação parece desempenhar um importante papel, em particular no desenvolvimento da retinopatia diabética. Neste contexto o presente estudo pretendeu aferir a putativa capacidade de um antidiabético, inibidor da DPP-IV, – Sitagliptina – de aliviar ou mesmo reverter esta sintomatologia microvascular. Posto isto, células endoteliais da retina de bovino - BREC – foram em concomitância tratadas durante 6 horas com duas concentrações de uma citocina pró-inflamatória – TNF – e de Sitagliptina, respectivamente, 5 ng/ml e 10 ng/ml ou 100 nM e 100 μM. Seguidamente, a formação de estruturas semelhantes a tubos, migração e proliferação de células endoteliais foram avaliadas, tendo tais funções sido preservadas ou, quando diminuídas devido ao TNF, reestabelecidas pela Sitagliptina. Em suma, resultados preliminares obtidos sugerem que a Sitagliptina exerce efeitos citoprotectores na microvasculatura da retina, quando instalada uma condição inflamatória, o que é indicativo dos benefícios anti-inflamatórios deste inibidor da DPP-IV na retina. No entanto, estudos adicionais são necessários tanto para confirmar estes resultados como para entender melhor os adjacentes mecanismos celulares e moleculares envolvidos neste efeito protector mediado pela Sitagliptina. Por conseguinte, tais dados podem contribuir para o desenvolvimento de novas e mais eficazes estratégias terapêuticas dirigidas para a prevenção ou centradas na minimização dos efeitos nefastos induzidos pela hiperglicemia crónica na microvasculatura da retina. Assim, um conhecimento mais aprofundado e detalhado destes inibidores da DPP-IV irão contribuir para um prognóstico mais aferido e eficaz e quiçá a almejada cura completa da Diabetes.
Diabetes is essentially a metabolic disruption caused either by the inability or by the decrease in pancreas insulin’s production capacity. On the other hand this apparently, in-a-few-words simple definition is in fact a process that could be as complex as hard to control. More importantly, it might be considered as the “Modern World Epidemic” becoming more severe as it extends and progresses. Thus the association between intensity and the overall duration of hyperglycemia periods increases the risk of microvascular complications such as, retinopathy, nephropathy, neuropathy and macrovascular ones. More so, Diabetes induces metabolic and physiological abnormalities in the retina in which inflammation seems to play a major role particularly in the development of diabetic retinopathy. In this context, the aim of this study was to verify if an antidiabetic DPP-IV inhibitor, Sitagliptin, could alleviate some of these symptoms. Therefore bovine retinal endothelial cells – BREC - where co-treated with two concentrations of an inflammatory cytokine TNF and Sitagliptin, respectively 5 ng/ml and 10 ng/ml or 100 nM and 100 μM for 6 hours. EC tube-like structures formation, migration and proliferation abilities seem as a result to be conserved by Sitagliptin and when impaired due to TNF reestablished by the first. In conclusion preliminary results obtained suggest that Sitagliptin exerts cytoprotective effects to the microvasculature of the retina, when an inflammatory condition is settled. What indicates that this DPP-IV inhibitor can have anti-inflammatory benefits to the retina. However additional studies are mandatory to both confirm these results and to further understand the underlined molecular and cellular mechanisms involved in this Sitagliptin-mediated-protective-action. This may contribute to the development of new and more effective therapeutic strategies directed to the prevention or focused in the minimization of the damaging-hyperglycemia-induced effects in the retina’s microvasculature. Nevertheless obtaining clear information about these mechanisms could contribute to an elevated knowledge, thorough diagnosis and in general putative cure for diabetes.

Dissertação de Mestrado Integrado em Medicina Veterinária
Os processos inflamatórios pós-parto com sede no útero, como a metrite, são um dos problemas mais frequentemente diagnosticados em bovinos de leite. Estão associadas a perdas económicas importantes, não só relacionadas com uma diminuição do desempenho reprodutivo, mas também com o seu efeito negativo na produção de leite. Como outras patologias do pós-parto na vaca leiteira, a metrite pós-parto (MPP), ou metrite puerperal, possui uma etiologia multifatorial. Ao longo do tempo têm sido identificados alguns fatores como sendo predisponentes desta afeção, apesar de haver alguma controvérsia sobre a sua associação à etiopatogenia da doença. Neste estudo pretendeu-se analisar a associação da MPP com a ocorrência prévia de Retenção Placentária (RP) e a presença de valores elevados de corpos cetónicos (CC) no momento do diagnóstico da metrite, além de outros parâmetros como a idade, paridade, momento de diagnóstico, condição corporal e tipo de parto (eutócico ou distócico). Foram incluídas neste trabalho um total de 42 vacas, sendo 19 as fêmeas diagnosticadas com metrite pós-parto. Incluiu-se no estudo fêmeas primíparas (n= 5) e multíparas (n= 37), com idades compreendidas entre os 3 e os 9 anos (idade média de 4 ±1,2 anos). As vacas eram provenientes de 5 explorações localizadas na Região Litoral Norte de Portugal, com maneio geral similar entre si. O diagnóstico de metrite foi realizado até aos 38 dias após o parto, após consulta solicitada pelo proprietário. O diagnóstico de metrite foi emitido após exame clínico e reprodutivo da vaca com base na existência de uma quebra na produção leiteira, diminuição na ingestão de matéria seca, e a presença de um odor pútrido ou de corrimento vaginal anómalo. As fêmeas do grupo controlo foram selecionadas dentro das mesmas explorações, entre animais que se encontravam no mesmo momento do pós-parto, tinham sensivelmente a mesma idade/paridade e um nível de produção de leite semelhante. Para os dois grupos foram registados os dados básicos do animal, bem como recolhida informação sobre a facilidade do parto (que foi classificado em eutócico/distócico) e a existência de retenção placentária (sim/não; RP) no último parto. A recolha de uma amostra de sangue para determinação de corpos cetónicos apenas foi possível em 21 animais (11 no grupo controlo e 10 no grupo metrite). A maior parte dos diagnósticos de metrite foram realizados nas duas primeiras semanas após o parto, tendo sido mais frequente em fêmeas com 2 e 3 partos (36,8% e 31,5% respetivamente) e menos comum nas fêmeas primíparas ou com quatro ou mais partos (15,8% em ambos os casos). A condição corporal dos animais incluídos no estudo era relativamente uniforme e dentro dos limites desejados no peri-parto. Em relação ao tipo de parto, foram registados 21% e 17% de partos distócicos respetivamente para os grupos metrite e controlo, não sendo estas diferenças estatisticamente significativas. A medição dos CC em ambos os grupos revelou valores médios semelhantes (P=0,704), embora os valores fossem numericamente mais baixos nas vacas controlo (0,84± 0,40 mmol/L) do que no grupo metrite (1,11± 0,91 mmol/L). A ocorrência prévia de retenção placentária foi registada em 14 (73,3%) e 5 (21,7%) animais nos grupos com diagnóstico de metrite e controlo, respetivamente. Não se encontraram influências significativas da idade, paridade, condição corporal, tipo de parto e presença de cetose no momento de diagnóstico sobre a ocorrência de MPP nos animais incluídos no estudo. No entanto, encontrou-se uma associação positiva entre a MPP e a ocorrência prévia de RP. A razão de probabilidades revelou que vacas com retenção placentária no último parto têm 10 vezes mais probabilidade de virem a apresentar um diagnóstico de MPP. Dados os encargos económicos que acompanham a ocorrência da MPP e o comprometimento do bem-estar das fêmeas afetadas, é importante a sensibilização do proprietário para a necessidade de programas preventivos que sejam eficazes no controlo da patologia e na monitorização mais apertada de animais que sofreram de complicações no peri-parto, como RP. A nutrição assume um papel determinante nesta prevenção, assim como a sinalização precoce das vacas em risco.
The inflammatory conditions of the postpartum uterus, such as metritis, are frequently diagnosed in dairy cattle. They are associated with serious economic losses, related not only with a decrease of the reproductive performance, but also with its adverse effect on milk production. Like in other postpartum conditions of dairy cows, postpartum metritis (PPM) presents a multifactorial etiology. Over time, some factors have been identified as predisposing to this disease, even though there is some controversy about its association with the pathogenesis of the disease. This study intent to analyze the association between PPM with retained placenta (RP) in the last calving and the presence of high levels of ketone bodies (CC) at the time of diagnosis of metritis, besides other parameters such as age, parity, body condition and type of delivery (eutocic or dystocic). Forthy-two cows were enrolled in this study, 19 of them having a diagnosis of PPM. It included either primiparous (n = 5) and multiparous (n = 37) females, within an age range between 3 and 9 years old (age average of 4 ±1,2 years). The cows belonged to 5 farms of similar management characteristics, from the North Coast of Portugal. Metritis diagnose was establish up to 38 days post-calving, following a call from the owner or caretaker that signalized the sick animal. The diagnosis of metritis was raised after the physical and reproductive examination of a cow based on the following clinical signs: inapetence, raised temperature, drop in milk production, decreased dry matter intake and the presence of a putrid odor, or abnormal vaginal discharge. Females used in the control group were selected within the same farm, between animals at the same postpartum time, similar same age/parity and similar milk production. For both groups, beside the basic animal data it was also recorded information on the calving easiness (scored as eutocic/distocic) and the occurrence of retained placenta (yes/no) on the last calving. The blood collection for measurement of ketone bodies was possible only once and limited to 21 animals (11 in the control group and 10 in the metritis group). The diagnosis of metritis was establish more often in the first two weeks after parturition, in females in the 2nd or 3rd calving (36.8% and 31.5% respectively) and it was less common in primiparous females or in cows with four or more calving (15.8% in both cases). The body condition score of the animals was relatively uniform and within the limits generally foreseen in the peripartum. Distocia was present in 21% and 17% of cows in the metritis and control groups, but the differences were not significant. Even though the values of ketone bodies were numerically lower for cows in the control group (0.84 ± 0.40 mmol / L) than the ones in the metritis group (1.11 ± 0.91 mmol / L), the groups did not differ (P = 0.704),. The occurrence of retained placenta in the previous calving was recorded in 14 (73.3%) and 5 (21.7%) animals for the groups metritis and control, respectively. The occurrence of PPM was not influenced by age, parity, and body condition score, type of delivery and presence of ketose at the time of diagnosis. However, a positive association between MPP and the prior occurrence of RP was found in this study. The odds ratio showed that cows with retained placenta in their last parturition were 10 times more likely to present a positive diagnosis for MPP. Given the major economic constraints associated to the occurrence of MPP and the negative impact on the affected cow´s welfare, it is important to alert the owner for the need to establish effective prevention programs to control the disease and also to put animals developing peri-parturient diseases under a closer monitorization. Nutrition plays a key role in this prevention, as well as an early detection of cows at risk.