Cardiac and orthopaedic diseases are significant causes of morbidity and mortality in dogs and are therefore critical areas for veterinary research. More information regarding the pathophysiology of these diseases, and the development of novel therapeutics are sorely required and adult stem cells (ASCs) are a promising source of cells for both investigation of these diseases in vitro and also potentially therapeutics in the longer term. ASCs are a readily available source of multipotent cells which bypass the ethical issues surrounding embryonic stem (ES) cells. ASCs have been described in several tissues of the body, and typically differentiate along specific cellular routes related to original source location. This thesis investigates whether ASCs can be isolated and cultured from the dog from two specific locations; cardiac, producing cardiac stem cells (CSCs); and the bone marrow, producing mesenchymal stem cells (MSCs). These cell sources will be extensively characterised at their baseline for morphology, culture behaviour and gene marker expression. Following characterisation each cell source will be subjected to differentiation techniques to examine canine ASC multipotent differentiation potential. CSCs were isolated from cultured atrial cardiac explant tissue taken from dogs post-mortem, with owners’ consent. These cells were able to survive successive passages in serum free media and formed large spherical cell clusters, termed ‘cardiospheres’. CSCs were capable of clonal expansion under controlled culture conditions, demonstrating their ability for self-renewal. Characterisation of these cells demonstrated the expression of CSC markers; c-Kit, GATA 4 and Flk-1 and no expression of cardiac lineage markers including cardiac troponin T and I, Nkx2.5, the cardiac ryanodine receptor and the β1-adrenergic receptor. Primary canine MSCs were isolated from bone marrow aspirates using ficoll separation and cultured on tissue culture plastic. Canine MSCs closely resembled MSCs described from other species, such as the human and mouse, and were found to express CD44 and STRO-1 and were negative for CD34 and CD45. CSCs and MSCs were exposed to published cardiac directed differentiation protocols and differentiation then analysed using cellular morphology and gene expression. Canine CSCs appeared to differentiate partially along cardiac lineages with upregulation of cardiac troponin T and Nkx2.5, and down regulation of c-Kit and endothelial lineage markers. Canine MSCs demonstrated some morphological changes during cardiac differentiation, and demonstrated up-regulation of Nkx2.5 and Flk-1 but no significant alteration in other markers examined. This suggested that cardiac directed differentiation was not as successful with canine MSCs compared to CSCs and conflicting with published data using rodent MSC models. Murine MSCs were used as a positive control cell line for cardiac directed differentiation, based upon published literature. Critically there were key marker expression differences between baseline murine and canine MSCs, including the expression of cardiac markers such as cardiac troponin T and I, and the Ryanodine receptor. Furthermore, expression analysis of cardiac genes changed with time in culture and passage number and no significant alteration was seen when cells were subjected to the cardiac differentiation protocol; thereby bringing into question the data regarding successful cardiac differentiation using murine MSCs. Canine MSCs were further differentiated toward a chondrocyte lineage to investigate the use of MSCs for orthopaedic research. Canine MSCs were successfully differentiated toward articular type cartilage, with demonstration of extracellular matrix secretions, an upregulation of collagen type II with downregulation of collagen type I and the development of SOX9 expression in differentiated cells. This thesis builds the groundwork for future ASC research in the dog. Successful isolation and culture of two ASC sources from the dog is demonstrated. Cardiac and cartilage directed differentiation was successful using primary sourced cells, but differentiation was found to be limited to highly specific routes for each stem cell source. The results presented here highlight the importance of analysing baseline stem cells extensively prior to differentiation and in particular, before making comparisons between cell populations isolated from different locations and species.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Nikkomycin Z (NikZ) is a chitin synthase inhibitor with antifungal efficacy against Coccidioides spp. and other endemic fungi. Dogs suffer a rate and range of natural coccidioidomycosis similar to humans and were considered an excellent model for initially testing NikZ against naturally acquired disease. Twelve dogs with coccidioidal pneumonia that had been present for an average of three months were treated with 250 mg (5 - 15 kg) or 500 mg (> 15 - 30 kg) twice daily for 60 days. Nine dogs completed the course of treatment and seven dogs had improvement in disease based on radiographs, clinicopathological parameters, physical examination findings, and subjective assessment by the owners; three dogs had resolution or near resolution of disease. Based on this small study, NikZ shows efficacy to treat naturally acquired coccidioidomycosis and merits further development for trials in humans.

Class of 2006 Abstract / Objectives: To evaluate the comparative efficacies of Tacrolimus and Pimecrolimus for the treatment of Keratoconjunctivitis Sicca (KCS), Pannus, and other autoimmune related ophthalmological disorders. Methods: A descriptive retrospective study of clinical data obtained through two separate veterinary studies. The improvement in Schirmer Tear Test (STT) between the Tacrolimus group and Pimecrolimus group is compared. Results: There were 27 canines in the Tacrolimus group, with a total of 50 affected eyes. The Pimecrolimus group had 14 total canines, but tear production was measured in only 8, for a total of 16 affected eyes. At baseline, there was no significant difference in tear production between the two groups. At first and second follow up, and in both eyes, Tacrolimus produced a significantly greater increase in tear production than Pimecrolimus (p=0.0093 and p=0.0292 respectively for the right eye first and second follow up; p=0.0425 and p=0.0065 respectively for the left eye first and second follow up). Conclusions: Treatment with Tacrolimus produced a greater increase in tear production that Pimecrolimus in canines with autoimmune keratitis.

Orientador: Fabrício Singaretti de Oliveira / Resumo: A raça Spitz Alemão Anão ganhou recente aumento de popularidade e algumas das particularidades oftalmológicas desta ainda são desconhecidas. Com o presente estudo objetivou-se conhecer parâmetros oftálmicos na raça. Estudaram-se 48 olhos de 24 cães, machos e fêmeas, com idades entre 1 e 7,5 anos, oriundos de canil de criação. Sob contenção física delicada, foram empregados teste lacrimal de Schirmer, tempo de ruptura do filme lacrimal, teste de Jones, estesiometria de Cochet-Bonnet, biomicroscopia com lâmpada em fenda, pressão intraocular, microscopia especular de não contato, flaremetria a “laser”, oftalmoscopia indireta e ultrassonografia. Aplicou-se estatística descritiva para análise dos dados. As médias e erros padrões encontrados foram, quanto ao teste lacrimal de Schirmer 24,310±0,448mm/min e ao tempo de ruptura do filme lacrimal 14,570±0,406s. Ao teste de Jones 8,420±1,571min sendo 62,5% dos olhos positivos. À estesiometria, a medida foi 3,667±0,157cm. À biomicroscopia, 43,75% dos olhos apresentaram pelos na carúncula, 25% cromodacriorreia, 25% discreto entrópio de canto medial, 10,42% distiquíase, 6,25% mácula corneal, 4,16% nódulo palpebral e 2,08% triquíase de canto medial. À tonometria de rebote, os valores obtidos média e erro padrão foram 17,960±0,697mmHg. À microscopia especular foi encontrada densidade de células endoteliais 1958,285±36,760células/mm². À hexagonalidade, 62,229±2,183% e para área das células endoteliais, 519,231±9,853mm². A espessura corneal ce... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Spitz German Klein breed has shown a substantial increase in popularity and ophthalmic particularities of this breed are still unknown. The present study aimed to establish ophthalmic parameters in German Spitz Klein. The research for this study was made through 48 eyes of 24 male and female dogs, aged between 1 and 7.5 years old, from a breeding kennel. Thru physical restraint it was used Schirmer tear test, break up time test, Jones test, Cochet-Bonnet esthesiometry, slit lamp biomicroscopy, rebound tonometry, specular microscopy of non-contact, laser flaremetry, ophthalmoscopy and ultrasonography. With the purpose of analyzing data this study was applied descriptive statistics. The means and standard errors found were, for the Schirmer tear test 24.310±0.448mm/min and the break up time test 14.570±0.406s. For the Jones test 8.420±1.571min being 62.5% of the positive eyes. For the esthesiometry was found 3.667±0.157cm. The biomicroscopy, it was found that 43.75% of the eyes presented hair on the caruncle, 25% chromodacryreia, 25% discrete medial entropion, 10.42% of distichiasis, 6.25% of corneal macula, 4.16% of palpebral nodule and 2.08% of medial trichiasis. For the rebound tonometry, the values obtained and standard error were 17.960±0.679mmHg. Furthermore, under the specular microscopy was found density of endothelial cells 1958.285±36.760cells/mm². The hexagonality, 62.229±2.183% and for endothelial cell area, 519.231±9.853mm². The central corneal thickness was 0.... (Complete abstract click electronic access below) / Mestre

Although dark coat color in dogs has been theorized as a risk factor for thermal stress, there is little evidence in the scientific literature to support that position. We utilized 16 non-conditioned Labradors (8 black and 8 yellow) in a three-phase test to examine effects of coat color on thermal status of the dog. Rectal, gastrointestinal (GI), surface temperature, and respiration rates measured in breaths per minute (bpm), were collected prior to (Baseline – phase 1) and immediately after a controlled 30-minute walk in an open air environment on a sunny day (Sunlight – phase 2). Follow up measurements were taken 15 minutes after walking (Cool down – phase 3) to determine post-exposure return to baseline. No effect of coat color was measured for rectal, gastrointestinal, surface temperature, or respiration (P > 0.05) in dogs following their 30-minute walk. Temperatures increased similarly across both coat colors (rectal 1.88 °C and 1.83 °C; GI 1.89 °C and 1.94 °C) for black and yellow dogs respectively during the sunlight phase (P > 0.05). All temperatures and respiration rates decreased similarly across coat colors for rectal (0.9°C and 1.0 °C) GI (1.5 °C and 1.3°C) for black and yellow dogs respectively (P>0.05). Similarly, sex did not impact thermal status across rectal, gastrointestinal, surface temperature or respiration rates measured (P > 0.05). These data contradict the commonly held theory that dogs with darker coat color may experience a greater thermal change when compared to dogs with a lighter coat color exposed to direct sunlight.

Canine cruciate ligament (CCL) rupture is a common veterinary orthopaedic problem, and has been associated with problems at the ipsilateral hip joint. However, the relationship between stifle and hip dysfunction has not been fully investigated. The purpose of this study was to establish whether CCL pathology is associated with a limitation in hip extension range large enough to affect gait. In a prospective, crosssectional study, passive and active hip extension range was determined in normal dogs as well as CCL deficient dogs. There was a significant reduction in ipsilateral passive hip extension range in the CCL deficient dogs (18.63º+/-7.57).There was a significant increase in hip extension range of the unaffected leg during gait compared to the normal group (7.03º+/-8.41). It was therefore concluded that canine CCL pathology directly affects hip extension range to a degree sufficient to affect normal gait.

Utilizamos nesta pesquisa 40 corações de cães adultos, 17 machos e 23 fêmeas, de idades variadas, que não portavam macroscopicamente nenhuma afecção cardíaca. Os corações tiveram as artérias coronárias injetadas, separadamente, com Neoprene Látex 450, corado com pigmento vermelho, e posteriormente dissecados. Em todas estas preparações verificamos que na vascularização dos ventrículos predominava a artéria coronária esquerda que fornecia os ramos interventriculares paraconal e subsinuoso. Já, a região ocupada pelo nó sinoatrial ficava mais frequentemente (17 vezes 42,5%) na dependência do ramo proximal atrial esquerdo ou de colateral deste vaso, oriundo do ramo circunflexo esquerdo, ou deste vaso associado ao ramo distal atrial direito (8 vezes 20%), procedente do ramo circunflexo direito. Com menor frequência (14 vezes 30%), a área tomada pelo nó sinoatrial, encontramos apenas colaterais do ramo circunflexo direito, mais exatamente somente o ramo distal atrial direito (10 vezes 25%), apenas o ramo proximal atrial direito (3 vezes 7,5%) ou ainda exclusivamente o ramo intermédio atrial direito (1 vez 2,5%). Em um único caso (1 vez 2,5%) no território do nó sinoatrial observamos apenas colateral do ramo circunflexo esquerdo, isto é o ramo distal atrial esquerdo. A análise destes resultados permite concluir, que nesta espécie não existe qualquer tipo de relação entre o tipo de vascularização dos ventrículos e a irrigação do nó sinoatrial. / In this research we analyzed 40 hearts of adult dogs, males and females with different ages, which had no cardiac disease. The hearts had the coronary arteries inject using Neoprene Latex 450, stained by red pigment and then were dissected. In the ventricular vascularization the left coronary artery was predominant and it gave rise to the branches paraconal interventricular and subsinuous. The region occupied by the sinoatrial node was most frequently (17 times 42.5%) in the dependence of the left atrial proximal branch or in the collateral of that vessel, coming from the left circumflex branch, or this vessel associated to the right atrial distal branch (8 times 20%). With less frequency (14 times 30%), in the area occupied by the sinoatrial node, we found only the collaterals of the right circumflex branch, rather only the right atrial distal branch (10 times 25%), only the right atrial proximal branch (3 times 7.5%) or exclusively through the right atrial intermediary branch (1 time 2.5%). In just one case (Once 2.5%) in the area of the sinoatrial node we observed only the collateral of the left circumflex branch, i.e. the left atrial distal branch. According to our results we concluded that in this specie there are no relation between the type of vascularization of the ventricles and the irrigation of the sinoatrial node.

A cinomose canina, causada pelo vírus Canine Distemper (CDV), é uma doença de grande importância não só no Brasil como no mundo. Isto se deve principalmente à sua vasta ocorrência, facilitada pelo tipo de transmissão que pode ser por contato com secreções respiratórias, oculares, por fezes e urina. Detectar rapidamente o vírus em animais contaminados é de extrema importância para que o controle aconteça de forma rápida e adequada, evitando o agravamento da doença, a disseminação e o aumento da mortalidade. O presente trabalho teve por objetivo desenvolver uma plataforma diagnóstica imunocromatográfica que possibilite a detecção rápida de biomoléculas relacionadas com esta doença. Os testes imunocromatográficos são por natureza plataformas de baixo custo e que fornecem resultados rápidos sem a necessidade de equipamentos para a interpretação. Essas plataformas são compostas por regiões diferentes de papéis ou membranas, constituídos de fibra de vidro (sample pad e conjugate pad), material celulósico (sample pad e absorbent pad) e nitrocelulose (região de detecção). Nesse trabalho, com o intuído de diminuir ainda mais o custo da plataforma, foi avaliada a possibilidade de substituição das diferentes regiões somente pelo do papel de filtro Whatman nº 1. Para isso, foi necessário a otimização de diversos reagentes e tratamentos que fossem capazes de modificar a estrutura do papel, deixando-o mais reativo frente às proteínas imobilizadas na região de detecção, como o periodato de sódio, divinilsulfona e tampão carbonato; e menos reativos, quando utilizado em outras regiões. Antes de iniciar os ensaios com o papel, os anticorpos foram avaliados por immunoblotting para verificar o funcionamento dos mesmos, em relação à sensibilidade e especificidade. Após a avaliação, os anticorpos foram imobilizados com sucesso no papel de filtro através do uso de tampão carbonado como reagente sensibilizador da celulose, levando à construção das regiões teste e controle. No conjugate pad o tratamento mais eficiente foi com a utilização de 5% de sacarose, 1% de BSA e 0,5% de Tween-20 em PBS, que garantiu a liberação satisfatória dos conjugados para a região de detecção. / Canine distemper, caused by the Canine Distemper virus (CDV), is a disease of great importance not only in Brazil, but also in the world. This is due mainly to its vast occurrence, facilitated by the transmission pathways, which may occur through contact with respiratory and/or ocular secretions, feces and urine. Rapid detection of the virus in contaminated animals is extremely important so that control takes place quickly and properly, avoiding disease worsening, its dissemination and mortality increase. The present work aimed in developing an immunochromatographic diagnostic platform that allows rapid detection of biomolecules related to this disease. Immunochromatographic tests are naturally low-cost platforms that provide fast results without need for equipment for interpretation. These platforms are composed by different regions of paper or glass fiber membranes (sample pad and conjugate pad), cellulosic material (sample pad and absorbent pad), and nitrocellulose (detection region). In this work, with the intention of further platform\'s cost reduction, the possibility of replacing the different regions was evaluated by using only the Whatman nº 1 filter paper. For this, it was necessary to optimize several reagents and treatments that were capable of modifying the paper structure, making it more reactive to the proteins immobilized in the detection region, such as sodium periodate, divinylsulfone, and carbonate buffer; and less reactive paper when used in other regions. Before initiating the paper assays, the antibodies were evaluated by immunoblotting for verifying their function, regarding sensitivity and specificity. With the results obtained within this test, new strategies had and have been drawn and will be followed in future experiments. After the evaluation, the antibodies were successfully immobilized on the filter paper with the use of carbonate buffer as the cellulose sensitizer reagent, leading to the construction of the test and control regions. In the conjugate pad the most efficient treatment was the use of 5% sucrose, 1% BSA and 0.5% Tween-20 in PBS, which ensured the satisfactory release of the conjugates to the detection region.

A cinomose canina, causada pelo vírus Canine Distemper (CDV), é uma doença de grande importância não só no Brasil como no mundo. Isto se deve principalmente à sua vasta ocorrência, facilitada pelo tipo de transmissão que pode ser por contato com secreções respiratórias, oculares, por fezes e urina. Detectar rapidamente o vírus em animais contaminados é de extrema importância para que o controle aconteça de forma rápida e adequada, evitando o agravamento da doença, a disseminação e o aumento da mortalidade. O presente trabalho teve por objetivo desenvolver uma plataforma diagnóstica imunocromatográfica que possibilite a detecção rápida de biomoléculas relacionadas com esta doença. Os testes imunocromatográficos são por natureza plataformas de baixo custo e que fornecem resultados rápidos sem a necessidade de equipamentos para a interpretação. Essas plataformas são compostas por regiões diferentes de papéis ou membranas, constituídos de fibra de vidro (sample pad e conjugate pad), material celulósico (sample pad e absorbent pad) e nitrocelulose (região de detecção). Nesse trabalho, com o intuído de diminuir ainda mais o custo da plataforma, foi avaliada a possibilidade de substituição das diferentes regiões somente pelo do papel de filtro Whatman nº 1. Para isso, foi necessário a otimização de diversos reagentes e tratamentos que fossem capazes de modificar a estrutura do papel, deixando-o mais reativo frente às proteínas imobilizadas na região de detecção, como o periodato de sódio, divinilsulfona e tampão carbonato; e menos reativos, quando utilizado em outras regiões. Antes de iniciar os ensaios com o papel, os anticorpos foram avaliados por immunoblotting para verificar o funcionamento dos mesmos, em relação à sensibilidade e especificidade. Após a avaliação, os anticorpos foram imobilizados com sucesso no papel de filtro através do uso de tampão carbonado como reagente sensibilizador da celulose, levando à construção das regiões teste e controle. No conjugate pad o tratamento mais eficiente foi com a utilização de 5% de sacarose, 1% de BSA e 0,5% de Tween-20 em PBS, que garantiu a liberação satisfatória dos conjugados para a região de detecção. / Canine distemper, caused by the Canine Distemper virus (CDV), is a disease of great importance not only in Brazil, but also in the world. This is due mainly to its vast occurrence, facilitated by the transmission pathways, which may occur through contact with respiratory and/or ocular secretions, feces and urine. Rapid detection of the virus in contaminated animals is extremely important so that control takes place quickly and properly, avoiding disease worsening, its dissemination and mortality increase. The present work aimed in developing an immunochromatographic diagnostic platform that allows rapid detection of biomolecules related to this disease. Immunochromatographic tests are naturally low-cost platforms that provide fast results without need for equipment for interpretation. These platforms are composed by different regions of paper or glass fiber membranes (sample pad and conjugate pad), cellulosic material (sample pad and absorbent pad), and nitrocellulose (detection region). In this work, with the intention of further platform\'s cost reduction, the possibility of replacing the different regions was evaluated by using only the Whatman nº 1 filter paper. For this, it was necessary to optimize several reagents and treatments that were capable of modifying the paper structure, making it more reactive to the proteins immobilized in the detection region, such as sodium periodate, divinylsulfone, and carbonate buffer; and less reactive paper when used in other regions. Before initiating the paper assays, the antibodies were evaluated by immunoblotting for verifying their function, regarding sensitivity and specificity. With the results obtained within this test, new strategies had and have been drawn and will be followed in future experiments. After the evaluation, the antibodies were successfully immobilized on the filter paper with the use of carbonate buffer as the cellulose sensitizer reagent, leading to the construction of the test and control regions. In the conjugate pad the most efficient treatment was the use of 5% sucrose, 1% BSA and 0.5% Tween-20 in PBS, which ensured the satisfactory release of the conjugates to the detection region.