Thesis (Ph. D.)--University of Wisconsin--Madison, 1955. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 70-75).

A rabbit lateral meniscectomy model was used to induce osteoarthritis. Separate studies were conducted to evaluate the progression of osteoarthritis and to identify possible biological markers. First, 21 male, New Zealand White rabbits were divided into 3 groups (n = 7 / group). A randomly selected left or right stifle underwent a lateral meniscectomy. The 3 groups were: corticosteroid administration, forced exercise and surgical control. An open field maze was used to assess mobility weekly. The rabbits were euthanitized 47 days after surgery. Histopathologic examination found that the lateral meniscectomy induced more severe lesions than in the non-surgical contralateral stifle. It also showed a significant sparing effect on erosion of cartilage in the corticosteroid group. The corticosteroid group, but not the exercise group, caused a significant increase in mobility (p = 0.008) compared to the surgical control. Secondly, synovial fluid was harvested from the 12 rabbits on days 0, 6, 26, 40, and 57 with surgery occurring on day 12. Trypan blue was used in the lavage fluid to estimate the volume of harvested synovial fluid. There was a significant increase in the volume harvested on day 26 (p < 0.001). Superoxide dismutase concentration in synovial fluid increased after surgery, although not significantly. These studies verify that the lateral meniscectomy model produce histopathologic lesions consistent with osteoarthritis. Furthermore, use of trypan blue appears to be a reliable concentration marker in a lavage sample to measure harvested synovial fluid. / Master of Science

Les transporteurs vésiculaires du glutamate (VGLUTs) sont impliqués dans la recapture du glutamate du cytosol vers les vésicules présynaptiques. Depuis leurs caractérisations récentes en 2000, leurs implications dans plusieurs maladies neurodégénératives ont été démontrées. Ils jouent ainsi un rôle primordial dans la transmission nerveuse glutamatergique. Deux colorants naturels, le Rose Bengale et le Bleu Trypan, restent les meilleurs inhibiteurs connus à ce jour, avec respectivement des CI50 de 25 et 50 nM. Dans un premier temps, nous avons conçu et optimisé une série d’analogues basée sur le synthon Rose Bengale (inhibiteur non-compétitif). Ce travail a notamment permis de mettre en évidence l’effet des formes tautomères (quinone et lactone) sur l’inhibition des VGLUTs. Ainsi la forme quinonique, présente à pH physiologique, a été confirmée comme étant la seule capable de bloquer la recapture du glutamate. Dans un second temps, nous nous sommes intéressés à la famille du Bleu Trypan (inhibiteur compétitif) et nous avons déterminé la structure minimale active avec l’objectif de rendre ces molécules plus « drug-like ». En effet, l’intérêt de ce projet est de développer de petites structures aisément radiomarquables pour une utilisation dans un contexte physio-pathologique. / Vesicular glutamate transporters (VGLUTs) are involved in the recapture and storage of glutamate from cytol to secretory synaptic vesicules. Since their recent characterization in 2000, their implication in several neurodegenerative disorders have been demonstrated. They play a crucial role in glutamatergic neurotransmission. Natural dyes, such as Rose Bengal and Tryptan Blue are the best known inhibitors with IC50 values of 25 and 50 nM, respectively. Firstly, we designed and optimized a series of analogues based on the synthon Rose Bengal (non-competitive inhibitor). This work has especially enabled to highlight the effect of tautomeric forms (quinone and lactone) on the inhibition of VGLUTs. Thus, the quinone form, present at physiological pH, was confirmed as the only able to block the reuptake of glutamate. Secondly, we have been interested in the family of Trypan Blue (competitive inhibitor) and we determined the minimal active structure in order to render these molecules more "drug-like". Indeed, the interest of this project is to develop small structures easily radiomarquable to use in a physiopathological context.

A remoção cirúrgica da catarata é uma intervenção cada vez mais praticada na medicina veterinária e, com ela, diversos estudos têm sido realizados com o intuito de reduzir as possíveis complicações de seu pós-operatório, tal como a opacidade da cápsula posterior, alteração ocasionada principalmente pela diferenciação e migração das células do epitélio lenticular para a cápsula posterior da lente. O objetivo deste estudo foi analisar a morfologia da cápsula anterior e seu epitélio pela coloração de rotina com hematoxilina-eosina; a composição molecular da cápsula pelas técnicas histoquímicas de PAS (avaliação de proteoglicanos) e picrosirius red (avaliação de colágeno IV); além da expressão de morte celular nas células epiteliais pelas técnicas de imunofluorescência com Beclina 1 (autofagia) e Caspase 3 (apoptose), em cães idosos com catarata diabética e não diabética do tipo hipermadura, submetidos ao uso ou não de azul de tripano a 0,1 % durante a cirurgia de remoção da catarata por facoemulsificação. Para o estudo, foram estudadas as células epiteliais e cápsula anterior da lente de 27 cães, sendo 21 fêmeas e 6 machos, de 8 a 12 anos de idade (média = 9,6 anos), de diversas raças, que foram primeiramente divididos em 2 grupos: GC (catarata hipermadura) e GCD (catarata diabética) e, depois, subdivididos em outros 4 grupos: os que utilizaram o corante: GAND (catarata hipermadura com azul de tripano) e GAD (catarata diabética com azul de tripano); e os que não foram submetidos ao uso do corante: GND (catarata hipermadura sem azul de tripano) e GD (catarata diabética sem azul de tripano). Os resultados das análises realizadas observaram que ambas as amostras, tanto as provenientes das cataratas hipermaduras, quanto as diabéticas, sofrem alterações em igual intensidade apesar do diferente processo de formação, ou seja, observou-se que houve positividade na expressão de morte celular do epitélio por apoptose e autofagia em igual intensidade e independente da utilização de azul de tripano a 0,1 % em todos os grupos; que a composição molecular da cápsula quanto à quantidade de proteoglicanos e colágeno IV também é semelhante e que, apesar da cápsula apresentar-se mais espessada do que o normal, ainda preserva sua relação anatômica com o epitélio. Conclui-se, portanto, que as lesões ocasionadas pela formação da catarata diabética e hipermadura no epitélio e em sua membrana basal (cápsula) são semelhantes apesar da diferente fisiopatologia de formação, mas que o epitélio e a cápsula ainda preservam sua relação anatômica e morfologia. Com os resultados, também se pode sugerir que cães que apresentem cataratas destes tipos, devam apresentar menor risco de ocorrência de opacidade na cápsula posterior no pós cirúrgico da catarata / Nowadays, surgical removal of cataracts in dogs is being considered a common intervention. Along with that, many studies have been realized aiming to reduce its post operatory complications, such as the posterior lens capsule opacity, which occurs due to epithelium cells differentiation and migration to the posterior pole. The objective of this study was to evaluate, by morphometry (hematoxilin-eosin), histochemistry (PAS and picrosirius red) and immunofluorescence (Caspase 3 and Beclin 1) analysis: the morphological alterations of the lens anterior capsule and its epithelium; the molecular components of the extracellular matrix; and the expression of lens epithelium cellular death (by autophagy and apoptosis), in elderly diabetic and non diabetic dogs with cataracts, after phacoemulsification surgery utilizing or not 0,1 % trypan blue. Twenty seven dogs, including 21 female and 6 male dogs, with ages varying from 8 to 12 years old (mean = 9.6 yo), of different breeds were studied. Animals were first divided into 2 groups: GC (hypermature cataracts) and GCD (diabetic cataracts); then, subdivided in 4 different groups: GAND (non diabetic dogs with trypan blue), GAD (diabetic dogs with trypan blue), GND (non diabetic dogs without trypan blue) and finally, GD (diabetic dogs without trypan blue). Results showed that both types of studied samples (diabetic and hypermature ones), are equally altered besides its different pathophysiology, since no statistical significant differences were observed in both capsule and epithelium thickness, type IV collagen and proteoglycans concentration and also, in the epithelium cells autophagy and apoptosis, observed by Caspase 3 and Beclin 1 expression in every studied group. In addition, 0,1 % trypan blue was not capable to demonstrate its toxicity rate, as cells were already under the referred death processes. In conclusion, our findings suggests that, besides the differences in both, hypermature and diabetic cataracts, pathophysiologies of formation, serious and similar alterations are observed with the same intensity in the anterior lens capsule and its epithelium in dogs, what must probably reduce posterior lens capsule opacity post phacoemulsification surgery

Background and motivation: The event of neural Ca2+ overload is known to have several deleterious effects resulting in cell death caused by ischaemia, hypoglycaemia, hypoxia and several neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and AIDS-related dementia. In vitro models for the investigation of the mechanisms involved in Ca2+ overload include brain slice preparations, neuronal cultures as well as acutely isolated neurons, mostly from the hippocampus and cortical brain areas. Additional models for investigating Ca2+ overload may bring about new knowledge to areas of the phenomenon that are still unresolved. Methodology: In this study, several theoretical Ca2+ overload-related interventions were combined aimed at inducing cell death in acutely isolated rat dorsal root ganglia. To elucidate the mechanism/s involved in the cell death observed following exposure to this intervention, the effects of several alterations to the intervention's composition were assessed. This examination was extended by the addition of several recognized and potential protective compounds to the intervention. Cell death was indicated by the trypan blue exclusion assay and recorded after 18 hours exposure to the interventions by counting live and dead neurons under a light microscope. Results and conclusions: The goal was to evaluate the possible application of dorsal root ganglia as a model for neural Ca2+ overload outside the brain. Since Ca2+w as required for cell death to be induced, it is concluded that the observed cell death was indeed primarily due to Ca2+ overload. Besides extracellular Ca2+, KC1-induced depolarization was also required for cell death to be induced, while the antagonists did not demonstrate significant protection against cell death. Based on the results, the mechanism of Ca2+ overload could not be defined beyond doubt, but the voltage activated Ca2+ channels are likely to be involved. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2005.

Suspensions of pancreatic islet cells from ob/ob-mice were incubated with Trypan Blue. Microscope photometry showed that apparently viable cells excluded the dye completely, whereas the nuclei of non-viable cells accumulated Trypan Blue by a saturable process. Alloxan rapidly increased the permeability of the plasma membrane in mouse 3-cells; the exclusion of Trypan Blue is a valid and useful measure of islet cell viability following alloxan exposure. The diabetogenic action of alloxan may be mediated by hydroxyl radicals. In several biological systems hydroxyl radicals are formed by an iron-catalyzed reaction between superoxide anion radicals and hydrogen peroxide. To test whether this applies to alloxan diabetogenicity, the effects of superoxide dismutase, catalase, scavengers of hydroxyl radicals, and metal ion chelators were tested (a) in a cell-free radical-generating system and (b) on islets and islet-cells exposed to alloxan In vitro. The effect of longtime-circulating superoxide dismutase injected prior to alloxan was tested on mice in vivo. Luminol chemiluminescence was used to monitor alloxan-dependent radical production. Accumulation of 8^Rb+ and exclusion of Trypan Blue were used as cell viability criteria in isolated mouse islets and islet-cells. Blood glucose was determined to monitor the development of diabetes in living animals. Superoxide dismutase, catalase, scavengers of hydroxyl radicals, and metal ion chelators inhibited the alloxan-dependent chemiluminescence and decreased the toxic effects on Rb+ accumulation or Trypan Blue exclusion in islets and islet-cells. Superoxide dismutase, linked to polyethylene glycol and injected 12 hours before alloxan, largely prevented the development of alloxan diabetes. Alloxan toxicity _in vitro and in vivo seems to depend on the formation of superoxide radicals and hydrogen peroxide which in turn form the noxious hydroxyl radical via an iron-catalyzed Haber-Weiss reaction. As free radicals and hydrogen peroxide can be formed by other chemicals and during inflammation, and inflammation may accompany the outbreak of human diabetes, studies on the beneficiary effects of superoxide dismutase and other scavengers of free radicals in other forms of diabetes seem warranted. / <p>S. 1-38: sammanfattning, s. 39-74: Härtill 6 uppsatser</p> / digitalisering@umu

Background and motivation: The event of neural Ca2+ overload is known to have several deleterious effects resulting in cell death caused by ischaemia, hypoglycaemia, hypoxia and several neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and AIDS-related dementia. In vitro models for the investigation of the mechanisms involved in Ca2+ overload include brain slice preparations, neuronal cultures as well as acutely isolated neurons, mostly from the hippocampus and cortical brain areas. Additional models for investigating Ca2+ overload may bring about new knowledge to areas of the phenomenon that are still unresolved. Methodology: In this study, several theoretical Ca2+ overload-related interventions were combined aimed at inducing cell death in acutely isolated rat dorsal root ganglia. To elucidate the mechanism/s involved in the cell death observed following exposure to this intervention, the effects of several alterations to the intervention's composition were assessed. This examination was extended by the addition of several recognized and potential protective compounds to the intervention. Cell death was indicated by the trypan blue exclusion assay and recorded after 18 hours exposure to the interventions by counting live and dead neurons under a light microscope. Results and conclusions: The goal was to evaluate the possible application of dorsal root ganglia as a model for neural Ca2+ overload outside the brain. Since Ca2+w as required for cell death to be induced, it is concluded that the observed cell death was indeed primarily due to Ca2+ overload. Besides extracellular Ca2+, KC1-induced depolarization was also required for cell death to be induced, while the antagonists did not demonstrate significant protection against cell death. Based on the results, the mechanism of Ca2+ overload could not be defined beyond doubt, but the voltage activated Ca2+ channels are likely to be involved. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2005.

A remoção cirúrgica da catarata é uma intervenção cada vez mais praticada na medicina veterinária e, com ela, diversos estudos têm sido realizados com o intuito de reduzir as possíveis complicações de seu pós-operatório, tal como a opacidade da cápsula posterior, alteração ocasionada principalmente pela diferenciação e migração das células do epitélio lenticular para a cápsula posterior da lente. O objetivo deste estudo foi analisar a morfologia da cápsula anterior e seu epitélio pela coloração de rotina com hematoxilina-eosina; a composição molecular da cápsula pelas técnicas histoquímicas de PAS (avaliação de proteoglicanos) e picrosirius red (avaliação de colágeno IV); além da expressão de morte celular nas células epiteliais pelas técnicas de imunofluorescência com Beclina 1 (autofagia) e Caspase 3 (apoptose), em cães idosos com catarata diabética e não diabética do tipo hipermadura, submetidos ao uso ou não de azul de tripano a 0,1 % durante a cirurgia de remoção da catarata por facoemulsificação. Para o estudo, foram estudadas as células epiteliais e cápsula anterior da lente de 27 cães, sendo 21 fêmeas e 6 machos, de 8 a 12 anos de idade (média = 9,6 anos), de diversas raças, que foram primeiramente divididos em 2 grupos: GC (catarata hipermadura) e GCD (catarata diabética) e, depois, subdivididos em outros 4 grupos: os que utilizaram o corante: GAND (catarata hipermadura com azul de tripano) e GAD (catarata diabética com azul de tripano); e os que não foram submetidos ao uso do corante: GND (catarata hipermadura sem azul de tripano) e GD (catarata diabética sem azul de tripano). Os resultados das análises realizadas observaram que ambas as amostras, tanto as provenientes das cataratas hipermaduras, quanto as diabéticas, sofrem alterações em igual intensidade apesar do diferente processo de formação, ou seja, observou-se que houve positividade na expressão de morte celular do epitélio por apoptose e autofagia em igual intensidade e independente da utilização de azul de tripano a 0,1 % em todos os grupos; que a composição molecular da cápsula quanto à quantidade de proteoglicanos e colágeno IV também é semelhante e que, apesar da cápsula apresentar-se mais espessada do que o normal, ainda preserva sua relação anatômica com o epitélio. Conclui-se, portanto, que as lesões ocasionadas pela formação da catarata diabética e hipermadura no epitélio e em sua membrana basal (cápsula) são semelhantes apesar da diferente fisiopatologia de formação, mas que o epitélio e a cápsula ainda preservam sua relação anatômica e morfologia. Com os resultados, também se pode sugerir que cães que apresentem cataratas destes tipos, devam apresentar menor risco de ocorrência de opacidade na cápsula posterior no pós cirúrgico da catarata / Nowadays, surgical removal of cataracts in dogs is being considered a common intervention. Along with that, many studies have been realized aiming to reduce its post operatory complications, such as the posterior lens capsule opacity, which occurs due to epithelium cells differentiation and migration to the posterior pole. The objective of this study was to evaluate, by morphometry (hematoxilin-eosin), histochemistry (PAS and picrosirius red) and immunofluorescence (Caspase 3 and Beclin 1) analysis: the morphological alterations of the lens anterior capsule and its epithelium; the molecular components of the extracellular matrix; and the expression of lens epithelium cellular death (by autophagy and apoptosis), in elderly diabetic and non diabetic dogs with cataracts, after phacoemulsification surgery utilizing or not 0,1 % trypan blue. Twenty seven dogs, including 21 female and 6 male dogs, with ages varying from 8 to 12 years old (mean = 9.6 yo), of different breeds were studied. Animals were first divided into 2 groups: GC (hypermature cataracts) and GCD (diabetic cataracts); then, subdivided in 4 different groups: GAND (non diabetic dogs with trypan blue), GAD (diabetic dogs with trypan blue), GND (non diabetic dogs without trypan blue) and finally, GD (diabetic dogs without trypan blue). Results showed that both types of studied samples (diabetic and hypermature ones), are equally altered besides its different pathophysiology, since no statistical significant differences were observed in both capsule and epithelium thickness, type IV collagen and proteoglycans concentration and also, in the epithelium cells autophagy and apoptosis, observed by Caspase 3 and Beclin 1 expression in every studied group. In addition, 0,1 % trypan blue was not capable to demonstrate its toxicity rate, as cells were already under the referred death processes. In conclusion, our findings suggests that, besides the differences in both, hypermature and diabetic cataracts, pathophysiologies of formation, serious and similar alterations are observed with the same intensity in the anterior lens capsule and its epithelium in dogs, what must probably reduce posterior lens capsule opacity post phacoemulsification surgery

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Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-09-12T20:54:45Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Marcio Tavares Costa.pdf: 1603225 bytes, checksum: 56077ca53540d1e03d5e586689091d43 (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-09-12T20:56:36Z (GMT) No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Marcio Tavares Costa.pdf: 1603225 bytes, checksum: 56077ca53540d1e03d5e586689091d43 (MD5) / Made available in DSpace on 2016-09-12T20:56:36Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Marcio Tavares Costa.pdf: 1603225 bytes, checksum: 56077ca53540d1e03d5e586689091d43 (MD5) Previous issue date: 2016-07-15 / Philodryas patagoniensis é uma serpente opistóglifa comum no Rio Grande do Sul. Sua peçonha, produzida na glândula Duvernoy, constitui-se basicamente por metaloproteinases. Compostos biologicamente ativos que, assim como outros extratos naturais, são alvos de pesquisas na busca por agentes farmacológicos. No entanto, em conjunto, os mesmos devem passar por testes para caracterizar seus efeitos toxicológicos. Estes exames permitem definir se o composto analisado é capaz de causar danos à saúde humana, diagnosticar seus possíveis locais de ação, além de apontar tratamentos em casos de intoxicações e envenenamentos. Assim, a presente dissertação visa avaliar os potenciais toxicológicos da peçonha de P. patagoniensis: citotoxicologia e genotoxicologia. Para tanto, espécimes de P. patagoniensis foram capturados para a extração da zootoxina, a qual teve sua concentração proteica determinada por método colorimétrico de Bradford. A determinação da toxicidade aguda foi realizada em Artemia salina. Para avaliar a citotoxicidade e a genotoxicidade utilizou-se o teste de exclusão de azul de Tripan e ensaio Cometa em leucócitos mononucleares humanos, respectivamente. Ao final, testou-se a presença de atividade DNase na peçonha via difusão radial em agarose, e sua capacidade de gerar estresse oxidativo em náuplios de A. salina. Os resultados indicaram uma concentração proteica média de 115,7 mg/mL nos pools de veneno extraídos, os quais apresentaram uma DL50 de 461 μg/mL em A. salina, dose que o caracteriza como tóxico para este modelo experimental. Ao testar a toxina nas concentrações de 55, 110, 220, 575 e 1150 μg/mL, sua capacidade citotoxicológica e genotoxicológica foi significativa nas duas maiores concentrações. A atividade DNase foi ausente, no entanto, o estresse oxidativo foi detectado pela presença de biomarcadores. Foi possível estabelecer uma relação direta entre a toxicidade aguda em A. salina, a citotoxicidade e a genotoxicidade apurada, pois as doses letais para as artêmias causaram danos significativos, enquanto que as subletais não obtiveram diferença expressiva. Entre as possíveis razões para esta toxicidade, o estresse oxidativo desencadeado pela zootoxina demonstra estar relacionado aos danos encontrados. / Philodryas patagoniensis is a common opisthoglyphous serpent in Rio Grande do Sul State. Its venom produced in Duvernoy’s gland consists mainly of metalloproteinases. Compounds biologically active which, like other natural extracts, are research targets in pursuit of pharmacological agents. However, together, should go through tests to characterize their toxicological effects. These tests allow you to define whether the analysed compound is able to cause damage to human health, diagnose its possible sites of action, in addition to point treatments in cases of intoxications and poisonings. Thus, the dissertation aims to evaluate the potential toxicity of P. patagoniensis venom: cytotoxicology and genotoxicology. Therefore, P. patagoniensis specimens were captured for the extraction of the zootoxin, which had its protein concentration determined by Bradford colorimetric method. Determination of acute toxicity was conducted in Artemia salina. To assess the cytotoxicity and genotoxicity used the exclusion of Trypan blue test and Comet assay in human mononuclear leukocytes, respectively. Finally, tested the presence of DNase activity in the venom via radial diffusion in agarose, and venom’s ability to generate oxidative stress in nauplii of A. salina. The results indicated a mean protein concentration of 115.7 mg/mL in pools of venom extracted, which showed an LD50 of 461 μg/mL in A. salina, dose that characterizes as toxic to this experimental model. When the toxin was tested at concentrations of 55, 110, 220, 575 and 1150 μg/ml, its cytotoxicology and genotoxicology capacity were significant in two largest concentrations. The DNase activity was absent, however, oxidative stress was detected by the presence of biomarkers. It was possible to establish a direct relationship between the acute toxicity in A. salina, cytotoxicity and genotoxicity found, because the lethal doses for brine shrimp caused significant damage, while sublethal did not obtain significant difference. Among the possible reasons for this toxicity, oxidative stress triggered by zootoxin shown to be related to the damage found.