As enfermidades reprodutivas representam um entrave à produção de bovinos leiteiros, à medida que a enfermidade diminui a fertilidade dos rebanhos e causa muitos prejuízos ligados ao custo de tratamento e perda produtiva. Entre estas as endometrites possuem papel de destaque. Existem diferentes técnicas para o diagnóstico destas enfermidades como a palpação uterina por via transretal, a vaginoscopia, o cultivo microbiológico de fluidos uterinos, a biópsia uterina, a citologia endometrial e a ultrassonografia que variam em grau de praticidade, especificidade e sensibilidade. O presente trabalho teve por finalidade avaliar e comparar a vaginoscopia, ultrassonografia, a histopatologia e suas possíveis associações para o diagnóstico da endometrite através da determinação da sensibilidade, da especificidade e do coeficiente de concordância kappa utilizando a citologia endometrial como padrão ouro. Para este fim foi necessário desenvolver um método adaptado para avaliação do exame histopatológico de útero bovino. Neste estudo, 106 bovinos do sexo feminino da raça Holandesa, foram submetidos ao exame ginecológico. Destes animais, os que não apresentaram secreção vaginal de aspecto catarral foram submetidos ao exame ultrassonográfico, à colheita de material para exames de citologia endometrial e a colheita de biópsia uterina. Os resultados demonstraram que a ultrassonografia constitui um método diagnóstico pratico e rápido, sendo da associação entre a determinação da presença de líquido uterino e a determinação do diâmetro cervical a maior eficiência diagnóstica neste contexto, com sensibilidade, especificidade e coeficiente de concordância kappa de 50%, 88% e 39% respectivamente. A histopatologia por sua vez também constituiu um bom método diagnóstico, no entanto menos prático que a ultrassonografia / Reproductive diseases are a great barrier to the dairy production, as it diminishes fertility and causes economical losses with treatment costs and lowering production. In this context, endometritis plays an important role. There are different diagnostic techniques for endometritis, rectal uterine palpation vaginoscopy, microbiological tests, uterine biopsies, endometrial cytology and ultrasonography, these tests vary in sensitivity and specificity. This work had as an objective evaluating and comparing vaginoscopic, ultrasongraphic and histopathological examinations, as well as their associations, for the endometritis diagnosis by determining their sensitivity, specificity and kappa agreement coefficient using endometrial cytology as gold standard. It was necessary developing an adaptive reading score to perform the uteris histological examination. In this study 106 female Holstein cows were submitted to a gynecological examination. The ones that had no vaginal catarrh were submitted to an ultrasonographic examination, to endometrial cytology examination and uterine biopsy. Results shows that ultrasonography is a practical and good diagnostic method, the association between the determination of intra-uterine fluid and cervical diameter was the most efficient ultrasonographic method, with 50%, 88% and 39% of sensitivity, specificity and agreement coefficient kappa respectively. Histopathological examination was good diagnostic method, even though not as practical as ultrasonography

Orientador: Priscylla Tatiana Chalfun Guimarães-Okamoto / Resumo: A doença renal crônica (DRC) em cães apresenta grande morbidade e alta taxa de mortalidade, sendo considerada a alteração renal mais comum, tendo a nefropatia glomerular como a alteração de maior prevalência. Este estudo tem por objetivo avaliar a prevalência das alterações glomerulares em conjunto com avaliação da razão proteína creatinina urinárias (RPC), dos níveis séricos de albumina e creatinina e pressão arterial sistólica comparando os parâmetros clínicos e laboratoriais entre o grupo de animais com a lesão predominante e o grupo abrangendo os demais tipos de lesões glomerulares que foram encontrados. Foram utilizados 24 cães com doença renal crônica que morreram. As lesões glomerulares foram classificadas segundo Cianciolo et al. (2013) por meio da análise histopatológica de tecidos renais corados pelas técnicas histoquímicas de hematoxilina e eosina, tricrômico de Masson, ácido periódico de Schiff e vermelho Congo. Foram inclusos animais com exames de urina com sedimento inativo, RPC, creatinina e albumina sérica. Para análise estatística foram utilizados os últimos valores obtidos antes do óbito, não excedendo o limite de quatro semanas. Não foi observado diferenças entre RPC, albumina e creatinina séricas e pressão arterial sistólica entre os animais com alteração membranosa e o grupo contendo os demais tipos de lesão glomerular encontrados em cães com DRC. Na DRC, independentemente do tipo de lesão glomerular, os parâmetros clínicos e laboratoriais avaliados neste... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

A antracnose da videira tem como agente causal o fungo Elsinoë ampelina Shear, o qual ataca tecidos tenros da parte aérea causando lesões necróticas e comprometendo o desenvolvimento da planta com consequente queda na quantidade e qualidade dos frutos produzidos. Cultivares de Vitis apresentam diferentes níveis de susceptibilidade à antracnose. Uma vez que características anatômicas e químicas podem atuar como mecanismos de resistência a patógenos, o presente estudo propõe comparar caracteres histológicos e bioquímicos em folhas jovens de Vitis labrusca \'Niagara Rosada\' (NR, susceptível), Vitis vinifera \'Thompson Seedless\' (TS, susceptível) e V. vinifera \'Pinot Noir\' (PN, resistente). As análises anatômicas foram realizadas em folhas sadias aos 4 e 11 dias após o brotamento (DAB) e em folhas lesionadas com 11 DAB, enquanto os compostos bioquímicos foram quantificados em folhas sadias aos 4 e 11 DAB. Os caracteres analisados foram: índice estomático, densidade de idioblastos cristalíferos, espessura da cutícula da face adaxial, espessura da parede periclinal externa e altura das células epidérmicas de ambas as faces, espessura do mesofilo total, do parênquima paliçádico e do parênquima lacunoso, o conteúdo de compostos fenólicos totais, flavonoides e de clorofila a, b e total, bem como a atividade de peroxidase e de polifenoloxidase. Os dados obtidos foram submetidos à análise estatística. A cultivar TS apresentou índice estomático maior nas duas idades avaliadas em comparação com o genótipo resistente PN, a qual apresentou maior espessura de cutícula nas duas idades e de mesofilo total aos 11 DAB. Os parâmetros quantitativos das amostras inoculadas apresentaram redução significativa em relação às não inoculadas, com exceção da área foliar de PN, a qual não foi significativamente menor. Para NR e TS, 100% das amostras inoculadas apresentaram várias lesões expandidas, enquanto PN apenas 60% das folhas analisadas apresentaram poucas lesões bem delimitadas e de formato circular. A cultivar resistente PN apresentou forte reação positiva a compostos fenólicos bem como o maior conteúdo desses compostos na idade 11 DAB, enquanto a cultivar susceptível TS apresentou reação muito fraca e a menor concentração. O conteúdo de flavonoides foi maior em PN em relação à TS nas duas idades avaliadas, e NR apresentou o valor mais baixo aos 4 DAB, quando é altamente susceptível. A atividade de polifenoloxidase aos 11 DAB foi maior na cultivar PN, intermediária em NR e mais baixa em TS. A análise de todos os parâmetros anatômicos e bioquímicos avaliados indica que a maior resistência da cultivar PN esteja relacionada ao menor índice estomático, à cutícula mais espessa, ao conteúdo mais elevado de compostos fenólicos totais e de flavonoides, bem como a maior atividade enzimática de polifenoloxidase. / Grapevine anthracnose is caused by the fungus Elsinoë ampelina Shear, which attacks tissues of the aerial part causing necrotic lesions, compromising plant growth with consequent decrease in quantity and quality of the fruits produced. Vitis cultivars show different levels of susceptibility to anthracnose. Anatomical and genetic characteristics can act as a resistance mechanism against pathogens, therefore, this study proposes to compare histological and biochemical characteristics in young leaves of Vitis labrusca \'Niagara Rosada\' (NR, susceptible), Vitis vinifera \'Thompson Seedless\' (TS, susceptible) and V. vinifera \'Pinot Noir\' (PN, resistant). The anatomical analyses were performed on healthy leaves at 4 and 11 days after budding (DAB) and at 11 DAB on lesioned leaves, while biochemical compounds were quantified on healthy leaves at 4 and 11 DAB. The features analyzed were: stomatal index, idioblasts density, adaxial face cuticle thickness, external periclinal wall thickness, epidermal cell height of both faces, total mesophyll thickness, palisade parenchyma and spongy parenchyma, total phenolic compounds content, flavonoids, chlorophyll a, b, and total, and peroxidase and polyphenoloxidase activity. The data were submitted to the statistical analysis. The TS cultivar had a higher stomatal index at both ages evaluated compared to the PN resistant genotype, which presented greater cuticle thickness at leaf ages and total mesophyll at 11 DAB. The quantitative parameters of the inoculated samples presented a significant reduction in relation to the non- inoculated, except for the PN leaf area, which was not significantly lower. For NR and TS, 100% of the inoculated samples presented several expanded lesions, whereas PN only 60% of the analyzed leaves showed few lesions well delimited and circular in shape. The resistant cultivar PN showed strong positive reaction to phenolic compounds as well as the highest content of these compounds at 11 DAB, while the TS susceptible cultivar presented very weak reaction and the lowest concentration. The content of flavonoids was higher in PN compared to TS at both ages evaluated, and NR presented the lowest flavonoid content at 4 DAB, when it was highly susceptible. The polyphenoloxidase activity at 11 DAB was higher in the PN cultivar, intermediate in NR, and lower in TS. The analyses of all the anatomical and biochemical parameters indicate that the higher resistance of the PN cultivar is related to the lower stomatal index, thicker cuticle, higher content of total phenolic compounds and flavonoids, as well as higher enzymatic activity of polyphenoloxidase.

A cercosporiose é a principal doença foliar do caquizeiro no Brasil. A doença está distribuída entre as principais regiões produtoras do país e é responsável pela queda precoce das folhas e maturação antecipada dos frutos. Informações referentes a este patossistema são escassas na literatura. O objetivo deste trabalho foi (I) desenvolver e validar uma escala diagramática para avaliar a severidade da cercosporiose; (II) caracterizar isolados obtidos de folhas de caquizeiros com sintomas de cercosporiose de diferentes regiões do Brasil através de análises filogenéticas, morfologia e testes de patogenicidade; e (III) estudar os processos infecção e colonização de isolado patogênico causador de cercosporiose em folhas de caquizeiro. A escala diagramática desenvolvida compreende seis diagramas com severidade variando entre 0,5 e 17,3%. O uso da escala diagramática melhorou a acurácia, precisão, concordância e reprodutibilidade das estimativas conduzidas por 10 avaliadores, mostrando-se, portanto, eficiente para avaliação de cercosporiose de caquizeiros. Para a caracterização de 37 isolados obtidos em folhas de caquizeiros com cercosporiose, coletadas nos estados de São Paulo, Paraná e Rio Grande do Sul, foram analisadas as sequências do espaçador interno transcrito (ITS), actina (ACT) e fator de elongação 1-α (TEF). Alta diversidade genética foi observada entre os isolados coletados. A rede de haplótipos gerada a partir da análise dos três genes de forma concatenada resultou na formação de 10 haplótipos. As colônias apresentaram coloração predominantemente esverdeada, topografia levantada com bordos inteiro, ausência de esporos e crescimento lento. Quanto ao processo de infecção, a penetração do patógeno ocorreu através dos estômatos, mas não foi observado tropismo positivo em direção aos estômatos. No processo de colonização dos tecidos foliares, compostos fenólicos foram acumulados no tecido infectado, porém não foram capazes de conter o processo de colonização. Os testes histoquímicos também revelaram o acúmulo de pectina nas células da extensão da bainha foliar e a presença de compostos lipofílicos nas áreas lesionadas. Foram observadas vesículas nos espaços intercelulares em regiões com a presença de hifas do patógeno, que possivelmente estão envolvidas na degradação da parede celular do hospedeiro. / Angular Leaf spot is the major foliar disease of persimmon in Brazil. The disease is widely spread among major producing areas in the country and is responsible for early defoliation and anticipated fruits ripening. Information about the pathosystem are limited. Therefore, this study aimed to (I) develop and validate a standard area diagram set (SADs) for assessing angular leaf spot severity; (II) characterize isolates obtained from persimmon leaves with angular leaf spot symptoms from different regions by means of phylogenetic analyses, morphological features and pathogenicity tests; and (III) study infection and colonization process of pathogen isolate that caused angular leaf spot in persimmon leaves. The SADs developed comprises six colour diagrams with severity ranging from 0.5 to 17.3%. The use of the SADs improved the accuracy, precision, agreement and inter-rater reliability of the estimates conducted by 10 raters, showed to be adequate for estimates of angular leaf spot in persimmon. To characterize 37 isolates obtained from persimmon leaves with angular leaf spot, collected from the states of São Paulo, Paraná and Rio Grande do Sul, were analyzed internal transcribed spacer (ITS), actin (ACT) and elongation factor 1-α (TEF) sequences. High genetic variability was observed among collected isolates. The haplotype network generated from combined analyzed of ITS, ACT and TEF, showed 10 distinct haplotypes. Colonies showed predominantly greenish color, raised elevation with circular form, absence of spores and slow growth. In the infection process, the germ tubes entered the leaf tissue through the stomata, but was not observed positive tropism to stomata. In the colonization process of foliar tissues, phenolic compounds were accumulated in the infected tissue; however, they did not prevent the colonization progress. Histochemical studies showed accumulation of pectin in the bundle sheath extension cells and the presence of lipophilic compounds in the injured areas. Vesicles were observed in the intercellular spaces in regions with the presence of hyphae of the pathogen, that are possibly involved in the degradation of the host cell wall.

The overall objective of my thesis research was to develop methodologies for assessing effects of mining effluents on pristine and sensitive northern rivers. I used a multi-trophic level approach in field studies to evaluate current monitoring methods and to determine whether metal mining activities had affected two otherwise pristine rivers that flow into the South Nahanni River, NWT; a World Heritage Site. Upstream reference conditions in the rivers were compared to sites downstream and further downstream of mines. The endpoints evaluated included concentrations of metals in river water, sediments and liver and flesh of slimy sculpin (Cottus cognatus); benthic algal and macroinvertebrate abundance, richness, diversity, and community composition; and various slimy sculpin measures. Elevated concentrations of copper (p=0.002)and iron (p=0.001) in liver tissue of sculpin from the Flat River were associated with high concentrations of mine-derived iron in river water and copper in sediments that were above national guidelines. In addition, sites downstream of the mine on the Flat River had increased algal abundances (p=0.002) and altered benthic macroinvertebrate communities ((p<0.001) whereas the sites downstream of the mine on Prairie Creek had increased benthic macroinvertebrate taxa richness (p=0.050) and improved sculpin condition (males: p=0.008; females: p=0.001). Biological differences in both rivers were consistent with mild enrichment of the rivers downstream of current and historical mining activity. Although the effects of mining activities on riverine biota in these northern rivers are currently limited, results of this research show that there is potential for effects to occur with proposed growth in mining activities.<p> Laboratory exposures were conducted using slimy sculpin, identified as a sentinel fish species in pristine northern rivers, to identify alternative methods for assessing toxicity of contaminants of concern in mining effluents. Ammonia was selected for the exposures based on effluent characteristics of northern mining effluents. Ammonia is known to be an important toxicant in aquatic environments. Although ammonia toxicity has been well studied in many fish species, effects of chronic exposure of slimy sculpin, a critical biomonitoring species for northern aquatic habitats, are not well known. Slimy sculpin were exposed to six concentrations of un-ionized ammonia relevant to concentrations found in northern mining effluents: control (0 ppm), 0.278 ppm, 0.556 ppm, 0.834 ppm, 1.112 ppm, and 1.668 ppm. An LC50 of 1.529 ppm was calculated from mortality data. Histopathological examination of gills indicated significant tissue damage, measured as lamellar fusion and epithelial lifting, at 0.834, 1.112, and 1.668 ppm. Using gill endpoints, NOEC and LOEC were calculated as 0.556 ppm and 0.834 ppm respectively. An EC50 of 0.775 ppm was determined for lamellar fusion and an EC50 of 0.842 ppm for epithelial lifting. Hemorrhage of gills was present in mortalities which occurred at 1.668 ppm of un-ionized ammonia. A significant decrease in liver somatic index (LSI) was seen in both male and female fish at 0.834 and 1.112 ppm, respectively. Gonadosomatic index (GSI) in female fish significantly increased at 1.668 ppm un-ionized ammonia with an associated significant increase in total whole body testosterone concentrations. GSI in male fish also significantly increased at 1.668 ppm but no differences were seen in testosterone concentrations. No significant differences were seen in gonad histopathological assessments or condition factor. Results from this study indicate that ammonia concentrations commonly reported in northern mine effluents hold potential to affect the health of slimy sculpin including acute, chronic, histological and endocrine endpoints. <p> Results from both the field study and laboratory exposures provide direction for future monitoring programs in pristine northern rivers and emphasize the importance of monitoring tools to detect change in these ecosystems. I recommend that monitoring of northern pristine rivers focus on a multi-trophic monitoring approach including indicators in algal and benthic macroinvertebrate communities due to their responsiveness. Laboratory exposures using slimy sculpin should be considered to obtain toxicological information for northern contaminants of concern. Gill histopathology endpoints may be a more sensitive indicator for detecting effects in slimy sculpin exposed to ammonia than traditional chronic endpoints. I also recommend monitoring of metal burdens in periphyton and benthic invertebrates for assessment of exposure to mine effluent and causal association in areas of low fish abundance.

The overall objective of my thesis research was to develop methodologies for assessing effects of mining effluents on pristine and sensitive northern rivers. I used a multi-trophic level approach in field studies to evaluate current monitoring methods and to determine whether metal mining activities had affected two otherwise pristine rivers that flow into the South Nahanni River, NWT; a World Heritage Site. Upstream reference conditions in the rivers were compared to sites downstream and further downstream of mines. The endpoints evaluated included concentrations of metals in river water, sediments and liver and flesh of slimy sculpin (Cottus cognatus); benthic algal and macroinvertebrate abundance, richness, diversity, and community composition; and various slimy sculpin measures. Elevated concentrations of copper (p=0.002)and iron (p=0.001) in liver tissue of sculpin from the Flat River were associated with high concentrations of mine-derived iron in river water and copper in sediments that were above national guidelines. In addition, sites downstream of the mine on the Flat River had increased algal abundances (p=0.002) and altered benthic macroinvertebrate communities ((p<0.001) whereas the sites downstream of the mine on Prairie Creek had increased benthic macroinvertebrate taxa richness (p=0.050) and improved sculpin condition (males: p=0.008; females: p=0.001). Biological differences in both rivers were consistent with mild enrichment of the rivers downstream of current and historical mining activity. Although the effects of mining activities on riverine biota in these northern rivers are currently limited, results of this research show that there is potential for effects to occur with proposed growth in mining activities.<p> Laboratory exposures were conducted using slimy sculpin, identified as a sentinel fish species in pristine northern rivers, to identify alternative methods for assessing toxicity of contaminants of concern in mining effluents. Ammonia was selected for the exposures based on effluent characteristics of northern mining effluents. Ammonia is known to be an important toxicant in aquatic environments. Although ammonia toxicity has been well studied in many fish species, effects of chronic exposure of slimy sculpin, a critical biomonitoring species for northern aquatic habitats, are not well known. Slimy sculpin were exposed to six concentrations of un-ionized ammonia relevant to concentrations found in northern mining effluents: control (0 ppm), 0.278 ppm, 0.556 ppm, 0.834 ppm, 1.112 ppm, and 1.668 ppm. An LC50 of 1.529 ppm was calculated from mortality data. Histopathological examination of gills indicated significant tissue damage, measured as lamellar fusion and epithelial lifting, at 0.834, 1.112, and 1.668 ppm. Using gill endpoints, NOEC and LOEC were calculated as 0.556 ppm and 0.834 ppm respectively. An EC50 of 0.775 ppm was determined for lamellar fusion and an EC50 of 0.842 ppm for epithelial lifting. Hemorrhage of gills was present in mortalities which occurred at 1.668 ppm of un-ionized ammonia. A significant decrease in liver somatic index (LSI) was seen in both male and female fish at 0.834 and 1.112 ppm, respectively. Gonadosomatic index (GSI) in female fish significantly increased at 1.668 ppm un-ionized ammonia with an associated significant increase in total whole body testosterone concentrations. GSI in male fish also significantly increased at 1.668 ppm but no differences were seen in testosterone concentrations. No significant differences were seen in gonad histopathological assessments or condition factor. Results from this study indicate that ammonia concentrations commonly reported in northern mine effluents hold potential to affect the health of slimy sculpin including acute, chronic, histological and endocrine endpoints. <p> Results from both the field study and laboratory exposures provide direction for future monitoring programs in pristine northern rivers and emphasize the importance of monitoring tools to detect change in these ecosystems. I recommend that monitoring of northern pristine rivers focus on a multi-trophic monitoring approach including indicators in algal and benthic macroinvertebrate communities due to their responsiveness. Laboratory exposures using slimy sculpin should be considered to obtain toxicological information for northern contaminants of concern. Gill histopathology endpoints may be a more sensitive indicator for detecting effects in slimy sculpin exposed to ammonia than traditional chronic endpoints. I also recommend monitoring of metal burdens in periphyton and benthic invertebrates for assessment of exposure to mine effluent and causal association in areas of low fish abundance.

Today great efforts are made to record and reduce waiting times in cancer care. Long and variable turnaround times (TATs) delay the start of treatment and waiting contributes to mental anguish. The purposes of the QI intervention were to establish an effective and streamlined histopathology process with shorter TATs, to extend customer collaboration and to build knowledge of internal processes in order to lay the foundation for a learning environment. The goal was to raise the proportion of reported tissue samples from 50% to 90% within a 15 day period, ending 31th December 2014. The study of the QI intervention intended to identify factors that affect the introduction of novel working methods. Both quantitative and qualitative methods were used to achieve the goals. Improvement knowledge was combined with lean-inspired methods, and two focus groups were arranged in which data were analysed using qualitative content analysis. The goal to report 90% of tissue samples within 15 days was not achieved for all sample types, but improved TATs were clearly noted. Customer collaboration and visualisation of the processes had a positive effect on staff. The study resulted in six key factors important working with QI interventions; competence, compliance, feedback, interaction, patient- and customer focus and resources. Having motivated and dedicated staff is a key success factor for improvement work, in contrast to a lack of resources, and people that oppose change. To achieve future ambitious goals requires continuous improvement initiatives that involve optimisation of both human resources and equipment. / Stort fokus riktas idag på att kartlägga och reducera väntetider inom cancervården. För långa och variabla svarstider fördröjer behandlingsstart och väntan innebär dessutom psykiskt lidande. Syftet med förbättringsarbetet var att etablera en effektiv och stabil patologiprocess med kortare svarstider, utöka kundsamverkan samt bygga kunskap om interna processer för att lägga grunden till en lärandemiljö. Målet var att senast den 31 december 2014 höja andelen besvarade vävnadsprover från 50 % till 90 % inom 15 dagar. Studien av förbättringsarbetet syftade till att identifiera faktorer som påverkar införandet av nya arbetssätt. Såväl kvantitativa som kvalitativa metoder användes för att uppnå målen; förbättringskunskap kombinerades med lean-inspirerade metoder och två fokusgrupper där data analyserades med kvalitativ innehållsanalys. Målet att höja andelen vävnadsprover som besvarades inom 15 dagar på 90 % uppnåddes inte för samtliga provtyper, men tydligt förbättrade svarstider noterades. Kundsamverkan och visualisering av processerna hade en positiv effekt på personalen. Studien resulterade i sex viktiga faktorer; kompetens, inställning, återkoppling/feedback, interaktion, patient- och kundfokus och resurser. Motiverad och engagerad personal är nyckelframgångsfaktorer i förbättringsarbeten i motsats till resursbrist och människor som motsätter sig förändring. För att på sikt uppnå högt uppsatta mål krävs fortsatta förbättringsinitiativ som involverar optimering av både personalresurser och instrumentering.

The effects of the toxic dinoflagellate, karenia brevis (Wilson clone), on larval survival and development of the northern quahog (=hard clam, Mercenaria mercenaria), eastern oyster (Crassostrea virginica) and bay scallop (Argopecten irradians) were studied in the laboratory. The effects of K. brevis on feeding activities of juveniles from these species plus the green mussel (Perna viridis) were also examined. Finally, adult bay scallops were exposed to K. brevis for two weeks to investigate possible cytotoxic effects. Survival of 3-day-old larvae was generally > 85% for all shellfish species at Karenia brevis densities of 100 cells . ml-1 or less, and not significantly different between whole and lysed culture. At 1,000 cells . ml-1, survival was significantly less in lysed culture than whole culture for both M. mercenaria and C. virginica. Survival of 7-day-old larvae in all species was not significantly affected at densities up to 1,000 cells . ml-1. At 5,000 cells . ml-1, however, survival was reduced to 37, 26 and 19% for A. irradians, M. mercenaria and C. virginica, respectively. Development of C. virginica and M. mercenaria larvae was protracted at K. brevis densities of 1,000 cells . ml-1. Clearance rates of juveniles were determined under static and flow-through conditions using whole and lysed cultures of K. brevis. The bay scallop was most sensitive, exhibiting a 79% reduction in clearance rate at 1,000 cells . ml-1 of whole culture. The eastern oyster was least responsive, showing a 38% reduction in clearance rate between the same treatments. The green mussel and the northern quahog displayed intermediate responses. Similar results were observed during longer (2 day) exposures to a continuous supply of K. brevis. Bay scallops showed a significant decline in clearance rate at 100 cells . ml-1 after 24 hr exposure; clearance rate of oysters was not affected by K. brevis at this concentration. No mortality was observed for any species during these brief exposures. Adult bay scallops exposed to K. brevis for two weeks showed degenerative and inflammatory changes in the digestive gland, including reduced thickness of the epithelium, increased size of digestive tubule lumens and hemocytic infiltration. The prospect for recovery of bay scallop populations in Florida may be hampered by recurring blooms of K. brevis.

Tumour cell dissemination is a major issue with the treatment of cancer, thus new therapeutic strategies which can control this process are needed. Antagonism of integrins highly expressed in tumours is one potential strategy. The integrins are transmembrane glycoprotein adhesive receptors. Two of the integrins, αVβ3 and αIIbβ3, are highly expressed in a number of tumours and induce bi-directional signalling through their interaction with extracellular matrix proteins, and growth factor receptors. Through this signalling they play an important role in a number of cellular processes that are involved in tumour dissemination such as tumour growth, migration, invasion, metastasis and angiogenesis. Dual αIIbβ3 and αVβ3 integrin antagonism will have a direct effect on β3-expressing tumour cells that leads to the inhibition of cell migration and dissemination. Furthermore, through targeting tumour cell interaction with endothelial cells and platelets, this will also lead to inhibition of angiogenesis and metastasis. The aim of this project was to characterise the expression of αVβ3 and αIIbβ3 integrin in a panel of tumour cell lines and in human tumour xenograft samples, and to develop and utilise cell-based models to investigate potential novel β3 antagonists. The expression of αV and β3 subunits was detected in xenograft tissue using immunoblotting techniques. A panel of cell lines of different tumour types including melanoma, prostate, breast, colon and non small cell lung carcinoma was then characterised for αVβ3 and αIIbβ3 integrin expression using immunoblotting and immunocytochemistry. Melanoma cell lines demonstrated the strongest αVβ3 expression. No αIIbβ3 integrin expression was seen in any of the cell lines evaluated. A selection of cell lines with varying αVβ3 expression were then used to develop a functional test for cell migration, the scratch wound healing assay. Migration of tumour cells that expressed αVβ3 integrin was inhibited by the known β3 antagonists, cRGDfV peptide and LM609 antibody. A panel of 12 potential novel β3 integrin antagonists was screened for cytotoxicity and activity in the validated scratch assay. ICT9055 was the most effective antagonist in inhibition of M14 cell migration as determined by the scratch assay, with an IC₅₀ of < 0.1 μM. Therefore the work presented in this thesis has established models and tools for evaluating potential novel β3 integrin antagonists, and identified a promising molecule to progress for further preclinical evaluation.

One in eight women in America will develop breast cancer at some point in their lives. Breast cancer is the second deadliest form of cancer for women in the United States. When a suspicious region of the breast is detected, the tissue is diagnosed by removing a sample, preparing an H&E section, and performing histopathology. This procedure is expensive, invasive, and can take days to return a diagnosis. An alternative to excision biopsies is to instead perform an optical biopsy. This work details endomicroscopes intended to perform optical biopsies in breast tissue. The work address two issues limiting current optical biopsy systems: insufficient resolution and inability to reject out of focus light. To improve the resolution of current endomicroscopes, ultra-slim objectives are developed using optical plastics and zero alignment fabrication techniques. These objectives can outperform current alternative endomicroscope objectives in terms of performance across the field of view and chromatic aberration correction, while remaining as narrow as a biopsy needle. Next, an endomicroscope which utilizes structured illumination to perform optical section is designed, tested, and evaluated on ex vivo breast biopsies. The new endomicroscope provides high contrast images by reducing out of focus background light. Finally, an achromatic, ultra-slim objective and the structured illumination endomicroscope are integrated to form an optical biopsy system with improved lateral resolution and axial response. This integrated system is a step forward for in vivo microscopy and cancer diagnoses.