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Immunohistological (IHC) stained images occupy a fundamental role in the pathologist's diagnosis and monitoring of cancer development. The manual process of monitoring such images is a subjective, time consuming process that typically relies on the visual ability and experience level of the pathologist. A novel and comprehensive system for the automated quantification of antibody inside stained cell nuclei in immunohistochemistry images is proposed and demonstrated in this research. The system is based on a cellular level approach, where each nucleus is individually analyzed to observe the effects of protein antibodies inside the nuclei. The system provides three main quantitative descriptions of stained nuclei. The first quantitative measurement automatically generates the total number of cell nuclei in an image. The second measure classifies the positive and negative stained nuclei based on the nuclei colour, morphological and textural features. Such features are extracted directly from each nucleus to provide discriminative characteristics of different stained nuclei. The output generated from the first and second quantitative measures are used collectively to calculate the percentage of positive nuclei (PS). The third measure proposes a novel automated method for determining the staining intensity level of positive nuclei or what is known as the intensity score (IS). The minor intensity features are observed and used to classify low, intermediate and high stained positive nuclei. Statistical methods were applied throughout the research to validate the system results against the ground truth pathology data. Experimental results demonstrate the effectiveness of the proposed approach and provide high accuracy when compared to the ground truth pathology data.

4 Abstract The main goal of my thesis was to create our own proposals for laboratory work in biology of unicellular organisms (Protista). I designed the laboratory tasks, so that they can be mastered by the pupils of sixth year of primary school. Creating the proposals for laboratory exercises was preceded by the work in the laboratory at Charles University in Prague, Faculty of Education, the Department of Biology and Environmental Studies. In the laboratory, I verified the methods of cultivation of selected representatives of ciliates (Paramecium caudatum), amoebae (Amoeba proteus) and euglenids (Euglena gracilis) and selected methods of dyeing their different organels. Key words: protists, Paramecium caudatum, Amoeba proteus, Euglena gracilis, cultivation, dying

Submitted by Maria Beatriz Vieira (mbeatriz.vieira@gmail.com) on 2017-04-27T12:46:24Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) tese_ireni_leitzke_carvalho.pdf: 1397252 bytes, checksum: ad9c13976120f5dcba14cf0b7f2d0ade (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-05-02T18:14:20Z (GMT) No. of bitstreams: 2 tese_ireni_leitzke_carvalho.pdf: 1397252 bytes, checksum: ad9c13976120f5dcba14cf0b7f2d0ade (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-05-02T18:15:55Z (GMT) No. of bitstreams: 2 tese_ireni_leitzke_carvalho.pdf: 1397252 bytes, checksum: ad9c13976120f5dcba14cf0b7f2d0ade (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-05-02T18:16:14Z (GMT). No. of bitstreams: 2 tese_ireni_leitzke_carvalho.pdf: 1397252 bytes, checksum: ad9c13976120f5dcba14cf0b7f2d0ade (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-12-09 / Sem bolsa / A calibração de metodologias capazes de identificar de forma rápida e eficiente a qualidade de um lote de sementes, mediante testes de viabilidade e de vigor como o teste de tetrazólio, cujo procedimento metodológico para a cultura do arroz ainda carece de ajustes. Assim, o objetivo deste trabalho foi estudar alternativas de preparo, pré-condicionamento e coloração das sementes de arroz para avaliação da viabilidade, e estabelecer classes de viabilidade, para estimar vigor de sementes de arroz. O trabalho foi conduzido em três etapas: a) ajuste de procedimentos de preparo das sementes de arroz para condução do teste de tetrazólio, para tanto foram utilizados sementes de quatro cultivares de arroz irrigado e duas de sequeiro, cada uma composta por dois lotes. Sendo testado quanto à necessidade da remoção da casca (pálea e lema) e corte das sementes; b) estudo de concentrações do sal de tetrazólio, período de pré-condicionamento e de coloração para avaliar a viabilidade de sementes de arroz. Nesta etapa foram utilizadas as mesmas cultivares da primeira etapa, cada uma composta por um lote, sendo utilizadas as metodologias que apresentaram os melhores resultados na etapa anterior. Foram avaliadas diferentes concentrações do Sal de Tetrazólio (0,1; 0,25; 0,5 e 1,0%), três tempos de coloração (0,5; 1 e 2h), cinco períodos de hidratação (0,5; 1; 2; 3 e 4h) das sementes e duas temperaturas de embebição (35 e 40 °C); c) estimativa do vigor das sementes pelo teste de tetrazólio, utilizando-se sementes de dez lotes da cultivar Irga 424. Foram estabelecidas quatro classes de viabilidade (sementes viáveis de alto vigor, viáveis de médio vigor, viáveis de baixo vigor e não viáveis e mortas). Os resultados indicaram que para a avaliação da viabilidade de sementes de arroz pelo teste de tetrazólio é recomendável a remoção da pálea e lema, sendo mais eficiente o corte longitudinal na metade distal do eixo embrionário. O período de execução do teste de tetrazólio para sementes de arroz pode ser reduzido para três horas, utilizando hidratação das sementes por 1h a 40°C, 1h para as atividades de corte e avaliação e 1h para coloração na concentração de 0,25%. Dessa forma, é possível concluir que as metodologias propostas nessa pesquisa permitem avaliar a viabilidade de sementes de arroz pelo teste de tetrazólio; para avaliação do vigor o teste se mostrou promissor, porém necessitando de aprimoramento metodológico. / Calibration methodologies able to identify quickly and efficiently the quality of a lot of seeds through viability and vigor tests is constantly searched by seed technology. One of the most widely used tests for this purpose is the tetrazolium test, which methodological procedure for the rice crop still needs adjustments. This research aimed to study the preparation of alternatives, preconditioning and staining of rice seeds to evaluate the viability and establishing classes of viability to estimate the vigor of rice seed. The experiment was conducted in three stages: a) adjustment preparation procedures of rice seeds for conducting the tetrazolium test for both irrigated and upland plant varieties, were used four irrigated rice plant variety and two of upland plant variety, each consisting of two lots. It is tested on the need for removal of the shell (palea and lemma) and cut the seeds; b) study tetrazolium salt concentrations, preconditioning period and staining to evaluate the viability of rice seeds. At this stage, we used the same cultivars of the first stage, each consisting of a lot being used methodologies that showed the best results in the previous step. They were evaluated different concentrations of tetrazolium salt (0.1, 0.25, 0.5 and 1.0%) three times of staining (0.5, 1 and 2h), five periods of hydration seeds (0,5; 1, 2, 3 and 4h) and two soaking temperatures (35 and 40 ° C); c) estimate of seed vigor by the tetrazolium test, using ten seed lots of plant variety Irga 424 were established four viability classes (viable seeds of high vigor, viable medium vigor, viable low vigor and no viable and dead). The results indicated for the evaluation of viability of rice seed by tetrazolium testing is recommended to remove palea and lemma thereof and slitting the distal half of the embryonic axis is more efficient. The tetrazolium test execution period for rice seeds can be reduced to three hours using seed hydration for 1h at 40 ° C, for 1h cutting and evaluation activities and staining for 1h at 0.25% concentration. Thus, it leads to conclude that the proposed methodologies in this research to allow to evaluate the viability of rice seeds by the tetrazolium test; in order to evaluate vigor the test showed promise but needing methodological improvement.

Made available in DSpace on 2016-04-28T20:18:28Z (GMT). No. of bitstreams: 1 2006-Juliana Solozabal Martins da Rocha.pdf: 3772089 bytes, checksum: 41414ee2cbb97d4f355ca0c9f47ec737 (MD5) Previous issue date: 2006-02-09 / The microfilariaes when found should be identified correctly, for obtaining a correct diagnosis, since Dipetalonema is apathogenic and the infection doesn't request treatment, while Dirofilariosis, known as Heartworm Disease, it can take the dog to the death. This work had as objective to contribute to the diagnosis using histochemical differentiation in twelve sanguine samples of microfilaremics dogs and aid in the recognition of the identified species with that staining, based on the individual acid fosfatase activity and to confirm the use of a commercial kit for this procedure. They were identified Dirofilaria immitis and Dipetalonema reconditum, making possible this technique in the taxonomy of those species. / As microfil?rias quando encontradas devem ser identificadas corretamente, para realiza??o de um diagn?stico preciso, j? que o Dipetalonema ? apatog?nico e a infec??o n?o requer tratamento, enquanto a Dirofilariose, conhecida como Doen?a do Verme do Cora??o, pode levar o c?o ? morte. Este trabalho teve como objetivo contribuir ao diagn?stico pela diferencia??o histoqu?mica em doze amostras sangu?neas de c?es microfilar?micos e auxiliar no reconhecimento das esp?cies identificadas por essa marca??o, baseada na atividade da fosfatase ?cida individual, confirmando a utiliza??o de um kit comercial para este procedimento. Foram identificadas histoquimicamente, Dirofilaria immitis e Dipetalonema reconditum, possibilitando esta t?cnica na taxonomia dessas esp?cies.

<p>Cyclooxygenase-2 (COX-2) catalyses prostaglandin synthesis from arachidonic acid during inflammation. COX-2 is expressed in the normal brain and is induced in neurological disorders. There is evidence that COX-2 is involved in secondary events leading to cell death in the brain. The first objective was to study the expression of COX-2 in the brain after kainate (KA)-induced limbic seizures and brain trauma caused by controlled cortical contusion (CCC) and fluid percussion injury (FPI). COX-2 mRNA and protein were strongly induced by limbic seizures in the hippocampus, amygdala and piriform cortex. CCC and FPI resulted in an upregulation of COX-2 mainly in the dentate gyrus and cortex, with differences in expression levels in these regions between the models. The second objective was to evaluate the effects of selective COX-2 inhibitors on delayed cell death. Limbic seizures induced cell death in parts of the hippocampus, amygdala and functionally connected regions. Treatment with the selective COX-2 inhibitor rofecoxib 8 h after KA injection significantly reduced hippocampal cell death. Pre-treatment with the COX-2 inhibitor nimesulide augmented acute seizures with increased mortality and thus the effect of nimesulide on delayed cell death could not be evaluated. Effects of rofecoxib on trauma-induced cell death were studied in the FPI model. FPI induced delayed cell death mainly in the ipsilateral cortex and bilaterally in the dentate gyrus. Rofecoxib treatment, starting directly after injury was caused, had no protective effect against cell death. </p><p>The results suggest that COX-2 inhibition may be both detrimental and beneficial and largely dependent on the time schedule of treatment. COX-2 inhibitors might thus be of value as a neuroprotective treatment approach, provided that the role of COX-2 and the time course of effects of its metabolites in the brain are elucidated.</p>

Cyclooxygenase-2 (COX-2) catalyses prostaglandin synthesis from arachidonic acid during inflammation. COX-2 is expressed in the normal brain and is induced in neurological disorders. There is evidence that COX-2 is involved in secondary events leading to cell death in the brain. The first objective was to study the expression of COX-2 in the brain after kainate (KA)-induced limbic seizures and brain trauma caused by controlled cortical contusion (CCC) and fluid percussion injury (FPI). COX-2 mRNA and protein were strongly induced by limbic seizures in the hippocampus, amygdala and piriform cortex. CCC and FPI resulted in an upregulation of COX-2 mainly in the dentate gyrus and cortex, with differences in expression levels in these regions between the models. The second objective was to evaluate the effects of selective COX-2 inhibitors on delayed cell death. Limbic seizures induced cell death in parts of the hippocampus, amygdala and functionally connected regions. Treatment with the selective COX-2 inhibitor rofecoxib 8 h after KA injection significantly reduced hippocampal cell death. Pre-treatment with the COX-2 inhibitor nimesulide augmented acute seizures with increased mortality and thus the effect of nimesulide on delayed cell death could not be evaluated. Effects of rofecoxib on trauma-induced cell death were studied in the FPI model. FPI induced delayed cell death mainly in the ipsilateral cortex and bilaterally in the dentate gyrus. Rofecoxib treatment, starting directly after injury was caused, had no protective effect against cell death. The results suggest that COX-2 inhibition may be both detrimental and beneficial and largely dependent on the time schedule of treatment. COX-2 inhibitors might thus be of value as a neuroprotective treatment approach, provided that the role of COX-2 and the time course of effects of its metabolites in the brain are elucidated.

Light microscopes are essential research tools in biology and medicine. Cell and tissue staining methods have improved immensely over the years and microscopes are now equipped with digital image acquisition capabilities. The image data produced require development of specialized analysis methods. This thesis presents digital image analysis methods for cell image data in 2D, 3D and time sequences. Stem cells have the capability to differentiate into specific cell types. The mechanism behind differentiation can be studied by tracking cells over time. This thesis presents a combined segmentation and tracking algorithm for time sequence images of neural stem cells.The method handles splitting and merging of cells and the results are similar to those achieved by manual tracking. Methods for detecting and localizing signals from fluorescence stained biomolecules are essential when studying how they function and interact. A study of Smad proteins, that serve as transcription factors by forming complexes and enter the cell nucleus, is included in the thesis. Confocal microscopy images of cell nuclei are delineated using gradient information, and Smad complexes are localized using a novel method for 3D signal detection. Thus, the localization of Smad complexes in relation to the nuclear membrane can be analyzed. A detailed comparison between the proposed and previous methods for detection of point-source signals is presented, showing that the proposed method has better resolving power and is more robust to noise. In this thesis, it is also shown how cell confluence can be measured by classification of wavelet based texture features. Monitoring cell confluence is valuable for optimization of cell culture parameters and cell harvest. The results obtained agree with visual observations and provide an efficient approach to monitor cell confluence and detect necrosis. Quantitative measurements on cells are important in both cytology and histology. The color provided by Pap (Papanicolaou) staining increases the available image information. The thesis explores different color spaces of Pap smear images from thyroid nodules, with the aim of finding the representation that maximizes detection of malignancies using color information in addition to quantitative morphological parameters. The presented methods provide useful tools for cell image analysis, but they can of course also be used for other image analysis applications.

Our understanding of subsurface flow depends on assumptions of how event characteristics and spatial scale affect the relationships between subsurface water velocity, discharge, water table dynamics, and runoff response. In this thesis, three chapters explore some of these patterns for a hillslope and small watershed in coastal British Columbia. In the first chapter, tracers were applied under natural and steady state conditions to determine the relationship between lateral tracer velocities and various hillslope and event characteristics; such as hillslope subsurface flow, rainfall intensity, water table level, hillslope length, and antecedent condition. The results showed that preferential flow made up a large percentage of the subsurface flow from the gauged hillslope. Flow velocities as measured by tracers were affected by slope length and boundary conditions. The flow velocity was most closely related to the rainfall intensity, and changes in flow velocity were large compared to the changes in the water table. In the second chapter, the preferential flow features that transmitted water during steady state were investigated by staining the soil with a food dye solution and excavating the soil. These data were used to explore the link between the topographical factors (slope and contributing area), the network of preferential features and soil properties. The contributing area appeared to be an indicator of the size of the preferential features and their connectivity. In the final manuscript chapter, water table level and stream discharge measurements were used to determine if areas within a watershed with runoff dominated by preferential flow could be grouped based on the observable physical information such as slope, contributing area, distance to stream, and vegetation. Preferential flow made the water table responses dynamic and thus, distinct zones could not be identified. Models of the water table – runoff were not able to predict the water table response for other sites with similar physical characteristics. Even though there was high variability in the results, the patterns and relationships revealed in this thesis conform to existing conceptual models of hillslope subsurface preferential flow. These patterns and relationships may be useful in developing or validating numerical models.

A number of fungi produce spalted wood, which is characterized by accumulation of black pigment in fine demarcation lines, often accompanied by discoloration or staining on the wood fibers. Specific spalting fungi were identified by molecular analysis. From a total of 19 isolates and 140 clones studied, 11 fungal species were identified. The two Chlorociboria species from North America were investigated and their anamorphs were unambiguously identified for the first time. Fungal pigment formation under the influence of moisture content and pH variation was investigated in sugar maple, American beech and agar inoculated with spalting fungi. Maximum pigment production occurred at treatment with pH 4.5 for sugar maple and beech inoculated with Trametes versicolor. Xylaria polymorpha produced external pigmentation in beech treated with buffer at pH 5 and sugar maple at pH 4.5. Fungal pigmentation by Trametes versicolor and Xylaria polymorpha was stimulated at low moisture content in both wood species tested. Melanin production by Inonotus hispidus and Polyporus squamosus was stimulated above 22-28% and 34-38% moisture content in beech and in sugar maple respectively. Fomes fomentarius and Polyporus brumalis produced maximum pigmentation in beech at 26 - 41% and in sugar maple at 59 - 96% moisture content. The variation of the moisture content and pH values of wood substrates can stimulate the intensity of pigmentation of specific fungi in wood. To investigate melanin synthesis from a variety of melanin precursors, experimental research on three spalting fungi tested their reaction to catechol and L-Dopa melanin precursors in wood and agar substrate. The results indicate multiple biosynthesis pathways for melanin assembly in Trametes versicolor, Xylaria polymorha and Inonotus hispidus, and catechol produced most pigmentation in all spalting fungi investigated. Microscopic analysis by light, fluorescence, electron and confocal microscopy also indicates a bi- or multi-modal activity of melanin production and assembly by several spalting fungi. Possible variations of melanin assembly were identified based on fungal and wood species. Immunofluorescence and immunogold labelling with Mab 6D2 melanin antibody confirmed the melanin nature of the pigments produced by Oxyporus populinus, Trametes versicolor, Xylaria polymorpha, Fomes fomentarius, and Inonotus hispidus.