Dissertations / Theses on the topic 'Hematoxylin'

This thesis aims at determining if computer-assisted analysis can be used to better understand pathologists’ perception of mitotic figures on Hematoxylin-Eosin (HE) stained breast histopathological digital slides. It also explores the feasibility of reproducible histologic nuclear atypia scoring by incorporating computer-assisted analysis to cytological scores given by a pathologist. In addition, this thesis investigates the possibility of computer-assisted diagnosis for categorizing HE breast images into different subtypes of cancer or benign masses. In the first study, a data set of 453 mitoses and 265 miscounted non-mitoses within breast cancer digital slides were considered. Different features were extracted from the objects in different channels of eight colour spaces. The findings from the first research study suggested that computer-aided image analysis can provide a better understanding of image-related features related to discrepancies among pathologists in recognition of mitoses. Two tasks done routinely by the pathologists are making diagnosis and grading the breast cancer. In the second study, a new tool for reproducible nuclear atypia scoring in breast cancer histological images was proposed. The third study proposed and tested MuDeRN (MUlti-category classification of breast histopathological image using DEep Residual Networks), which is a framework for classifying hematoxylin-eosin stained breast digital slides either as benign or cancer, and then categorizing cancer and benign cases into four different subtypes each. The studies indicated that computer-assisted analysis can aid in both nuclear grading (COMPASS) and breast cancer diagnosis (MuDeRN). The results could be used to improve current status of breast cancer prognosis estimation through reducing the inter-pathologist disagreement in counting mitotic figures and reproducible nuclear grading. It can also improve providing a second opinion to the pathologist for making a diagnosis.

När ett vävnadspreparat har genomgått en otillräcklig dehydrering måste det åtgärdas innan provet kan gå vidare i den histotekniska processen. I dagsläget finns det två åtgärdande metoder på Patologilaboratoriet, Region Jönköpings län. Den ena metoden går ut på att provet placeras i smält paraffin för mer stadga. Den andra metoden går ut på att provet rehydreras för att sedan dehydreras på nytt. Syftet med denna studie var att utvärdera en tredje metod, ”dumpingmetoden” som går ut på att vävnadspreparaten dehydreras på nytt direkt utan att först bli rehydrerade.  Denna metod utförs i nuläget redan på vissa hud- och obduktionspreparat på Patologilaboratoriet. ”Dumpingmetoden” skulle kunna förenkla arbetet på laboratoriet då den är snabbare, kräver färre kemikalier och är automatiserad till skillnad från befintliga metoder. Resultaten från studien visade att ett otillräckligt dehydrerat vävnadspreparat aldrig kan bli lika bra som om det hade hanterats korrekt från början. Det är dock trots allt möjligt att åtgärda ett otillräckligt dehydrerat preparat i den graden att det blir bedömbart. ”Dumpingmetoden” förenklade snittningen av alla preparat som testades. Hos kolon- och tonsillpreparat verkade ”dumpingmetoden” ge bättre kvalitet av hematoxylin- och eosinfärgningen än de befintliga metoderna. För hudpreparat gav dock metoden där vävnadspreparat rehydrerats för att sedan dehydreras på nytt bäst kvalité av infärgningen.
A tissue that has been insufficiently dehydrated has to be fixed before it can continue through the histotechnological process. Currently, there are two established measures at the Pathology Laboratory, Department of Laboratory Medicine, Region Jönköping County. One of the methods is to put the sample in melted paraffin to give the tissue more support. The other method is to rehydrate the tissue followed by a new dehydration. The purpose of this study was to evaluate a third method called the “dumping method”. The “dumping method” involves a directly dehydration of the tissue, excluding rehydration. This method is currently being used on some of the skin and autopsy samples at the Pathology Laboratory. The “dumping method” would ease the work at the Laboratory since it is faster, requires fewer chemicals and can be automated, unlike the current available methods. The results from the study showed that an insufficiently dehydrated tissue can’t be restore to its original state. Never less it is possible to restore the tissue sufficiently for diagnosis. The “dumping method” eased the sectioning of all the tissue samples included in this study. For colon and tonsil tissues, the “dumping method” ensured higher quality of the haematoxylin and eosin staining than the current available methods. For skin tissues, however, the method of rehydration followed by a new dehydration resulted in the highest staining quality

Real-time on-site histopathology review of biopsy tissues at the point-of-procedure has great potential for significant clinical value and improved patient care. For instance, on-site review can aid in rapid screening of diagnostic biopsies to reduce false-negative results, or in quantitative assessment of biospecimen quality to increase the efficacy of downstream laboratory and histopathology analysis. However, the only currently available rapid pathology method, frozen section analysis (FSA), is too time-and labor-intensive for use in screening large quantities of biopsy tissues and is too destructive for maximum tissue conservation in multiple small needle core biopsies. In this work we demonstrate the spectrally-compatible combination of the nuclear stain DRAQ5 and the anionic counterstain eosin as a dual-component fluorescent staining analog to hematoxylin and eosin intended for use on fresh, unsectioned tissues. Combined with optical sectioning fluorescence microscopy and pseudo-coloring algorithms, DRAQ5 and eosin ("D&E") enables very fast, non-destructive psuedohistological imaging of tissues at the point-of-acquisition with minimal tissue handling and processing. D&E was validated against H&E on a one-to-one basis on formalin-fixed paraffin-embedded and frozen section tissues of various human organs using standard epi-fluorescence microscopy, demonstrating high fidelity of the staining mechanism as an H&E analog. The method was then applied to fresh, whole 18G renal needle core biopsies and large needle core prostate biospecimen biopsies using fluorescence structured illumination optical sectioning microscopy. We demonstrate the ability to obtain high-resolution histology-like images of unsectioned, fresh tissues similar to subsequent H&E staining of the tissue. The application of D&E does not interfere with subsequent standard-of-care H&E staining and imaging, preserving the integrity of the tissue for thorough downstream analysis. These results indicate that this dual-stain pseudocoloring method could provide a real-time histology-like image at the time of acquisition and valuable objective tissue analysis for the clinician at the time of service.

Peripheral neuropathies are a set of common diseases that affect the peripheral nervous system, causing damage to vital connections between various parts of the body and the brain and spinal cord. Different clinical conditions are known to selectively impact various size nerve fibers, which often makes it difficult to diagnose which peripheral neuropathy a patient might have. The nerve conduction velocity diagnostic test provides clinically useful information in the diagnosis of some peripheral neuropathies. This method is advantageous because it tends to be minimally invasive yet it provides valuable diagnostic information. However, this test does not determine characteristics of peripheral nerve fiber size distributions, and therefore does not show any detailed information regarding the nerve fibers within the nerve trunk. Being able to determine which nerve fibers are contributing to the evoked potential within a nerve trunk could provide additional information to clinicians for the diagnosis of specific pathologies of the peripheral nervous system, such as chronic inflammatory demyelinating polyneuropathy or early diabetic peripheral neuropathy. In this study, three rat sciatic nerves are sectioned and stained with hematoxylin and eosin in order to measure the nerve fiber diameters within the nerve trunk. Stained samples are viewed using brightfield microscopy and images are analyzed using ImageJ. Histograms were created to show the frequency of various nerve fiber diameters. The nerve fiber diameters measured during this research are consistent with the range of previously published diameter values and will be used to support continuing research for a novel method to characterize peripheral nerve fiber size distributions using group delay.

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This study aimed to determine the histological features of the endometrium of biches, as well as the cell proliferation at specific moments of diestrus, 10, 20, 30, 40, 50 and 60 days post-ovulation, correlating the endometrial thickness with the uterine cell proliferation and the metabolic state (weight, blood glucose and plasma cholesterol) of the animals. Therefore, the right and left uterine horns of 26 clinically healthy bitches submitted to ovariohysterectomy were histologically analyzed 10, 20, 30, 40, 50 and 60 days post-ovulation. The hematoxylin-eosin and AgNOR staining techniques were performed. All parameters were evaluated by the Shapiro-Wilk normality assumption and the samples subjected to ANOVA and post-hoc Tukey test (p<0.05). The correlation between endometrial thickness and uterine cell proliferation, weight, blood glucose and plasma cholesterol of animals was observed using the Pearson method (p<0.05). The software used was BioEstat® version 5.0. It was concluded that the endometrial thickness did not differs between post-ovulation days and was not correlated with the uterine cell proliferation, weight, blood glucose or serum cholesterol of the bitches. However, there was greater cell proliferation at 40 days post-ovulation compared to 60 days.
O presente estudo teve o objetivo de determinar as características histológicas do endométrio de cadela, assim como a sua proliferação em momentos específicos do diestro nos dias 10, 20, 30, 40, 50 e 60 pós-ovulação, correlacionando a espessura do endométrio com o peso, glicemia e colesterol das cadelas.Para tanto, foram analisados histologicamente os cornos uterinos direito e esquerdo de 26 cadelas clinicamente sadias ovariohisterectomizadas aos 10, 20, 30, 40, 50 e 60 dias após a ovulação. Foram realizadas a coloração de hematoxilina-eosinae também a coloração pelo método de AgNOR. Todos os parâmetros foram avaliados pelo pressuposto de normalidade de Shapiro-Wilk e as amostras submetidas a ANOVA seguida de Tukey (p<0,05). A correlação entre espessura de endométrio e peso, glicemia e colesterol plasmático foi verificada pelo método de Pearson (p<0,05). O programa utilizado foi o BioEstat® versão 5.0. Conclui-se que a espessura do endométrio não difere entre os dias pós-ovulação e que não tem correlação com o peso, glicemia ou o colesterol sérico das cadelas. Porém, há uma maior proliferação celular aos 40 dias pós-ovulação em relação aos 60 dias.

Introdução: A moléstia de Hirschsprung (MH) se caracteriza pela ausência de neurônios intramurais em segmentos variáveis do intestino grosso, levando a suboclusão intestinal. Na forma mais frequente o reto-sigmoide está comprometido. A biopsia retal é o método histológico de escolha no diagnóstico da MH. O método da hematoxilina e eosina (HE) é classicamente utilizado na prática histopatológica. Nessa técnica, um fragmento de parede total do reto é processado através de parafinização, para posteriormente ser seccionado e corado por HE. Esta coloração evidencia células neurais em intestinos normais e troncos nervosos hipertrofiados nos casos de MH. É uma técnica muito simples, ainda hoje muito utilizada no diagnóstico da doença, necessitando de fragmentos grandes de reto para um maior acerto no diagnóstico. Este detalhe torna o diagnóstico do recém-nascido mais difícil. Outro método de coloração utilizado no diagnóstico da MH é o método histoquímico de pesquisa de atividade de Acetilcolinesterase (AChE). Nesta técnica é necessário apenas um pequeno fragmento de mucosa e submucosa que será congelado e depois processado. A pesquisa de AChE, nos casos de MH mostrará a presença desta enzima em quantidade aumentada, corando troncos e ou fibrilas de cor acastanhado. Este método já vem sendo utilizado pelo Instituto da Criança - HCFMUSP há mais de 30 anos e possui um acerto diagnóstico superior a 90%. Porém, por ser uma técnica mais elaborada, pouquíssimos centros no Brasil a utilizam no diagnóstico da MH. Um outro método mais recente, e que também pode ser realizado em fragmentos menores, é a marcação imunohistoquímica da calretinina, que permite a visualização dos neurônios do plexo submucoso e das fibrilas finas na região da lâmina própria em não doentes. Esta técnica também apresenta maior complexidade e, portanto, não é utilizada. A possibilidade de realizar o diagnóstico da MH através da coloração HE em fragmentos menores poderia ser uma alternativa para os serviços que não dispõe de técnicas mais especificas. Objetivos: Avaliar a concordância dos resultados obtidos pelo método de coloração HE e da calretinina com a pesquisa de atividade de AChE em fragmentos de mucosa e submucosa no diagnóstico da Moléstia de Hirschsprung. Métodos: Para este trabalho foram selecionados 50 casos arquivados em nosso laboratório. O material encontrava-se emblocado em parafina. Foram feitos 60 níveis de cada fragmento para o HE e mais 3 níveis para a calretinina. Essas lâminas foram analisadas em microscópio, fotografadas e classificadas como positivas para MH quando não foram encontradas células neurais e houve a presença de troncos nervosos, e em negativas nos casos de visualização dos neurônios. Foi realizado estudo cego por dois pesquisadores. Os resultados da leitura das lâminas foram comparados com o da AChE. Resultados: Dos 50 casos avaliados pela técnica do HE, apenas 5 discordaram do diagnóstico realizado pela AChE, com um valor de Kappa de 0,800 e acurácia 90%. Na comparação entre a calretinina e a AChE 8 casos discordaram, com um valor de Kappa de 0,676 e acurácia de 84%. Conclusões: A concordância obtida entre os métodos da AChE e HE foi satisfatória. Tornando possível a utilização do método do HE em 60 níveis de fragmento de mucosa e submucosa como alternativa para o diagnóstico da MH. A técnica imunohistoquímica da Calretinina não apresentou a concordância esperada com a pesquisa de atividade de AChE em nosso estudo
Introduction: Hirschsprung disease (HD) is characterized by the absence of intramural neurons in variable segments of the large intestine, leading to intestinal subocclusion. In the most frequent form the rectum-sigmoid is compromised. Rectal biopsy is the histological method of choice in the diagnosis of HD. The hematoxylin and eosin (HE) method is classically used in histopathological practice. In this technique, a full-thickness rectum wall fragment is processed through paraffinization, to be later sectioned and stained by HE. This staining shows neural cells in normal intestines and hypertrophied nerve trunks in cases of HD. It is a very simple technique, still used today in the diagnosis of the disease, requiring large fragments of the rectum for a better diagnosis. This detail makes the diagnosis of the newborn more difficult. The staining histochemical methods more used are the research of acetylcholinesterase activity (AChE) and staining of calretinin. However, these techniques are not available in all centers and the possibility of diagnosing HD through HE staining in smaller fragments could be valuable alternative for services that do not have more specific techniques. Objectives: To evaluate the concordance of the results obtained by the HE staining and the calretinin method with the investigation of AChE activity in fragments of mucosa and submucosa in the diagnosis of Hirschsprung\'s disease. Methods: For this study, 50 cases from our laboratory were selected. The material was embedded in paraffin. Sixty levels of each fragment were made for HE and other 3 levels for calretinin. These slides were analyzed under microscope, photographed and classified as positive for HD when no nerve cells were found and there were nerve trunks present, and in negative in cases of visualization of the neurons. A blind study was carried out by two researchers. The results of reading the slides were compared with that of AChE. Results: Of the 50 cases evaluated by the HE technique, only 5 disagreed with the diagnosis performed by AChE, with a Kappa value of 0.800 and accuracy of 90%. In the comparison between calretinin and AChE, 8 cases disagreed, with a Kappa value of 0.676 and an accuracy of 84%. Conclusions: The concordance of results from AChE and HE methods was satisfactory, allowing the possibility of the use of the HE method in fragments of mucosa and submucosa as valid alternative for the diagnosis of HD. The immunohistochemical technique of Calretinin did not show a good agreement with the AChE activity in our study

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Universidade Federal de Minas Gerais
The influence of toxic aluminum (Al) on root development and absorption of water and nutrients has been identified as a major cause of low productivity of many soils. The theoretical and experimental study on the tolerance of varieties of sugarcane to adverse conditions of extreme acidity and, especially, high Al saturation, is still inconclusive The objectives of this study were: to develop and propose an experimental protocol for the study tolerance of varieties of sugarcane to Al in solution and to study the sensitivity of nine varieties of sugarcane (RB928064, RB966928, RB937570, RB855156, RB855453, RB935744, RB867515, RB925345, SP813250) to Al stress solution through a series of analyzes: (a) review the allocation of the Al at the tip roots, (b) observation of the root apices and (c) root growth; identify the main changes in the root; identify the critical dose to the Al root growth and identify the most efficient method for the classification of varieties on the Al in solution. The experiment was conducted in laboratory conditions in a completely randomized design with three replications. After the germination period, the mini-stalks of sugarcane were acclimated in tanks with nutrient solution. After six days, the mini-cuttings were transferred to containers with nutrient solution and increasing doses of Al (0, 130, 380, 500, 800, 1000, 1400, 2000, 3000 and 4000 μmol L -¹), where they remained for six days and subsequently were removed for analyzes. The development of the experimental protocol was efficient for the evaluation of tolerance and its main factors were germination method, selecting the range of Al concentration in solution and the methods of tolerance assessment. The varieties differed in Al tolerance according to the analyzes. The main changes of the tip root were mucilage production, loss of epidermal cells of the root cap and its disintegration, and brown staining of root tips. The root elongation rate (TAR) depended on the interaction between variety and concentration of Al, and, for the varieties studied, the critical value ranged from 130 to 3000 μmol L-¹. The TAR method was considered more efficient to evaluate the tolerance of varieties of sugarcane to Al in solution and in accordance with this method the varieties were classified as tolerance in the following order: RB928064 > RB935744 > RB855453 = RB966928 > RB925345 = RB867515 > SP813250 > RB855156 > RB937570.
A influência da toxidez por alumínio (Al) sobre o desenvolvimento do sistema radicular e a absorção de água e de nutrientes tem sido apontada como uma das principais causas da baixa produtividade de muitos solos. O referencial teórico e experimental sobre a tolerância de variedades de cana-de-açúcar a condições adversas de acidez extrema e, principalmente, à alta saturação por Al, ainda é inconclusivo Os objetivos deste estudo foram: desenvolver e sugerir um protocolo experimental para o estudo da tolerância de variedades de cana-de-açúcar ao Al em solução; estudar a sensibilidade de 9 variedades de cana-de-açúcar (RB928064, RB966928, RB937570, RB855156, RB855453, RB935744, RB867515, RB925345 e SP813250) ao estresse por Al em solução através de um conjunto de análises: (a) exame da alocação do Al na extremidade das raízes; (b) observação do ápice radicular e (c) crescimento radicular; identificar as principais alterações na raiz; identificar a dose crítica de Al ao crescimento radicular e identificar o método mais eficiente para a classificação das variedades quanto ao Al em solução. O experimento foi conduzido em condições de laboratório em delineamento inteiramente casualizado, com três repetições. Após o período de germinação, os mini-toletes de cana-de-açúcar foram aclimatados em tanques com solução nutritiva. Após seis dias, os mini-toletes foram transferidos para recipientes com solução nutritiva e doses crescentes de Al (0, 130, 380, 500, 800, 1000, 1400, 2000, 3000 e 4000 μmol L-¹), onde permaneceram por seis dias e posteriormente foram retirados para análises. O desenvolvimento do protocolo experimental foi eficiente para a avaliação da tolerância e seus principais fatores foram método de germinação, seleção da faixa de concentração de Al em solução e os métodos de avaliação da tolerância. As variedades apresentaram diferenças quanto à tolerância ao Al de acordo com as análises realizadas. As principais alterações dos ápices radiculares foram a produção de mucilagem, perda de células epidérmicas da coifa, desintegração da coifa e coloração parda. A taxa de alongamento radicular (TAR) dependeu da interação entre variedade e concentração de Al, sendo que, para as variedades estudadas, o valor crítico variou de 130 a 3000 μmol L-1. O método de TAR foi considerado o mais eficiente para a avaliação da tolerância de variedades de cana-de-açúcar ao Al em solução e de acordo com esse método as variedades foram classificadas quanto a tolerância na seguinte ordem: RB928064 > RB935744 > RB855453 = RB966928 > RB925345 = RB867515 > SP813250 > RB855156 > RB937570.

Pasture plants already adapted to acidic soil conditions are required as part of an integrated approach (with lime amelioration) to managing acid soils on the Tablelands of New South Wales, Australia. The objective of this thesis is to evaluate the usefulness of Austrodanthonia species for this purpose. The material evaluated in this study was collected during a previous survey of the distribution of Austrodanthonia on the Central, Southern and Monaro Tablelands of New South Wales. It was hypothesised that the genus Austrodanthonia has a wide range of tolerance to acid soils. A series of experiments that provided information on the growth and physiology of Austrodanthonia in relation to soil acidity, with a view to the identification and eventual domestication of the most promising plant material have been conducted through pot, hydroponics and field investigations. Firstly, soils were acidified or limed to obtain a range of soil pH and Al concentrations. This experiment showed that adding aluminium sulfate and calcium carbonate followed by washing excess salts with water is a simple, rapid and convenient method for adjusting soil pH for pot experiments. The pH of the amended soils remained relatively unchanged eight months after treatment. The experimental set-up also resulted in a wide range of soluble Al (2-52 mg/kg) across the soils. The relative Al-tolerance of 183 accessions from 15 Austrodanthonia species was tested in a pot experiment using a range of soil pH. Emergence, survival and growth of all accessions were drastically reduced by high soil acidity (pH 3.9, P < 0.001). About 11% of plants emerged at pH 3.9, whereas at pH 4.4 and 5.3, ~72% of plants emerged. Accessions exhibited large variation within and between species in their tolerance to soil acidity. From the species/accessions tested, 49 accessions from eight species were selected for further study (on the basis of being more acid tolerant). Hydroponic experiments conducted in the glasshouse evaluated: (i) formulation of nutrient solution with a stable pH, (ii) effectiveness of the formulation using tap water and deionised water and (iii) estimation of free ion activities of Al and Mn in the nutrient solution and their effects on Austrodanthonia growth. These experiments showed that a NO3-N/NH4-N ratio of 9:4 is the most appropriate ratio to obtain a stable pH 4.0 without affecting plant growth; that there was little difference between tap water and deionised water on the ionic effects of Al and Mn, and plant-size did not play a role on accession survival and that accessions of Austrodanthonia could grow well within a wide range of pH (3.5-5.5), Al (50-250 �M) and Mn (100-2000 �M). Growth of Austrodanthonia accessions declined under high acidity (pH < 3.5) and Al (300 �M), but tolerated high concentrations of Mn (2000 �M). Root-tips stained with hematoxylin grouped accessions in a similar way to the pot and hydroponic experiments for most of the accessions tested. The intensity of root staining with hematoxylin and the differential distribution of Al in the shoots and roots provided an indication that different tolerance mechanisms may be involved with Austrodanthonia accessions. It appears that both exclusion and internal mechanisms may operate for Al- and Mn-tolerance. A field experiment was conducted at Carcoar (33037�S, 149013�E, elevation 800 m) using gradients in soil pH and Al available on-site to grow selected accessions of Austrodanthonia. The accessions exhibited a range of responses to soil acidity. The accession responses to acidity from the pot and hydroponic experiments were similar to those obtained in the field, especially where Al was present as a low Al-challenge. Overall, this study shows that Austrodanthonia exhibits a wide range of acid tolerance between species and accessions within species. Among the species tested, A. duttoniana and A. fulva appeared to have the greatest commercial potential, because of their productivity and acid tolerance. The variability that exists in the accessions may be exploitable in breeding and selection programs for improved cultivars.

Pasture plants already adapted to acidic soil conditions are required as part of an integrated approach (with lime amelioration) to managing acid soils on the Tablelands of New South Wales, Australia. The objective of this thesis is to evaluate the usefulness of Austrodanthonia species for this purpose. The material evaluated in this study was collected during a previous survey of the distribution of Austrodanthonia on the Central, Southern and Monaro Tablelands of New South Wales. It was hypothesised that the genus Austrodanthonia has a wide range of tolerance to acid soils. A series of experiments that provided information on the growth and physiology of Austrodanthonia in relation to soil acidity, with a view to the identification and eventual domestication of the most promising plant material have been conducted through pot, hydroponics and field investigations. Firstly, soils were acidified or limed to obtain a range of soil pH and Al concentrations. This experiment showed that adding aluminium sulfate and calcium carbonate followed by washing excess salts with water is a simple, rapid and convenient method for adjusting soil pH for pot experiments. The pH of the amended soils remained relatively unchanged eight months after treatment. The experimental set-up also resulted in a wide range of soluble Al (2-52 mg/kg) across the soils. The relative Al-tolerance of 183 accessions from 15 Austrodanthonia species was tested in a pot experiment using a range of soil pH. Emergence, survival and growth of all accessions were drastically reduced by high soil acidity (pH 3.9, P < 0.001). About 11% of plants emerged at pH 3.9, whereas at pH 4.4 and 5.3, ~72% of plants emerged. Accessions exhibited large variation within and between species in their tolerance to soil acidity. From the species/accessions tested, 49 accessions from eight species were selected for further study (on the basis of being more acid tolerant). Hydroponic experiments conducted in the glasshouse evaluated: (i) formulation of nutrient solution with a stable pH, (ii) effectiveness of the formulation using tap water and deionised water and (iii) estimation of free ion activities of Al and Mn in the nutrient solution and their effects on Austrodanthonia growth. These experiments showed that a NO3-N/NH4-N ratio of 9:4 is the most appropriate ratio to obtain a stable pH 4.0 without affecting plant growth; that there was little difference between tap water and deionised water on the ionic effects of Al and Mn, and plant-size did not play a role on accession survival and that accessions of Austrodanthonia could grow well within a wide range of pH (3.5-5.5), Al (50-250 �M) and Mn (100-2000 �M). Growth of Austrodanthonia accessions declined under high acidity (pH < 3.5) and Al (300 �M), but tolerated high concentrations of Mn (2000 �M). Root-tips stained with hematoxylin grouped accessions in a similar way to the pot and hydroponic experiments for most of the accessions tested. The intensity of root staining with hematoxylin and the differential distribution of Al in the shoots and roots provided an indication that different tolerance mechanisms may be involved with Austrodanthonia accessions. It appears that both exclusion and internal mechanisms may operate for Al- and Mn-tolerance. A field experiment was conducted at Carcoar (33037�S, 149013�E, elevation 800 m) using gradients in soil pH and Al available on-site to grow selected accessions of Austrodanthonia. The accessions exhibited a range of responses to soil acidity. The accession responses to acidity from the pot and hydroponic experiments were similar to those obtained in the field, especially where Al was present as a low Al-challenge. Overall, this study shows that Austrodanthonia exhibits a wide range of acid tolerance between species and accessions within species. Among the species tested, A. duttoniana and A. fulva appeared to have the greatest commercial potential, because of their productivity and acid tolerance. The variability that exists in the accessions may be exploitable in breeding and selection programs for improved cultivars.

INTRODUÇÃO: A biópsia do neuroepitélio olfatório (NeuO) oferece perspectivas para aplicações terapêuticas tanto em doenças do olfato como doenças neurodegenerativas. Uma coleta bem sucedida desse tecido in vivo ainda não é rotina, devido á carência de estudos sobre a distribuição do NeuO em conchas superior (CS) e média (CM). Neste trabalho, descrevemos a distribuição do NeuO na CS e CM em cadáveres a partir da retirada integral dessas estruturas e posterior análise histológica por coloração de hematoxilina e eosina (HE) e imunoistoquímica. Além disso, também analisamos a influência do sexo, idade e lateralidade no grau de presença do NeuO nas CS e CM. CASUÍSTICA E MÉTODOS: Estudo anatômico prospectivo realizado de março de 2006 a janeiro de 2008. A CS e a CM foram endoscopicamente retiradas de um total de 25 cadáveres frescos com menos de 12 horas de óbito. Cada concha foi seccionada na metade de seu comprimento ântero-posterior. Assim, cada um dos 25 cadáveres deu origem a oito fragmentos de mucosa de regiões anatômicas distintas, totalizando 200 lâminas para análise tanto por coloração de HE como por reação de imunoistoquímica. Nas lâminas coradas por HE, classificamos a distribuição do NeuO em graus 0, 1, 2, 3, 4, sendo que a análise foi realizada por 3 patologistas de forma cega. Para imunoistoquímica, só obtivemos positividade com a proteína S-100. A concordância entre os três patologistas foi avaliada aos pares utilizando-se o coeficiente de Kappa. A distribuição do NeuO foi analisada de acordo com a idade, sexo, tempo de óbito, simetria entre as fossas nasais e acurácia da imunoistoquímica. RESULTADOS: Pela HE na CS, o NeuO esteve presente em 82,9% das vezes e, na CM, em 17,1%. Na CS, o NeuO foi detectado em 82,9 % das lâminas, 4,9 vezes a prevalência na CM, que foi de 17,1 % das lâminas (p < 0,001). Pela imunoistoquímica, foi possível encontrar NeuO em um total de 15 fragmentos. Desses, 10 (20%) eram da metade posterior da CS e cinco (7,6%) da metade anterior da CS. Pelo cálculo da razão de prevalência, temos que a chance de encontrar NeuO é 4,9 vezes maior na CS do que na CM (IC95%: 3,3 7,4). Dos 15 fragmentos com marcação positiva para proteína S-100, sete corresponderam aos que tinham uma distribuição grau 3 (>50% e 75%) pela HE e outros sete aos que tinham uma distribuição grau 4 (acima de 75%). Somente um fragmento teve marcação positiva para imunoistoquímica no grupo 2 (entre 26 e 50%) na HE. A proteína S-100 apresentou uma sensibilidade de 13,5% e especificidade de 100% para detecção de NeuO. Não houve diferença estatisticamente significante na prevalência de NeuO quando os fragmentos foram divididos de acordo com o sexo, idade de óbito e lado da fossa nasal. No entanto, ao analisarmos a presença de NeuO de acordo com o grau de distribuição entre cada lado, não se percebe uma concordância. CONCLUSÕES: A quantidade total de NeuO foi simetricamente distribuída entre as fossas nasais, mas não houve uma concordância entre os lados quanto à maneira como o NeuO está distribuído. O NeuO apresenta maior probabilidade de ser encontrado na metade posterior de CS. A HE é um método eficaz para distinção entre NeuO e epitélio respiratório, devido a grande concordância entre três patologistas distintos.
INTRODUCTION: Olfactory neuroepithelium (ON) biopsy provides perspectives for several therapeutic applications, both in disorders of olfaction and in neurodegenerative diseases. Successful in vivo collection of ON is still not routine, due to a dearth of studies on ON distribution in the superior and middle turbinate (ST and MT respectively). This study describes the distribution of ON in cadaver ST and MT as determined by complete endoscopic removal of turbinates and histological analysis with hematoxylin and eosin (H&E) and immunohistochemical staining. We also analyzed the influence of gender, age, and naris side on the extent to which ON is present in the superior and middle turbinate. CASE SELECTION AND METHODS: We conducted a prospective anatomical study from March 2006 to January 2008. The superior and middle turbinates of 25 fresh cadavers (less than 12 hours post-mortem) were removed endoscopically. Each turbinate was halved into anterior and posterior fragments. Eight anatomically distinct fragments were therefore obtained from each of the 25 cadavers for a total of 200 specimens, which were analyzed through H&E staining and immunohistochemistry. Hematoxylin and eosin-stained slides were subjected to blind examination by three independent pathologists; ON distribution was graded on a fivepoint numeric scale (grade 0, 1, 2, 3, or 4). Immunohistochemistry was only positive through S-100 staining. Pairwise agreement between pathologists was assessed by means of the Kappa coefficient. The distribution of ON was analyzed regarding age, gender, time elapsed between death and specimen harvesting, symmetry between nares, and accuracy of immunohistochemistry results. RESULTS: In H&E-stained slides, olfactory neuroepithelium was present in 82.9% of ST and 17.1% of MT specimens; prevalence in the superior turbinate was therefore 4.9-fold greater (p < 0.001). Immunohistochemical analysis was able to identify ON in 15 fragments, 10 of which (20%) were from the posterior half of the superior turbinate; the remaining five specimens (7.6%) were from the anterior ST. According to prevalence ratio, the odds of finding ON are 4.9 times greater in superior turbinate than in the middle turbinate (CI, 95%; 3.37.4). Of the 15 immunohistochemistry-positive fragments, seven were assigned distribution grade 3 (>50% and 75% presence of ON) on H&E staining seven others were graded 4 (>75% presence of ON). A single immunohistochemistrypositive fragment was found to have grade 2 ON distribution (i.e., it contained 26% to 50% olfactory neuroepithelium) on H&E staining. S-100 staining showed a sensitivity of 13.5% and specificity of 100% for ON detection. There was no statistically significant difference in ON prevalence when fragments were compared according to gender, age at time of death, and naris side. However, when we analyzed ON presence according to the degree of ON distribution in each side, we found no concordance. CONCLUSIONS: Total ON was distributed symmetrically between nares, but we found no concordance between sides in the manner in which ON is distributed. ON is most likely to be found in the posterior half of the superior turbinate. Hematoxylin and eosin (H&E) staining is an effective method for distinguishing ON from respiratory epithelium, as shown by high inter-rater agreement among three independent pathologists

碩士
靜宜大學
化粧品科學系
104
In this study, we focus on the effects of commonly used hair dyes , such as Basic red 51, Basic red 76, Basic blue 99 and Hematoxylin on melanogenesis in B16-F10 cells. Firstly, we use MTT assay to confirm the non-cytotoxic concentrations of these agents. Subsequently, these agents were used to test the effects on melanin production, tyrosinase activity and the expressions of microphthalmia-associated transcription factor (MITF) and tyrosinase.The results showed that: Under non-cytotoxic concentrations, Basic red 51, Basic red 76, Basic blue 99 generate no significant role for B16-F10. However Hematoxylin exhibits significantly increased melanin production. Our results indicated that Hematoxylin has abilities to increase the melanin productions of 14.7 %. Hematoxylin has ability to increase the tyrosinase activity 15.2 %. Hematoxylin has ability to increase tyrosinase activity approximately 1.29 fold of control in tyrosinase zymography and also protein levels of MITF and tyrosinase. Therefore, Hematoxylin can evidently increase the production of melanin in B16-F10 Cells.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Emily Zsáková Supervisor: Assoc. Prof. Přemysl Mladěnka, PharmD., Ph.D. Title of diploma thesis: Interference of reducing agents with the hematoxylin assay for determination of cupric chelation Copper is an essential trace element of living organisms. Disorders of its homeostasis affect various pathological conditions. In excessive amounts, it is toxic due to its ability to form free radicals. Vitamin C, trolox (a water soluble form of vitamin E), glutathione and hydroxylamine possess reducing properties that protect our body from oxidative tissue damage. Oxidized glutathione (GSSG) was also tested for comparison. The object of study was to test the chelating ability of these substances. A simple and fast spectrophotometric method was used. The combination of reducing properties of copper and chelating agents could lead to an improvement in chelation therapy either in heavy metal poisoning, treatment of Wilson's disease, or other diseases. We found that all substances were chelators/copper-chelating agents in vitro in a slightly competitive environment, using hematoxylin as an indicator. However, by using the bathocuproine assays to verify chelating properties, none of the substances...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany Candidate: Zuzana Lomozová Supervisor: PharmDr. Jana Karlíčková, Ph.D. Title of Thesis: Interactions of tamarixetin and isorhamnetin with copper Keywords: tamarixetin, isorhamnetin, chelation, reduction, copper, bathocuproine, hematoxylin Copper is a biogenic trace element important for proper function of human organism. It is an essential part of several enzymes and is involved in metabolic processes in the body. Excess or lack of serum copper can lead to pathological conditions. Copper chelating agents are used to treat copper toxic effects. Flavonoids are polyphenolic substances belonging to secondary metabolites of various plants. They are part of the human diet and have a positive impact on our health. They exhibit antioxidant and anti-inflammatory effects and are able to chelate transient metals, especially iron and copper. Chelation therapy is currently used in Wilson's disease in which copper is overloaded. In the future, the chelating effects of flavonoids could be used to treat neurodegenerative diseases, cardiovascular diseases or cancer. In this diploma thesis, interactions of two flavonoids (namely tamarixetin and isorhamnetin) with copper ions in different buffers were tested....

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany and Ecology Candidate: Kamila Zemanová Supervisor: PharmDr. Jana Karlíčková, Ph.D. Title of Thesis: Interaction of phenylpropionic acids with copper Copper is an essential trace element which is essential for our body. It has a significant effect on the correct functioning of important organs and it plays an important role in the transfer of electrons at the major enzymatic pathways as a prostetic group. On the other hand, excess or deficiency of copper in the human body can cause many diseases. Phenylpropionic acids are group of substances which can have antioxidant, anti-inflammatory and anticancer effects. They are involved in the scavenging of free radicals and reactive oxygen species. In this diploma thesis, I tested copper chelating activity of six phenylpropionic acids at different pHs by spectrofotometric methods using a hematoxylin and a bathocuproinedisulfonic acid disodium salt as an indicator. 3-(3,4-dihydroxyphenyl)propionic acid showed the highest chelating potential but only using a hematoxylin. KEYWORDS: Copper, Phenylpropionic acids, Antioxidants, Chelating activity, Hematoxylin, Bathocuproin

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany Candidate: Hana Němcová Supervisor: PharmDr. Jana Karlíčková, Ph.D. Title of Thesis: Comparison of interactions of equol and desmethylangolensin with iron and copper Iron and copper are essential trace elements, which are important for our body. Both elements have a significant effect on the correct function of organs and make part of many enzymes. They are able to accept or donate electrons - conversion between oxidized (Fe3+, Cu2+) and reduced (Fe2+, Cu+) forms. If these metals are excess in organism, they are accumulated in the cells and mediate the creation of free radicals, that destroy cell structures. This deficiency is treated with chelators, which facilitate the excretion of metals from the body. Isoflavonoids are polyfenolic substances, which can have antioxidant effects and they are involved in the scavening of free radicals. Isoflavonoids can have also a pro-oxidative effect, because they are able to reduce metal ions. In this study were tested interactions (chelation and reduction) between the metabolites of isoflavonoids (equol and desmethylangolensin) and ions of iron and copper. Both metabolites only weakly chelate metal ions, but they significantly reduce cupric ions. KEYWORDS: Iron,...