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Amaral, Telmo. "Analysis of breast tissue microarray spots". Thesis, University of Dundee, 2010. https://discovery.dundee.ac.uk/en/studentTheses/0a83915d-2f11-4b89-9c24-8dc3c15346f2.
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Tissue microarrays (TMAs) are a high-throughput technique that facilitates the survey of very large numbers of tumours, important both in clinical and research applications. However, the assessment of stained TMA sections is laborious and still needs to be carried manually, constituting a bottleneck in the pathologist?s work-flow. This process is also prone to perceptual errors and observer variability.Thus, there is strong motivation for the development of automated quantitative analysis of TMA image data. The analysis of breast TMA sections subjected to nuclear immunostaining begins with the classification of each spot as to the maintype of tissue that it contains, namely tumour, normal, stroma, or fat. Tumour and normal spots are then assigned a so-called quickscore composed of a pair or integer values, the first reflecting the proportion of epithelial nuclei that are stained, and the second reflecting the strength of staining of those nuclei. In this work, an approach was developed to analyse breast TMA spots subjectedto progesterone receptor immunohistochemistry. Spots were classified into their four main types through a method that combined a bag of features approachand classifiers based on either multi-layer perceptrons or latent Dirichlet allocation models. A classification accuracy of 74.6 % was achieved. Tumour and normal spots were scored via an approach that involved the computation of global features formalising the quickscore values used by pathologists, and the use of Gaussian processes for ordinal regression to predict actual quickscores based on global features. Mean absolute errors of 0.888 and 0.779 were achieved in the prediction of the first and second quickscore values, respectively. By setting thresholds on prediction confidence, it was possible to classify and score fractions of spots with substantially higher accuracies and lower mean absolute errors. Amethod for the segmentation of TMA spots into regions of different types was also investigated, to explore the generative nature of latent Dirichlet allocation models.
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Nguyễn, Hoài Nam. "Méthodes et algorithmes de segmentation et déconvolution d'images pour l'analyse quantitative de Tissue Microarrays". Thesis, Rennes 1, 2017. http://www.theses.fr/2017REN1S104/document.
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Ce travail de thèse a pour objectif de développer les méthodes originales pour l'analyse quantitative des images de Tissue Microarrays (TMAs) acquises en fluorescence par des scanners dédiés. Nous avons proposé des contributions en traitement d'images portant sur la segmentation des objets d'intérêts (i.e. des échantillons de tissus sur la lame de TMA scannée), la correction des artefacts d'acquisition liés aux scanners en question ainsi que l'amélioration de la résolution spatiale des images acquises en tenant compte des modalités d'acquisition (imagerie en fluorescence) et la conception des scanners. Les développements permettent d'envisager une nouvelle plateforme d'analyse de TMAs automatisée, qui représente aujourd'hui une forte demande dans la recherche contre les cancers. Les TMAs (ou “puces à tissus”) sont les lames histologiques sur lesquelles de nombreux échantillons tissulaires venant de différents donneurs sont déposés selon une structure de grille afin de faciliter leur identification. Pour pouvoir établir le lien entre chaque échantillon et ses données cliniques correspondantes, on s'intéresse non seulement à segmenter ces échantillons mais encore à retrouver leur position théorique (les indices de ligne et de colonne) sur la grille TMA car cette dernière est souvent très déformée pendant la fabrication des lames. Au lieu de calculer directement les indices de ligne et de colonne (des échantillons), nous avons reformulé ce problème comme un problème d'estimation de la déformation de la grille de TMA théorique à partir du résultat de segmentation en utilisant l'interpolation par splines ''plaques minces''. Nous avons combiné les ondelettes et un modèle d'ellipses paramétriques pour éliminer les fausses alarmes, donc améliorer les résultats de segmentation. Selon la conception des scanners, les images sont acquises pixel par pixel le long de chaque ligne, avec un change de direction lors du balayage entre les deux lignes. Un problème fréquent est le mauvais positionnement des pixels dû à la mauvaise synchronisation des modules mécaniques et électroniques. Nous avons donc proposé une méthode variationnelle pour la correction de ces artefacts en estimant le décalage entre les pixels sur les lignes consécutives. Cette méthode, inspirée du calcul du flot optique, consiste à estimer un champ de vecteurs en minimisant une fonction d'énergie composée d'un terme d'attache aux données non convexe et d'un terme de régularisation convexe. La relaxation quadratique est ainsi utilisée pour découpler le problème original en deux sous-problèmes plus simples à résoudre. Enfin, pour améliorer la résolution spatiale des images acquises qui dépend de la PSF (point spread function) elle-même variant selon le faisceau laser d'excitation, nous avons introduit une méthode de déconvolution d'images en considérant une famille de régulariseurs convexes. Les régulariseurs considérés sont généralisés du concept de la variation parcimonieuses (Sparse Variation) combinant la norme L1 de l'image et la variation totale (Total Variation) pour rehausser les pixels dont l'intensité et le gradient sont non-nuls. Les expériences montrent que l'utilisation de cette régularisation produit des résultats déconvolution d'images très satisfaisants en comparaison avec d'autres approches telles que la variation totale ou la norme de Schatten de la matrice HessienneThis thesis aims at developing dedicated methods for quantitative analysis of Tissue Microarray (TMA) images acquired by fluorescence scanners. We addressed there issues in biomedical image processing, including segmentation of objects of interest (i.e. tissue samples), correction of acquisition artifacts during scanning process and improvement of acquired image resolution while taking into account imaging modality and scanner design. The developed algorithms allow to envisage a novel automated platform for TMA analysis, which is highly required in cancer research nowadays. On a TMA slide, multiple tissue samples which are collected from different donors are assembled according to a grid structure to facilitate their identification. In order to establish the link between each sample and its corresponding clinical data, we are not only interested in the localization of these samples but also in the computation of their array (row and column) coordinates according to the design grid because the latter is often very deformed during the manufacturing of TMA slides. However, instead of directly computing array coordinates as existing approach, we proposed to reformulate this problem as the approximation of the deformation of the theoretical TMA grid using “thin plate splines” given the result of tissue sample localization. We combined a wavelet-based detection and a ellipse-based segmentation to eliminate false alarms and thus improving the localization result of tissue samples. According to the scanner design, images are acquired pixel by pixel along each line, with a change of scan direction between two subsequent lines. Such scanning system often suffers from pixel mis-positioning (jitter) due to imperfect synchronization of mechanical and electronic components. To correct these scanning artifacts, we proposed a variational method based on the estimation of pixel displacements on subsequent lines. This method, inspired from optical flow methods, consists in estimating a dense displacement field by minimizing an energy function composed of a nonconvex data fidelity term and a convex regularization term. We used half-quadratic splitting technique to decouple the original problem into two small sub-problems: one is convex and can be solved by standard optimization algorithm, the other is non-convex but can be solved by a complete search. To improve the resolution of acquired fluorescence images, we introduced a method of image deconvolution by considering a family of convex regularizers. The considered regularizers are generalized from the concept of Sparse Variation which combines the L1 norm and Total Variation (TV) to favors the co-localization of high-intensity pixels and high-magnitude gradient. The experiments showed that the proposed regularization approach produces competitive deconvolution results on fluorescence images, compared to those obtained with other approaches such as TV or the Schatten norm of Hessian matrix
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Sievertzon, Maria. "Transcript profiling of small tissue samples using microarray technology". Doctoral thesis, Stockholm Department of Biotechnology, Royal Institute of Technology, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-158.
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Cheng, Yabin. "Tissue microarray based biomarker study in human cutaneous melanoma". Thesis, University of British Columbia, 2014. http://hdl.handle.net/2429/46655.
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Cancer therapy recently experienced remarkable advances with better understanding of cancer pathogenesis and introduction of new intervention strategies. Biomarkers reflective of the presence of tumor cells, or linked with clinical outcomes, have potential to improve the management of cancers. The purpose of this thesis study is to identify novel biomarkers of human cancers based on tissue microarray (TMA) technology and to determine their value for clinical application in cancer management using melanoma as the model. Melanoma arises from uncontrolled proliferation of melanocytes. Although melanoma accounts for only 4% of all skin cancer, it is responsible for 80% of deaths related to skin malignancies.
To discover novel biomarkers of melanoma, we constructed a TMA using biopsies from 707 patients with various stages of melanocytic lesions. Using immunohistochemistry and TMA, multiple biomarker candidates were evaluated, and many were found to have significant prognostic value, including expression loss of Fbw7. To further improve the clinical value of these markers, various combinations of individual markers were evaluated, leading to the identification of KAI1 and p27 that together showed much stronger prognostic value than when used as individual markers. Moreover, since there has been a dearth of reliable prognostic markers to offer prognostic information on specific melanoma stages, we identified the AJCC-stage specific prognostic markers, including BRAF protein expression as a prognostic marker for thin melanomas.
In that significant prognostic value was found for Fbw7 protein in melanoma, we performed in vitro experiments on this protein in detail. Our data showed that the alpha isoform of Fbw7, located in the cell nucleus, was the dominant form expressed in melanoma. Knock-down of Fbw7α promoted melanoma cell migration, and the MAPK signaling pathway was required for Fbw7 function in melanoma. These findings indicate loss of Fbw7 to be an independent melanoma prognostic marker, and important for the development of malignant behaviors of melanoma cells.
This study has demonstrated that the combination of TMAs of cancers with the corresponding clinical database represents a powerful technological platform for biomarker discovery. TMA/clinical database combination-based investigations should be applicable for the investigation of other types of human cancers as well.
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Xie, Dan y 謝丹. "Application of high-throughput tissue microarray technology in cancer research". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B30283619.
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Mckenzie, Gavin Medical Sciences Faculty of Medicine UNSW. "The analysis of signalling pathways in sporadic colorectal carcinoma using tissue microarrays". Publisher:University of New South Wales. Medical Sciences, 2008. http://handle.unsw.edu.au/1959.4/43370.
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Colorectal carcinoma arises through sequential genetic changes whereby an adenoma develops from normal colonic epithelium and then becomes a carcinoma. Critical to this process is two forms of mutually exclusive genomic instability ?? chromosomal instability (CIN) and microsatellite instability (MSI). The colorectal tumours that develop from each of these pathways have distinct pathological and molecular differences. Most MSI+ colorectal carcinomas are associated with the CpG island methylator phenotype (CIMP) - an epigenetic phenomenon where a specific and consistent group of genes are silenced through promoter methylation. However, over half of fall CIMP+ colorectal tumours are microsatellite stable (MSS). It is well known that the WNT/β-catenin signalling pathway is instrumental in the initiation and development of CIN type tumours but it is less clear whether this pathway has any significant involvement in MSI+ or methylated tumours. The role of the PI3K1AKT signalling pathway in the development of solid human tumours has only recently been established and the affects of abnormal PI3K/AKT signalling in sporadic colorectal carcinomas is yet to be fully elucidated. The objective of this thesis was to investigate the involvement of the WNT/β-catenin and PI3K/AKT signalling pathways in the CIN, MSI+ and methylated subgroups of sporadic colorectal carcinoma. To achieve this, the expression patterns of β-catenin, p-AKT and PTEN were identified by immunohistochemistry on sections from tissue microarrays consisting of cores from a large group of sporadic colorectal carcinomas. Each of these proteins is an integral part of the constitutive activation of WNT/β-catenin or PI3K/AKT signalling and their expression patterns were correlated with the clinical, pathological and molecular characteristics of the different subgroups of colorectal carcinoma. Increased nuclear β-catenin expression, an indicator of activated WNT signalling, is associated with MSS and the pathological features of CIN type tumours and inversely associated with the pathological and molecular features of MSI+ and CIMP+ tumours. In all forms of sporadic colorectal carcinoma, nuclear β-catenin expression was not an indicator of overall survival. PTEN was not associated with any particular subgroup of sporadic colorectal carcinoma, but decreased cytoplasmic expression was indicative of overall worse outcome, especially in MSS or CIN type tumours. While the identification of nuclear β-catenin in sporadic colorectal carcinomas is not a satisfactory prognostic marker, the immunohistochemical detection of absent PTEN expression may prove useful in identifying poor outcome in individuals with sporadic MSS colorectal carcinoma.
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Jernås, Margareta. "Microarray analysis of gene expression in human adipocytes and adipose tissue /". Göteborg : Institute of Medicine, Dept. of Molecular and Clinical Medicine, Sahlgrenska Academy, Göteborg University, 2008. http://hdl.handle.net/2077/9583.
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PANSINI, P. F. "Potencial Prognóstico da Survivina em Carcinoma Epidermóide da Cavidade Bucal". Universidade Federal do Espírito Santo, 2017. http://repositorio.ufes.br/handle/10/7104.
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Previous issue date: 2017-02-15O carcinoma epidermóide de cabeça e pescoço (CECP) é o sexto tipo de câncer mais comum em todo o mundo. Nos últimos anos, tem sido sugerida a participação da survivina na progressão tumoral em CECP. Este estudo teve como objetivo avaliar a survivina como potencial biomarcador de progressão tumoral em CECB. Foram utilizados no estudo dados clínicos e amostras biológicas de 115 indivíduos com carcinoma epidermóide da cavidade bucal. Lâminas contendo tecidos tumorais coradas pelo método hematoxilina e eosina foram usadas para as análises histopatológicas para avaliar o infiltrado linfocitário tumoral, padrão de invasão tumoral, gradação tumoral, invasão vascular, linfática e perineural. Tissue Microarrays foram construídos para realizar a análise imunohistoquímica da expressão da proteína survivina utilizando o anticorpo primário monoclonal de coelho anti-survivina. Para avaliar as associações entre as variáveis estudadas foram utilizados os testes Qui-Quadrado e o Exato de Fisher. A comparação das médias dos segmentos foi obtida pelo teste T de amostras independentes. As curvas de sobrevida foram calculadas pelo modelo de Kaplan-Meier e confirmadas pelo modelo multivariado de Cox. Nossos resultados mostraram existir correlação entre o infiltrado linfocitário tumoral alto, tamanho do tumor primário T1/T2 (p = 0,001) e estadiamento clínico I e II (p = 0,005). O padrão de invasão tumoral tipo IV foi correlacionado com o tamanho do tumor primário T3/T4 (p = 0,006) e estadiamento clínico avançado (estádio III e IV) (p = 0,028). Invasão perineural foi associada com o tamanho do tumor primário T1/T2 (p = 0,035). A expressão nuclear da survivina na porção mediana do tumor mostrou associação com a metástase em linfonodos regionais (p = 0,004) e o estadiamento clínico (p = 0,041). A análise regressiva multivariada confirmou que as variáveis tamanho do tumor primário (p = 0,004) e acometimento linfonodal (p= 0,06) são fatores prognósticos independentes para sobrevida global, enquanto o etilismo influencia na sobrevida livre de doença (p = 0,048). Com este estudo pode-se concluir que a elevada expressão da survivina está correlacionada com o comportamento tumoral mais agressivo, estadiamento clínico avançado, presença de mestástase linfonodal, podendo ser considerada como indicador de prognóstico em pacientes com CECB. A variável histopatológica padrão de invasão tumoral mostrou que sua correlação com tamanho do tumor primário e estadiamento clínico avançado podendo estar relacionada ao pior prognóstico dos pacientes em CECB.
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Foster, Cheryl June. "Identifying a prognostic test in follicular lymphoma using a tissue microarray and immunohistochemistry". Thesis, Kingston, Ont. : [s.n.], 2008. http://hdl.handle.net/1974/1296.
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Habibi, Golareh. "Y-box binding protein-1 (YB-1) is a bio-marker of aggressiveness in breast cancer and is a potential target for therapeutic intervention". Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/911.
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Early detection is one of the most important factors for successful treatment of cancer. Currently, scientists are searching for molecular markers that can help identify and predict outcome and chance of recurrence in patients. In this study, we demonstratet he potential impact of Y-Box binding protein-1 (YB-1) as a marker of aggressiveness and cancer recurrence in breast malignancies by screening one of the largest tissue microarrays in North America.
YB-1 is an oncogenic transcription/translation factor, which is over-expressed in the majority of malignancies, including breast cancer. In the cohort of 4049 primary breast tumours, we show that YB-1 is a strong marker of aggressiveness, poor survival and cancer recurrence in all subtypes of human breast cancer with a particularly high frequency of expression in the ER negative basal-like and HER-2 breast cancer subtypes. This suggests that targeting YB-1 may provide a new avenue for therapeutic intervention in these breast cancers that are currently challenging to treat. Cox regression multivariate analysis indicates that YB-1 is second only to nodal status as a strong independent prognostic marker for poor outcome and relapse compared to established clinico-pathological biomarkers, including tumour size, age, grade, ER and HER-2 status. This finding suggests that YB-1 has great potential to be in a priority list of biomarkers for identifying the patients with a higher risk of relapse and poor outcome. Subsequently, we find an association between YB-1 and urokinase Plasminogen Activator (uPA) expression in the basal-like subtype. We then show that YB-1 is involved in the regulation of uPA expression. More importantly, silencing YB-1 or uPA results in a significant reduction in cancer cell invasion. As there are no commercially available YB-linibitors we examine the efficacy of BMS-536924, a small molecule inhibitor for activated IGF-1R/IR on SUM149 cells. We demonstrate that activated IGF-1R is associated with poor survival in primary breast tumours and, that BMS-536924 reduces uPA expression through inhibition YB-1 in SUM149 cells.
We therefore conclude that YB-1 is a bio-marker for poor survival and relapse. We also indicate that YB-1 has potential use as a molecular marker in a clinical setting. Inhibiting YB-1 may provide an ideal opportunity for targeted therapy in breast cancer.
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Carlberg, Konstantin. "In Situ RNA Quality Control : A spatial heat map of RNA integrity with single cell resolution". Thesis, KTH, Skolan för bioteknologi (BIO), 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-176873.
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The quality of RNA is of great importance in gene expression studies. It is mostly measured using the RNA integrity number (RIN). Lately it has been shown that samples with low RIN and different fragmentation patterns could affect quality of sequencing data. For such low RIN samples a new approach has been developed by Illumina called the DV200 metric, which is the percentage of fragments >200 nucleotides. For samples with low RIN, DV200 has proved to be a better method to predict if good quality data from RNA sequencing can be generated. However, neither RIN nor DV200 provide spatial infromation on the RNA integrity. Thus, tissues with areas of heterogeneous RNA integrity, where regions of good quality RNA sequencing data could be generated from are missed. We have designed a method to spatially evaluate the RNA integrity in tissue, which we named in situ RNA QC. The method uses three probes with three different fluorophores, each bound to three specific cDNA regions synthesized from the high abundant and well conserved 18S rRNA. With the help of in-house technology from the Spatial Transcriptomics (ST) group at SciLifeLab, we enable creation of heat maps over the RNA integrity to show spatial fragmentation patterns of RNA in tissue. This could reveal the regional quality of transcripts in situ, which is crucial knowledge when selecting samples for further RNA sequencing. The assay has been tried using different tissue fixation methods in order to show a proof of concept that formalin gives shorter cDNA fragments than acetone. The generated heat-map provides a visual overview of RNA integrity in situ; hence this method could be used to select samples for sequencing by evaluation the spatial quality of RNA. For instance from fresh frozen and formalin fixated paraffin embedded (FFPE) tissue (biobanks contain large number of longterm storage FFPE samples). With this assay we will be able to determine which samples are suitable for sequencing.
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Neves-Silva, Rodrigo 1986. "Estudo para validação do uso do tissue microarray como método para análise imunoistoquímica de ameloblastomas = A validation study for the use of tissue microarray as a method for immunohistochemical analysis of ameloblastomas". [s.n.], 2015. http://repositorio.unicamp.br/jspui/handle/REPOSIP/289468.
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Orientador: Alan Roger dos Santos SilvaTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: Tumores odontogênicos (TOs) são neoplasias originadas do órgão de desenvolvimento dentário ou de seus remanescentes. Neste contexto, o ameloblastoma é um dos TOs mais relevantes devido a sua elevada frequência e alto potencial de agressividade clínica. O presente estudo se propôs a realizar uma análise clinicopatológica retrospectiva de ameloblastomas e, posteriormente, validar o uso da técnica do tissue microarray (TMA) para a análise imunoistoquímica de ameloblastomas. Material e métodos: O estudo clinicopatológico se baseou numa amostra de 869 TOs, oriundos de dois centros de Patologia Oral brasileiros, sendo 177 (20,36%) casos de ameloblastomas, dos quais foram selecionados 40 (22,59%) casos representativos de ameloblastomas multicísticos que foram distribuídos em dois blocos do TMA montados em triplicata contendo cilindros de 1.0mm e 2.0mm (Sakura Co., Tokyo, Japan). A validação da análise imunoistoquímica foi realizada para proteínas expressas em citoplasma (citoqueratina 14, citoqueratina 19 e Bcl-2) e em núcleo (Ki-67); analisada por meio semiquantitativo e também por microscopia óptica digital (Aperio Scanscope CS® Slide Scanner; Aperio Technologies Inc.; Vista, CA; USA) e quantificada por meio dos algoritmos PixelCount® e NuclearV9® (Aperio Technologies Inc.; Vista, CA; USA). Resultados: Os 40 casos de ameloblastoma multicístico afetaram predominantemente pacientes do gênero masculino (62,5%), a média de idade no momento do diagnóstico foi de 39,92 anos (variando de 15 a 71 anos), a maioria (92,5%) dos casos ocorreu na região posterior da mandíbula. A concordância obtida para a expressão imunoistoquímica entre os diferentes diâmetros do TMA e os cortes convencionais mostrou melhores resultados para os cilindros de 2.0mm montados em triplicata nas análises digital e semiquantitativa. Em conclusão, o presente estudo demonstrou originalmente que a técnica do TMA pode ser usada com rigor científico para estudos baseados na análise imunoistoquímica de marcadores de citoplasma e núcleo celular em ameloblastomas multicísticos
Abstract: Odontogenic tumors (OTs) are neoplasms originating from the tooth developing apparatus or its remnants. In this context, ameloblastoma is one of the most relevant OTs due to its high frequency and clinical potential of aggressiveness. The present study performed a retrospective, clinicopathological analysis of ameloblastomas and subsequently validated the use of the tissue microarray (TMA) for immunohistochemical analysis of ameloblastomas. Materials and methods: The clinicopathologic study was based on a sample of 869 OTs from two Brazilian Oral Pathology Centers. Of these, 177 (20.36%) cases were ameloblastomas, of which 40 (22.59%) cases of multicystic ameloblastomas were selected and divided into two TMA blocks mounted in triplicate containing cores of 1.0mm and 2.0mm (Sakura Co., Tokyo, Japan). The validation of the immunohistochemical analysis was performed for proteins expressed in the cytoplasm (cytokeratin 14, cytokeratin 19, and Bcl-2) and nuclei (Ki-67), analyzed in a semiquantitative way as well as by digital optical microscopy (Aperio Scanscope CS® Slide Scanner; Aperio Technologies Inc., Vista, CA, USA), and quantified by PixelCount® and NuclearV9® algorithms (Aperio Technologies Inc., Vista, CA, USA). Results: Forty cases of ameloblastoma affected predominantly male patients (n=25; 62.5%). The mean age at diagnosis was 39.92 years (ranging from 15 to 71 years), and the majority (92.5%) of the cases occurred in the posterior mandible. The concordance obtained for the immunohistochemical expression in different TMA core diameters and whole sections showed best results for 2.0mm TMA assembled in triplicates both for semiquantitative and digital analyses. In conclusion, this study originally demonstrated that the TMA technique could be used with scientific rigor in studies based on immunohistochemical analysis of cytoplasmic and nuclear markers in multicystic ameloblastomas
Doutorado
Estomatologia
Doutor em Estomatopatologia
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Das, Gupta Paromita Clinical School Prince of Wales Hospital Faculty of Medicine UNSW. "Gene profiling in soft tissue sarcoma: predictive value of EGFR in sarcoma tumour progression and survival". Publisher:University of New South Wales. Clinical School - Prince of Wales Hospital, 2007. http://handle.unsw.edu.au/1959.4/43259.
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Despite improvements in the clinical management of soft tissue sarcomas (STS), 50% of patients will die of metastatic disease that is largely unresponsive to conventional chemotherapeutic agents. The aims of this study were to identify genes and pathways that are dysregulated in progressive and metastatic STS. In addition to this, cell lines from fresh tumours were initiated and established, thus increasing the repository of cell lines available for functional studies. Recent advances in the understanding of the molecular biology of STS have thus far not resulted in the use of molecular markers for clinical prognostication. Identifying novel genes and pathways will lead to molecular diagnostic methods to better stratify prognostic groups and could identify cellular targets for more efficacious treatments. Gene expression profiling of sarcoma cell lines of increasing metastatic potential revealed over-expression of genes involved in the epidermal growth factor (EGF) and transforming growth factor beta (TGFb) pathways. Factors involved in invasion and metastasis such as integrins and MMPs were over-expressed in the cell lines with higher metastatic potential. The developmental Notch pathway and cell cycle regulators were also dysregulated. NDRG1 was significantly over-expressed in the high grade sarcoma cell line, a novel finding in sarcomas. The expression of EGFR, NDRG1 and other genes from the above pathways was validated using quantitative RT-PCR in real time (qRT-PCR). A tissue microarray (TMA) comprising STS of varying tumour grades was constructed for high throughput assessment of target proteins. EGFR, its activated form and its signal transducers were investigated using immunohistochemistry (IHC). Activated EGFR (HR 2.228, p < 0.001) and phosphorylated Akt (HR 2.032, p = 0.003) were found to be independent predictors of overall survival and both correlated with tumour grade. Of the several STS cultures initiated and maintained, two of these cell lines were fully characterised in terms of cytogenetics, telomerase and alternate lengthening of 5 telomeres (ALT) status, KIT and TP53 mutation and the expression of certain biomarkers using both qRT-PCR and IHC. In summary, transcript profiling identified several potential biomarkers of tumour progression and metastasis in STS. Crucially, activated EGFR and pAkt were found in a cohort of STS samples to correlate with clinical outcome, identifying them as potential diagnostic and therapeutic targets in the treatment of STS. Activated EGFR can be used as a diagnostic marker for patient selection, as well as for target effect monitoring. Furthermore, the cell lines established in this project will serve as valuable tools in future preclinical studies.
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Strömberg, Sara. "Antibody-based Profiling of Expression Patterns using Cell and Tissue Microarrays". Doctoral thesis, Uppsala University, Department of Genetics and Pathology, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8680.
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In this thesis, methods to study gene and protein expression in cells and tissues were developed and utilized in combination with protein-specific antibodies, with the overall objective to attain greater understanding of protein function.
To analyze protein expression in in vitro cultured cell lines, a cell microarray (CMA) was developed, facilitating antibody-based protein profiling of cell lines using immunohistochemistry (IHC). Staining patterns in cell lines were analyzed using image analysis, developed to automatically identify cells and immunohistochemical staining, providing qualitative and quantitative measurements of protein expression. Quantitative IHC data from CMAs stained with nearly 3000 antibodies was used to evaluate the adequacy of using cell lines as models for cancer tissue. We found that cell lines are homogenous with respect to protein expression profiles, and generally more alike each other, than corresponding cancer cells in vivo. However, we found variability between cell lines in regards to the level of retained tumor phenotypic traits, and identified cell lines with a preserved link to corresponding cancer, suggesting that some cell lines are appropriate model systems for specific tumor types.
Specific gene expression patterns were analyzed in vitiligo vulgaris and malignant melanoma. Transcriptional profiling of vitiligo melanocytes revealed dysregulation of genes involved in melanin biosynthesis and melanosome function, thus highlighting some mechanisms possibly involved in the pathogenesis of vitiligo. Two new potential markers for infiltrating malignant melanoma, Syntaxin-7 and Discs large homolog 5, were identified using antibody-based protein profiling of melanoma in a tissue microarray format. Both proteins were expressed with high specificity in melanocytic lesions, and loss of Syntaxin-7 expression was associated with more high-grade malignant melanomas.
In conclusion, the combination of antibody-based proteomics and microarray technology provided valuable information of expression patterns in cells and tissues, which can be used to better understand associations between protein signatures and disease.
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Qiao, Guibin. "Molecular prognostic study of non-small cell lung cancer using high-throughput tissue microarray and immunohistochemistry". [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=975693859.
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Zerati, Marcelo. "Estudo de fatores prognósticos moleculares no carcinoma renal de células claras pela técnica de tissue microarray". Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/5/5153/tde-23082011-143257/.
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INTODUÇÃO: O carcinoma renal (CR) é uma doença agressiva, e sua incidência vem aumentando. A variante de células claras (CRCC) é a mais comum e apresenta comportamento biológico mais agressivo. Os recentes avanços no conhecimento da biologia molecular do tumor demonstram que a oncogênese dos diversos tipos histológicos é regida por mecanismos celulares diversos. Os modelos prognósticos atuais vêm procurando incorporar os recentes avanços da biologia molecular, com o intuito de melhorar sua capacidade de predizer a evolução e o desfecho destes pacientes. OBJETIVOS: Correlacionar a imunoexpressão dos marcadores selecionados com: 1) sobrevida global e, 2) com parâmetros prognósticos estabelecidos (estadio clínico TNM, tamanho tumoral, grau nuclear de Fuhrman, invasão microvascular e invasão de gordura perirrenal) em portadores de CRCC não metastático. MÉTODOS: Neste estudo de coorte retrospectivo, avaliamos 99 pacientes portadores de CRCC não metastático, quanto à expressão imunoistoquímica das seguintes proteínas: CA-IX, EGF-R, Ki-67, p53, PTEN, VEGF e VEGF-R. Os parâmetros analisados foram: Sobrevida global, estadio TNM, tamanho tumoral, grau nuclear de Fuhrman, invasão microvascular e invasão de gordura perirrenal. Utilizamos um tissue microarray construído exclusivamente para esta finalidade e realizamos a leitura da imunoexpressão por técnica digital utilizando o software Photoshop®. RESULTADOS: O tempo de seguimento médio foi de 7,9 anos. Com relação à sobrevida global, não observamos sua correlação com nenhum dos marcadores avaliados. Quanto à correlação da expressão dos marcadores com os parâmetros prognósticos convencionais, observamos que a expressão do EGF-R se correlacionou com estadio T (p= 0,049) e invasão da gordura perirrenal (p=0,020); e o VEGF-R se correlacionou com grau de Fuhrman (p=0,022) e invasão microvascular (p=0,005). Nos demais marcadores, não foi observada correlação significativa. CONCLUSÃO: Os fatores prognósticos moleculares EGF-R e VEGF-R apresentam-se como ferramentas úteis para avaliação do risco de prognóstico desfavorável em portadores de carcinoma renal de células claras não metastáticoINTODUCTION: Renal cell carcinoma is an aggressive disease and its incidence is rising. The clear cell variant is the most common, and also the most aggressive. Recent advances in the understanding of the tumors molecular biology indicate that the oncogenesis of each histologic subtype is controlled by distinct cellular mechanisms. Current prognostic models are gradually incorporating the advances in molecular biology, in the hope to improve their predictive capacity. OBJECTIVES: To correlate the immunoexpression of selected markers with 1) overall survival, and 2) with established prognostic parameters (clinical TNM stage, tumor size, Fuhrman nuclear grade, microvascular invasion and perirenal fat invasion) in patients with non-metastatic ccRCC. METHODS: This is a retrospective cohort study, we evaluated 99 patients with non-metastatic clear cell renal cell carcinoma, as to the expression of the following proteins: CA-IX, EGF-R, Ki-67, p53, PTEN, VEGF e VEGF-R. The analyzed parameters where: overall survival, TNM stage, tumor size, Fuhrman nuclear grade, microvascular invasion, perirenal fat invasion. We utilized a custom built tissue microarray, and the immunoexpression was digitally quantified using the Photoshop® software. RESULTS: The mean follow-up time was 7,9 years. We found no correlation between the expression of the studied molecular markers and overall survival. As for the conventional prognostic parameters, we found the expression of EGF-R to correlate with T stage (p= 0,049) and perirenal fat invasion (p= 0,020), and VEGF-R to correlate with Fuhrman nuclear grade (p= 0,022) and microvascular invasion (p= 0,022). None of the other markers showed correlation with the studied parameters. CONCLUSIONS: The expression of EGF-R and VEGF-R may be useful tools in the prognostic evaluation of unfavorable risk in patients with non metastatic clear cell renal cell carcinoma
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Barros, Érika Aparecida Felix de. "Aneuploidia dos Cromossomos 3, 7, 9 e 17 no câncer de próstata localizado tratado com cirurgia: Análise e correlação prognóstica". Universidade Nove de Julho, 2014. http://bibliotecadigital.uninove.br/handle/tede/1151.
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Prostate cancer (PCa) is the most common non-skin solid tumor in man in western countries. The prognosis is defined by the measurement of PSA, stage and Gleason score, however, none of these factors classics, even when combined, show good performance in prognosis definition, therefore molecular markers are being studied. Cytogenetic abnormalities, represented by chromosomal deletions and gains, are characteristic of oncogenesis and potentially can be used as a prognostic marker. Objectives: The study aimed to identify the cytogenetic alterations of aneuploidy in chromosomes 3 , 7 , 17 and 9p21 locus by FISH technique located in PCa underwent radical prostatectomy. As a secondary objective correlate the changes found with biochemical recurrence after surgical treatment and the classical prognostic factors of prostate adenocarcinoma. Methodology: For this case-control study 112 patients with clinically localized PCa who underwent radical prostatectomy were followed up postoperatively than ten years were analyzed. Samples of the primary tumor of each patient were available for tissue microarray matrix and cytogenetic changes were assessed by FISH technique. Results: Among the patients for the 9p21 locus, 6.4 % had lost one of the two alleles. In relation to the results obtained for chromosomes 3, 7 and 17 find deletion of one allele of 2.3, 1.2 and 1.8% of the cases respectively. No association between aneuploidy of these chromosomes with the Gleason score, pathological stage, serum PSA level or risk group . We observed that the loss of the 9p21 locus was associated with shorter time to recurrence (p 0.038 ). Conclusions: We found a low occurrence of aneuploidy detected by probes CEP 3 , 7 and 17 and LSI 9p in our series of tumors. We observed that the loss of the 9p21 locus was associated with worse prognosis in CaP located treated with surgery.
Introdução: O câncer de próstata (CaP) é o tumor sólido não cutâneo mais comum do homem em países ocidentais. O prognóstico é feito pela dosagem do PSA, estádio e escore de Gleason, porém, nenhum destes fatores clássicos, mesmo quando avaliados em conjunto, apresentam bom desempenho na determinação prognóstica, por isso marcadores moleculares estão sendo estudados. Alterações citogenéticas, representadas por ganhos e deleções cromossômicas, são características da oncogênese e potencialmente podem ser empregadas como marcador de prognóstico. Objetivos: O estudo teve como objetivo principal identificar as alterações citogenéticas de aneuploidia nos cromossomos 3, 7, 17 e lócus 9p21 pela técnica de FISH no CaP localizado submetido à prostatectomia radical. Como objetivo secundário correlacionamos as alterações encontradas com a recidiva bioquímica após tratamento cirúrgico e com os fatores prognósticos clássicos do adenocarcinoma de próstata. Metodologia: Para este estudo caso controle foram analisados 112 pacientes com diagnóstico de CaP clinicamente localizado submetidos à prostatectomia radical com seguimento pós operatório superior a dez anos. As amostras do tumor primário de cada paciente foram disponibilizadas em matriz de microarranjo tecidual e as alterações citogenéticas foram avaliadas através da técnica de FISH. Resultados: Dos pacientes avaliados para o lócus 9p21, 6,4% apresentaram perdas de um dos dois alelos. Em relação aos resultados obtidos para os cromossomos 3, 7 e 17 encontramos deleção de um dos alelos em 2,3; 1,2 e 1,8% dos casos respectivamente. Não observamos associação entre aneuploidia desses cromossomos com o escore de Gleason, estádio patológico, nível sérico de PSA ou grupo de risco. Observamos que a perda do lócus 9p21 esteve associado a menor tempo para recidiva (p 0,038). Conclusões: Encontramos baixa ocorrência de aneuploidia detectadas pelas sondas CEP 3, 7 e 17 e LSI 9p em nossa série de tumor. Observamos que a perda do lócus 9p21 esteve associado a pior prognóstico no CaP localizado tratado com cirurgia.
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Fujimoto, Masakazu. "Stromal plasma cells expressing immunoglobulin G4 subclass in non-small cell lung cancer". Kyoto University, 2015. http://hdl.handle.net/2433/199170.
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Hinterberger, Marc Lorenz. "D2-40 and calretinin : a tissue microarray analysis of 341 malignant mesotheliomas with emphasis on sarcomatoid differentiation /". [S.l.] : [s.n.], 2009. http://opac.nebis.ch/cgi-bin/showAbstract.pl?sys=000281124.
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Guo, Dongli. "Expression of Wnt signaling targets and their clinico-pathological significance in colorectal neoplasm a tissue microarray study /". Click to view the E-thesis via HKUTO, 2006. http://sunzi.lib.hku.hk/hkuto/record/B38610541.
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Tawfik, El-Mansi M. M. "Molecular analysis and application of tissue microarray technology to the histopathological and immunohistochemical analysis of cervical adenocarcinoma". Thesis, University of Edinburgh, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.662741.
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A tissue microarray method yielded staining of good quality and is feasible for morphological and immunohistochemical studies in cervical adenocarcinoma. Analysis of two TMA cores achieved 100% representation for morphological studies and greater then 97% representation for immunohistochemical studies. Tissue array sections were immunostained with 8 antibodies, carcinoembryonic antigen (CEA). Cytokeratin7 (CK7), Cytokeratin20 (CK20), oestrogen receptor (ER), progesterone receptor (PgR), phosphatase and tensin homolog deleted on chromosome ten (PTEN), MIB-1 proliferation marker, and p53 suppressor gene utilizing the power vision technique for ER only and Envision technique for all other antibodies. Our findings support that all of these 8 antibodies are of potential biomarkers of a panel of antibodies for diagnosis of cervical adenocarcinomas. HPV DNA was extracted from paraffin-embedded, formalin-fixed tissues of 161 specimens of 139 patients with excluding 22 patients with the second samples and 16 normal cervical tissues. HPV DNA was detected by PCR test using type specific primers from the E6 gene and E7 gene of HPV type 16 and HPV type 18. HPV DNA was identified in 87 cases (62.6%) in which, HPV16 was positive for 65 (47%) patients and HPV18 was positive for 41 (29%) patients. Genotyping by RFLP and PCR revealed that, HPV type 16 was the most frequent type of infection comprising 46 cases (33%), followed by HPV type 18 in 22 cases (16%), and both HPV type 16 and HPV type 18 in 19 cases (14%). HPV typing in all cases of 16 normal cervical biopsies revealed negative with both HPV type 16 and HPV type 18. Our findings support that HPV 16, along with HPV 18, may play a possible role in the pathogenesis of adenocarcinoma of the uterine cervix.
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Schreiber, Melanie [Verfasser] y Guido [Akademischer Betreuer] Sauter. "Tissue microarray based identification of molecular cancer therapy targets in clinical routine / Melanie Schreiber. Betreuer: Guido Sauter". Hamburg : Staats- und Universitätsbibliothek Hamburg, 2013. http://d-nb.info/1045024171/34.
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Mayer, Lisa [Verfasser]. "Immunhistochemische Evaluation der Proteinexpression verschiedener Somatostatin-Rezeptoren beim muskelinfiltrierenden Harnblasenkarzinom mittels der Tissue Microarray Technik / Lisa Mayer". Tübingen : Universitätsbibliothek Tübingen, 2021. http://d-nb.info/1232725730/34.
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Wang, Yemin. "Role of tumour suppressor ING3 in melanoma pathogenesis". Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/3850.
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The type II tumour suppressor ING3 has been shown to modulate transcription, cell cycle control, and apoptosis. To investigate the putative role of ING3 in melanoma development, we examined the expression of ING3 in 58 dysplastic nevi, 114 primary melanomas, and 50 metastatic melanomas with tissue microarray and immunohistochemistry. Overall ING3 was reduced in metastatic melanomas compared with dyslastic nevi and primary melanomas. Reduced nuclear ING3 staining also correlated with melanoma progression, increased cytoplasmic ING3 level, tumour location at sun-exposed sites, and a poorer disease-specific 5-year survival of patients with primary melanoma. Multivariate analysis revealed that nuclear ING3 staining can independently predict patient outcome in primary melanomas. In melanoma cells, ING3 expression was rapidly induced by UV irradiation. Using stable clones of melanoma cells overexpressing ING3, we showed that ING3 significantly promoted UV-induced apoptosis. Unlike its homologues ING1b and ING2, ING3-enhanced apoptosis upon UV irradiation was independent of functional p53. Furthermore, ING3 did not affect the expression of mitochondrial proteins but increased the cleavage of Bid and caspases. Moreover, ING3 upregulated Fas expression and ING3-mediated apoptosis was blocked by inhibiting caspase-8 or Fas activation. Knockdown of ING3 expression decreased UV-induced apoptosis remarkably, suggesting that ING3 plays a crucial role in cellular response to UV radiation. To explore how ING3 is deregulated in advanced melanomas, we examined ING3 expression in metastatic melanoma cells and found that ING3 was downregulated due to a rapid protein turnover in these cells. Further studies demonstrated that ING3 undergoes degradation via the ubiquitin-proteasome pathway. We also demonstrate that ING3 interacts with the SCF (Skp1/Cul1/Roc1/Skp2) E3 ligase complex. Knockdown of Cul1 or Skp2 significantly stabilized ING3 in melanoma cells. In addition, lysine residue 96 is essential for ING3 ubiquitination as its mutation to arginine completely abrogated ING3 turnover and enhanced ING3-stimulatd apoptosis upon UV irradiation. Taken together, ING3 is deregulated in melanomas as a result of both nucleus-to-cytoplasm shift and rapid degradation. The level of ING3 in the nucleus may be an important marker for human melanoma progression and prognosis. Restoration of ING3 expression significantly sensitizes melanoma cells to UV radiation through the activation of Fas/caspase-8 pathway.
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Sahadevan, Kanagasabai. "The development of tissue microarray to investigate the role of fibroblast growth factor signalling regulators in prostate cancer". Thesis, University of Newcastle upon Tyne, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.440559.
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Weitbrecht, Timo Konstantin [Verfasser]. "Expression von Cytokeratin 18 in menschlichen Tumoren und Normalgeweben: Eine Tissue-Microarray-Studie an 11.952 Tumoren / Timo Konstantin Weitbrecht". Hamburg : Staats- und Universitätsbibliothek Hamburg Carl von Ossietzky, 2020. http://d-nb.info/1229625615/34.
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Schneider, Jana Verfasser] y Guido [Akademischer Betreuer] [Sauter. "Thyreoglobulin Expression in humanen Tumor- und Normalgeweben : Eine Tissue Microarray-Studie an 3448 Tumoren / Jana Schneider ; Betreuer: Guido Sauter". Hamburg : Staats- und Universitätsbibliothek Hamburg, 2020. http://nbn-resolving.de/urn:nbn:de:gbv:18-106511.
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Schneider, Jana [Verfasser] y Guido [Akademischer Betreuer] Sauter. "Thyreoglobulin Expression in humanen Tumor- und Normalgeweben : Eine Tissue Microarray-Studie an 3448 Tumoren / Jana Schneider ; Betreuer: Guido Sauter". Hamburg : Staats- und Universitätsbibliothek Hamburg, 2020. http://d-nb.info/1216629714/34.
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Junttila, S. (Sanna). "Studies of kidney induction in vitro using gene expression profiling and novel tissue manipulation technique". Doctoral thesis, Oulun yliopisto, 2014. http://urn.fi/urn:isbn:9789526206608.
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Abstract For decades, the mammalian kidney has served as a model system for studying developmental processes, such as induced epithelialization, branching morphogenesis, and cell differentiations. The possibility to recapitulate and follow the renal organogenesis ex vivo in organ culture set-ups has provided a large amount of molecular and cellular information about sequential events during development. However, certain limitations remain when combining traditional organ culture set-ups with modern molecular technology. This thesis seeks to address these disadvantages. In the experimental part of the thesis, the traditional organ culture set-ups were studied, modified, and optimized to meet the needs of functional genetic screening. First, the traditional transfilter- induced nephrogenesis was characterized with a panel of nephron segment specific markers to reveal the differentiation level of in vitro developing mouse renal tissue. A comprehensive genome wide time course microarray analysis was also performed to in vitro- induced metanephric mesenchyme. Next, to improve the accessibility of genetic tools into the three- dimensional organ in culture, the classic kidney culture set-ups were modified to tolerate dissociation and re-aggregation before the induction of nephrogenesis. This step was achieved with the aid of preservative growth factors offering a 24- hour window to manipulate the genetic and cellular composition of the explant. The dissociation and re-aggregation per se had not particular effect on the progress of the nephron differentiation. Demonstrations of the addition and removal of cells, as well as a virus vector mediated gene knock in and knock down are presented. The gene expression data, together with the novel organ manipulation and culture techniques presented in this thesis, provide a useful guide and specific tools to further characterize the details of nephron development and differentiation in functional mannerTiivistelmä Nisäkkäiden munuainen on toiminut vuosikymmeniä mallielimenä tutkittaessa kehitysbiologisia tapahtumasarjoja, kuten epitelisaatiota, haaroittumismorfologiaa sekä solujen erilaistumista. Munuaisaihioita voidaan viljellä laboratorio-olosuhteissa, jolloin kehityksen aikaisia muutoksia päästään seuraamaan lähes reaaliaikaisesti. Perinteisten kudosviljelytekniikoiden tarjoamat mahdollisuudet solujen molekulaariseen muokkaukseen ovat kuitenkin varsin rajalliset. Tässä väitöskirjassa esitettävät tulokset pyrkivät osaltaan vähentämään näitä rajoitteita. Väitöskirjan kokeellisessa osassa tarkastellaan lähemmin klassista munuaiskudosviljelyä sekä esitetään siihen tehtyjä optimointeja, joiden avulla kudosviljelyä pyritään hyödyntämään geenien toiminnan tutkimuksessa. Aluksi perinteisellä tavalla reikäisen kalvon läpi indusoitu nefroni karakterisoitiin tarkasti hyödyntäen useita erilaistumista osoittavia merkkimolekyylejä. Lisäksi samalla tekniikalla tuotettujen munuaiskudosviljelmien geeniekspressiota tutkittiin mikrosiruanalyysillä. Klassisia kudosviljelytekniikoita muokattiin soveltuvammaksi moderneille geneettisille työkaluille. Munuaiskudos hajotettiin ensin solususpensioksi, jonka jälkeen solut muodostivat uudelleen kolmiulotteisen, kudosmaisen rakenteen. Hyödyntämällä suojaavia kasvutekijöitä, hajotus kyettiin tekemään jo ennen nefronien muodostumisen alkua. Näin saavutettin 24 tunnin aikaikkuna indusoimattoman kudoksen geneettiselle muokkaukselle. Väitöskirjassa esitelläänkin demonsrtaatiot solujen lisäämisestä ja poistamisesta sekä virusvälitteisestä geenin aktivoinnista ja hiljennyksestä hyödyntäen uutta kudosmanipulaatio ja –vilejelytekniikkaa. Nefronin kehityksen aikaisen geeniekspression kartoitus sekä tässä tutkimuksessa kehitetyt uudet kudosmanipulaatio ja -viljelytekniikat tarjoavat yhdessä työkaluja molekyylitason yksityiskohtaiseen tutkimiseen
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Matilda, Rentoft. "The use of formalin fixed paraffin embedded tissue and global gene expression profiling for increased understanding of squamous cell carcinoma of the tongue". Doctoral thesis, Umeå universitet, Patologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-54005.
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Head and neck cancer is the 6th most common malignancy worldwide, with tumours of the tongue being one of the most prevalent sites. Despite advances in surgery and radiotherapy, the five-year survival has not changed during the last decades and remains at approximately 50%. Identification of novel biomarkers for more personalized treatment is important for increasing survival in these patients. One of the most commonly used methods in the search for new biomarkers is microarray analysis. A substantial limitation with this technique is the requirement for fresh frozen samples from which high quality RNA can be extracted. This becomes particularly problematic when attempting to discover differences associated with individual sub-types or rare cancers. Recent developments, including the DASL microarray platform, have provided the possibility of analysing RNA of poorer quality from formalin fixed paraffin embedded (FFPE) samples. FFPE is the standard way of preserving tissue from patients and millions of samples are stored around the world. In this thesis we have evaluated the use of FFPE samples and global gene expression profiling for increasing basic knowledge in a subgroup of oral cancer patients with tumours of the tongue. As confirmation of microarray results using qPCR is of outmost importance for conclusive data evaluation, we first aimed at finding a housekeeping gene stably expressed across malignant and non-malignant FFPE oral tissue. TUBA6, which belongs to the tubulin family was detected as being the most stable out of eight possible genes and was thus used for qPCR normalization throughout the following studies. We have performed three separate microarray experiments. Initially only a focused DASL array covering 502 cancer related genes was available and we used it to analyze a smaller cohort of patients and controls (n=36). A similar cohort (n=29) was also analyzed for expression of 836 micoRNAs. In 2009 a whole genome DASL array was launched, covering over 20,000 genes, and all tongue tumour samples available between 1997 and 2010 (n=87) were analysed using this array. Similar to other research groups we observed very high replicate reproducibility using both DASL arrays. When using the microRNA array and the whole genome DASL array an effect of sample quality on the detected expression level of individual genes was noticed. While the expression of some genes severely decreased with a decrease in sample quality others were not changed. This will impair normalization, leading to a residual non-biological variation within the data. Based on our findings we have presented some recommendations for minimizing the effect of sample quality and maximizing the level of biologically relevant information obtained from these experiments, e.g. ensuring that samples in groups to be compared are of the same quality range. For the microRNA data we also introduced an additional normalization step to the standard normalizations. We could show that lists of differentially expressed genes generated when taking these precautions were enriched for genes involved in cancer related processes and contained for tongue carcinoma previously identified changes. A number of differentially expressed genes, novel for tongue carcinoma, were also confirmed in high quality fresh frozen samples, including BCL2A1 (apoptosis), CXCL10 (immune response), SLC2A6 (energy transport) and miR-424 (angiogenesis). In conclusion microarrays can be used to analyze FFPE samples but should be performed with care. Standard normalization methods will not remove the variation introduced by samples being of different quality, leading to spurious results. Taking a few precautions, however, led to the identification of differentially expressed genes relevant in tumour development and maintenance. The recommendations we make can facilitate design of future studies using FFPE samples. The genes we identified as being differentially expressed in tumour tissue now need to be further evaluated for their potential as biomarkers in tongue carcinoma.
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Agnarsdóttir, Margrét. "Biomarker Discovery in Cutaneous Malignant Melanoma : A Study Based on Tissue Microarrays and Immunohistochemistry". Doctoral thesis, Uppsala universitet, Institutionen för immunologi, genetik och patologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-146436.
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The incidence of cutaneous malignant melanoma has increased dramatically in Caucasians the last few decades, an increase that is partly explained by altered sun exposure habits. For the individual patient, with a localized disease, the tumor thickness of the excised lesion is the most important prognostic factor. However, there is a need to identify characteristics that can place patients in certain risk groups. In this study, the protein expression of multiple proteins in malignant melanoma tumors was studied, with the aim of identifying potential new candidate biomarkers. Representative samples from melanoma tissues were assembled in a tissue microarray format and protein expression was detected using immunohistochemistry. Multiple cohorts were used and for a subset of proteins the expression was also analyzed in melanocytes in normal skin and in benign nevi. The immunohistochemical staining was evaluated manually and for part of the proteins also with an automated algorithm. The protein expression of STX7 was described for the first time in tumors of the melanocytic lineage. Stronger expression of STX7 and SOX10 was seen in superficial spreading melanomas compared with nodular malignant melanomas. An inverse relationship between STX7 expression and T-stage was seen and between SOX10 expression and T-stage and Ki-67, respectively. In a population-based cohort the expression of MITF was analyzed and found to be associated with prognosis. Twenty-one potential biomarkers were analyzed using bioinformatics tools and a protein signature was identified which had a prognostic value independent of T-stage. The protein driving this signature was RBM3, a protein not previously described in malignant melanoma. Other markers included in the signature were MITF, SOX10 and Ki-67. In conclusion, the protein expression of numerous potential biomarkers was extensively studied and a new prognostic protein panel was identified which can be of value for risk stratification.
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Mattedi, Romulo Loss. "Carcinomas uroteliais de bexiga: aspectos anatomopatológicos e imuno-histoquímicos. Pesquisa de metaloproteinases de matriz utilizando a técnica de tissue microarray (TMA)". Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/5/5144/tde-30082011-160347/.
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OBJETIVOS: Estudar variáveis anatomopatológicas relacionadas à progressão tumoral em carcinomas uroteliais primários de bexiga e sua associação com a imunoexpressão de metaloproteinases de matriz (MMPs) - 2, -9 e -14 no epitélio e no estroma dos tumores primários e nas metástases linfonodais. MÉTODOS: Sessenta e um casos de carcinomas uroteliais musculoinvasivos ou localmente avançados primários da bexiga operados no Hospital das Clínicas da Faculdade de Medicina da USP e no Instituto do Câncer do Estado de São Paulo, sendo 34 casos com metástase para linfonodo regional, foram caracterizados quanto ao gênero, idade, tamanho, focalidade, grau histológico, tipo/configuração neoplásica, tipo papilífero da neoplasia, padrão arquitetural de invasão tumoral, grau de atipia nuclear, componente sarcomatoide, diferenciações escamosa e glandular, variante histológica, invasões linfovascular e perineural, carcinoma in situ, estádio do tumor primário, metástase para linfonodo regional, tamanho da metástase e extensão extranodal. Amostras teciduais de 1,0 mm foram dispostas em micromatrizes teciduais (TMA) para pesquisa imuno-histoquímica (IH) das enzimas MMP-2, MMP-9 e MMP-14. A expressão IH das MMPs foi graduada em uma escala semiquantitativa de 0 (ausência de expressão) até 20 (maior expressão). As associações entre a imunoexpressão das MMPs de forma global, no epitélio e estroma do tumor primário e na metástase linfonodal com as variáveis anatomopatológicas foram avaliadas através do teste do qui-quadrado de Pearson, sendo consideradas significativas ao nível de p<0,05. RESULTADOS: Trinta e seis, 57 e 60 casos do tumor primário foram positivos para MMP-2, MMP-9 e MMP-14, respectivamente. Nas metástases linfonodais, 20, 27 e 26 casos foram positivos para MMP-2, MMP-9 e MMP-14, respectivamente. A imunoexpressão global de MMP-2 no tumor primário mostrou-se associada com o padrão arquitetural de invasão (p=0,022) e sua expressão no estroma com o grau de atipia nuclear (p=0,032) e a porcentagem de componente sarcomatoide (p=0,003). A imunoexpressão global de MMP-9 no tumor primário mostrou-se associada com diferenciação escamosa (p=0,033). O padrão arquitetural de invasão relacionou-se com a expressão de MMP-9 no epitélio (p=0,043) e no estroma (p=0,044). A expressão de MMP-9 no estroma mostrou-se associada com o grau de atipia nuclear (p=0,031), componente sarcomatoide (p=0,036) e com a porcentagem desse componente no tumor primário (p=0,013). O estádio tumoral agrupado pT2+pT3 vs pT4 demonstrou associação com a MMP-9 expressa no epitélio (p=0,049). Para a MMP-14, o padrão arquitetural de invasão demonstrou associação significativa com a imunoexpressão global (p=0,022) e no epitélio tumoral (p=0,045). A porcentagem de componente sarcomatoide relacionou-se ainda com a expressão estromal de MMP-14 (p<0,001). Considerando a imunoexpressão de MMPs apenas na metástase linfonodal, houve associação significativa da MMP-9 com o tipo de variante histológica do tumor (p=0,021) e da expressão de MMP-14 com a porcentagem de componente sarcomatoide no tumor primário (p=0,017). CONCLUSÃO: O estudo da imunoexpressão de MMP-2, MMP-9 e MMP-14 em amostras organizadas em TMA, tanto no epitélio como no estroma dos carcinomas uroteliais de bexiga e nas metástases de linfonodos regionais, demonstrou associações estatísticas com características anatomopatológicas reconhecidas como de prognóstico ruim para essas neoplasias, indicando a ação dessas enzimas na transição epitélio-mesênquima e nas etapas de progressão e metástase dos carcinomas uroteliaisOBJECTIVES: To study morphological features related to tumor progression in urothelial carcinoma of the urinary bladder and its association with immunohistochemical (IHC) expression of matrix metalloproteinases (MMPs) -2, -9 and -14 in epithelial and stromal cells of primary tumor and regional lymph node metastases. METHODS: Sixty-one cases of muscle-invasive or locally advanced urothelial carcinomas of the bladder operated on Clinic\'s Hospital of Faculty Medicine Sao Paulo University and the Cancer Institute of the State of Sao Paulo, with 34 cases showing regional lymph nodes metastases, were characterized regarding gender, age, tumor size, multifocality, histological grade, neoplastic type/configuration, papillary type, architectural pattern of invasive tumor, nuclear atypia, sarcomatoid component, squamous and glandular diffentiation, histological variants, lymphovascular and perineural invasion, carcinoma in situ, tumor stage, metastases to regional lymph nodes, metastases size and extranodal extension. Tissue samples of 1.0 mm were arranged in tissue microarrays blocks (TMA) for IHC detection of MMP-2, MMP-9 and MMP-14. The grading of expression of MMPs was determined to a semiquantitative scale from 0 (absence) to 20 (higher expression). The associations between the IHC global expression of MMPs, in epithelium and in stromal cells of the primary tumor and in the lymph node metastases with the morphological features were obtained through Pearson\'s chi-square (significant at p<0.05). RESULTS: Thirty-six, 57 and 60 cases of primary tumor were positive for MMP-2, MMP-9 and MMP-14 respectively. In the lymph nodes metastases, 20, 27 and 26 cases were positive for MMP-2, MMP-9 and MMP-14 respectively. The global IHC expression of MMP-2 in primary tumor has been associated with the architectural pattern of invasion (p=0.022). The expression in stromal cells were correlated with the degree of nuclear atypia (p=0.032) and the percentage of sarcomatoid component (p=0.003). The IHC expression of MMP-9 in primary tumor has been associated with squamous differentiation (p=0.033). The architectural pattern of invasion was related to the expression of MMP-9 in epithelium (p=0.043) and in the stroma (p=0.044). Expression of MMP-9 in the stroma was associated with the degree of nuclear atypia (p=0.031), sarcomatoid component (p=0.036) and the percentage of this component in primary tumor (p=0.013). The grouped tumoral stage pT2+pT3 vs pT4 showed association with MMP-9 expressed in epithelium (p=0.049). For MMP-14, the architectural pattern of invasion showed significant association with global IHC expression (p=0.022) and tumor epithelium (p=0.045). The percentage of sarcomatoid component related to the estromal expression of MMP-14 (p<0.001). Considering the IHC expression of MMPs in lymph nodes metastases, there was a significant association between MMP-9 with the type of histological variants (p=0.021) and the expression of MMP-14 with the percentage of sarcomatoid component in primary tumor (p=0.017). CONCLUSION: The study of IHC expression of MMP-2, MMP-9 and MMP-14 in bladder carcinoma samples arranged in TMA, both in epithelium and in stromal cells and regional lymph nodes metastases, demonstrated significant association with morphological features recognized as prognostically important for these tumors. These findings herald the importance of action of these enzymes in epithelialmesenchymal transition, providing basis for the understanding of tumoral progression and metastases in urothelial carcinoma
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Vidale, Mariana Marras [UNESP]. "Avaliação da prliferação e apoptose com a utilização de arranjos em matriz de amostra tecidual (Tissue Microarray - TMA) em mastocitomas cutâneos caninos". Universidade Estadual Paulista (UNESP), 2010. http://hdl.handle.net/11449/89270.
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Os mastócitos são células provenientes do precursor CD34 da medula óssea, o mastocitoma (MCT) pertence ao grupo das neoplasias de células redondas e possui etiologia desconhecida, é a neoplasia cutânea mais comum em cães, sem predileção por raça, sexo ou idade. Seu comportamento biológico é bastante agressivo e de alta frequência. A graduação histopatológia proposta Patnaik et al., (1984), é o critério mais utilizado para a graduação histopatológica da neoplasia sendo o grau 1 uma neoplasia bem diferenciada, o grau 2 moderadamente diferenciada e o grau 3 pouco diferenciada ou anaplásica. O presente trabalho teve como objetivos a avaliação do ínidice proliferativo e apoptotico dos MCT em lâmina de Microarranjo de tecido (TMA) utilizando a técnica de imunoistoquiímica e TUNEL. Para tal utilizou-se 166 casos de MCT compondo um arranjo em matriz de amostra tecidual, ou tissue microarray (TMA) com 190 amostras, sendo avaliado o padrão de marcação da proteína Kit, a proliferação celular com o anticorpo primário Ki67 e apoptose com o anticorpo primário caspase-3 clivada e pela técnica da Terminal Deoxynucleotidyl Transferase Mediated dUTP Nick end Labeling Assey (TUNEL). Quando os casos foram avaliados quanto à imunoexpressão de c-KIT, os tumores (únicos e múltiplos) com expressão citoplasmática da proteína KIT tiveram uma taxa proliferativa maior e uma menor taxa apoptótica quando comparados aos com expressão membranosa. Os MCTs grau 3 tiveram um maior índice proliferativo (imunoexpressão de Ki67) quando comparados aos tumores de graus 1 e 2. Não foi observada correlação entre a imunomarcação do anticorpo primário caspase-3 clivada e a técnica de TUNEL, sendo que a técnica de TUNEL se mostrou mais eficaz na avaliação da apoptose que a imunomarcação para caspase-3 clivada
Mast cells are from precursor CD34 cells bone marrow, the mast cell tumor (MCT) belongs to the group of round cell malignancies and has unknown etiology, is the most common skin cancer in dogs without distinction of race, sex or age. Its biological behavior is very aggressive and high frequency. The histopathological grading proposal from Patnaik et al., (1984), is the most commonly used criterion for the histopathological grade of the tumor is a tumor grade 1 are well-differentiated, grade 2 are moderately differentiated and grade 3 are poorly differentiated or anaplastic. This study aimed to evaluate the proliferative and apoptotic index of MCT in blade tissue microarray (TMA) using the TUNEL technique and immunohistochemistry. To this end we used 166 cases of MCT composing an tissue microarray (TMA) with 190 samples, and evaluated the pattern of protein labeling kit, cell proliferation, with the primary antibody Ki67 and apoptosis with primary antibody and cleaved caspase-3 by the technique of terminal deoxynucleotidyl transferase dUTP Nick End Labeling Mediated Assey (TUNEL). When the cases were evaluated for immunoexpression of c-KIT, tumors (single and multiple) with cytoplasmic KIT protein expression had a higher proliferative rate and a lower apoptotic rate when compared to those with membranous expression. Grade 3 MCTs had a greater proliferative index (Ki67 immunostaining) when compared with tumor grades 1 and 2. No correlation was observed between the immunostaining of primary antibody caspase-3 cleavage and TUNEL technique, and TUNEL technique was more effective in assessing apoptosis that immunostaining for cleaved caspase-3
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Fonseca, Felipe Paiva 1986. "Análise clinicopatológica de 493 casos de tumores de glândulas salivares e construção de blocos de parafinas utilizando a técnica de tissue microarray". [s.n.], 2012. http://repositorio.unicamp.br/jspui/handle/REPOSIP/288414.
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Orientador: Pablo Agustin VargasDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: Tumores de glândulas salivares correspondem à cerca de 3 a 6% de todos os tumores de cabeça e pescoço, apresentando uma ampla variação quanto à freqüência dos diferentes tipos histológicos e seus respectivos cursos clínicos. Desta forma, a determinação de novos marcadores moleculares que estejam relacionados com o comportamento biológico destas neoplasias se faz necessário e o uso da técnica de tissue microarray (TMA) ou micro-arranjo tecidual representa uma ferramenta altamente eficaz para a identificação destes marcadores. Sendo assim, o objetivo do presente estudo é avaliar as características clinicopatológicas de 493 neoplasias de glândulas salivares e descrever os princípios técnicos de construção de blocos de micro-arranjo tecidual, assim como suas vantagens e desvantagens para o estudo destes tumores. Para isto, os prontuários de um centro de patologia médica e de um centro de patologia oral compreendidos entre os anos de 2001 e 2011 foram revisados e os dados clinico patológicos coletados, enquanto que a construção dos blocos de TMA foi realizada por meio de equipamento manual de arranjo tecidual em matriz, onde três áreas tumorais representativas foram selecionadas e incluídas no bloco receptor. Após a obtenção dos resultados, foi observado que o adenoma pleomórfico e o carcinoma mucoepidermóide representaram as neoplasias benigna e maligna de glândulas salivares mais freqüentes e após a construção de 12 blocos de TMA foi possível obter boa representatividade utilizando-se cilindros de 1,0, 2,0 ou 3,0 mm, especialmente em neoplasias sólidas. Portanto, a distribuição dos tumores de glândulas salivares na amostra estudada está de acordo com os achados relatados anteriormente na literatura e a técnica de TMA apresenta-se como uma metodologia de alto rendimento e baixo custo no estudo de tumores de glândulas salivares
Abstract: Salivary gland tumors account for 3 to 6% of the head and neck tumors, with a broad variation in the incidence of their different histological subtypes and their respective clinical courses. For this reason, the determination of new molecular markers truly associated to the biological behavior of these neoplasias becomes necessary and the use of tissue microarray (TMA) technique represents a high-throughput laboratory tool for identifying such markers. The aim of the present study is to evaluate the clinic-pathological features of 493 salivary gland neoplasias and to describe the technical principles for construction of TMA blocks, as well as their advantages and disadvantages regarding the study of salivary gland tumors. For this, medical charts of a general pathology service and of an oral pathology service from 2001 to 2011 were reviewed and the clinic-pathological data acquired, whereas TMA blocks were constructed using a manual tissue arrayer by selecting three representative neoplastic areas to be included in the recipient block. Following the acquisition of the results, it was observed that pleomorphic adenoma and mucoepidermoid carcinoma represented the most frequent benign and malignant neoplasias, respectively, and that after the building of 12 TMA blocks it was possible to obtain high representative cores by using 1.0, 2.0 and 3.0 mm cylinders, especially in solid neoplasias. Hence, the distribution of salivary gland tumors in the sample studied is in agreement with the findings reported previously in the literature and the TMA technique presents as a high-throughput and low-cost methodology in salivary gland tumors study
Mestrado
Patologia
Mestre em Estomatopatologia
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Vidale, Mariana Marras. "Avaliação da prliferação e apoptose com a utilização de arranjos em matriz de amostra tecidual (Tissue Microarray - TMA) em mastocitomas cutâneos caninos /". Botucatu : [s.n.], 2010. http://hdl.handle.net/11449/89270.
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Orientador: Renée Laufer AmorimBanca: Noeme Sousa Rocha
Banca: Antonio Carlos Alessi
Resumo: Os mastócitos são células provenientes do precursor CD34 da medula óssea, o mastocitoma (MCT) pertence ao grupo das neoplasias de células redondas e possui etiologia desconhecida, é a neoplasia cutânea mais comum em cães, sem predileção por raça, sexo ou idade. Seu comportamento biológico é bastante agressivo e de alta frequência. A graduação histopatológia proposta Patnaik et al., (1984), é o critério mais utilizado para a graduação histopatológica da neoplasia sendo o grau 1 uma neoplasia bem diferenciada, o grau 2 moderadamente diferenciada e o grau 3 pouco diferenciada ou anaplásica. O presente trabalho teve como objetivos a avaliação do ínidice proliferativo e apoptotico dos MCT em lâmina de Microarranjo de tecido (TMA) utilizando a técnica de imunoistoquiímica e TUNEL. Para tal utilizou-se 166 casos de MCT compondo um arranjo em matriz de amostra tecidual, ou tissue microarray (TMA) com 190 amostras, sendo avaliado o padrão de marcação da proteína Kit, a proliferação celular com o anticorpo primário Ki67 e apoptose com o anticorpo primário caspase-3 clivada e pela técnica da Terminal Deoxynucleotidyl Transferase Mediated dUTP Nick end Labeling Assey (TUNEL). Quando os casos foram avaliados quanto à imunoexpressão de c-KIT, os tumores (únicos e múltiplos) com expressão citoplasmática da proteína KIT tiveram uma taxa proliferativa maior e uma menor taxa apoptótica quando comparados aos com expressão membranosa. Os MCTs grau 3 tiveram um maior índice proliferativo (imunoexpressão de Ki67) quando comparados aos tumores de graus 1 e 2. Não foi observada correlação entre a imunomarcação do anticorpo primário caspase-3 clivada e a técnica de TUNEL, sendo que a técnica de TUNEL se mostrou mais eficaz na avaliação da apoptose que a imunomarcação para caspase-3 clivada
Abstract: Mast cells are from precursor CD34 cells bone marrow, the mast cell tumor (MCT) belongs to the group of round cell malignancies and has unknown etiology, is the most common skin cancer in dogs without distinction of race, sex or age. Its biological behavior is very aggressive and high frequency. The histopathological grading proposal from Patnaik et al., (1984), is the most commonly used criterion for the histopathological grade of the tumor is a tumor grade 1 are well-differentiated, grade 2 are moderately differentiated and grade 3 are poorly differentiated or anaplastic. This study aimed to evaluate the proliferative and apoptotic index of MCT in blade tissue microarray (TMA) using the TUNEL technique and immunohistochemistry. To this end we used 166 cases of MCT composing an tissue microarray (TMA) with 190 samples, and evaluated the pattern of protein labeling kit, cell proliferation, with the primary antibody Ki67 and apoptosis with primary antibody and cleaved caspase-3 by the technique of terminal deoxynucleotidyl transferase dUTP Nick End Labeling Mediated Assey (TUNEL). When the cases were evaluated for immunoexpression of c-KIT, tumors (single and multiple) with cytoplasmic KIT protein expression had a higher proliferative rate and a lower apoptotic rate when compared to those with membranous expression. Grade 3 MCTs had a greater proliferative index (Ki67 immunostaining) when compared with tumor grades 1 and 2. No correlation was observed between the immunostaining of primary antibody caspase-3 cleavage and TUNEL technique, and TUNEL technique was more effective in assessing apoptosis that immunostaining for cleaved caspase-3
Mestre
36
Gry, Marcus. "Global expression analysis of human cells and tissues using antibodies". Doctoral thesis, Stockholm : Bioteknologi, Kungliga Tekniska högskolan, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-9116.
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Timoszczuk, Luciana Maria Sevo. "Análise de proteínas cuja expressão é controlada por miRNA e relacionada à progressão do adenocarcinoma de próstata por imuno-histoquimica em tissue microarray". Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/5/5153/tde-13122012-162235/.
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Introdução: O Câncer de Próstata (CaP) é o tumor mais comum do homem e a segunda causa de óbito por câncer no Brasil. MicroRNA (miRNA) é uma classe de pequenos RNA regulatórios não codificantes de proteínas que tem papel fundamental no controle da expressão dos genes. São responsáveis pelo controle de processos fundamentais na célula e estão envolvidos na tumorigênese em humanos. Previamente demonstramos alterações no perfil de expressão dos miRNA 100, let7c e 218 comparando carcinomas localizados e metastáticos. A caracterização de perfis de expressão de suas proteínas alvo no CaP é crucial para a compreensão dos processos envolvidos na carcinogênese, dando-nos a oportunidade do descobrimento de novos marcadores diagnósticos, prognósticos e mais importante identificação de alvos para o desenvolvimento de terapias inovadoras. Objetivo: Analisar a expressão das proteínas controladas pelo miR-let7c (Ras, c-Myc e Bub1), miR-100 (Smarca5 e Retinoblastoma) e miR-218 (Laminina 5 3) e a atividade proliferativa (Ki-67) no câncer de próstata com a técnica de imuno-histoquímica utilizando microarranjos teciduais representativos de CaP localizado e suas metástases linfonodais e ósseas. Correlacionar os níveis de expressão dos miRNA com suas proteínas alvo. Analisar a expressão dos miRNA, proteínas e atividade proliferativa com os fatores prognósticos do câncer de próstata e com a evolução da doença. Material e Métodos: A imunoexpressão de Smarca5, Retinoblastoma, Laminina, Ras, c- Myc, Bub1 e Ki-67 foi avaliada através de IH pela técnica de microarranjo tecidual caracterizando três estágios do CaP, sendo 112 casos de CaP localizado, 19 metástases linfonodais e 28 metástases ósseas. As imagens obtidas foram submetidas a um software de análise de imagem digital MacBiophotonics ImageJ do National Institutes of Health, EUA, onde a intensidade de luminescência foi quantificada densitometricamente. O perfil de expressão dos miR-let7c, 100 e 218 foi analisado utilizando o bloco de parafina de 61 pacientes dos 112 pacientes com carcinoma localizado, que foram submetidos a analise protéica por IH. O processamento dos miRNA envolveu três etapas: extração do miRNA com kit específico, geração do DNA complementar e amplificação do miRNA por PCR quantitativo em tempo real (qRT-PCR) cujo controle endógeno foi RNU-43 (Applied Biosystems). Os resultados foram analisados usando o método 2-CT. Como controle, utilizamos amostras de tecido com hiperplasia prostática benigna (HPB). Avaliamos a relação entre a expressão dos miRNA e suas proteínas alvo, com o escore de Gleason, estadiamento patológico e evolução da doença considerando recidiva bioquímica, níveis de PSA>0,4 ng/mL, em uma média de seguimento de 77,5 meses. A análise estatística foi realizada através do software SPSS 19.0, utilizamos o test T de Student, Mann-Whitney, Kruskal-Wallis e qui-quadrado. O valor de p foi considerado estatisticamente significante quando inferior na 0,05 em todos os cálculos. Resultados: Observamos uma diminuição de expressão de Ras (p=0,017) e Laminina (p<0,0001) conforme a progressão tumoral do CaP localizado a metástase linfonodal e óssea. Houve um aumento de expressão de Rb (p=0,0361) e aumento da atividade proliferativa avaliada pelo Ki- 67 (p<0,0001). Encontramos ainda uma tendência a relação entre a positividade de expressão de c-Myc com estadiamento patológico pT3 (p=0,070). Todos os miRNA se mostraram superexpressos no CaP localizado. Laminina apresentou uma média de intensidade de expressão maior quanto maior a expressão de miR-218 (p=0,038). Porém os demais miRNA não apresentaram relação de expressão com suas proteínas alvo. Também não houve relação entre a expressão de miRNA e expressão das proteínas por IH com a recidiva bioquímica. Conclusões: Apesar de confirmarmos os nossos achados de superexpressão dos miRNA 100, let7c e 218 no CaP localizado, não houve correlação entre esses e a imunoexpressão de suas proteínas alvo. Demonstramos que houve alteração de imunoexpressão de Ras, Laminina 5 3, Retinoblastoma e Ki-67 de acordo com a progressão tumoral no CaP. E uma maior expressão de c-Myc por IH mostrou uma significância tendência a relacionar-se com tumores não confinados estadiados pT3Introduction: Prostate cancer (PCa) is the most common tumor in men and the second leading cause of cancer death in men in Brazil. MicroRNA (miRNA) is a class of small non-coding RNA that plays a key role in the control of gene expression. They are responsible for the control of key processes in the cell and are involved in tumorigenesis in humans. Previously, we demonstrated alterations in the expression profile of miRNA 100, 218 and let7c comparing localized and metastatic carcinomas. The characterization of expression profiles of their target proteins in PCa is crucial to understanding the processes involved in carcinogenesis, giving us the opportunity to discover new diagnostic or prognostic markers, and most importantly to find new targets for the development of innovative therapies. Objective: To analyze the expression of proteins controlled by miR-let7c (Ras, c- Myc and Bub1), miR-100 (Smarca5 and Retinoblastoma) and miR- 218 (Laminin 5 3) and proliferative activity (Ki-67) in prostate cancer with immunohistochemistry using tissue microarrays representing localized PCa, lymph node and bone metastases. To correlate the expression levels of miRNAs with their target proteins. To analyze the expression of miRNAs, proteins and proliferative activity with prognostic factors of prostate cancer and disease progression. Methods: The immunoexpression of Smarca5, Retinoblastoma, Laminin, Ras, c-Myc, Bub1 and Ki-67 was evaluated by IHC by tissue microarray technique featuring three stages of PCa, with 112 cases of localized PCa, 19 lymph node metastases and 28 bone metastases. The images obtained from IHC were submitted to analysis using the digital image software MacBiophotonics ImageJ from the National Institutes of Health, USA, where the intensity of luminescence was quantified densitometrically. We studied the expression profile of the miRNAs in the paraffin blocks of 61 patients out of the 112 patients with localized carcinoma, who underwent protein analysis by IHC. The processing of miRNA involved three steps: extraction of miRNA, generation of complementary DNA and amplification of the miRNA by quantitative real time PCR (qRT-PCR). To analyze the data we used a control endogenous RNU-43. The results were analyzed using the 2-CT formula. As control, we used the tissue from five patients with benign prostate hyperplasia (BPH) submitted to surgery. The relationship between the expression of miRNAs and their target proteins were analyzed as well as their expression with Gleason score, pathological stage and disease progression considered as PSA>0.4 ng/mL in a mean follow-up of 77.5 months. The statistical analysis was performed using SPSS 19.0 software, we used the Student t test, Mann-Whitney test, Kruskal- Wallis and chi-square. The value was considered statistically significant when p0.05. Results: There was a decrease in the expression of Ras (p=0.017) and Laminin (p<0.0001) according to PCa progression from localized to lymph node and bone metastases. There was an increase in the expression of Retinoblastoma (p=0.0361) and an increase in proliferative activity assessed by Ki-67 (p<0.0001). We also found a relationship between the positivity of c-Myc expression with pT3 staged tumors (p=0.070). All miRNAs showed overexpression in PCa samples. Laminin showed a higher expression together with higher expression of miR-218 (p=0.038). The other miRNAs did not show a relationship with protein expression by IHC. There was no correlation between the expression of miRNAs and protein expression by IHC with biochemical recurrence. Conclusions: Although our findings confirm the overexpression of miR-100, 218 and let7c in localized PCa, there was no correlation between their expression and the protein of their target using immunohistochemistry. We demonstrated that there was a change in immunostatining of Ras, Laminin 5 3, Retinoblastoma and Ki- 67 according to tumor progression. The increased expression of c- Myc per IHC showed a significant tendency to relate to tumor unconfined staged pT3
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Barrezueta, Luis Fernando Mesias [UNIFESP]. "Imuno-expressão das proteínas da família BCL-2 (BCL-2. BCL-XL, BAX, BAK, BAD) em câncer gátrico, preparados em arranjo em matriz (TMA)". Universidade Federal de São Paulo (UNIFESP), 2009. http://repositorio.unifesp.br/handle/11600/9724.
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Previous issue date: 2009-01-28Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Em casos de carcinoma gástrico, para contribuir ao conhecimento do processo de carcinogênese: Objetivo: Estudar a expressão das proteínas da família Bcl-2 (BcI-2, Bcl-xl, Bak, Bad, Bax). Correlacionar a expressão destas proteínas com 0 índice apoptótico mediante a expressão da proteína Caspase 3 clivada, com 0 índice mit6tico mediante a expressão da proteína Ki-67 e com a expressão da proteína p53. Método: Técnica de arranjo em matriz de amostras teciduais (TMA): em 87 amostras de adenocarcinomas gástricos (grupo teste) e de mucosa gástrica não tumoral (grupo controle) foi avaliada a imuno-expressão das proteínas da família BcI-2 (BeI-2, Bcl-xl, Bak, Bad, Bax), da proteína p53, da proteína caspase 3 e da proteína Ki-67. Resultados: Todas as proteínas examinadas foram observadas nos adenocarcinomas e mucosa não tumoral, porem com diferenças de expressão em relação à porcentagem de positividade e intensidade. Observamos: i) Houve associação entre 0 tamanho do tumor e a proteína p53. ii) Houve associação da proteína Bad no adenocarcinoma com a idade dos pacientes. iii) Associação das proteínas Bax, Bad e Ki-67 com 0 adenocarcinoma de tipo intestinal. iv) As proteínas Bcl-xl, Bak, Bad, p53 e Ki-67 apresentaram diferenças estatisticamente significantes entre a imuno-expressão no tumor e na mucosa não tumoral. v) Associação das proteínas Bax, Bak e Bad na mucosa não tumoral. vi) Não houve correlação da imunoexpressão das proteínas com a sobrevida dos pacientes. Conclusão: A expressão aumentada da proteína Bcl-xl nos adenocarcinomas, com evidente diferença de expressão entre 0 grupo teste e 0 grupo controle, esta relacionada com 0 efeito anti-apoptótico da proteína. A expressão reduzida das proteínas Bak e Bad e a expressão aumentada das proteínas p53 e Ki-67 nos adenocarcinomas demonstram 0 desequilíbrio entre morte e proliferação celular, permitindo 0 crescimento descontrolado das células neoplásicas.
Purpose: To study the immunoexpression of Bcl-2 family proteins (Bcl-2, Bcl-xl, Bax, Bak, Bad) and to evaluate the correlation between the immunoexpression of these proteins with the cleaved caspases 3, Ki-67 and p53 immuno-expression. Methods: A TMA paraffin block was constructed with gastric carcinoma tissue (test group) and normal gastric adjoining mucosa (control group) of 87 patients. The TMA block was submitted to immunohistochemistry for Bcl-2, Bcl-xl, Bax, Bak, Bad, p53 and-cleaved Caspase 3. Results: All studied proteins were present in tumor and normal gastric adjoining mucosa, but with different intensity and amount of positive cells. i) There was an association between tumor size and p53 expression. ii) association between Bad expression in the tumor and patient’s age. iii) Intestinal type adenocarcinoma was positively correlated with the expression of Bax, Bad and Ki-67. iv) The protein Bcl-xl, Bak, Bad, p53 and Ki-67 showed statistically significant differences between the immuno-expression in tumor and normal gastric adjoining mucosa. v) There was an association between the proteins Bax, Bak and Bad expression in the normal gastric adjoining mucosa. vi) No correlation between patient’s survival rates and the expression of the proteins was observed. Conclusions: The higher expression of Bcl-xl protein in adenocarcinoma, the difference of Bcl-xl expression between test group and control group, might be related with the anti-apoptotic effect of this protein. The lower expression of Bak and Bad and the increased expression of p53 protein and Ki-67 protein in adenocarcinomas demonstrate the imbalance between death and cellular proliferation, which allows the uncontrolled tumor cell proliferation.
FAPESP: 04/09932-4
FAPESP: 06/54187-0
TEDE
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Hemdan, Tammer. "Prognostic and Predictive Factors in Bladder Cancer". Doctoral thesis, Uppsala universitet, Urologkirurgi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-282607.
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Bladder cancer is a potentially curable malignancy; however in regards to the state of current therapy regimens, a plateau has been reached in both the non-muscle and muscle invasive types. To obtain effective treatment, and consequently a decreased mortality, it has become imperative to test and understand aspects affecting therapy response. The aim of this thesis is to illustrate a better understanding of clinical factors affecting therapy response using new drug combinations and new tumor markers alongside established risk criteria. In Paper I we reported the 5 year follow up from a multicenter, prospectively randomized study and we evaluated the 5-year outcomes of BCG alone compared to a combination of epirubicin and interferon-a2b in the treatment of patients with T1 bladder cancer. Treatment, tumor size and tumor status at second resection were independent variables associated with recurrence. Concomitant Cis was not predictive of failure of BCG therapy. Independent factor for treatment failure was remaining T1 stage at second resection. In Paper II &III we investigated the validity of emmprin, survivin and CCTα proteins as biomarkers for response and survival before neoadjuvant cisplatin chemotherapy. Bladder tumor specimens were obtained before therapy from a total of 250 patients with T1-T4 bladder cancer enrolled in 2 randomized trials comparing neoadjuvant chemotherapy before cystectomy with a surgery only arm. Protein expression was determined by immunohistochemistry (IHC). Patients in the chemotherapy cohort with negative emmprin and CCTα expression had significantly better overall survival (OS) than those with positive expression. In Paper IV primary end point was examining STMN1 as prognostic factor in bladder cancer. Analysis was performed on three bladder cancer patient cohorts using IHC, western blot and a bladder cancer cell line. High levels of STMN1, expression correlated to shorter disease-specific survival and the growth and migration of the cells were significantly reduced when transfecting the cells with STMN1 siRNA. Conclusion Risk assessment and predictors of outcomes could help in individualized treatment and follow up. Biomarkers will become more important for treatment choices in bladder cancer management.
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Gong, Ting. "Computational Dissection of Composite Molecular Signatures and Transcriptional Modules". Diss., Virginia Tech, 2009. http://hdl.handle.net/10919/77302.
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This dissertation aims to develop a latent variable modeling framework with which to analyze gene expression profiling data for computational dissection of molecular signatures and transcriptional modules.
The first part of the dissertation is focused on extracting pure gene expression signals from tissue or cell mixtures. The main goal of gene expression profiling is to identify the pure signatures of different cell types (such as cancer cells, stromal cells and inflammatory cells) and estimate the concentration of each cell type. In order to accomplish this, a new blind source separation method is developed, namely, nonnegative partially independent component analysis (nPICA), for tissue heterogeneity correction (THC). The THC problem is formulated as a constrained optimization problem and solved with a learning algorithm based on geometrical and statistical principles.
The second part of the dissertation sought to identify gene modules from gene expression data to uncover important biological processes in different types of cells. A new gene clustering approach, nonnegative independent component analysis (nICA), is developed for gene module identification. The nICA approach is completed with an information-theoretic procedure for input sample selection and a novel stability analysis approach for proper dimension estimation. Experimental results showed that the gene modules identified by the nICA approach appear to be significantly enriched in functional annotations in terms of gene ontology (GO) categories.
The third part of the dissertation moves from gene module level down to DNA sequence level to identify gene regulatory programs by integrating gene expression data and protein-DNA binding data. A sparse hidden component model is first developed for this problem, taking into account a well-known biological principle, i.e., a gene is most likely regulated by a few regulators. This is followed by the development of a novel computational approach, motif-guided sparse decomposition (mSD), in order to integrate the binding information and gene expression data.
These computational approaches are primarily developed for analyzing high-throughput gene expression profiling data. Nevertheless, the proposed methods should be able to be extended to analyze other types of high-throughput data for biomedical research.Ph. D.
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Rasiah, Krishan Kumar St Vincent's UNSW. "The identification of novel biomarkers in the development and progression of early prostate cancer". Awarded by:University of New South Wales. St Vincent's, 2006. http://handle.unsw.edu.au/1959.4/24187.
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ABSTRACT The morphological premalignant changes in prostate epithelium such as high grade prostatic intraepithelial neoplasia (HGPIN) precede invasive prostate cancer (PC) by several decades. The overall aim of this project was to identify patterns of gene expression in HGPIN and early PC which increase our understanding of the early biology of PC and identify genes and pathways that correlate with an aggressive phenotype. A comprehensive tissue cohort of premalignant prostate lesions was collected in a tissue microarray (TMA) platform that was utilised for high-throughput validation of target genes. Using this unique resource, the expression of the tumour suppressor gene PTEN was assessed using immunohistochemistry in an initial candidate gene approach based on mouse models implicating PTEN in carcinogenesis. No significant difference in expression of PTEN was detected in premalignant and benign epithelium. A transcript profiling approach was undertaken by integrating laser capture microdissection, linear RNA amplification and oligonucleotide microarrays to perform a screen of matched patient samples of normal, HGPIN and PC cells. The expression patterns of two genes encoding secreted proteins, neuropeptide Y (NPY) and macrophage inhibitory cytokine (MIC-1) were validated using immunohistochemistry on TMAs representing the progression model of early PC. Increased expression of these proteins in PC was confirmed to occur early in the disease process and altered expression of NPY and MIC-1 was associated with worse clinical outcome. Further analysis of global gene expression patterns using a structured network knowledge base identified a notable aberration in the expression of extracellular matrix and extracellular matrix associated proteins in HGPIN and provided novel evidence for the role of this class of molecules in the development of PC. In summary, contrary to current dogma based on work in animal models, altered PTEN expression is unlikely to represent an important event in the development of malignancy in the human prostate. In contrast, the expression patterns and prognostic value of NPY and MIC-1 in HGPIN support their further evaluation as biomarkers for the development and progression of PC. The aberrant expression of genes and networks of genes detected in HGPIN will assist in further identification of biological pathways which may be targeted in therapeutic strategies against the development and progression of PC.
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Geiges, Annabella [Verfasser] y Arnulf [Akademischer Betreuer] Stenzl. "Evaluation von Alterationen innerhalb der mTOR-Regulation entlang der Progression zum metastasierten Prostatakarzinom – Immunhistochemische Untersuchungen mittels Tissue-Microarray-Technik / Annabella Christina Geiges ; Betreuer: Arnulf Stenzl". Tübingen : Universitätsbibliothek Tübingen, 2019. http://d-nb.info/1196633770/34.
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Larsson, Karin. "Generation and characterization of antibodies for proteomics research". Doctoral thesis, Stockholm : Skolan för bioteknologi, Kungliga Tekniska högskolan, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-11425.
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Griffith, Obi Lee. "Identification of gene expression changes in human cancer using bioinformatic approaches". Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/689.
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The human genome contains tens of thousands of gene loci which code for an even greater number of protein and RNA products. The highly complex temporal and spatial expression of these genes makes possible all the biological processes of life. Altered gene expression by mutation or deregulation is fundamental for the development of many human diseases. The ultimate aim of this thesis was to identify gene expression changes relevant to cancer. The advent of genome-wide expression profiling techniques, such as microarrays, has provided powerful new tools to identify such changes and researchers are now faced with an explosion of gene expression data. Processing, comparing and integrating these data present major challenges. I approached these challenges by developing and assessing novel methods for cross-platform analysis of expression data, scalable subspace clustering, and curation of experimental gene regulation data from the published literature. I found that combining results from different expression platforms increases reliability of coexpression predictions. However, I also observed that global correlation between platforms was generally low, and few gene pairs reached reasonable thresholds for high-confidence coexpression. Therefore, I developed a novel subspace clustering algorithm, able to identify coexpressed genes in experimental subsets of very large gene expression datasets. Biological assessment against several metrics indicates that this algorithm performs well. I also developed a novel meta-analysis method to identify consistently reported genes from differential expression studies when raw data are unavailable. This method was applied to thyroid cancer, producing a ranked list of significantly over-represented genes. Tissue microarray analysis of some of these candidates and others identified a number of promising biomarkers for diagnostic and prognostic classification of thyroid cancer. Finally, I present ORegAnno (www.oreganno.org), a resource for the community-driven curation of experimentally verified regulatory sequences. This resource has proven a great success with ~30,000 sequences entered from over 900 publications by ~50 contributing users. These data, methods and resources contribute to our overall understanding of gene regulation, gene expression, and the changes that occur in cancer. Such an understanding should help identify new cancer mechanisms, potential treatment targets, and have significant diagnostic and prognostic implications.
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Baneshi, Mohammad Reza. "Statistical models in prognostic modelling with many skewed variables and missing data : a case study in breast cancer". Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/4191.
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Prognostic models have clinical appeal to aid therapeutic decision making. In the UK, the Nottingham Prognostic Index (NPI) has been used, for over two decades, to inform patient management. However, it has been commented that NPI is not capable of identifying a subgroup of patients with a prognosis so good that adjuvant therapy with potential harmful side effects can be withheld safely. Tissue Microarray Analysis (TMA) now makes possible measurement of biological tissue microarray features of frozen biopsies from breast cancer tumours. These give an insight to the biology of tumour and hence could have the potential to enhance prognostic modelling. I therefore wished to investigate whether biomarkers can add value to clinical predictors to provide improved prognostic stratification in terms of Recurrence Free Survival (RFS). However, there are very many biomarkers that could be measured, they usually exhibit skewed distribution and missing values are common. The statistical issues raised are thus number of variables being tested, form of the association, imputation of missing data, and assessment of the stability and internal validity of the model. Therefore the specific aim of this study was to develop and to demonstrate performance of statistical modelling techniques that will be useful in circumstances where there is a surfeit of explanatory variables and missing data; in particular to achieve useful and parsimonious models while guarding against instability and overfitting. I also sought to identify a subgroup of patients with a prognosis so good that a decision can be made to avoid adjuvant therapy. I aimed to provide statistically robust answers to a set of clinical question and develop strategies to be used in such data sets that would be useful and acceptable to clinicians. A unique data set of 401 Estrogen Receptor positive (ER+) tamoxifen treated breast cancer patients with measurement for a large panel of biomarkers (72 in total) was available. Taking a statistical approach, I applied a multi-faceted screening process to select a limited set of potentially informative variables and to detect the appropriate form of the association, followed by multiple imputations of missing data and bootstrapping. In comparison with the NPI, the final joint model derived assigned patients into more appropriate risk groups (14% of recurred and 4% of non-recurred cases). The actuarial 7-year RFS rate for patients in the lowest risk quartile was 95% (95% C.I.: 89%, 100%). To evaluate an alternative approach, biological knowledge was incorporated into the process of model development. Model building began with the use of biological expertise to divide the variables into substantive biomarker sets on the basis of presumed role in the pathway to cancer progression. For each biomarker family, an informative and parsimonious index was generated by combining family variables, to be offered to the final model as intermediate predictor. In comparison with NPI, patients into more appropriate risk groups (21% of recurred and 11% of non-recurred patients). This model identified a low-risk group with 7-year RFS rate at 98% (95% C.I.: 96%, 100%).
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Landman, Maria Luísa de Lima. "Expressão de marcadores imunoistoquímicos em neoplasias melanocíticas de equinos por microarranjo de tecidos". Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/10/10133/tde-13032012-161024/.
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O objetivo deste estudo foi verificar o comportamento morfológico e a expressão das proteínas S-100, Melan-A, HMB-45, Ki-67, PCNA e p53, em 25 neoplasias melanocíticas de equinos. Informações clínicas (gênero, raça, pelagem, idade e localização da lesão) e morfológicas (características celulares, pigmentares, nucleares, de nucléolo, de melanófagos, presença de invasão e necrose) dos animais foram coletadas. Para a expressão das proteínas por imunoistoquímica foi confeccionado um bloco de microarranjo de tecidos das amostras teciduais, juntamente com os controles positivos das reações. A avaliação da expressão das proteínas S100, HMB-45 e Melan-A foi baseada em um escore, e a das proteínas Ki-67, PCNA e p53 foi feita por contagem de células. Animais SRD (16/25, 64%), de raça Lusitana (6/25, 24%), Árabe (2/25, 8%) e Sueca (1/25, 4%) fizeram parte deste estudo, todos tordilhos e a maioria machos (18/25, 72%). A idade dos animais variou de 4 a 24 anos (média de 13 anos). A região perianal (13/25, 52%) foi a que mais apresentou neoplasias. Na análise morfológica houve predomínio de neoplasias com celularidade moderada (52%) e intensa (40%), distribuição difusa e em feixes (52%), ausência de figuras de mitose (96,0%) e predomínio de células epitelióides e fusiformes no mesmo tumor (80%). A atipia nuclear era discreta (48%) e moderada (44%), com núcleos de formato arredondado e alongado em um mesmo tumor (76%) e cromatina dispersa (60%). Os nucléolos eram múltiplos e, em sua maioria, proeminentes (88%). Observou-se predomínio de células tumorais de pigmentação intensa (68%), distribuição difusa e localização em derme (100%). A maioria dos casos apresentou alta celularidade de macrógafos (64%) e distribuição difusa (96%). Quanto à expressão de proteínas para melanócitos por imunoistoquímica, 44% dos casos apresentaram expressão moderada a forte de S100, 56% apresentaram expressão fraca de HMB-45 e 64% apresentaram expressão negativa de Melan-A. Houve positividade de 72% dos casos para pelo menos dois dos anticorpos citados acima. Os anticorpos de proliferação celular Ki-67 e PCNA tiveram média de acima. Os anticorpos de proliferação celular Ki-67 e PCNA tiveram média de positividade de 0,0005% e 15,7%, respectivamente. A análise da expressão de p53 teve média de 6,1% de positividade. Houve associação estatisticamente positiva entre a celularidade dos macrófagos com S100 e com p53. Em conclusão: 1. os dados clínicos obtidos reproduzem o comportamento biológico das neoplasias melanocíticas em equinos, exceto pela idade dos animais; 2. as neoplasias equinas se assemelham a nevos azuis celulares em humanos e melanocitomas em cães; 3. o microarranjo de tecidos mostrou-se uma maneira econômica, rápida e com menos variáveis técnicas; 4. a utilização de um painel de anticorpos de melanócitos é pertinente na diferenciação entre tumores melanocíticos e não melanocíticos, reproduzindo o painel diagnóstico utilizado em literatura humana e canina; 5. o índice de proliferação celular encontrado sugere que os dois anticorpos (Ki-67 e PCNA) podem ser usados na contagem de células em atividade mitótica e 6. a proteína p53 tem maior relação com a parada do ciclo celular que a observada em outros estudos em equinos, podendo indicar um comportamento biológico diferente do apresentado em cães e humanos.The aim of this study was to evaluate the morphological behavior, and expression of the following proteins: S-100, Melan-A, HMB-45, Ki-67, PCNA and p53, in 25 equine melanocytic neoplasms. Clinical (gender, breed, coat color, age and lesions location) and morphological (cellular, pigment, nuclear, nucleoli, melanophages, invasion and necrosis) data were collected. A tissue microarray block, embedded in paraffin, with equine tissue samples and positive controls, was elaborated for protein expression through immunohistochemistry. The evaluation of S100, HMB-45 and Melan-A was based on a score, and for Ki-67, PCNA and p53 it was based on cellular count. Breeds were: Mixed breed (16/25, 64%), Lusitano (6/25, 24%), Arab (2/25, 8%) and Swedich (1/25, 4%). All animals were gray and the majority males (18/25, 72%). Age varied from 4 to 24 years old (mean=13 years). The perianal region (13/25, 52%) was the most common location. Morphological analysis have shown neoplasms with predominantly moderate (52%) and intense (40%) cellularity, diffuse and fascicles distribution (52%), no mitoses figures (96%) and predominance of epithelioid and spindle cells in the same tumor (80%). There was discrete (48%) and moderate (44%) nuclear atypia, round and elongated nucleus in the same tumor (76%), and disperse chromatin (60%). Nucleoli were multiple and prominent in the majority of cases (88%). Tumor cells with diffuse and intense pigmentation, with dermal location (100%) were predominant. High cellularity of macrophages (64%) with diffuse distribution (96%) was mostly seen. The protein expression for melanocytes have shown 44% of moderate to strong expression for S100 protein, 56% of weak expression for HMB-45 protein and 64% of negative expression for Melan-A protein. It was found positivity for more than two antibodies in 72% of equine melanocytic neoplasms. The proliferation antibodies Ki-67 and PCNA had mean positivity of 0,0005% and 15,7%, respectively. The p53 expression had mean positivity of 6,1%. Macrophages cellularity was statistically associated with S100 and p53. In conclusion: 1. clinical data obtain reproduce the biological behavior of equine Macrophages celllularity was statistically associated with S100 and p53. In conclusion: 1. clinical data obtain reproduce the biological behavior of equine melanocytic neoplasms, excepting the animals age; 2. equine melanocytic neoplasms assemble to human cellular blue nevi and dogs melanocytoma; 3. the tissue microarray was shown to be an economic, rapid and less variable technique; 4. using a panel for antibodies for melanocytes is relevant to differentiate melanocytic and not melanocytic tumors, reproducing the diagnosis panel used in human and canine literature; 5. the proliferation index found suggests that both antibodies (Ki-67 and PCNA) could be used in mitotic activity cell count; and 6. p53 protein has more relation with cellular cycle stop than in other equine studies, probably indicating a different biological behavior than the presented in humans and dogs.
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Lindholm, Andreas. "Ezrin som prognosmarkör för rektalcancer". Thesis, Malmö högskola, Fakulteten för hälsa och samhälle (HS), 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:mau:diva-26644.
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Rektalcancer drabbar cirka 2000 personer i Sverige per år. Trots förbättringar i preoperativ utredning och behandling, utgör fortfarande lokalrecidiv (lokalt återfall i bäckenet) ett allvarligt problem. I dagsläget överlever inte majoriteten av patienterna som får ett lokalrecidiv. För närvarande finns det inga kliniskt introducerade prognostiska vävnadsmarkörer för risken att utveckla ett lokalrecidiv. Identifieringen av en sådan markör skulle kunna leda till att en individuell behandlingsplan skapas för patienten, både primärt och postoperativt samt för uppföljning. Patienter med högre risk att utveckla lokalrecidiv skulle identifieras i ett tidigare skede. Proteinet ezrin har rapporterats vara en potentiell tumörmarkör vid olika cancerformer. Association mellan ett högt uttryck av ezrin och dålig prognos har påvisats. Syftet med studien var att undersöka om ezrin är en prognosmarkör för rektalcancer. Därigenom skulle eventuellt en förbättrad individbaserad behandlingsplan kunna skapas. Detta genom framställning av vävnadsklotsar med tekniken tissue microarray för att därefter utföra immunhistokemisk färgning för detektion av förekomsten av ezrin. Patienterna utgör en kontrollgrupp omfattande patienter som inte har utvecklat lokalrecidiv. I en case-only studie av Jörgren et al. (2010), med patienter som utvecklat lokalrecidiv fem år från primäroperation, visades att ezrin skulle kunna vara användbar som en prognosmarkör för rektalcancer. I 19,4 % (59/304) bedömdes färgintensiteten i vävnadscytoplasman som svag, som måttlig i 56,9 % (173/304) och som intensiv i 23,7 % (72/304). Intensiteten av cytoplasmafärgningen för ezrin ska analyseras multivariat och dess eventuella prognostiska betydelse värderas.Yearly, about 2000 persons in Sweden are diagnosed with rectal cancer. Although advances have been made in tumour detection and its treatment, local recurrence still represents a severe matter. A majority of the patients diagnosed with local recurrence will not survive. There are no clinically available prognostic tissue markers for rectal cancer patients concerning the risk of local recurrence at the moment. The identification of a prognostic marker could lead to the development of an individualized treatment plan for the patient, both primarily and postoperatively as well as in follow-up. Several published papers have shown that the protein ezrin could be a potential tumour marker in various tumours. Association between high ezrin expression and poor prognosis has been demonstrated. The aim of this study was to further explore if ezrin may function as a prognostic marker for rectal. This by manufacturing tissue blocks utilizing the technique of tissue microarray that were stained immunohistochemically for ezrin. By analysing the expression of ezrin in the tumour cell cytoplasms microscopically by determining the colour intensity. Patients in the study form a control group consisting of only patients that did not develop a local recurrence. The ezrin expression of the control group will be compared to the results from the study of Jörgren et al. (2010) on rectal cancer patients who developed local recurrence within 5 years of surgery, where it was shown that ezrin expression could be used as a prognostic marker for rectal cancer. In 19,4% (59/304) the expression intensity of the tumour cells cytoplasm was assessed as weak, as moderate in 56,9% (173/304) and as intense in 23,7% (72/304). The intensity of the ezrin expression will be analysed multivariately, and the prognostic value further evaluated.
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Brentel, Sahra. "Protokollutveckling för caldesmon samt en jämförelse med SMMS-1 av dess effektivitet som en myoepitelial markör på fibroadenom". Thesis, Malmö högskola, Fakulteten för hälsa och samhälle (HS), 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:mau:diva-26142.
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Immunohistokemi (IHK) är en metod som används för att undersöka närvaron av specifika biomarkörer i vävnad. Metoden utnyttjar antikroppars affinitet till antigen och dess närvaro visualiseras. Caldesmon är ett protein associerat med cytoskelettet som förekommer i glatta muskelceller och i icke-muskelceller. Det används inom IHK som en glatt muskelmarkör men även som en markör för myoepiteliet. Syftet med studien var att utveckla ett nytt protokoll för caldesmon och att jämföra den med SMMS-1, även den en markör för glatt muskulatur och myoepiteliet. Jämförelsen utfördes på fibroadenom vävnad, en bröstcancerform som utvecklats ur epitelialvävnad och stroma, då den innehåller mycket myoepitelial celler. För att effektivisera processen användes tissue microarray tekniken, där vävnad från flera olika preparat (klotsar) stansades ut och sammanfördes till en ny klots. Instrumentet Ventana Benchmark Ultra användes för att utveckla ett nytt protokoll för caldesmon. Det nya protokollet användes sedan vid jämförelsen med SMMS-1. Resultatet visade att SMMS-1 gav en tydligare infärgning av det myoepiteliala lagret då det enbart färgade in det, medan caldesmon även färgade in stroma. Då fibroadenom vävnad innehöll mycket stroma var det svårare att urskilja det myoepiteliala lagret från dess omgivning med caldesmon infärgningen.Immunohistochemistry (IHC), is a method used for examining the presence of specific biomarkers in tissue. It uses antibodies affinity for antigens and their presence can then be visualized. Caldesmon is a protein that is associated with the cytoskeleton in smooth muscle and non-smooth muscle cells. It is used within IHC as a marker for smooth muscle but also as a marker for the myoepithelium. The intent of this study was to develop a new protocol for caldesmon and to compare it with SMMS-1, also a marker for smooth muscle and the myoepithelium. The comparison was performed on tissue from fibroadenoma, a type of breast cancer that evolved from epithelial tissue and stroma, due to its high content of myoepithelial cells. For optimal effectiveness, the tissue microarray technique was used, where tissue from several blocks are extracted and brought together into a single new block. A Ventana Benchmark Ultra was used to develop a new protocol for the new antibody, caldesmon. The new protocol was used in the comparison with SMMS-1 on fibroadenoma tissue. The results were that SMMS-1 had a clearer and sharper staining of the myoepithelial layer, due to the fact that it only stained the layer. While caldesmon also stained the stroma in the tissue. Because fibroadenom tissue contained so much stroma the staining done with caldesmon made it harder to separate the myoepithelial layer from its surroundings.
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Boeuf, Stéphane. "Comparative study of gene expression during the differentiation of white and brown preadipocytes". Phd thesis, Universität Potsdam, 2002. http://opus.kobv.de/ubp/volltexte/2005/51/.
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EinleitungSäugetiere haben zwei verschiedene Arten von Fettgewebe: das weiße Fettgewebe, welches vorwiegend zur Lipidspeicherung dient, und das braune Fettgewebe, welches sich durch seine Fähigkeit zur zitterfreien Thermogenese auszeichnet. Weiße und braune Adipozyten sind beide mesodermalen Ursprungs. Die Mechanismen, die zur Entwicklung von Vorläuferzellen in den weißen oder braunen Fettzellphenotyp führen, sind jedoch unbekannt. Durch verschiedene experimentelle Ansätze konnte gezeigt werden, daß diese Adipocyten vermutlich durch die Differenzierung zweier Typen unterschiedlicher Vorläuferzellen entstehen: weiße und braune Preadipozyten. Von dieser Hypothese ausgehend, war das Ziel dieser Studie, die Genexpression weißer und brauner Preadipozyten auf Unterschiede systematisch zu analysieren.
Methoden
Die zu vergleichenden Zellen wurden aus primären Zellkulturen weißer und brauner Preadipozyten des dsungarischen Zwerghamsters gewonnen. „Representational Difference Analysis“ wurde angewandt, um potentiell unterschiedlich exprimierte Gene zu isolieren. Die daraus resultierenden cDNA Fragmente von Kandidatengenen wurden mit Hilfe der Microarraytechnik untersucht. Die Expression dieser Gene wurde in braunen und weißen Fettzellen in verschiedenen Differenzierungsstadien und in braunem und weißem Fettgewebe verglichen.
Ergebnisse
12 Gene, die in braunen und weißen Preadipozyten unterschiedlich exprimiert werden, konnten identifiziert werden. Drei Komplement Faktoren und eine Fettsäuren Desaturase werden in weißen Preadipozyten höher exprimiert; drei Struktur Gene (Fibronectin, Metargidin und a Actinin 4), drei Gene verbunden mit transkriptioneller Regulation (Necdin, Vigilin und das „small nuclear ribonucleoprotein polypeptide A“) sowie zwei Gene unbekannter Funktion werden in braunen Preadipozyten höher exprimiert. Mittels Clusteranalyse (oder Gruppenanalyse) wurden die gesamten Genexpressionsdaten charakterisiert. Dabei konnten die Gene in 4 typischen Expressionsmuster aufgeteilt werden: in weißen Preadipozyten höher exprimierte Gene, in braunen Preadipozyten höher exprimierte Gene, während der Differenzierung herunter regulierte Gene und während der Differenzierung hoch regulierte Gene.
Schlußfolgerungen
In dieser Studie konnte gezeigt werden, daß weiße und braune Preadipozyten aufgrund der Expression verschiedener Gene unterschieden werden können. Es wurden mehrere Kandidatengene zur Bestimmung weißer und brauner Preadipozyten identifiziert. Außerdem geht aus den Genexpressionsdaten hervor, daß funktionell unterschiedliche Gruppen von Genen eine wichtige Rolle bei der Differenzierung von weißen und braunen Preadipozyten spielen könnten, wie z.B. Gene des Komplementsystems und der extrazellulären Matrix.
Introduction
Mammals have two types of adipose tissue: the lipid storing white adipose tissue and the brown adipose tissue characterised by its capacity for non-shivering thermogenesis. White and brown adipocytes have the same origin in mesodermal stem cells. Yet nothing is known so far about the commitment of precursor cells to the white and brown adipose lineage. Several experimental approaches indicate that they originate from the differentiation of two distinct types of precursor cells, white and brown preadipocytes. Based on this hypothesis, the aim of this study was to analyse the gene expression of white and brown preadipocytes in a systematic approach.
Experimental approach
The white and brown preadipocytes to compare were obtained from primary cell cultures of preadipocytes from the Djungarian dwarf hamster. Representational difference analysis was used to isolate genes potentially differentially expressed between the two cell types. The thus obtained cDNA libraries were spotted on microarrays for a large scale gene expression analysis in cultured preadipocytes and adipocytes and in tissue samples.
Results
4 genes with higher expression in white preadipocytes (3 members of the complement system and a fatty acid desaturase) and 8 with higher expression in brown preadipocytes were identified. From the latter 3 coded for structural proteins (fibronectin, metargidin and a actinin 4), 3 for proteins involved in transcriptional regulation (necdin, vigilin and the small nuclear ribonucleoprotein polypeptide A) and 2 are of unknown function. Cluster analysis was applied to the gene expression data in order to characterise them and led to the identification of four major typical expression profiles: genes up-regulated during differentiation, genes down-regulated during differentiation, genes higher expressed in white preadipocytes and genes higher expressed in brown preadipocytes.
Conclusion
This study shows that white and brown preadipocytes can be distinguished by different expression levels of several genes. These results draw attention to interesting candidate genes for the determination of white and brown preadipocytes (necdin, vigilin and others) and furthermore indicate that potential importance of several functional groups in the differentiation of white and brown preadipocytes, mainly the complement system and extracellular matrix.
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Lundberg, Emma. "Bioimaging for analysis of protein expression in cells and tissues using affinity reagents". Doctoral thesis, Stockholm : School of biotechnology, Royal institute of technology, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4862.
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