Dissertations / Theses on the topic 'Immunohistochemistry - Diagnosis'

Primary scalp alopecia is classically divided into cicatricial (scarring) and non-cicatricial (non-scarring). Challenging cases are assessed with a scalp biopsy. The use of both horizontal and vertical sections (HoVert sections) has dramatically improved the accuracy of histopathological diagnosis. In this work, we have used immunostaining to address diagnostic difficulties, which persist despite all currently available tools. We performed an immunostain panel (CD3, CD4, CD8 and CD20) in order to distinguish pattern hair loss from alopecia aerate in cases which do not have the usual peribulbar lymphocytic infiltrate and showed that CD3+ T-lymphocytes within the empty fibrous follicular tracts favor a diagnosis of alopecia areata. We performed CD123 in order to distinguish lichen planopilaris from alopecia lupus erythematosus in cases with only a superficial lymphocytic infiltrate and an uninvolved interfollicular epidermis and showed that clusters of CD123+ plasmacytoid dendritic cells favor a diagnosis of lupus erythematosus. We performed cytokeratin 15 in order to assess whether the loss of the follicular bulge stem cells has diagnostic value in cicatricial alopecia and demonstrated that the loss of cytokeratin 15+ bulge stem cells is identified in lichen planopilaris, frontal fibrosing alopecia, and lupus erythematous, so cytokeratin 15 has no diagnostic value. We have attempted to integrate the new concepts and our findings into the traditional classifications of alopecia and proposed a new diagnostic algorithm. In conclusion, immunostaining combined with HoVert grossing advances the accuracy of histopathological diagnosis of primary scalp alopecia.
L’alopécie primitive du cuir chevelu est habituellement classée en cicatricielle et non-cicatricielle. Dans les cas difficiles, la biopsie du cuir chevelu peut aider au diagnostic. L’utilisation de coupes, à la fois verticales et horizontales sur le même spécimen (technique HoVert), a radicalement amélioré le diagnostic histopathologique. Dans ce travail, nous avons utilisé l’immunohistochimie pour évaluer les difficultés diagnostiques qui persistent malgré tous les outils actuels. Nous avons utilisé les CD3, CD4, CD8 et CD20 pour différencier l’alopécie androgénique de la pelade dépourvue de l’infiltrat lymphocytaire péribulbaire habituel et nous avons démontré que la présence de lymphocytes CD3+ dans les travées folliculaires fibreuses est en faveur de la pelade. Nous avons utilisé le CD123 pour différencier le lichen plan pilaire du lupus érythémateux alopécie avec infiltrat lymphocytaire superficiel et sans atteinte de l’épiderme interfolliculaire et nous avons démontré que la présence d’amas de cellules dendritiques plasmacytoïdes CD123+ est en faveur du lupus érythémateux. Nous avons utilisé la cytokératine 15 pour évaluer si la perte des cellules souches du bulge a une valeur diagnostique dans l’alopécie cicatricielle et nous avons démontré que cette perte s’observait de manière identique dans le lichen plan pilaire, l’alopécie frontale fibrosante comme dans le lupus érythémateux et n’avait donc aucune valeur diagnostique. Nous avons tenté d’intégrer les nouveaux concepts et nos données dans les classifications traditionnelles des alopécies et nous avons élaboré un nouvel algorithme diagnostique. L’association des immunomarquages avec la technique HoVert ouvre de nouvelles perspectives dans le diagnostic histopathologique des alopécies primaires du cuir chevelu.
Doctorat en Sciences médicales (Médecine)
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Background: Autoimmune bullous dermatoses present with overlapping clinical features that require histopathological correlation. Immunofluorescence is the most routinely used reliable investigation for diagnosis but requires specialised equipment and is technically sophisticated. Collagen IV immunohistochemistry is reported as a reliable test for the diagnosis of epidermolysis bullosa acquisita whereby It stains the roof of a subepidermal blister and would be expected on the floor in bullous pemphigoid. This technique could be performed as an easily accessible alternative to direct immunofluorescence and has been used anecdotally at our hospital. Aim: To investigate whether collagen IV immunohistochemistry can be used as a reliable histopathological confirmation of bullous pemphigoid. Methods: Two major investigations: 1. A systematic literature search was undertaken of all studies describing the use of collagen IV immunohistochemistry and those comparing it with immunofluorescence in the diagnosis of bullous pemphigoid. 2. A retrospective study of patients diagnosed with bullous pemphigoid over 12 years seen at Groote Schuur Hospital was performed. Patient records that had results for both direct immunofluorescence and collagen IV immunohistochemistry were selected. The positive percentage agreement was calculated. Results: 1. Two studies were found that investigated the use of collagen IV immunohistochemistry in bullous pemphigoid. All reported 33 (100%) cases demonstrated collagen IV at the floor of a subepidermal blister. Of these, 25/25 cases were in agreement with direct immunofluorescence and 7/8 with indirect immunofluorescence which were used as reference standard investigations. 2. In this study, collagen IV was positive in 96% (79/82) of cases and direct immunofluorescence was positive in 85% (72/82) of cases. A positive percentage agreement of 80.5% suggested a strongly positive test accordance. Limitations: 1. The literature search was limited to articles written in english only. 2. The retrospective design and the lack of controls without bullous pemphigoid made it impossible to calculate sensitivity and specificity as well as the kappa statistic. Conclusion: Collagen IV immunohistochemistry is a valid, simple and widely available test which demonstrates accordance with routinely used direct immunofluorescence in the confirmation of bullous pemphigoid. Through clinical and histomorphological correlation, it may be a useful test in resourcelimited settings without facilities for direct immunofluorescence. However, larger controlled studies are warranted to confirm this.

This work describes 5 groups of tumours seen in routine neuropathological practice in Southampton between 1980 and 1986. The clinical and light microscopic findings in a total of 44 tumours are described. A panel of monoclonal and polyclonal antibodies was used in immunohistochemical studies on each group, and this made important contributions to the diagnosis and characterisation of these tumours. Six primary central nervous system lymphomas were shown to be 5 follicle centre cell lymphomas (Kiel-classification) and 1 T-cell lymphoma. 15 lymphomas causing spinal cord compression were typed as 11 follicle centre cell lymphomas, 3 T-cell lymphomas and 1 lymphoblastic lymphoma. T-cell lymphomas appear to be more likely to be localised in the spine and to have a better prognosis. In all these tumours admixed reactive cell populations were also identified immunohistochemically. Ten primitive neuroectodermal tumours of the cerebrum were examined and immunohistochemistry assisted in their distinction from lymphoma and metastatic carcinoma. It also showed evidence of differentiation in apparently poorly differentiated parts of the tumours. Immunohistochemical studies facilitated the distinction of pleomorphic xanthoastrocytoma from a monstrocellular astrocytoma and a meningeal malignant fibrous histiocytoma. The recognition of this first entity has important prognostic implications. In 10 cerebellar haemangioblastomas a panel of antibodies was used to investigate the histogenesis of the stromal cell component of these tumours. Endothelial and stromal cells were found to be antigenically distinct and neurone specific enolase activity was found in the latter. The implications of this finding are discussed. The studies confirm the important and increasing role played by immunohistochemistry in our understanding of central nervous system neoplasia.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
A leishmaniose cutÃnea (LC) à a forma mais comum da leishmaniose, com 1 milhÃo de novos casos por ano. Apresenta um amplo espectro clÃnico, podendo ser confundida com outras doenÃas que acometem a pele. Desse modo, à de fundamental importÃncia o diagnÃstico rÃpido e preciso da doenÃa. O diagnÃstico parasitolÃgico à o mais utilizado na rotina laboratorial, entretanto o exame direto geralmente apresenta baixa sensibilidade. Em virtude disso, torna-se necessÃrio buscar tÃcnicas alternativas para o diagnÃstico das leishmanioses. Neste trabalho, comparou-se o teste do press imprint smear com o exame histolÃgico e a imuno-histoquÃmica, no diagnÃstico direto de leishmaniose cutÃnea. Pacientes com suspeita clinica de LC foram submetidos à coleta de duas biÃpsias da borda das lesÃes, com punch de 3 mm, para a realizaÃÃo dos testes: press imprint smear, exame histolÃgico e imuno-histoquÃmica. Este Ãltimo foi considerado o teste padrÃo ouro. O press imprint smear consistiu em esmagar o fragmento de biÃpsia entre duas lÃminas de vidro sob pressÃo, de tal modo que o extrato (suco) tissular se espalha na superfÃcie das duas lÃminas. Os esfregaÃos foram corados com Giemsa, para pesquisa de amastigotas. Outro fragmento foi fixado em formalina a 10%. Desta amostra, uma lÃmina foi utilizada no exame histolÃgico, corada com hematoxilina-eosina, e outra para o teste de imuno-histoquÃmica. Ao total, foram incluÃdos 78 pacientes com diagnÃstico clinico-epidemiolÃgico de LC. A tÃcnica do press imprint smear e o exame histolÃgico foram realizados na amostra de todos os pacientes. Entretanto, a imuno-histoquÃmica foi realizada em apenas 73 amostras. Setenta e dois (98,6%) foram positivos para Leishmania na imuno-histoquÃmica, com sensibilidade de 98,6%; o press imprint smear foi positivo em 48 pacientes (61,5%), com sensibilidade de 58,3%. No exame histolÃgico foram identificadas amastigotas em 35 pacientes (44,9%), apresentando sensibilidade de 45,8%. Os resultados mostraram que o press imprint smear apresentou maior sensibilidade do que o exame histolÃgico, para o diagnÃstico da leishmaniose cutÃnea. AlÃm disso, à uma tÃcnica de baixo custo e fÃcil execuÃÃo.
The cutaneous leishmaniasis (CL) is the most common form of leishmaniasis, with 1 million new cases per year. Presents a broad clinical spectrum, which may be confused with other diseases that affect the skin. Thus, it is of fundamental importance to rapid and accurate diagnosis of the disease. Parasitological diagnosis is the most used test in laboratory routine, however direct examination generally has low sensitivity. As a result, it becomes necessary to find alternative techniques for the diagnosis of leishmaniasis. This study compared the test press imprint smear with histological examination and immunohistochemistry, for the direct diagnosis of leishmaniasis. Patients with clinical suspicion of LC underwent a sampling from two samples of biopsies from the edge of the lesions, with a punch of 3 mm for the tests: press imprint smear, histological examination and immunohistochemistry. The latter was considered the gold standard test. The press imprint smear consisted in crushing the biopsy fragment between two glass plates under pressure so that the extract (juice) tissue spreads on the surface of the two blades. The smears were stained with Giemsa for the detection of amastigotes. Another fragment was fixed in 10% formalin. From this sample, a blade was used in histology, stained with hematoxylin-eosin, and one for the immunohistochemical test. In total, 78 patients were included with clinical and epidemiological diagnosis of LC. The press imprint smear technique and histologic examination were conducted on the sample of all patients. However, immunohistochemistry was performed in only 73 samples. Seventy-two (98,6%) were positive for leishmania in immunohistochemistry, with sensitivity of 98,6%; the press imprint smear was positive in 48 patients (61,5%), with sensitivity of 58,3%. In the histological examination were identified amastigotes in 35 patients (44,9%), with sensitivity of 45,8%. The press imprint smear was more sensitive than histological examination for the diagnosis of cutaneous leishmaniasis. In addition, a technique is inexpensive and easy to perform.

>Magister Scientiae - MSc
Cancer is a disease most often associated with poor prognosis. During the development of the disease, cells acquire genetic mutations which result in changes in bio-molecules (DNA and protein), thus altering normal functioning of cells. These bio-molecules can thus serve as biomarkers for the diagnosis of cancer and can also facilitate the early detection of cancer. Antibodies labelled with organic fluorophores are typically used in immunohistochemistry techniques to screen cancerous tissue for the presence of biomarkers. More recently, researchers started to use cancer specific peptides (e.g LYP-1, RGD,) rather than antibodies for this purpose. Advantages of peptides include high affinity to their binding target, rapid accumulation at target sites and the ability to evade the immune system. Fluorescent nanocrystals or quantum dots are emerging as nanoparticles that can replace organic fluorophores. Several properties of quantum dots make these nanoparticles an ideal application in the detection of cancer related biomarkers. These include size tunable fluorescence emission, resistance to photobleaching as well as high quantum yields that result in bright emission of fluorescence. The aim of this research project was to investigate the specific binding of selected peptides to cancer cells using functionalized quantum dots. Since the cost of synthetic peptides are so high, the aim of this study was also to express these peptides in E.coli bacterial cells. Cancer targeting peptides were identified from literature and oligonucleotides with sequences encoding these peptides were designed. Four oligonucleotides encoding the peptides p6.1, p.L, MV and NL1.1 were successfully cloned using the pET21b plasmid vector. However, the peptides were not successfully expressed in E.coli. Cancer targeting peptides namely p.C, p.H, p.L, p6.1 and Frop-1 were chemically synthesized and obtained from GL biochem (Shanghai). These peptides were conjugated to quantum dots (Qdot 525) using 1-ethyl-3-(3-dimethylamino) carbodiimide HCl (EDC) chemistry. The peptidequantum dot conjugates were applied to cancer cells to achieve specific binding. The Kmst-6 noncancerous cell line served as a control. The binding of the peptide-quantum dot conjugates was analyzed using flow cytometry and fluorescence microscopy. The p.H peptide revealed the highest binding affinity to cancer cells as indicated by fluorescence intensity. This was followed by the p.C peptide which showed differential binding amongst the cancer cell lines. The Frop-1 peptide displayed the lowest binding affinity, while the binding affinity of the peptides to Kmst-6 cell lines was very low. This study demonstrated that the cancer targeting peptides used in this study bind to cancer cells and that the specificity with which these peptides bind to the cells depends on the cell types and the peptide

The amyloidoses are biochemically heterogeneous diseases with patholophysiologic deposits of various proteins. Amyloid deposits can occur either localized to one organ or tissue or as part of a systemic disease with deposits in many different tissue. The clinical course, prognosis and therapy are different for each type of amyloidosis and therefore a type specific diagnosis is demanded as early as possible. We describe a method for typing of the most common systemic amyloidoses based on Western blot analysis combined with specific

in- house antibodies, using subcutaneous fat biopsies. We found that the method is reliable and easy to perform and the tissue sample needed is obtained by minor surgery.

In the aortic intima amyloid deposits are often associated with atherosclerosis plaques. In our study we also investigated the prevalence of intimal amyloid from 10 patients age 58-94, amyloid deposits were present in 50% of the cases.

Amyloidos är ett sjukdomstillstånd där proteiner som normalt är lösliga i kroppen felveckas och formar långa olösliga fibriller som ansamlas i vävnader och organ såsom t.ex. hjärta, hjärna och lever. Det finns cirka 25 proteiner som kan ge upphov till amyloidos. Man kan skilja på två huvudgrupper av amyloidos, systemisk och lokaliserad. Vid lokal amyloidos kan inlagringar förekomma i specifika vävnader vid framför allt vissa åldersberoende sjukdomar som t.ex. Alzheimers sjukdom. Vid systemisk amyloidos förekommer inlagringar i praktiskt taget alla vävnader. Symtomatologin vid systemisk amyloidos är variabel och sjukdomsbilden kan vara svårtolkad men tidig och specifik diagnostik ger möjlighet till riktad terapi mot den bakomliggande sjukdomen. Syftet med denna studie var att utvärdera en Western blot metod som använts för typning av vanligaste formerna av systemisk amyloidos. De slutsatser som nåtts är att denna metod är snabbt, pålitligt och enkel att utföra. Diagnos erhölls med finnålsbiopsi av bukfettvävnad som är enkel, snabb och billig metod med liten risk för patienternas hälsa. Vi lyckades också med hjälp av immunhistokemisk infärgning titta på prevalens av amyloid i aortas intima.

In 2012, more than 1.6 million new cases of breast cancer were diagnosed and about half a million women died of breast cancer. The incidence has increased in the developing world. The mortality, however, has decreased. This is thought to partly be the result of advances in diagnosis and treatment. Studying tissue samples from biopsies through a microscope is an important part of diagnosing breast cancer. Recent techniques include camera-equipped microscopes and whole slide scanning systems that allow for digital high-throughput scanning of tissue samples. The introduction of digital pathology has simplified parts of the analysis, but manual interpretation of tissue slides is still labor intensive and costly, and involves the risk for human errors and inconsistency. Digital image analysis has been proposed as an alternative approach that can assist the pathologist in making an accurate diagnosis by providing additional automatic, fast and reproducible analyses. This thesis addresses the automation of conventional analyses of tissue, stained for biomarkers specific for the diagnosis of breast cancer, with the purpose of complementing the role of the pathologist. In order to quantify biomarker expression, extraction and classification of sub-cellular structures are needed. This thesis presents a method that allows for robust and fast segmentation of cell nuclei meeting the need for methods that are accurate despite large biological variations and variations in staining. The method is inspired by sparse coding and is based on dictionaries of local image patches. It is implemented in a tool for quantifying biomarker expression of various sub-cellular structures in whole slide images. Also presented are two methods for classifying the sub-cellular localization of staining patterns, in an attempt to automate the validation of antibody specificity, an important task within the process of antibody generation.  In addition, this thesis explores methods for evaluation of multimodal data. Algorithms for registering consecutive tissue sections stained for different biomarkers are evaluated, both in terms of registration accuracy and deformation of local structures. A novel region-growing segmentation method for multimodal data is also presented. In conclusion, this thesis presents computerized image analysis methods and tools of potential value for digital pathology applications.

Hirschsprung’s disease is a common pathology in pediatric surgery. Besides, long-term outcome of surgically-treated patients remains a crucial issue. The management of Hirschsprung’s disease has remarkably advanced over the years, but difficulties persist particularly in the developing countries (such as Vietnam), where essential diagnostic procedures, such as preoperative histopathological exploration techniques/ facilities (mainly for acetylcholinesterase staining), or adequate postoperative management and follow-up requirements are unavailable.We, therefore, contemplated to work-out a relevant histo-diagnostic approach to overcome these constraints that limit our diagnostic approaches, namely, in Vietnam, and we introduced a “less-demanding” diagnostic approach, namely calretinin immunohistochemical staining which is known to be adequate for formalin-fixed tissues (and thus not necessitating frozen section equipment). We thus used calretinin immunohistochemistry in a prospective study on a large cohort of Vietnamese HD cases. Results showed that rectal suction biopsy using calretinin immunohistochemistry provides an effective histopathological diagnostic tool that can replace AChE and provides a valuable evaluating approach for both preoperative and postoperative management.In addition, we also studied long-term outcome in operated patients and impact of postoperative morbidities on their quality of life. Indeed, a long-term multidisciplinary management with dedicated procedures such as anorectal manometry is essentially required for patients with severe defecation disorders.
Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)
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Orientadores: Patricia Sabino de Matos, Konradin Metze
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: O diagnóstico diferencial entre algumas lesões da tireóide é, muitas vezes, difícil de ser determinado, mesmo diante de diversas amostras de tecido. O propósito deste estudo foi o de investigar até que ponto a análise de textura da cromatina em cortes histológicos de rotina contribui para o diagnóstico diferencial entre lesões foliculares da glândula tireóide. Entraram no estudo cortes histológicos corados com hematoxilina-eosina de 12 nódulos adenomatosos hiperplásicos (NA), 18 adenomas foliculares (AF), 24 carcinomas foliculares mínimamente invasores (CFMI) e 22 variantes foliculares do carcinoma papilífero (VFCP). As amostras de tecido utilizadas foram analisadas morfologicamente por meio de estudo histológico e estudo imunoistoquímico pela expressão de diferentes proteínas ou marcadores. Os marcadores utilizados foram HBME-1, CK-19 e galectina-3. Em cada caso 100 núcleos foram aleatoriamente escolhidos, digitalizados e segmentados. A estrutura dos núcleos foi descrita por morfometria, densitometria e por variáveis derivadas da matriz de co-ocorrência. Sexo, idade do paciente e metástases foram informações pesquisadas. Os resultados da análise de imagem computadorizada foram comparados com aqueles obtidos por meio de estudo imunoistoquímico, na distinção das lesões foliculares benignas e malignas da tireóide. Os dados foram comparados por análise de variância (nível de significância p<0,05). As amostras sugerem que o HBME-1 foi um marcador mais sensível quanto à malignidade. Entretanto, o uso dos três marcadores, num painel, pode ser útil em casos específicos, como por exemplo, na diferenciação de lesões foliculares, com referência especial à variante folicular do carcinoma papilífero. Dados descritos como fatores prognósticos, nas lesões foliculares malignas, não se mostraram significantes para discriminar o carcinoma folicular da variante folicular do carcinoma papilífero. As variáveis de morfometria não apresentaram grande valor discriminante, exceto, pelo P5FF4 (quinto percentil do fator de forma) que estabeleceu diferenças significantes entre AF e VFCP e entre CFMI e VFCP. Variáveis densitométricas demonstraram diferenças significantes entre NA e AF, NA e CFMI, AF e VFCP, AF e CFMI e entre CFMI e VFCP. Variáveis derivadas da matriz de co-ocorrência demonstraram diferenças entre os subgrupos, mas somente o P5Energy (quinto percentil da variável ?energia?), demonstrou diferença entre AF e CFMI. Ainda a variável r2Mink (grau de aproximação da dimensão fractal de Minkowski) diferenciou lesões benignas de malignas (NA e CFMI, NA e VFCP, AF e CFMI, AF e VFCP). A análise de textura pôde auxiliar na discriminação entre lesões benignas e malignas da tireóide principalmente quando se tratava da discriminação entre NA ou AF e VFCP, na qual a análise histológica, baseada em características nucleares pode trazer grandes dificuldades. Por outro lado, o diagnóstico entre AF e CFMI é difícil e não se baseia em características nucleares, mas no achado de invasão capsular ou vascular. Entretanto, nestes casos, as variáveis 'energia' e r2Mink, contribuíram para o diagnóstico diferencial. Portanto, a análise de imagem computadorizada pode ser considerada como uma ferramenta que auxilia no diagnóstico diferencial de lesões foliculares da tireóide, somando-se à interpretação morfológica e imunoistoquímica
Abstract: The differential diagnosis between some thyroid lesions is often difficult to determine, even with permanent sections. The purpose of this study was to find out if chromatin texture analysis contributes to the differential diagnosis of thyroid follicular lesions. We selected slides stained with H-E (hematoxilin-eosin) of 12 hyperplastic adenomatous nodules (AN), 18 follicular adenomas (FA), 24 minimally invasive follicular carcinomas (MIFC) and 22 follicular variants of papillary carcinomas (FVPC). A total of 100 nuclei of each case were digitalized and segmented. The utility of a combination of immunostaining including galectin-3, HBME-1 and CK-19 in the routine differentiation of the histologies of thyroid malignancies was evaluated. The nuclei structure was described by morfometry, densitometry and by variables derived by co-occurrence matrix. Sex, age of the patient and metastasis were clinical and morphologic parameters searched in this study. These data, described as prognostic factors for follicular malignant tumors, were compared by variance analysis (significance value p<0,05) and did not differentiate significantly among the groups. The best marker, in terms of sensitivity and specificity, was HBME-1. The combination CK-19, HBME-1 and galectin-3, in a panel, may be useful in specific cases, for example, for differentiate folicullar lesions, with special reference for folicullar variant of papillary carcinoma. Morphometric variables did not show significant discriminant value, except, the P5FF4 (5th percentile of the form factor) which demonstrated significant differences between FA and FVPC and between MIFC and FVPC. Densitometric variables demonstrated significant differences between AN and FA, AN and MIFC, FA and FVPC, FA and MIFC and even between MIFC and FVPC. The variables derived from co-occurrence matrix demonstrated differences among the sub- groups, but only P5 Energy (fifth percent of energy variable) showed significant differences between FA and MIFC. Also, the variable r2Mink (Goodnesss of fit of the Minkowski - fractal dimension) was able to differentiate benign and malignant lesions such as: AN and MIFC, AN and FVPC, FA and MIFC, FA and FVPC. Texture analysis may help to differentiate benign and malignant follicular lesions of the thyroid, specially when one is trying to differentiate AN or FA and FVPC. In this situation the histological analysis based on nuclear characteristics may not be definitive; owing to the fact that all the nuclear criteria may not be always present in most cases. On the other hand, the differential diagnosis of FA and MIFC is difficult and it is not based on nuclear characteristics, but on the finding of capsular or vascular invasion. However, in these cases, the variables energy and r2Mink contributed to the differential diagnosis. Therefore, the computerized image analysis may be considered as a tool that helps the differential diagnosis of follicular thyroid lesions, added to the morphological and immunohistochemical analysis
Doutorado
Anatomia Patologica
Doutor em Ciências Médicas

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CAPES
O Câncer de colo uterino é o quarto mais comum em mulheres no mundo. Sabe-se que a carcinogênese cervical precede da infecção e persistência do Papilomavírus humano (HPV), o qual tem a capacidade de se integrar ao genoma do hospedeiro introduzindo os genes E6 e E7. O produto desses genes, as oncoproteínas E6 e E7 perturbam o ciclo celular promovendo superexpressão da proteína p16INK4a e as metaloproteinases de matriz (MMP), que são moléculas capazes de promover a progressão das lesões cervicais uterinas. Nesse intuito, o presente estudo avaliou a expressão da oncoproteína E6, p16INK4a, metaloproteinase de matriz 3 (MMP-3) e inibidor tissular de metaloproteínase 2 (TIMP-2) nos diferentes graus de lesões e câncer cervical, associando com a presença do HPV e seus genótipos. Foram utilizadas 86 amostras de escovado cervical e de fragmentos de biopsias de mulheres que foram diagnosticadas citologicamente com lesões cervicais intraepiteliais e invasivas. Todas as amostras foram genotipadas e submetidas a técnica de imunohistoquímica para a identificação da oncoproteína E6-HPV16/18, p16INK4a, MMP-3 e TIMP-2. O perfil genotípico identificou o HPV16 (44,4%) como o mais prevalente, seguido por HPV31 (30,3%), HPV58 (15,1%), HPV18 (6,1%) e HPV33 (4,1%). A reação imunohistoquímica para E6 se mostrou mais intensa nos casos de invasão, assim como p16INK4a. Entretanto MMP-3 e o TIMP-2 se mostraram mais reativos em lesões pré-malignas. Portanto, este estudo mostra maior prevalência do HPV-16 em mulheres atendidas no Hospital das Clínicas – UFPE e sugere que a oncoproteína E6 e a proteína p16INK4a são biomarcadores úteis para aumentar a precisão do diagnóstico precoce de câncer de colo do útero. Contudo, em lesões pré-malignas os biomarcadores clinicamente úteis seriam MMP-3 e TIMP-2.
Cervical cancer is the fourth most common in women worldwide. Cervical carcinogenesis is known to precede the infection and persistence of human papillomavirus (HPV), which has the ability to integrate into the host genome by introducing the E6 and E7 genes. The product of these genes, E6 and E7 oncoproteins disrupt the cell cycle promoting overexpression of p16INK4a protein and matrix metalloproteinases, molecules which are able to promote the progression of cervical uterine injury. To that end, the present study aimed to evaluate the expression of the oncoprotein E6, p16INK4a, matrix metalloproteinase 3 and the tissue inhibitor of metalloproteinase 2 in varying degrees of injuries and cervical cancer, associated with the presence of HPV and its genotypes. 86 cervical brush samples were used and biopsies fragments of women who were diagnosed cytologically with intraepithelial cervical lesions and invasive. All samples were genotyped and subjected to immunohistochemistry to identify the oncoprotein E6, HPV16 / 18, p16INK4a, MMP-3 and TIMP-2. The genotype profile identified HPV16 (44.4%) was the most prevalent, followed by HPV31 (30.3%), HPV58 (15.1%), HPV18 (6.1%) and HPV33 (4.1%). The immunohistochemical reaction for E6 was more reactive in the case of invasion, as well as p16INK4a. However MMP-3 and TIMP-2 were more reactive in premalignant lesions. Therefore, this study showed a higher prevalence of HPV-16 in women treated at the Hospital - UFPE and suggests that the E6 oncoprotein and p16INK4a protein biomarkers are useful to improve the accuracy of early diagnosis of cervical cancer. However, in premalignant lesions would be clinically useful biomarkers MMP-3 and TIMP-2.

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2010.
Committee Chair: Nie, Shuming; Committee Member: Bao, Gang; Committee Member: Murthy, Niren; Committee Member: Varma, Vijay; Committee Member: Wang, Zhong Lin. Part of the SMARTech Electronic Thesis and Dissertation Collection.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Propósito: É propósito deste trabalho estudar a agressividade da lesão de células gigantes periférica da cavidade bucal (LCGP) através de aspectos clínicos, radiográficos, histopatológicos, histoquímicos e imunohistoquímicos, objetivando melhor conhecer o perfil clínico do paciente e, sobretudo, avaliar o comportamento biológico dessa lesão. Material e Método: Foram estudados retrospectivamente 61 casos de LCGP, procedentes da Faculdade de Odontologia de Araçatuba - UNESP e da Faculdade de Odontologia da Universidade de Passo Fundo - FO-UPF. Primeiramente, realizou-se confirmação diagnóstica histopatológica e avaliação da presença de tecido ósseo imaturo nas lesões. Após, os casos selecionados foram agrupados com base em critérios clínico-radiográficos, em difererentes graus (agressividade nula, leve, moderada, severa e extrema) e seus dados clínico-demográficos e imaginológicos foram avaliados e correlacionados. Em seguida, utilizando-se 15 casos do total estudado (05 casos com agressividade clínico-radiográfica nula, 05 com agressividade moderada e 05 com agressividade severa), investigou-se a proliferação celular das LCGPs por meio da contagem e aferição da área de AgNORs (regiões de organização nucleolar marcadas pela prata) e avaliação da expressão de PCNA (antígeno de proliferação nuclear celular), Ki-67 (Kiel-67) e p53 (proteína p53). Resultados: Dentre os resultados clínico-radiográficos obtidos destacam-se os que dizem respeito à idade dos pacientes (prevalência entre 31 e 40 anos), sexo (prevalência em mulheres), raça (prevalência em brancos) e localização anatômica (prevalência na região mandibular anterior). A maioria das lesões se mostrou como nódulo indolor, de superfície lisa, dois meses de evolução, consistência fibrosa, formato arredondado ou ovóide, coloração púrpura e base séssil...
Purpose: To study the aggressiveness of peripheral giant cell lesion of buccal cavity (PGCL) by means of clinical, radiographic, histopathological, histochemical and immunohistochemical aspects in order to determine the patient's profile and to evaluate the biologic behavior of this lesion. Material and Method: 61 cases of PGCL of patients from Faculdade de Odontologia de Araçatuba - UNESP and Faculdade de Odontologia da Universidade de Passo Fundo - FOUPF were studied retrospectively. A corroborative diagnostic and the evaluation of immature bony tissue in the lesions were accomplished. These cases were divided into different groups according a classification based on grades of clinical-radiographic aggressiveness. The data of each group were evaluated and correlated and 15 cases were selected (5 cases with null clinical-radiographic aggressiveness, 5 with moderate aggressiveness and 5 with severe aggressiveness). An investigation of the cellular proliferation of PGCL was accomplished by count and measurement of the area of AgNORs (nucleolar organization regions marked by silver) and evaluation of PCNA (proliferating cell nuclear antigen), Ki-67 (Kiel-67) and p53 (protein p53). Results: The following conclusions were drawn by the clinical-radiographic results: in respect to the patient's age (prevalence between 31 and 40 years old), sex (prevalence in women), race (prevalence in white people), anatomical location (prevalence in the mandibular anterior region). Clinically the lesions were predominantly observed as painless tissue growths of smooth surface, two months of evolution, fibrous consistence, nodular form, purple coloration and sessile basis of implantation. In the greater number of cases the radiographic image of the subjacent bony tissue to the PGCL... (Complete abstract, click electronic address below)

Orientador: Eliane Maria Ingrid Amstalden
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: O osteossarcoma destaca-se por ser o sarcoma mais comum dentre os tumores ósseos malignos, na espécie canina, e pelo seu alto potencial de agressividade. Do ponto de vista histológico é um tumor de padrão celular heterogêneo, que se caracteriza pela produção de matriz osteóide e/ou osso imaturo por osteoblastos. Apenas dois estudos sistemáticos, relativos ao osteossarcoma canino, foram identificados na literatura nacional. Um deles refere-se a dados epidemiológicos e o outro aborda achados clínico-morfológicos. Estudos objetivando a avaliação dos aspectos histogenéticos e do índice de proliferação celular não foram ainda descritos na literatura nacional. A proposta deste estudo foi determinar a diferenciação dos diversos elementos celulares constituintes do osteossarcoma canino e avaliar o índice de proliferação celular destes tumores, correlacionando-os com seus diferentes subtipos morfológicos, utilizando-se de imunomarcadores, tais como: vimentina, osteocalcina, proteína S -100, 1A4, HHF35, AE1/AE3, CD31, CD34 e Ki-67. Foi realizado estudo retrospectivo de 65 casos de cães portadores de osteossarcoma, os quais foram avaliados clinicamente quanto ao sexo, raça, idade e localização topográfica das lesões. Os tumores foram classificados quanto ao subtipo histológico em osteoblásticos pouco produtivos ou osteoblásticos produtivos (de acordo com a quantidade de osteóide produzido), condroblásticos, fibroblásticos, do tipo células gigantes, telangiectásicos e indiferenciados. Cães machos, da raça Rottweiler, com idade média de 7,4 anos, foram mais freqüentemente afetados por osteossarcoma. Em 77,4 % dos casos as lesões ocorreram no esqueleto apendicular (fêmur). Os subtipos histológicos identificados foram: osteoblásticos (47,7%), fibroblásticos (23,1%), condroblásticos (21,5 %), do tipo células gigantes (4,7%), telangiectásicos (1,5%) e indiferenciados (1,5%). Em 98,2 % dos osteossarcomas houve reatividade para vimentina. Imunorreatividade à osteocalcina ocorreu em células osteoblásticas, condroblásticas e fibroblásticas de 91% dos osteossarcomas. Proteína S-100 foi expressa em 79,7% dos tumores, tendo ocorrido em todos os tumores condroblásticos. Proporção significativa de osteossarcomas reagiu positivamente aos anticorpos 1A4 e HHF35. Todos os osteossarcomas foram negativos para AE1/AE3e para CD31 e CD34. A imunomarcação com Ki-67 revelou alto índice de proliferação celular em osteossarcomas osteoblásticos pouco produtivos. Os osteossarcomas caninos são constituídos por diversificada população de células, representadas por elementos osteoblásticos, condroblásticos, miofibroblásticos e mioblásticos, os quais possivelmente, originam-se de uma única célula mesenquimal pluripotente ou de osteoblastos imaturos, que sofrem diferenciação diversificada. Osteossarcomas osteoblásticos pouco produtivos apresentaram índice expressivo de proliferação celular, o qual pode estar correlacionado a um caráter de agressividade mais acentuado
Abstract: Osteosarcoma is the most common of all malignant canine bone tumors and has a high aggressiveness potential. From the histological point of view it is a tumor with a heterogeneous cell pattern that is characterized by the production of bone matrix and/or immature bone by osteoblasts. Systematic studies with a clear objective to evaluate histogenetic aspects and the cellular proliferation index in canine osteosarcomas have not yet been published. The objective of this study was to determine the differentiation of the constituting cellular elements and evaluate the cellular proliferation index of these tumors correlating them with their different morphological subtypes, using markers such as: vimentin, osteocalcin, S-100 protein, 1A4, HHF53, factor VIII, AE1/AE3, CD31, CD34 and Ki-67. A retrospective study was performed using 65 cases of canine osteosarcoma, clinically evaluated according to sex, breed, age and topography of the lesions. Tumors were classified according to their histological subtype: moderately productive osteoblastic tumor, productive tumor (both according to the production of osteoid matrix), chondroblastic tumor, fibroblastic tumor, giant cell tumor, telangiectatic tumor and undifferentiated tumors. Males from the Rottweiler breed with an average age of 7,4 years were more frequently affected by osteosarcoma. In 77,4% of cases, the lesions were on the appendicular skeleton (femur). The Identified histological subtypes were: osteoblastic (47,7%), fibroblastic (23,1%), chondroblastic (21,5 %), giant cell (4,7%), telangiectatic (1,5%) and undifferentiated (1,5%). In 98,2% of all tumors there was vimentin reactivity. Immunoreactivity to osteocalcin occurred in osteoblastic, chondroblastic and fibroblastic cells of 91% of all osteosarcomas. The S-100 protein was expressed in 79,7% of all tumors, and in 100% of the chondroblastic tumors. A significant proportion of osteosarcomas reacted positively to the 1A4 and HHF35 antibodies. All osteosarcomas were negative for AE1/AE3 and CD31 and CD34 testing. Marking essays with Ki-67 revealed a high cell proliferation index in moderately productive osteoblastic osteosarcomas. Canine osteosarcomas are constituted by a diverse cell population, composed of osteoblastic, chondroblastic, myofibroblastic and mioblastic elements, which are probably originated from a single pluripotent mesenchymal cell or from immature osteoblasts that go thru a differentiation process. Moderately productive osteoblastic osteosarcoma presented a considerably high cell proliferation index that may relate to a more aggressive behavior
Doutorado
Ciencias Biomedicas
Doutor em Ciências Médicas

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease caused by a virus of the family Bunyaviridae, genus Phlebovirus. It is responsible for extensive outbreaks of disease in livestock in Africa with significant mortality and economic impact. Virus neutralization is considered the gold standard for confirming Rift Valley fever virus (RVFV) infection but the procedure is time consuming and expensive. Real-time reverse transcription-polymerase chain reaction (rRT-PCR), histopathology, and immunohistochemistry (IHC) are the diagnostic methods most often used in South Africa to confirm or exclude a diagnosis of RVF in necropsied animals. Validated estimates of diagnostic accuracy of these tests, in naturally infected livestock, however, have not been published. The objective of this study was to estimate the diagnostic sensitivity and specificity of rRT-PCR, histopathology, and IHC using Bayesian latent class methods in the absence of a gold standard. A secondary objective was to estimate stratum-specific values based on species, age, degree of specimen autolysis, and the presence/absence of tissue pigments. The Sensitivity (Se) and Specificity (Sp) of qRT-PCR were 97.4% (95% credibility interval (CI): 95.2% - 98.8%) and 71.7% (95% CI: 65% - 77.9%) respectively. The extraordinary analytical sensitivity of PCR makes this test very susceptible to false positive reactions, and thus reduced specificity. This is more likely during large-scale epidemics due to crosscontamination of specimens at necropsy facilities or testing laboratories. The Se and Sp of histopathology were 94.6% (95% CI: 91% - 97.2%) and 92.3% (95% CI: 87.6% - 95.8%) respectively. Single cases of RVF could be confused with acute poisoning with plants, bacterial septicaemias, and viral diseases such as infectious bovine rhinotracheitis and Wesselsbron disease. Most of these conditions, however, can be excluded using histological examination of the liver, special stains, bacterial culture, and toxicological or serological investigations. The Se and Sp of IHC were 97.6% (95% CI: 93.9% - 99.8%) and 99.4% (95% CI: 96.9% - 100%) respectively. Immunohistochemistry is highly specific because characteristic positive immunolabelling of the cytoplasm of hepatocytes can be correlated with the presence of hepatocellular injury typical for RVFV infection. False negative results are sometimes obtained with IHC because of reader error or loss of the antigenic epitopes due to advanced autolysis. Scant positive immunolabelling might be missed or viral proteins might be absent from sections of liver with advanced hepatocellular damage. The stratified analysis suggested differences in test accuracy in foetuses and severely autolysed specimens. The Sp of histopathology in foetuses (83.0%) was 9.3% lower than the value obtained for the sample population (92.3%). Lesions in some foetuses are more subtle and the typical eosinophilic intranuclear inclusions are often difficult to detect. In severely autolysed specimens, the Se of IHC decreased by 16.1% and the Sp of rRT-PCR by 17.4%. There is no plausible biological explanation for this decrease in the Sp of rRTPCR since the RNA of RVFV is resistant to degradation in autolysed tissues. Conversely, the antibody used to detect RVFV using IHC detects epitopes raised against nucleoproteins of the virus and it is possible that viral proteins become too widely dispersed and/or degraded in autolysed tissues to detect by light microscopy. It is possible that the marked decrease in Se of histopathology and IHC in severely autolysed specimens caused an apparent decrease in Sp of rRT-PCR, due to the latent class method. In conclusion, the high estimated Sp (99.4%) of IHC and the low Sp of rRT-PCR (71.3%) suggests that the definitive diagnosis or exclusion of RVF should not rely on a single PCR test and that IHC would be an effective confirmatory test for rRT-PCR positive field cases necropsied during an epidemic. Immunohistochemistry results from severely autolysed specimens, however, should be interpreted with caution and aborted foetuses in areas endemic for RVF should be screened using a variety of tests. The diagnostic Se and Sp of histopathology was much higher than expected confirming the value of routine post mortem examinations and histopathology of liver specimens. The most feasible RVF testing option in areas that do not have suitably equipped PCR laboratories, and where disease is often not detected in livestock until after human cases have been diagnosed, would be routine histopathology screening with IHC confirmation. Key Words: Rift Valley fever; Rift Valley fever virus; Bayesian; latent-class model; real-time reverse transcription-polymerase chain reaction; immunohistochemistry; histopathology; diagnosis; sensitivity; specificity.
Dissertation (MSc)--University of Pretoria, 2014.
gm2014
Paraclinical Sciences
unrestricted

O linfoma canino é uma das neoplasias mais prevalentes em cães, diagnosticado frequentemente através de exame citológico de aspirados. A imunofenotipagem para diferenciação entre linfomas de células B e T é importante para definição prognóstica e tratamento, entretanto, dificilmente é realizada em materiais provenientes de aspirados citológicos. A citologia em meio líquido (CML) é uma metodologia automatizada de produção de esfregaços citológicos, que permite a conservação de todo o material da aspiração, e utilização do material residual para, por exemplo, produção de emblocado celular (EC) e imunocitoquímica. Este trabalho teve como objetivo aplicar a CML e EC com imunocitoquímica em amostras de linfonodos caninos suspeitos para linfoma. Além disso, pretendeu caracterizar morfologicamente as células linfoides neste tipo de preparado, comparando ao esfregaço convencional. Para isto, foram selecionados 54 cães com linfadenomegalia, 10 deles sorologicamente positivos para leishmaniose canina. Comparou-se a CML com a citologia convencional, quanto às características técnicas e testou-se dois métodos diferentes de EC: Bouin e agarose com formalina. Aplicou-se painel imunocitoquímico de anticorpos anti-CD3, anti-CD79a, anti-Pax-5 e anti-Ki67, para imunofenotipagem e determinação da proliferação celular. Adicionalmente, realizaram-se análises estatísticas para avaliação do desempenho e concordância interobservador comparada entre citologia convencional, CML e o uso conjunto de CML e imunocitoquímica do emblocado celular. Como resultado, as células linfoides apresentaram-se preservadas, com melhor definição de cromatina e nucléolos, porém com tamanho reduzido e pior definição citoplasmática, na CML. O EC de Bouin revelou grupos densos e bem definidos, que permitiram a aplicação da imunocitoquímica. Por outro lado, os emblocados de agarose, apresentaram-se frouxos, com células marcadamente dispersas, revelando-se inadequados para preparados de células linfoides. O anticorpo anti-Pax-5 mostrou-se mais adequado do que o anti-CD79a para marcação de células B em emblocados, por produzir menos fundo e pela marcação nuclear ser mais distinguível do que a citoplasmática. A concordância interobservadores da CML foi moderada (k= 0,434), enquanto a da citologia convencional foi boa (k=0,762). Ambas as técnicas exibiram a mesma acurácia e, com aplicação da CML, houve redução do percentual de amostras insatisfatórias. A CML em conjunto com EC e imunocitoquímica apresentou maior sensibilidade (75%), especificidade (99%) e acurácia (89,47%). Como conclusão, a CML em conjunto com EC e imunocitoquímica pode ser aplicada para diagnóstico de linfoma canino, com maior sensibilidade, especificidade e acurácia
Canine lymphoma is one of the most commonly treated neoplasia in dogs, frequently diagnosed through cytological smears. Differentiating B lymphomas from T lymphomas is important for prognosis and treatment, however, immunophenotyping is rarely applied to cytological preparations. Liquid-based cytology (LBC) is an automated method for producing cytological smears, allowing preservation of the residual material for other analysis, as cell block (CB) immunocytochemistry. The aim of this study was to apply LBC and cell block immunocytochemistry in the diagnosis of lymphoid samples from dogs suspected of lymphoma. Besides, we intended to characterize the lymphoid cells regarding to its morphology, comparing to conventional cytology. To accomplish these goals, 54 dogs were selected, being 10 of them with canine leishmaniasis. LBC smears were compared to conventional smears, with production of two types of CB: bouin and formalin with agarose. Also, an immunocytochemistry panel with anti-CD3, anti-CD79a, anti-Pax-5 and anti-Ki-67 was applied, for immunophenotyping and cellular proliferation determination. As results, lymphoid cells were well preserved, with better nuclear definition, but smaller size and worst cytoplasmic definition, in LBC. Bouin CBs revealed cohesive groups, which allowed immunocytochemistry. In the other hand, agarose CBs were loose, with disperse cells, inadequate for lymphoid aspirates. Anti-Pax-5 antibody was more adequate than anti-CD79a, once it produced less background and the nuclear marking was more distinct than cytoplasmic. CML interrater agreement was moderate (k=0,434), while conventional cytology agreement was good (k=0,762). Both techniques exhibited the same accuracy, and, with CML, there was reduction of unsatisfactory results. LBC with cell block immunocytochemistry presented the higher sensitivity (75,00%), specificity (99,99%) and accuracy (89,47%). As conclusion, LBC with immunocytochemistry CB can be applied for canine lymphoma diagnosis, with higher sensitivity, specificity and accuracy

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Quarenta e sete cães machos e não castrados foram divididos em três grupos de acordo com a faixa etária em: animais adultos jovens (grupo A), animais de meia idade (grupo B) e idosos (grupo C). Dez animais com alterações prostáticas visíveis à ultra–sonografia foram reunidos no grupo D, independentemente da idade. Os animais foram eutanasiados, submetidos à ultrasonografia transretal e excisão da glândula. Foram colhidas amostras para exame histopatológico, transcrição reversa e reação em cadeia da polimerse (RT-PCR) e imunoistoquímico. A histopatologia revelou que todos os cães dos grupos B, C e D apresentavam afecção prostática, sendo a hiperplasia prostática (HPB) a mais freqüente. A maioria dos animais apresentava mais de um tipo de afecção simultaneamente, especialmente HPB e prostatite não bacteriana. A RT-PCR foi realizada para as enzimas CPSE, PAP e PSA, obtendo-se amplificação das amostras para CPSE e PAP, mas não para o PSA. Não houve diferença significativa na quantidade de âmplicons de CPSE entre as amostras do grupo A, B e C, mas esse valor foi significativamente maior no grupo D. A quantidade de âmplicons para a PAP não variou entre os grupos. Tampouco houve correlação entre a quantidade de âmplicons para as diferentes enzimas ou entre esses valores e as dimensões prostáticas na ultra-sonografia. Observou-se imunorreação fraca e pouco extensa (++) nos tecidos prostáticos dos animais dos grupos A e B para o PSA e negativa nos grupos C e D. O tecido prostático de todos os animais apresentou marcação positiva (+++) para a PAP na parte apical do citoplasma das células epiteliais. A CPSE apresentou maiores possibilidades de utilização clínica para avaliação da próstata canina, devendo-se investigar melhor sua atividade diante de afecções prostáticas distintas.
Forty seven intact male dogs were divided in three groups according to the age: young adults dogs (A group), middle aged dogs (B group) and old dogs (C group). Ten animals with prostatic alterations noted in ultrasonographic exam had been grouped separately (D group). The animals were submitted to euthanasia, transrectal ultrasonography and prostate gland extirpation. Samples were collected to hystopatology, immunohistochemistry and reverse transcriptase and polymerase chain reaction (RT-PCR). The histopathological exam revealed that all samples from B, C and D groups had a prostatic affection in which benign prostatic hyperplasia (BPH) associated to non-bacterial prostatitis was more often. The RT-PCR was performed to evaluate CPSE, PAP and PSA expression, the results showed positive amplification of all samples to CPSE and PAP, but not to PSA. No difference was found between the amplicons quantity of CPSE to A, B and C groups, but this quantity was higher in D group. There was no statistic difference between the amplicons levels of PAP among the groups. Non correlation was found between CPSE amplicons levels and PAP, neither between these quantities and prostatic dimensions obtained by ultrasonography. The immunoreactions was weak and little extensive (++) in prostatic tissue of animals from A and B groups to PSA and the staining was negative in samples from C and D groups. All samples presented positive staining (+++) to PAP in apical cell surface of the prostatic epithelium. CPSE was better than PAP and PSA for canine prostate diagnosis Further more researches are needed to assess the activity of CPSE in different pathology of canine prostate.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Frog farming is an expanding activity in South America. In Brazil it can be considered consolidated, especially in the state ofGoiás. In the last few years it turned into a superintensive activity and consequently favored the emergence of diseases that pose a threat to the technical and economic viability of farms of aquatic organisms. Several diseases with high morbidity and mortality have been observed in tadpoles and frogs. Especially in the state of Goiás, two diseases affect the production cycle in the frog farms. The first manifests itself in 30 days tadpoles leading to high mortality and has been associated to the presence of Ranavirus. The second affects frogs in all stages from post-molt to the point of slaughtering. In post-metamorphic phase the animals are cachectic and 10-50 % of the populationdevelops nervous signs such as incoordination and abnormal postures chronic condition. Despite the board's neurological symptoms, some frogs are able to feed and continue to grow slowly, with few recovering normal condition. This clinical manifestation may be attributed to the anatomy of the vestibular system and hearing. Afferent bilateral vestibular signals from semicircular canal and otolith organs are essential for the stabilization of gaze, the control of posture and locomotion as well as the cognitive aspects of balance, spatial orientation and vegetative homeostasis. Considering the relevance of the topic, the objective of this study is to contribute to the elucidation of Vestibular Syndrome in Rana catesbeiana focusing in the presence of Ranavirus through the use of methods of diagnosis and histopathological techniques , real-time PCR , immunohistochemistry and electron microscopy. In the first study, we developed a method of detecting Ranavirus by real-time PCR for diagnosis of clinically healthy and diseased frogs with nerve signals in order to monitor the spread of this agent for this and other species of animals that inhabit the frog farms around the state of Goiás 91 samples were collected randomly frog farms in the countryside of the stateof Goiás of these, 50 were from frogs without clinical symptoms and 41 box of frogs with nerve sequelae characterized by lack of coordination , posture with abnormal lordosis, scoliosis and various degrees of head tilt to the left or right side. Based on these results, it is concluded that the frequency of ranavirus frogs with neurological symptoms is higher than the frequency of ranavirus frogs without apparent clinical symptoms, indicating association of clinical findings with the presence of the virus. The detection of ranavirus in animals living near the frog farms in the state of Goiás indicates that the virus is spread in the sampled detection in fish and frog farms reveals need for monitoring thespread of the virus to prevent cases of mass mortality in other species. In the second study, samples were collected randomly from 60 bullfrogs from frog farms in the state of Goiás in order to identify the agent involved in chronic, vestibular syndrome similar to clinical problems through diagnostic techniques such as transmission electron microscopy, histopathology and immunohistochemistry. Of these, 30 bullfrogs had typical clinical picture and 30 bullfrogs had become asymptomatic. At histopathology, inflammatory type of lesions with necrosis and lymphocytic infiltration were seen in the posterior regions of the cerebrum and cerebellum, with meningoencephalitis and inclusion bodies, areas of gliosis, and necrotic foci with abundant macrophages and cellular debris associated with inflammatory infiltrates. The choroid plexus showed abundant inflammatory infiltrates and lesions similar to those described above. Theselesions were also observed in the region of the semicircular canals and the central and peripheral vestibular apparatus to the front and cross cuts. These specimens subjected to staining with specific anti - iridovirus revealed the presence of positive staining in the choroid plexus region. This finding associated with the lesions described in anatomohistopatologia confirms the presence of Ranavirus. When analyzing the images obtained by electron microscopy transmission of samples from kidney and liver of bullfrog it was possible to visualize viral particles with similar characteristics to that of ranavirus, with icosahedral symmetry and size ranging from 120 - 300nm . Thus, it is concluded that the lesions of inflammatory type, described in anatomohistopatologia in the posterior central nervous system bullfrogs region, extending to the middle and inner ear, nervous symptoms associated with vestibular character indicate the presence of Ranavirus. Regarding immunohistochemistry, the presence of positive staining with specific anti- Iridovirus in the affected cells of the choroid plexus of bullfrog, associated to lesions described in anatomohistopatologia region confirms the presence of ranavirus and the presence of viral particles in the liver and kidney bullfrog, associated with inflammatory lesions in the posterior type central nervous system region, extending to the middle and inner ear, described in the present study also confirms the presence of Ranavirusin frog farms of the state of Goiás.
A ranicultura é uma atividade em expansão na América do Sul podendo ser considerada consolidada no Brasil, particularmente no Estado de Goiás. Nos últimos anos, transformou-se em uma atividade superintensiva e como consequência favoreceu o aparecimento de doenças que constituem uma ameaça para a viabilidade técnica e econômica dos criatórios de organismos aquáticos. Diversas doenças com alta morbidade e mortalidade têm sido observadas emgirinos e rãs. Especialmente no estado de Goiás, duas patologias afetam o ciclo produtivo nos ranários. A primeira se manifesta em girinos de até 30 dias levando à alta taxa de mortalidade e já foi relacionada à presença de Iridovírusdo gênero Ranavirus. A segunda acomete rãs em todas as fases desde a pós-metamorfose até animais no ponto de abate. Na fase pósmetamórfica os animais apresentam-se caquéticos e de 10 a 50% da população desenvolve um quadro crônico caracterizado clinicamente por sinais nervosos como falta de coordenação e posturas anormais. Apesar do quadro com sintomatologia nervosa, algumas rãs conseguem se alimentar e continuam crescendo lentamente, sendo que poucas recuperam a condição normal. Essamanifestação clínica pode ser atribuída à própria anatomia do sistema vestibular e do aparelho auditivo. Sinais aferentes vestibulares bilaterais de canal semicircular e órgãos otolíticos são essenciais para a estabilização do olhar, o controle da postura e locomoção, bem como para os aspectos cognitivos de equilíbrio, orientação espacial e homeostase vegetativa. Considerando a relevância do tema, objetivou-se como presente estudo contribuir para elucidação da Síndrome Vestibular em Rana catesbeiana com foco na presença de Ranavirus por meio do emprego de métodos de diagnóstico como técnicas histopatológicas, PCR em tempo real, microscopia eletrônica e imunohistoquímica. No primeiro estudo, foi desenvolvido um método de detecção de Ranavirus por PCR em tempo real para diagnóstico em rãs clinicamente sadias e em rãs enfermas com sinais nervosos, com o objetivo de monitorar a disseminação desse agente para essa e outras espécies de animais que habitam o entorno dos ranários do estado de Goiás. Foram colhidas 91 amostras distribuídas aleatoriamente em ranários da zona rural do estado de Goiás. Dessas, 50 eram provenientes de rãs sem sintomatologia clínica e 41 de rãs com quadro de sequelas nervosas caracterizado por falta de coordenação, postura anormal com lordose, diversos graus de escoliose e desvio da cabeça para os lados esquerdo ou direito. Diante dos resultados obtidos, conclui-se que a frequência de Ranavirusem rãs com sintomatologia nervosa é superior à frequência de Ranavirus em rãs sem sintomatologia clínica aparente, indicando associação do quadro clínico com a presença do vírus. A detecção de Ranavirusem animais que habitam o entorno dos ranários do estado de Goiás indica que o vírus encontra-se disseminado nos ranários amostrados e a detecção em peixes revela necessidade de monitoramento da disseminação do vírus para evitar casos de mortalidade em massa em outras espécies. No segundo estudo, foram colhidas aleatoriamente amostras de 60 rãs da espécie Rana catesbeiana provenientes de ranários do estado de Goiás com o objetivo de identificar o agente envolvido nos quadros clínicos crônicos, similares à síndrome vestibular, por meio de técnicas de diagnóstico como microscopia eletrônica de transmissão, anatomohistopatologia e imunohistoquímica. Dessas, 30 apresentavam quadro clínico característico e 30 apresentavam-se assintomáticas. À anatomohistopatologia foram identificadas lesões do tipo inflamatória com necrose e infiltrado linfocitário nas regiões posteriores do encéfalo e cerebelo, com meningoencefalite e corpúsculos de inclusão, regiões de gliose, e focos necróticos com abundantes macrófagos e restos celulares associados a infiltrados inflamatórios. O plexo coroide apresentou infiltrado inflamatório abundante e lesões semelhantes às descritas anteriormente. Essas lesões também foram observadas na região dos canais semicirculares e no aparelho vestibular central e periférico aos cortes frontal e transversal. A leitura das lâminas submetidas à coloração com anticorpo específico anti-iridovírus revelou a presença de marcação positiva na região do plexo coroide. Essa constatação associada às lesões descritas na anatomohistopatologia confirma a presença de Ranavirus. Ao analisar as imagens obtidas por microscopia eletrônica de transmissão de amostras de rim e fígado foi possível visualizar partículas virais com características semelhantes à dos Ranavirus, ou seja, com simetria icosaédrica e tamanho variando de 120-300nm. Desse modo, conclui-se que as lesões do tipo inflamatórias, descritas na anatomohistopatologia, na região do sistema nervoso central posterior, com extensão para o ouvido interno e médio, associadas à sintomatologia nervosa de caráter vestibular indicam a presença de Ranavirus. Em relação à imunohistoquímica, a presença de marcação positiva com anticorpo específico antiiridovirus em células afetadas da região do plexo coroide, associada às lesões descritas na anatomohistopatologia confirma a presença de Ranavirus e a presença de partículas virais em fígado e rim, associada às lesões de tipo inflamatórias na região do sistema nervoso central posterior, com extensão para o ouvido interno e médio, descritas no presente estudo, também confirma a presença de Ranavirus em rãs de criação de ranários do estado de Goiás

Orientador: Wilter Ricardo Russiano Vicente
Banca: Lúcia Daniel Machado da Silva
Banca: Diana Magalhães de Oliveira
Banca: Paulo Henrique Franceschini
Banca: Julieta Rodini Engracia de Moraes
Resumo: Quarenta e sete cães machos e não castrados foram divididos em três grupos de acordo com a faixa etária em: animais adultos jovens (grupo A), animais de meia idade (grupo B) e idosos (grupo C). Dez animais com alterações prostáticas visíveis à ultra-sonografia foram reunidos no grupo D, independentemente da idade. Os animais foram eutanasiados, submetidos à ultrasonografia transretal e excisão da glândula. Foram colhidas amostras para exame histopatológico, transcrição reversa e reação em cadeia da polimerse (RT-PCR) e imunoistoquímico. A histopatologia revelou que todos os cães dos grupos B, C e D apresentavam afecção prostática, sendo a hiperplasia prostática (HPB) a mais freqüente. A maioria dos animais apresentava mais de um tipo de afecção simultaneamente, especialmente HPB e prostatite não bacteriana. A RT-PCR foi realizada para as enzimas CPSE, PAP e PSA, obtendo-se amplificação das amostras para CPSE e PAP, mas não para o PSA. Não houve diferença significativa na quantidade de âmplicons de CPSE entre as amostras do grupo A, B e C, mas esse valor foi significativamente maior no grupo D. A quantidade de âmplicons para a PAP não variou entre os grupos. Tampouco houve correlação entre a quantidade de âmplicons para as diferentes enzimas ou entre esses valores e as dimensões prostáticas na ultra-sonografia. Observou-se imunorreação fraca e pouco extensa (++) nos tecidos prostáticos dos animais dos grupos A e B para o PSA e negativa nos grupos C e D. O tecido prostático de todos os animais apresentou marcação positiva (+++) para a PAP na parte apical do citoplasma das células epiteliais. A CPSE apresentou maiores possibilidades de utilização clínica para avaliação da próstata canina, devendo-se investigar melhor sua atividade diante de afecções prostáticas distintas.
Abstract: Forty seven intact male dogs were divided in three groups according to the age: young adults dogs (A group), middle aged dogs (B group) and old dogs (C group). Ten animals with prostatic alterations noted in ultrasonographic exam had been grouped separately (D group). The animals were submitted to euthanasia, transrectal ultrasonography and prostate gland extirpation. Samples were collected to hystopatology, immunohistochemistry and reverse transcriptase and polymerase chain reaction (RT-PCR). The histopathological exam revealed that all samples from B, C and D groups had a prostatic affection in which benign prostatic hyperplasia (BPH) associated to non-bacterial prostatitis was more often. The RT-PCR was performed to evaluate CPSE, PAP and PSA expression, the results showed positive amplification of all samples to CPSE and PAP, but not to PSA. No difference was found between the amplicons quantity of CPSE to A, B and C groups, but this quantity was higher in D group. There was no statistic difference between the amplicons levels of PAP among the groups. Non correlation was found between CPSE amplicons levels and PAP, neither between these quantities and prostatic dimensions obtained by ultrasonography. The immunoreactions was weak and little extensive (++) in prostatic tissue of animals from A and B groups to PSA and the staining was negative in samples from C and D groups. All samples presented positive staining (+++) to PAP in apical cell surface of the prostatic epithelium. CPSE was better than PAP and PSA for canine prostate diagnosis Further more researches are needed to assess the activity of CPSE in different pathology of canine prostate.
Doutor

Resumo: Propósito: É propósito deste trabalho estudar a agressividade da lesão de células gigantes periférica da cavidade bucal (LCGP) através de aspectos clínicos, radiográficos, histopatológicos, histoquímicos e imunohistoquímicos, objetivando melhor conhecer o perfil clínico do paciente e, sobretudo, avaliar o comportamento biológico dessa lesão. Material e Método: Foram estudados retrospectivamente 61 casos de LCGP, procedentes da Faculdade de Odontologia de Araçatuba - UNESP e da Faculdade de Odontologia da Universidade de Passo Fundo - FO-UPF. Primeiramente, realizou-se confirmação diagnóstica histopatológica e avaliação da presença de tecido ósseo imaturo nas lesões. Após, os casos selecionados foram agrupados com base em critérios clínico-radiográficos, em difererentes graus (agressividade nula, leve, moderada, severa e extrema) e seus dados clínico-demográficos e imaginológicos foram avaliados e correlacionados. Em seguida, utilizando-se 15 casos do total estudado (05 casos com agressividade clínico-radiográfica nula, 05 com agressividade moderada e 05 com agressividade severa), investigou-se a proliferação celular das LCGPs por meio da contagem e aferição da área de AgNORs (regiões de organização nucleolar marcadas pela prata) e avaliação da expressão de PCNA (antígeno de proliferação nuclear celular), Ki-67 (Kiel-67) e p53 (proteína p53). Resultados: Dentre os resultados clínico-radiográficos obtidos destacam-se os que dizem respeito à idade dos pacientes (prevalência entre 31 e 40 anos), sexo (prevalência em mulheres), raça (prevalência em brancos) e localização anatômica (prevalência na região mandibular anterior). A maioria das lesões se mostrou como nódulo indolor, de superfície lisa, dois meses de evolução, consistência fibrosa, formato arredondado ou ovóide, coloração púrpura e base séssil... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Purpose: To study the aggressiveness of peripheral giant cell lesion of buccal cavity (PGCL) by means of clinical, radiographic, histopathological, histochemical and immunohistochemical aspects in order to determine the patient's profile and to evaluate the biologic behavior of this lesion. Material and Method: 61 cases of PGCL of patients from Faculdade de Odontologia de Araçatuba - UNESP and Faculdade de Odontologia da Universidade de Passo Fundo - FOUPF were studied retrospectively. A corroborative diagnostic and the evaluation of immature bony tissue in the lesions were accomplished. These cases were divided into different groups according a classification based on grades of clinical-radiographic aggressiveness. The data of each group were evaluated and correlated and 15 cases were selected (5 cases with null clinical-radiographic aggressiveness, 5 with moderate aggressiveness and 5 with severe aggressiveness). An investigation of the cellular proliferation of PGCL was accomplished by count and measurement of the area of AgNORs (nucleolar organization regions marked by silver) and evaluation of PCNA (proliferating cell nuclear antigen), Ki-67 (Kiel-67) and p53 (protein p53). Results: The following conclusions were drawn by the clinical-radiographic results: in respect to the patient's age (prevalence between 31 and 40 years old), sex (prevalence in women), race (prevalence in white people), anatomical location (prevalence in the mandibular anterior region). Clinically the lesions were predominantly observed as painless tissue growths of smooth surface, two months of evolution, "fibrous" consistence, nodular form, purple coloration and sessile basis of implantation. In the greater number of cases the radiographic image of the subjacent bony tissue to the PGCL... (Complete abstract, click electronic address below)
Orientador: Norberto Perri Moraes
Coorientador: Marcelo Macedo Crivelini
Banca: Glauco Issamu Miyahara
Banca: Soluete Oliveira da Silva
Banca: Norberto Perri Moraes
Mestre

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As les?es orais do l?quen plano e do l?pus eritematoso exibem apar?ncia cl?nica e histopatol?gica similar, o que dificulta o diagn?stico. A presente pesquisa teve por objetivo investigar crit?rios de diagn?stico diferencial das les?es orais das duas doen?as por meio de PAS e imunoistoqu?mica. Pacientes portadoras de les?es orais de l?quen plano (n=21) e de l?pus eritematoso (n=23) foram submetidas ? bi?psia incisional das les?es, e o diagn?stico cl?nico foi confirmado por HE e imunofluoresc?ncia direta. Ap?s a confirma??o diagn?stica, os esp?cimes foram submetidos ? colora??o por PAS e processamento imunoistoqu?mico. As imagens histol?gicas foram avaliadas no programa Image ProPlus 5.4.1, sendo quantificadas a express?o imunoistoqu?mica de CD1a, CD4, CD8, CD20, caspase-3, triptase e a espessura da membrana basal. A express?o imunoistoqu?mica de CD1a foi significativamente maior no grupo l?quen plano, tanto para epit?lio, quanto para conjuntivo. A express?o de CD4 e CD20 em epit?lio n?o diferiu significativamente entre l?quen plano e l?pus eritematoso; entretanto, no tecido conjuntivo foi significativamente maior para o grupo l?quen plano. A express?o de CD8 foi maior no l?quen, tanto para epit?lio quanto para conjuntivo. A express?o da caspase-3 n?o diferiu significativamente entre os grupos, enquanto a express?o da triptase foi maior no epit?lio das les?es de l?pus eritematoso. A espessura da membrana basal n?o exibiu diferen?a estatisticamente significativa entre os grupos. Os resultados permitem concluir que (1) a express?o imunoistoqu?mica de CD1a, CD4, CD8, CD20, caspase-3 e triptase, bem como a espessura da membrana basal em PAS n?o constituem crit?rios definidores para diagn?stico diferencial entre les?es orais de l?quen plano e de l?pus eritematoso; (2) a express?o imunoistoqu?mica de CD4, CD8 e CD20 ? maior nas les?es orais de l?quen plano do que nas de l?pus, o que sugere que, no l?quen, o infiltrado seja mais rico em linf?citos; (3) a triptase est? mais expressada no epit?lio das les?es orais de l?pus eritematoso, enquanto o CD1a prevalece tanto em epit?lio quanto em conjuntivo das les?es orais de l?quen plano; (4) as les?es orais de l?quen plano e l?pus eritematoso parecem diferir no que se refere ao ant?geno que suscita a resposta inflamat?ria, mas partilham a apoptose de c?lulas epiteliais e inflamat?rias em sua patogenia

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CAPES
For investigation of Mycobacterium avium subsp. paratuberculosis infection 34 buffaloes properties or ranches in Northeastern Brazil were visited for paratuberculosis diagnosis. Investigations included herd evaluations, inspection of facilities and pastures, obtainment of flock history, clinical examination of suspicious animals and collecting samples for diagnosis. Samples were obtained from 26 farms or ranches, including two slaughterhouses and a quarantine area in six states. Approximately 15,600 buffalos, including males and females of the Murrah, Mediterranean and Jafarabadi breeds as well as their crossbreeds, were evaluated for meat, dairy and mixed properties with semi-intensive or extensive regimes. For diagnostic purposes, necropsies, histopathological and immunohistochemical exams and Ziehl-Neelsen tests of fecal smears and scraped intestinal mucosa were performed. Polymerase chain reaction (PCR) methods were applied to samples of feces, milk, mesenteric nodes and intestines. This exams allowed us to identify eigth new Johne?s disease outbreaks, which, together with those previously identified by our staff, allow us to infer that the disease is being dispersed in the Brazilian Northeast, similar to what is occurring with bovine herds in other areas of the country. The increase in the number of positive farms is a consequence of the ignorance of farmers, inadequate health management, free trade of ruminants and lack of a official control program in the country. This study alerts for risk of commercialization of dairy products for human consumption, reinforces the importance of research on paratuberculosis in Brazil and contributes to the understanding of the factors that work together to increase the number of paratuberculosis cases in the Brazilian Northeast.
Com o objetivo de identificar focos e estudar a epidemiologia e o diagn?stico da paratuberculose em b?falos na regi?o Nordeste do Brasil foram realizadas visitas e ou examinados material provenientes de 34 propriedades com suspeita cl?nica da doen?a. Obtivemos material biol?gico de sete estados da regi?o Nordeste do Brasil (Maranh?o, Cear?, Para?ba, Pernambuco, Alagoas e Bahia). Dos rebanhos foi obtido o hist?rico, realizou-se avalia??o cl?nica e inspe??o das instala??es e pastagens. Para a confirma??o do diagn?stico foi coletado material para exames laboratoriais em 26 propriedades ou cria??es de b?falos, entre estes, dois matadouros e um quarenten?rio. Foram rebanhos cujo somat?rios de b?falos era de aproximadamente 15.600 b?falos, das ra?as Murrah, Mediterr?neo, Jafarabadi e seus mesti?os, com aptid?o para corte, leite ou mista, em propriedades com regimes semi-intensivo, extensivo ou extrativista. Foram realizadas 22 necropsias e coleta de material para exames histopatol?gicos e imuno-histoqu?micos, al?m de colora??o de Ziehl-Neelsen em esfrega?os de fezes, raspados de mucosa intestinal e fragmentos de linfonodo mesent?rico e de intestino que apresentavam les?es sugestivas da doen?a. Para a realiza??o da rea??o em cadeia da polimerase (PCR) foram utilizadas amostras de fezes, leite, linfonodos mesent?ricos e intestinos. Estes exames permitiram identificar oito focos de paratuberculose, o que nos permite inferir que a doen?a est? se dispersando na regi?o Nordeste do Brasil a exemplo do que est? acontecendo em outras regi?es do pa?s com o rebanho bovino. O desconhecimento da doen?a, o manejo inadequado, o com?rcio n?o regulamentado de b?falos e a falta de um programa de controle voltado para a realidade da regi?o facilitam a dispers?o do agente e s?o fatores que contribuem para o aumento do n?mero de focos no pa?s. Os resultados desta pesquisa contribuem com a epidemiologia e o diagn?stico da doen?a em b?falos e auxilia na compreens?o dos fatores que colaboram para a ocorr?ncia crescente do n?mero de casos desta doen?a.

Estudo dos processos linfoproliferativos cutâneos de células B objetivando demonstrar a difícil distinção diagnóstica entre os linfomas cutâneos e pseudolinfomas. Foram avaliados 38 casos de processos linfoproliferativos cutâneos de células B. Foi realizada revisão de prontuários médicos e exames anátomo-patológicos. Foram estudados 25 casos de linfomas cutâneos, 7 de pseudolinfomas e 6 casos onde não foi possível a distinção diagnóstica entre as entidades em estudo. As características clínicas, histológicas e imunoistoquímicas foram descritas para cada grupo. A análise estatística foi realizada demonstrando a similaridade entre os linfomas cutâneos e os pseudolinfomas
Study of cutaneous B-cell lymphoproliferative process with the purpose of demonstrating the difficulty of distinguishing between cutaneous lymphomas and pseudolymphomas. 38 cases of cutaneous B-cell lymphoproliferative processes were evaluated. A review of medical records and histophatologic material was performed. The study comprised 25 cases of cutaneous lymphomas, 7 cases of pseudolymphomas and 6 cases where a diagnosis distinguishing between the entities in study was not possible. It described clinical, histophatologic and immunohistochemical characteristics of each group. A statistical analysis showing the similarity between lymphomas and pseudolymphomas was performed

Kidney transplants from both living-related (LRD) and living unrelated (LURD) donors have superior function and survival than transplants from cadaver donors. This may be unsurprising as kidneys from living donors are procured under optimal conditions, from healthy donors with minimal ischaemia times. In contrast, cadaver kidneys are obtained from traumatised donors and may experience extended periods of cold ischaemic storage before transplantation. An immunohistochemical analysis has been performed on biopsies obtained before, and immediately after transplantation, to investigate the potential causes of early inflammatory events associated with cadaver renal transplantation that may influence subsequent graft outcome. An immunohistochemical analysis of biopsies obtained before transplantation demonstrated upregulated expression of endothelial E-selectin and proximal tubular expression of ICAM-1, VCAM-1 and HLA Class II antigens in cadaver donor kidneys. Analysis of donor parameters demonstrated that traumatic physiological events experienced in intensive care around the time of brain death were significantly associated with the induction of proinflammatory antigens. Antigen induction in cadaver donor kidneys before transplantation was significantly associated with early acute rejection. Furthermore, in cadaveric kidneys with long cold ischaemia times, glomerular neutrophil infiltration and deposition of activated platelets expressing P-selectin on intertubular capillaries were detected following reperfusion, in association with impaired short and long term graft function. Expression of inflammatory mediators were absent in all LRD renal allografts before and after reperfusion. A clinical trial was performed to determine whether ischaemia/reperfusion injury may be ameliorated by reflushing cadaver kidneys after cold storage to remove harmful products that may have accumulated in the vessel lumen. Reflushing did not prevent the inflammatory events observed after reperfusion or improve graft function. Therefore, a novel, oxygen free radical scavenger (lec-SOD) was obtained to assess its potential efficacy in preventing ischaemia/reperfusion injury. Lec-SOD bound with high affinity to macro- and microvascular endothelial cells under cold hypoxic conditions following incorporation into Marshall's preservation solution, significantly inhibiting cold hypoxia induced cell death, adhesion molecule induction and neutrophil adhesion. Furthermore, preservation of kidneys with lec- SOD for 18 hr in an experimental model of chronic renal allograft rejection, significantly attenuated neutrophil infiltration and MHC Class I induction day 1 post-transplant, with improved long term renal function. The results presented in this Thesis demonstrate that donor factors and cold ischaemia/ reperfusion injury elicit an early inflammatory response that may influence graft outcome of cadaver kidneys. Refinements in donor management and organ preservation may limit the deleterious effects of ischaemia/reperfusion injury in cadaver renal allografts, increasing graft survival to that observed in living donor transplantation.

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2010.
Committee Chair: Griffin, Paul; Committee Member: Butera, Robert; Committee Member: Halpern, Michael; Committee Member: Nichols, Richard; Committee Member: Vidakovic, Brani. Part of the SMARTech Electronic Thesis and Dissertation Collection.

No setor de Ginecologia do Hospital das Clínicas da FMUSP, 67 mulheres com leiomiomas do útero e idade de 24 a 39 anos, nuligestas foram estudadas. 31 receberam goserelin a cada 28 dias por 6 meses (grupo I) e 36 não (grupo II). Do grupo I, 16 apresentaram redução volumétrica menor ou igual a 36% (subgrupo Ia) e 15, maior ou igual a 36% (subgrupo Ib). Após a miomectoma, os nódulos foram encaminhados para anatomopatológico. Um único leiomioma de cada mulher foi submetido ao estudo eimuno-histoquímico para avaliação das concentrações de receptores de estrógeno, progesterona, vasos sanguíneos, colágeno, AgNOR e da celularidade. Concluiu-se que o análogo do GnRH está relacionado à diminuição da concentração de receptores de estrógeno. Não apresentou influência uniforme para progesterona, vasos sanguíneos, colágeno e celularidade
From 1994 to 1998, a total of 67 women with leiomyomas in the uterus, aging from 24 to 39, nuliparous and avid for pregnancy were studied in the Department of Gynaecology and Obstetrics of Hospital das Clínicas of Medical School of the University of São Paulo. From these, 31 received Goserelin 3,6mg at each 28 days for six months (group I) and 36 did not received medication (group II or control group). From the pacients who received medication, 16 presented volumetric reduction equal to or less than 36% (subgroup Ia) and the other 15 reduction larger than 36% (subgroup Ib). All women were submitted to myomectomy and the nodes were sent to anatomicopathological study. Only one leiomyoma of each woman was submitted to histochemical and immunohistochemical study to measure the concentrations of receptors of estrogen and progesterone, blood vessels, collagen, AgNOR and cellularity. It was observed that the group that presented larger volumetric reduction after using this medication showed variations of the concentration of receptors of estrogen (p0,001), progesterone (p=0.019), blood vessels (p=0.060), collagen (p=0.048), AgNOR (p=0.321) and number of cells (p=0.221), in comparison to the subgroup Ia and the group II (control group). As a result , it was observed that the GnRH analogue is related to the decrease of the concentration of receptors of estrogen, however it did not present uniform influence in the receptors of progesterone, blood vessels, collagen, and cellularity of this tumor

The aim of this study was to evaluate new methods to improve detection and investigation of the effects of chronic or subclinical infection with Trypanosoma evansi in various mammalian species. Some of the more resistant host species, including pigs and buffaloes, are present in large feral populations in the northern parts of Australia, the area where T. evansi is most likely to gain entry to the country. Existing tests are not sufficiently reliable to detect all cases of disease and they cannot distinguish acute from chronic infections. Furthermore, the tests have different sensitivities in different host species. Surveillance for trypanosomiasis in Australia is problematic because of the need to work in remote parts of northern Australia where provision of a cold-chain for traditional blood and serum storage is difficult. An existing dried blood storage system was modified by treating cotton lint filter paper (Whatman #903) with a commercial post coating buffer (TropBio, Queensland). This treatment increased the longevity of antibodies to T. evansi in serum and blood stored on the paper (detected using an antibody-detection ELISA) compared to samples stored on plain paper, especially when the papers were stored under humid conditions and at high ambient temperatures. Attempts were made to improve the diagnostic utility and repeatability of antibody-ELISAs through the use of 2 recombinant T. brucei antigens (PFRA and GM6) and to optimize a competitive ELISA using RoTat 1.2 variable surface antigen and its monoclonal antibody. Antibody-detection using the two recombinant proteins was not sufficiently specific to enable their use for the detection of T. evansi. The RoTat 1.2 cELISA had good sensitivity and specificity (75% and 98% respectively) when used to test serum from cattle and buffaloes experimentally infected with T. evansi and uninfected animals. However, the test was not able to detect anti-T. evansi antibodies in serum from wallabies, pigs, a dog or a horse that were experimentally infected with T. evansi. The inability of the cELISA to detect anti-T. evansi antibodies may be due to the small number of samples tested or the lack of RoTat 1.2 specific antibodies in the animals tested. The feasibility of using an enzymatic test to detect trypanosome aminotransferase or antibodies to this enzyme was evaluated. Prior publications suggested that the detection of TAT was an appropriate diagnostic tool for the detection of T. evansi infection in camels. However, the results from this study did not support the use of this test for the detection of T. evansi infection in cattle or buffaloes with low to moderate parasitaemia. Trypanosomiasis is an immunological disease that affects most of the body’s organs, with more severe disease developing over time. Attempts were made to determine key cytokine and biochemical patterns that would distinguish infected from uninfected animals and acute from chronic infections. The results from this study showed that there was no specific pattern in serum cytokines or serum biochemistry that could be used to distinguish infected from uninfected animals, or different stages of disease. Immunohistochemistry was used on tissues from buffaloes and mice experimentally infected with T. evansi and T. brucei gambiense respectively to characterise the cellular immune response that was present. The immune response was predominantly cell mediated, with CD3+ T lymphocyte and macrophage infiltration occurring in most tissues. In end stage disease there was often suppression of the immune system with disruption of the architecture of the spleen and a decrease in B lymphocytes in the circulation. Trypanosomes were rarely visible in the tissues and were only seen in those animals with high parasitaemia. Lesions generally became more severe over time, but there was a large variation between animals, which suggests that immunohistochemistry is unsuitable as a diagnostic tool.

Para avaliar a hipótese da presença de inflamação em artérias pulmonares periféricas de pacientes com hipertensão pulmonar (HP) decorrente de cardiopatias congênitas, foram quantificadas células inflamatórias através de marcação imunohistoquímica em biópsias de 26 pacientes e comparadas com 11 controles sem cardiopatia. Detectou-se quantidades semelhantes de células inflamatórias nos dois grupos, mas com predomínio de linfócitos T no grupo controle e de macrófagos jovens no grupo HP. Esses achados podem estar relacionados com a redução do estímulo dependente de macrófagos para diferenciação e maturação de linfócitos T nos cardiopatas e/ou a deficiência imunológica primária nesses pacientes
To evaluate the hypothesis of increased inflammation in peripheral pulmonary arteries from patients with pulmonary hypertension secondary to congenital cardiac shunts, we quantified the inflammatory cells with the aid of immunohystochemistry in 26 biopsies (HP group), comparing them to 11 patients with no cardiac disease. Similar quantities of inflammatory cells were observed in the two groups, with a predominance of T-lymphocytes in the controls and of young macrophages in the HP group. These findings could be related to a reduction of macrophagic stimulus to the differentiation and maturation of T-lymphocytes and/or to a primary immunological deficiency in patients with congenital cardiac shunts

The aim of this thesis is to in various ways explore protein expression in human normal tissue and in cancer and to apply that knowledge in biomarker discovery. In Paper I the prognostic significance of RNA-binding motif protein 3 (RBM3) is explored in malignant melanoma. To further evaluate the prognostic significance of RBM3 expression was assessed in 226 incident cases of malignant melanoma from the prospective populationbased cohort study Malmö Diet and Cancer Study using tissue microarray technique (TMA). RBM3 was shown to be down regulated in metastatic melanoma and high nuclear expression in the primary tumor was an independent marker of prolonged over all survival. As a tool to facilitate clinical biomarker studies the Human Protein Atlas has created a tissue dictionary as an introduction to human histology and histopathology. In Paper II this work is introduced. A cancer diagnosis can be a complex process with difficulties of establishing tumor type in localized disease or organ of origin in generalized disease. Immunohistochemically assisted diagnosis of cancer is common practice among pathologists where its application combined with known protein expression profiles of different cancer types, can strengthen or help dismiss a suspected diagnosis. In Paper III the diagnostic performance of 27 commonly used antibodies are tested in a predominantly metastatic, multicancer cohort using TMA technique. Overall these 27 diagnostic markers showed a low sensitivity and specificity for its intended use, highlighting the need for novel, more specific markers. Breast, ovarian, endometrial and ovarian cancers affect predominantly women. Differential diagnostics between these cancer types can be challenging. In Paper IV an algorithm, based on six different IHC markers, to differentiate between these cancer types is presented. A new diagnostic marker for breast cancer, namely ZAG is also introduced. In Paper V the transcriptomic landscape of the adrenal gland is explored by combining a transcriptomic approach with a immunohistochemistry based proteomic approach. In the adrenal gland we were able to detect 253 genes with an elevated pattern of expression in the adrenal gland, as compared to 31 other normal human tissue types analyzed. This combination of a transcriptomic and immunohistochemical approach provides a foundation for a deeper understanding of the adrenal glands function and physiology.

Despite recent advances, prognosis in metastatic prostate cancer remains poor. As with other cancers, tumor heterogeneity is an increasingly evident contributor in prostate tumorigenesis and developed resistance. Using in vitro and in vivo model systems, we examined novel diagnostic and therapeutic strategies in prostate cancer. In these studies, combination treatment with amuvatinib, a receptor tyrosine kinase inhibitor, and erlotinib, an epidermal growth factor inhibitor, was assessed for its ability to differentially modulate growth signaling in pathway diverse LNCaP (PTEN⁻) and DU-145 (PTEN⁺) human prostate cancer cell and mouse xenograft models. Our results suggest both individual mechanistic signaling activities, as well as benefits of the combination therapy though modulations of MAPK (pERK) and 4EBP1/cyclin D1 in growth signaling divergent PTEN+ and PTEN- prostate cancer cells. Additionally, despite the importance preanalytical tissue preservation on downstream diagnostic assays, exact protocols are not well defined and highly variable clinically and, as such, critical diagnostic information is lost. We show that a novel 2+2 fixation method induces target- and cell-specific alterations in immunostain intensity and efficacy. Importantly, cyclin D1 is increasingly utilized for as a clinical prognostic/diagnostic marker and demonstrated improved immunohistochemical staining efficacy with 2+2 fixation compared with treatment-matched xenograft protein alterations as assessed by western analysis. Finally, pentoxifylline (PTX) is a clinically utilized and well tolerated PDE inhibitor that has shown promise as a radio-/chemo-sensitization and anti-cancer agent against a variety of cancers. In these studies, we demonstrate that PTX induces cell and tumor growth inhibition in LNCaP prostate cancer cells. Mechanistically, PTX induces transient cellular signaling modulations of both the AMPK metabolic and AKT/mTOR growth pathways, while inducing autophagy. Also, PTX sensitizes LNCaP prostate cancer to cytotoxicity induced by first line chemotherapy docetaxel, inducing significant cellular apoptosis and reducing effective docetaxel concentrations by >10 fold for equivalent toxicity in viability assays. These findings nominate PTX as an adjunct therapy for the treatment of prostate cancer. In summary, these studies characterize the targeted signaling modulation by combination erlotinib and amuvatinib therapy, as well as pentoxifylline, for their use as therapies for prostate cancer. A novel fixation protocol was also assessed for improved diagnostic tissue preservation of critical signaling proteins. Further understanding in these areas will aid and expand the development of effective diagnostics, as well as emphasize the benefits of these and similar therapeutics for the treatment of prostate cancer.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
O diagnóstico da hanseníase neural pura baseia-se em dados clínicos e laboratoriais do paciente, incluindo a histopatologia de espécimes de biópsia de nervo e detecção de DNA de Mycobacterium leprae (M. leprae) pelo PCR. Como o exame histopatológico e a técnica PCR podem não ser suficientes para confirmar o diagnóstico, a imunomarcação de lipoarabinomanana (LAM) e/ou Glicolipídio fenólico 1 (PGL1) - componentes de parede celular de M. leprae foi utilizada na primeira etapa deste estudo, na tentativa de detectar qualquer presença vestigial do M. leprae em amostras de nervo sem bacilos. Além disso, sabe-se que a lesão do nervo na hanseníase pode diretamente ser induzida pelo M. leprae nos estágios iniciais da infecção, no entanto, os mecanismos imunomediados adicionam severidade ao comprometimento da função neural em períodos sintomáticos da doença. Este estudo investigou também a expressão imuno-histoquímica de marcadores envolvidos nos mecanismos de patogenicidade do dano ao nervo na hanseníase. Os imunomarcadores selecionados foram: quimiocinas CXCL10, CCL2, CD3, CD4, CD8, CD45RA, CD45RO, CD68, HLA-DR, e metaloproteinases 2 e 9. O estudo foi desenvolvido em espécimes de biópsias congeladas de nervo coletados de pacientes com HNP (n=23 / 6 BAAR+ e 17 BAAR - PCR +) e pacientes diagnosticados com outras neuropatias (n=5) utilizados como controle. Todas as amostras foram criosseccionadas e submetidas à imunoperoxidase. Os resultados iniciais demonstraram que as 6 amostras de nervos BAAR+ são LAM+/PGL1+. Já entre as 17 amostras de nervos BAAR-, 8 são LAM+ e/ou PGL1+. Nas 17 amostras de nervos BAAR-PCR+, apenas 7 tiveram resultados LAM+ e/ou PGL1+. A detecção de imunorreatividade para LAM e PGL1 nas amostras de nervo do grupo HNP contribuiu para a maior eficiência diagnóstica na ausência recursos a diagnósticos moleculares. Os resultados da segunda parte deste estudo mostraram que foram encontradas imunoreatividade para CXCL10, CCL2, MMP2 e MMP9 nos nervos da hanseníase, mas não em amostras de nervos com outras neuropatias. Além disso, essa imunomarcação foi encontrada predominantemente em células de Schwann e em macrófagos da população celular inflamatória nos nervos HNP. Os outros marcadores de ativação imunológica foram encontrados em leucócitos (linfócitos T e macrófagos) do infiltrado inflamatório encontrados nos nervos. A expressão de todos os marcadores, exceto CXCL10, apresentou associação com a fibrose, no entanto, apenas a CCL2, independentemente dos outros imunomarcadores, estava associada a esse excessivo depósito de matriz extracelular. Nenhuma diferença na frequência da imunomarcação foi detectada entre os subgrupos BAAR+ e BAAR-, exceção feita apenas às células CD68+ e HLA-DR+, que apresentaram discreta diferença entre os grupos BAAR + e BAAR- com granuloma epitelioide. A expressão de MMP9 associada com fibrose é consistente com os resultados anteriores do grupo de pesquisa. Estes resultados indicam que as quimiocinas CCL2 e CXCL10 não são determinantes para o estabelecimento das lesões com ou sem bacilos nos em nervo em estágios avançados da doença, entretanto, a CCL2 está associada com o recrutamento de macrófagos e com o desenvolvimento da fibrose do nervo na lesão neural da hanseníase.
The diagnosis of pure neural leprosy (PNL) is based on clinical and laboratory data, including the histopathology of nerve biopsy specimens and detection of M. leprae DNA by polymerase chain reaction (PCR). Given that histopathological examination and PCR methods may not be sufficient to confirm diagnosis, immunolabeling of lipoarabinomanan (LAM) and/or phenolic glycolipid 1 (PGL1) M. leprae wall components were utilized in the first step of this investigation in an attempt to detect any vestigial presence of M. leprae in AFB- nerve samples. Furthermore, its well known that nerve damage in leprosy can be directly induced by Mycobacterium leprae in the early stages of infection; however, immunomediated mechanisms add gravity to the impairment of neural function in symptomatic periods of the disease. Therefore, this study also investigated the immunohistochemical expression of immunomarkers involved in the pathogenic mechanisms of leprosy nerve damage. These markers selected were CXCL10, CCL2 chemokines and CD3, CD4, CD8, CD45RA, CD45RO, CD68, HLA-DR, metalloproteinases 2 and 9 in nerve biopsy specimens collected from leprosy (23) and nonleprosy patients (5) suffering peripheral neuropathy. Twenty-three PNL nerve samples (6 AFB+ and 17 AFB-PCR+) were cryosectioned and submitted to LAM and PGL1 immunohistochemical staining by immunoperoxidase; 5 nonleprosy nerve samples were used as controls. The 6 AFB-positive samples showed LAM/PGL1 immunoreactivity. Among the 17 AFB- samples, only 8 revealed LAM and/or PGL1 immunoreactivity. In 17 AFB-PCR+ patients, just 7 had LAM and/or PGL1-positive nerve results. In the PNL cases, the detection of immunolabeled LAM and PGL1 in the nerve samples would have contributed to enhanced diagnostic efficiency in the absence of molecular diagnostic facilities. The results of the second part of this study showed that CXCL10-, CCL2-, MMP2- and MMP9-immunoreactivities were found in the leprosy nerves but not in nonleprosy samples. Immunolabeling was predominantly found in recruited macrophages and Schwann cells composing the inflammatory cellular population in the leprosy-affected nerves. The immunohistochemical expression of all the markers, but CXCL10, was associated with fibrosis; however, only CCL2 was, independently from the other markers, associated with this excessive deposit of extracellular matrix. No difference in the frequency of the immunolabeling was detected between the AFB+ and AFB- leprosy subgroups of nerves, exception made to some statistical tendency to difference in regard to CD68+ and HLA-DR+ cells in the AFB- nerves exhibiting epithelioid granuloma. MMP9 expression associated with fibrosis is consistent with previous results of this research group. The findings conveys the idea that CCL2 and CXCL10 chemokines at least in advanced stages of leprosy nerve lesions are not determinant for the establishment of AFB+ or AFB- leprosy lesions, however, CCL2 is associated with macrophage recruitment and fibrosis.

Introdução: Apesar dos avanços nas técnicas de diagnóstico e tratamento, o câncer de próstata avançado ainda é uma condição letal. Terapêuticas mais eficazes são necessárias para reduzir as taxas de morbi-mortalidade associadas à doença. A Proteína-78 regulada pela glicose (GRP78), uma proteína de choque térmico envolvida na apresentação de antígenos, foi recentemente descrita como sendo um possível marcador molecular para o câncer de próstata. Ainda mais, a resposta imune a essa proteína mostrou correlação com o desenvolvimento de doença hormônio-independente e com pior sobrevida para a doença. Objetivos: Neste estudo, avaliou-se a hipótese de que a GRP78 poderia ser usada como marcador molecular em câncer de próstata no desenvolvimento de um sistema de receptor-ligante, através do uso da tecnologia de apresentação de fagos. Casuística e métodos: Inicialmente, foram clonados dois peptídeos que apresentam afinidade à proteína regulada pela GRP78 (os peptídeos WIFPWIQL e WDLAWMFRLPVG) no vetor fUSE5, criando-se fagos com capacidade teórica de ligação à mesma proteína. Posteriormente foi testada a capacidade de ligação desses fagos à GRP78 na membrana de células prostáticas malignas em solução, em xeno-tumores in vivo e em metástases ósseas de câncer de próstata humano. Resultados: Demonstrou-se que ambos os fagos se ligam especificamente à GRP78 in vitro, em comparação à proteínas com seqüência semelhante (proteínas de choque térmico 70 e 90) e não semelhante (albumina sérica bovina). Em seguida, mostrou-se que esses fagos se ligam com afinidade pelo menos 30 vezes maior à células de câncer de próstata que o fago controle, e que os fagos são internalizados por essas células. Posteriormente, mostrou-se que os fagos rastrearam xeno-tumores prostáticos quando injetados in vivo num modelo animal de câncer de próstata. Finalmente, mostrou-se que os fagos ligam-se especificamente à GRP78 expressa em metástases ósseas de adenocarcinoma prostático humano. Conclusões: Os fagos criados apresentam capacidade de ligação específica à GRP78 in vitro, em células em suspensão e in vivo. A estratégia e o sistema de receptor-ligante definidos no presente estudo podem ter implicacões relevantes no desenvolvimento de terapias dirigidas para o tratamento do câncer de próstata.
Introduction: Despite the advances in diagnosis and treatment, advanced prostate cancer remains a lethal condition. Improved methods of therapy are needed to reduce the morbidity and mortality rates associated with this disease. The Glucose-regulated protein-78 (GRP78), a stress-responsive heat-shock protein involved in antigen presentation, was recently described as a possible molecular marker for prostate cancer. Moreover, immune response against this protein was shown to have correlation with the development of androgen-independent prostate cancer and shorter overall survival. Objectives: We hipothesized that GRP78 could be used as a molecular marker for prostate cancer in the development of a receptor-ligand system, by using phage display technology. Patients and methods: We initially cloned two GRP78-targeting peptides (WIFPWIQL and WDLAWMFRLPVG) into a fUSE5-based phage. We then tested binding capacity of the phage to GRP78 in vitro, to GRP78 expressed in intact prostate cancer cell membranes, to a prostate cancer xenograft and to human bone metastases. Results: We showed that both phage created bound specifically to GRP78 in vitro, in comparison to related (Heat-shock proteins 70 and 90) and unrelated control proteins (bovine serum albumin). Next, we showed that these phage bound at least 30 times more to prostate cancer cells than the control phage, and were also internalized into these cells. Both GRP78-binding phage showed a strong homing in vivo to a human prostate cancer xenograft in a mouse model. Finally, we showed that both phage bound specifically to GRP78 expressed in human prostate cancer bone metastases. Conclusions: Both phage are capable of binding specifically to GRP78 in vitro, in the context of intact prostate cancer cells and in vivo. The strategy and the ligand-receptor system we have defined in this study may have relevant implications in the development of targeted therapies for the treatment of prostate cancer.

Urinary bladder cancer is a heterogeneous disease appearing in different forms, e.g. non-muscle invasive and muscle invasive. For all variants, the expression of proteins is interesting to analyze for diagnostic, predictive, prognostic and drug targeting purposes, since it reflects the altered gene expression causing the cancer. Since urothelial cells of the bladder are in direct contact with urine it is likely that this body fluid contains cancer-related proteins. In Paper I, unbiased analysis of proteins in urine from urinary bladder cancer patients and controls, using label-free quantification by mass spectrometry, was applied and four interesting proteins APOE, FGB, LRG and SERPINA1 were selected and further analyzed with western and dot blot. In Paper II, two more proteins, POLR1E and TOP2A, were validated as relevant proteins in bladder cancer urine. In Paper III and IV, the proteins GAL1 and STMN1 were investigated for their prognostic and therapeutic target potential in bladder cancer. In Paper II, III and IV, the expression of seven of the proteins were analyzed on tissue microarrays representing tumour tissue from 360 patients with different tumour stages. For the proteins identified by the urine screening approach, their protein expressions were confirmed in bladder cancer tissue. The expression level in tissue of five of the proteins, APOE, FGB, POLR1E (Paper II), GAL1 (Paper III) and STMN1 (Paper IV), increased with tumour stage, showing diagnostic relevance and three of the proteins, SERPINA1 (Paper II), STMN1 (Paper IV) and GAL1 (Paper III) had prognostic potential in urinary bladder cancer. In addition, GAL1 and STMN1 were demonstrated to be highly expressed in metastatic disease and inhibition of STMN1 reduced cell growth (Paper III and IV), indicating that these proteins are promising drug targets in urinary bladder cancer. In conclusion, the approach of this thesis has generated several candidate protein biomarkers in urine and tissue, validated with independent methods, which have the potential to improve the care for bladder cancer patients.

Objectifs: Notre travail avait pour objectif la prédiction du risque de récidive du cancer de prostate localisé à partir d’une caractérisation multimodale de ce cancer comprenant des facteurs anatomopathologiques, des biomarqueurs tissulaires et des paramètres quantitatifs originaux issus de l’IRM pré-thérapeutique, ainsi que l'élaboration d'une méthodologie pour l'évaluation des corrélations existantes entre ces différents facteurs. Matériel et méthodes : La valeur pronostique de la nouvelle classification ISUP, de l'infiltration lympho-vasculaire, de l'expression des marqueurs Ki67, Survivin et Caveolin-1 a été évaluée sur de larges cohortes multicentriques de patients avec cancer de prostate traité par prostatectomie radicale, en analyse multivariée avec calcul des C-index correspondants. L'intérêt, notamment pronostique, de la radiomique, correspondant à l'extraction et l'étude de caractéristiques quantitatives d'imagerie, a été évalué dans le cancer de prostate par une revue systématique de la littérature. L'intérêt de descripteurs texturaux issus de l'IRM pour la prédiction du risque de récidive biologique a été évalué dans une cohorte monocentrique de patients traités par radiothérapie externe. Dans une étude préliminaire basée sur 8 patients, une méthodologie a été proposée pour la reconstruction 3D de la pièce de prostatectomie et son recalage avec l'imagerie pré-opératoire. Ce travail a alors permis une mise en correspondance des données biologiques tumorales et les données quantitatives de l'imagerie. Des classifieurs pour l'identification et la caractérisation tumorale ont été construits et validés. Résultats : Nos travaux ont confirmé que la nouvelle classification ISUP, l'infiltration lymphovasculaire, et l'expression de Ki-67, Survivin, et Caveolin-1 sur la pièce de prostatectomie étaient associées à des caractéristiques pathologiques défavorables dans le CaP et au risque de récidive biologique après prostatectomie radicale. Cependant, leurs valeurs pronostiques sont limitées comparé aux facteurs pronostiques standards. La radiomique est un domaine prometteur pour l'identification et la caractérisation tumorale dans le CaP mais son intérêt pronostique reste à démontrer. Nos travaux confirment sa valeur pronostique pour les patients traités par radiothérapie externe. Avec une erreur moyenne de recalage de 5 mm, notre méthodologie de recalage nous a ensuite permis de proposer une mise en correspondance des données biologiques tumorales basées sur le score de Gleason et l'expression de marqueurs tissulaires. Cette mise en correspondance a permis l'élaboration de classifieurs permettant de prédire la présence de tumeurs et estimer leur score de Gleason avec des résultats satisfaisants. L'évaluation de l'expression immunohistochimique de marqueurs tissulaires reste limitée. Conclusion : Ces travaux confirment la valeur pronostique de marqueurs issus de l'étude quantitative de l'imagerie pré-opératoire, ainsi que de l'analyse anatomopathologique et immunohistochimique de la pièce de prostatectomie. Une méthodologie de mise en correspondance et de corrélation de ces différents marqueurs a été proposée avec des résultats préliminaires justifiant la poursuite des travaux sur une cohorte de patients plus importante
Objective: The aim of this work was the prediction of prostate cancer recurrence based on a multimodal characterization of this cancer including pathological markers, tissue biomarkers, and quantitative parameters from Magnetic Resonance Imaging (MRI), and to propose a method to assess correlations between these factors. Material and methods: The prognostic values of the new ISUP groups, the lymphovascular invasion, the expressions of Ki67, Survivin et Caveolin-1 were assessed in large multicentric and international cohorts of patients with prostate cancer treated with radical prostatectomy. A review of the literature investigated the use and the performance of radiomics and texture analysis to predict prostate cancer recurrence. The prognostic value of MRI features including Haralick’s texture features to predict biochemical recurrence after prostate cancer radiotherapy was assessed in a retrospective cohort of 83 patients. In a preliminary study including eight patients, a method to correlate in-vivo observations from pre-operative imaging with biological findings from radical prostatectomy using quantitative analysis was proposed. Résultats: Our studies confirmed the new ISUP groups, the lymphovascular invasion, the expressions of Ki67, Survivin and Caveolin-1 were associated with adverse pathologic features and prostate cancer recurrence after radical prostatectomy. However, these factors did not add clinically relevant information to established models. Our review of the literature revealed radiomics was promising for tumor identification and characterization but that few studies assessed its prognostic value. We demonstrated T2-w Haralick’s features were predictors of biochemical recurrence after prostate cancer radiotherapy. With a mean error of 4.90mm, our method to register prostate whole mount histology to in-vivo MRI resulted in the construction of classifiers to predict the presence of tumor, Gleason score, and Ki67 expression. Conclusion : Our work confirm the prognostic value of different markers from tissue, pathological pre-operative imaging analyses. Our method of registration and correlation of these markers leads to promising preliminary results that justify further evaluation with larger cohorts of patients

INTRODUÇÃO: Os microRNAs (miRNAs) são uma classe de pequenas moléculas não codificadoras de proteínas que regulam a expressão gênica durante a etapa de tradução. Esta regulação é feita pelo pareamento de bases com o mRNA-alvo (RNA mensageiro), resultando na supressão da tradução ou na clivagem do mRNA. A depender se os miRNAs têm como alvo genes supressores de tumor ou oncogenes, eles podem atuar como supressores tumorais ou oncogenes. A imunoistoquímica triplo negativa, no câncer de mama, é, comumente, utilizada como substituto clínico para identificação dos tumores basaloides, que se caracterizam pela expressão de genes epiteliais basais, sendo associados a menores taxas de sobrevida livre de doença e sobrevida global. O câncer de mama triplo negativo faz com que seja necessária a descoberta de marcadores moleculares que possam servir de alvos terapêuticos ou, pelo menos, que sirvam como marcadores preditivos da resposta aos quimioterápicos. OBJETIVO: avaliar a expressão de microRNAs, por PCR em tempo real, no carcinoma mamário ductal invasivo (CDI) triplo negativo. MÉTODOS: Foram avaliados materiais em parafina de tumor de 31 pacientes com as seguintes características: carcinoma invasivo de mama, receptores de estrogênio e de progesterona negativos e HER 2 negativo, bem como tecido mamário histologicamente normal. Foram utilizados kit para extração de RNA de amostras fixadas e parafinadas - miRNeasy FFPE; kit para síntese de cDNA - miScript II RT; kit miScript SYBR Green PCR e miScript miRNA PCR Arrays para análise de 84 sequências de miRNA de câncer humano. Foram avaliados dados clínicos, como idade, paridade, amamentação, status menopausal; variáveis histológicas, como tamanho do tumor, status linfonodal, invasão linfática; características imunoistoquímicas, como expressão de Ki-67, EFGR e CK 5/6. O seguimento das pacientes buscou verificar a ocorrência e o tempo de aparecimento de recidiva loco regional, metástase à distância e óbito. Para análise estatística foi utilizado o software miScript miRNA PCR Array Data Analysis, que utiliza o método de quantificação relativa DeltaCt. RESULTADOS: A análise comparativa dos 31 casos de CDI triplo negativo com os 18 casos de parênquima mamário normal definiu microRNAs hiperexpressos, sendo eles: miR-96-5p (fold-regulation(FR) = 9,68, p = 0,000008), miR-21-5p (FR = 4,47, p = 0,00), miR-7-5p (FR = 5,8, p = 0,00137) , miR-182-5p (FR= 7,92, p = 0,000001), miR-210-3p (FR = 11,83, p = 0,000048), miR-18a-5p (FR = 9,51, p = 0,000034), miR-155-5p (FR= 4,40 , p = 0,00019) e miR-93-5p (FR= 4,15, p = 0,000023). Aponta, ainda, microRNAs com hipoexpressão, a saber: miR-204-5p (FR = -10,26, p = 0), miR-205-5p (FR= -4,07, p = 0,019822), miR-125b-5p (FR= -4,29, p=0) e let 7c-5p (FR= -4,91, p=0). CONCLUSÃO: a expressão de microRNAs no carcinoma ductal invasivo triplo negativo permite diferenciá-lo do tecido normal
INTRODUCTION: MicroRNAs (miRNAs) are a class of small non-coding protein molecules that regulate gene expression during the translation stage. This adjustment is made by base pairing with the mRNA (messenger RNA) target resulting in suppression of translation or cleavage of the mRNA. Depending on whether miRNAs target tumor suppressor genes or oncogenes, they can act as tumor suppressors or oncogenes. The triple negative immunohistochemistry in breast cancer is commonly used as a substitute for clinical identification of basaloid tumors, which are characterized by the expression of basal epithelial genes and are associated with lower rates of disease-free survival and overall survival. The triple negative breast cancer makes necessary the discovery of molecular markers that may serve as therapeutic targets or at least as predictive markers of response to chemotherapy. OBJECTIVE: evaluate the expression of microRNAs by RT-PCR in triple negative breast invasive ductal carcinoma (IDC). METHODS: Paraffin embedded tumor material from 31 patients with the following characteristics were evaluated: invasive breast carcinoma, negative estrogen and progesterone receptor, negative HER 2, and histologically normal breast tissue. Were used: Kit for RNA extraction from fixed and paraffin embedded samples - miRNeasy FFPE; cDNA synthesis kit - miScript II RT; miScript SYBR Green PCR Kit and miScript miRNA PCR Arrays for analysis of 84 miRNA sequences of human cancer. Clinical data such as age, parity, breastfeeding, menopausal status; histological variables such as tumor size, lymph node status, lymphatic invasion; immunohistochemical characteristics, such as expression of Ki-67, EFGR and CK 5/6 were evaluated. The follow-up of patients aimed to verify the occurrence and time of appearance of loco regional recurrence, distant metastasis and death. For statistical analysis the miScript miRNA PCR Array Data Analysis software, which uses the method of relative quantification DeltaCt, was used. RESULTS: A comparative analysis of 31 cases of triple negative IDC with 18 cases of normal breast parenchyma defined microRNAs overexpressed, as follows: miR-96-5p (fold-regulation (FR) = 9.68, p = 0.000008), miR -21-5p (FR = 4.47, p = 0.00), 5p, miR-7 (FR = 5.8, p = 0.00137), miR-182-5p (FR = 7.92, p = 0.000001), miR-210-3p (FR = 11.83, p = 0.000048), miR-18a-5p (FR = 9.51, p = 0.000034), miR-155-5p (FR = 4.40, p = 0.00019) and miR-93-5p (FR = 4.15, p = 0.000023). Furthermore, microRNAs with reduced expression, as follows: miR-204-5p (FR = -10.26, p = 0), miR-205-5p (FR = -4.07, p = 0.019822), miR -125b-5p (FR = -4.29, p = 0) and Let-7c 5p (FR = -4.91, p = 0). CONCLUSION: the expression of microRNAs in triple negative invasive ductal carcinoma allows to differentiate it from normal tissue

HP, uma bactéria gram-negativa envolvida na patogênese do tecido gastroduodenal, foi classificada como carcinogênico classe I em 1994. Seus mecanismos são pouco entendidos, mas a presença da ilha de patogenicidade (PAI) é fator importante de virulência. PAI sugere aumento da proliferação celular, diminuindo a apoptose. A detecção do HP e da PAI (cagA) foi feita por PCR. Casos positivos são associados aos resultados histopatológicos, proliferação celular e apoptose. Detectamos HP em 37,0% (111/300), sendo 40,5% (45/111) dos pacientes cagA+. A relação proliferação celular/apoptose (P/A) mostra aumento em pacientes infectados por HP/cagA até 8,8 vezes maior no surgimento de doença gástrica grave
HP, a gram-negative bacterium involved in pathogenesis of gastroduodenal tissues, was classified as type I carcinogen in 1994. The mechanisms involved in carcinogenesis point to the pathogenicity island (PAI) as an important virulent factor. PAI suggests an increase in cell proliferation, attenuating apoptosis. HP detection and PAI were performed by using PCR. Positive results were associated to the histological findings, cell proliferation and apoptosis. We detected HP in 37,0% (111/300) and 40,5% (45/111) of the patients. A significant increase index appears between proliferation and apoptosis in those infected by HP/cagA+ with higher risk of 8,8 for the development of gastric cancer

碩士
國立臺灣大學
獸醫學研究所
96
Based on the previous studies, histological examination has a limitation to make a definite diagnosis of pathogens of central nervous diseases (CNS). To develop convenient diagnostic tools with high potential for rapid and specific diagnosis for this purpose, non-biotin horseradish peroxidase (non-biotin HRP) immunohistochemistry, loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions, and polymerase chain reaction (PCR) are employed in this study. A total of 84 pig cases with miscellaneous CNS diseases were obtained from archival paraffin sections which have been stored up to 20 years. In non-suppurative meningoencephalitis, including 23 pigs with hog cholera, 14 pigs with pseudorabies, 5 pigs with Salmonella sp. infection, 5 pigs with Toxoplasma gondii infection, 13 pigs with unidentified diseases. In suppurative meningoencephalitis, including 24 pigs with Streptococcus suis type 2 meningitis. All cases with any of 5 pathogenic infections were successfully diagnosed by non-biotin HRP immunohistochemistry. DNA from 26 suspected pseudorabies specimens were successful extracted. Among them, 4 samples were amplified and the target nucleic acid was detected by PCR, and 8 samples were amplified and the target nucleic acid was detected by LAMP. The preliminary results demonstrate that non-biotin HRP immunohistochemistry, LAMP, and PCR possess varying potential to detect pathogens of swine CNS diseases for infectious and retrospective studies.

A dissertation submitted to the Faculty of Health Sciences, University of Witwatersrand, in fulfilment of the requirements for the degree of Master of Science (Medicine) in the branch of Anatomical Pathology Johannesburg, 2012
Objective To determine the effectiveness of the cell block technique for immunocytochemical diagnosis by comparing cytomorphologic preservation and immunocytochemistry (ICC) stains in paired cell block and conventional fine needle aspiration (FNA) samples. Study Design This was a prospective study. Material for both conventional smears and cell blocks were collected simultaneously during fine needle aspiration of 50 lesions comprising lymph node, lung and liver masses. Grading of cellularity, morphological preservation, architectural preservation, immunocytochemical staining intensity and presence of background staining were compared on paired FNA smears and cell block samples derived from the same case. Each arm of the paired analysis was performed blindly without knowledge of the grading outcome of the other. The Kappa statistic (Κ) was used to measure inter-rater agreement. Results The fifty samples evaluated included FNAs from the lung, 24/50 (48%); liver, 23/50 (46%) and lymph node, 3/50 (6%). The immunocytochemistry stains consisted of 44/50 (88%) CK7, 44/50 (88%) CK20, 18/50 (36%) TTF1, 10/50 (20%) synaptophysin, 10/50 (20%) Hepar-1 and 7/50 (14%) AE1/3. There was no overall agreement in preservation of cytomorphological detail and ICC staining between the two methods. The Papanicolaou stained conventional FNA smears fared better then cell block for the vi evaluation of nuclear and cytomorphologic characteristics; cells in the cell block were poorly preserved in many cases. The ICC stains worked better on the cell block samples due to lack of background and aberrant staining. Conclusion Conventional FNA smears and cell blocks complement each other. Our results indicate that it would be optimal to use both modalities in the diagnostic work-up of mass lesions amenable to FNA diagnosis; the former to assess morphology, and the latter for optimal immunocytochemistry results. In resource constrained settings, the cost implications of performing both conventional and blocked smears on all FNA material warrants further evaluation.

by Wang Wen.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.
Includes bibliographical references (leaves 113-124).
Abstracts in English and Chinese.
ABSTRACT --- p.i
FLOWCHART --- p.vi
ACKNOWLEDGEMENT --- p.x
ABBREVIATIONS --- p.xi
INDEX FOR FIGURES --- p.xii
INDEX FOR TABLES --- p.xv
TABLE OF CONTENTS --- p.xvi
Chapter 1. --- INTRODUCTION --- p.1
Chapter 1.1 --- PATELLAR TENDINOSIS --- p.1
Chapter 1.1.1 --- Introduction --- p.1
Chapter 1.1.2 --- Epidemiology of Patellar Tendinosis --- p.3
Chapter 1.1.3 --- Etiology of Patellar Tendinosis --- p.3
Chapter 1.1.4 --- Manifestations of Patellar Tendinosis --- p.4
Chapter 1.1.5 --- Imaging Examination on Patellar Tendinosis --- p.4
Chapter 1.1.6 --- Clinical Diagnosis of Patellar Tendinosis --- p.6
Chapter 1.1.7 --- Management of Patellar Tendinosis … --- p.6
Chapter 1.2 --- ANATOMY AND HISTOLOGY OF PATELLAR TCNDON --- p.7
Chapter 1.3 --- STRUCTURE AND METABOLISM OF TENDON --- p.9
Chapter 1.3.1 --- Tenocytes --- p.9
Chapter 1.3.2 --- Extra-cellular Matrix --- p.11
Chapter 1.3.2.1 --- Collagen --- p.11
Chapter 1.3.2.2 --- Proteoglycans --- p.12
Chapter 1.4 --- ROLES OF GROWTH FACTORS TENDON HEALING AND REPAIR --- p.14
Chapter 1.4.1 --- Platelet-Derived Growth Factor --- p.14
Chapter 1.4.2 --- Transforming Growth Factor-beta --- p.15
Chapter 1.5 --- HISTOPATHOLOGY OF PATELLAR TENDINOSIS --- p.16
Chapter 1.6 --- STUDY PLAN --- p.17
Chapter 1.6.1 --- Characterization on Hypercellularity --- p.18
Chapter 1.6.2 --- Characterization on Disorganization and Loosening of Collagen --- p.18
Chapter 1.6.3 --- Characterization on Inflammatory Trace --- p.20
Chapter 1.6.4 --- Characterization on Growth Factors in Tendinosis --- p.21
Chapter 1.7 --- OBJECTIVES --- p.22
Chapter 2. --- MATERIALS AND METHODS --- p.27
Chapter 2.1 --- HUMAN TISSUES --- p.27
Chapter 2.1.1 --- Patellar Tendinosis Tissues --- p.27
Chapter 2.1.1.1 --- Diagnosis of patellar tendinosis --- p.27
Chapter 2.1.1.2 --- Recruitment of patients --- p.27
Chapter 2.1.4 --- Healthy Patellar Tendon tissues --- p.28
Chapter 2.2 --- TISSUES COLLECTION AND PREPARATION --- p.28
Chapter 2.3 --- HISTOLOGICAL STUDY ON HUMAN SPECIMENS --- p.28
Chapter 2.3.1 --- Haematoxyline and Eosin Staining --- p.29
Chapter 2.3.2 --- Safranin O Staining --- p.29
Chapter 2.3.2.1 --- Reagents preparation --- p.29
Chapter 2.3.2.2 --- Experimental procedure --- p.30
Chapter 2.3.5 --- Polarization Microscopy --- p.30
Chapter 2.4 --- IMMUNOHISTOCHEMICAL STAINING --- p.30
Chapter 2.4.1 --- Reagents Preparation --- p.31
Chapter 2.4.2 --- Experimental Procedure --- p.33
Chapter 2.5 --- IMAGE ANALYSIS --- p.35
Chapter 2.5.1 --- Equipment --- p.35
Chapter 2.5.2 --- Procedures --- p.35
Chapter 2.6 --- IN SITU ZYMOGRAPHY --- p.37
Chapter 2.6.1 --- Reagents Preparation --- p.37
Chapter 2.6.2 --- Experimental Procedure --- p.38
Chapter 2.7 --- STATISTIC ANALYSIS.… --- p.39
Chapter 3. --- RESULTS --- p.42
Chapter 3.1 --- HUMAN SAMPLES --- p.42
Chapter 3.1.1 --- Patellar tendinosis patients --- p.42
Chapter 3.1.2 --- Healthy control group --- p.43
Chapter 3.2 --- HISTOLOGICAL STUDY ON HUMAN SPECIMENS --- p.43
Chapter 3.2.1 --- Gross Morphology --- p.43
Chapter 3.2.2 --- Haematoxyline and Eosin Staining --- p.44
Chapter 3.2.3 --- Safranin O Staining --- p.44
Chapter 3.2.4 --- Polarization Microscopy --- p.44
Chapter 3.3 --- IMAGE ANALYSIS --- p.45
Chapter 3.3.1 --- Immunohistochemistry of PCNA --- p.45
Chapter 3.3.2 --- Immunohistochemistry of hsp47 --- p.46
Chapter 3.3.3 --- Immunohistochemistry of Procollogen Type I --- p.47
Chapter 3.3.4 --- Immunohistochemistry of MMP1 --- p.47
Chapter 3.3.5 --- Immunohistochemistry of TIMP1 --- p.48
Chapter 3.3.6 --- Immunohistochemistry of COX-2 --- p.49
Chapter 3.3.7 --- Immunohistochemistry of TGFP --- p.49
Chapter 3.3.8 --- Immunohistochemistry of PDGFbb --- p.50
Chapter 3.3.9 --- Immunohistochemistry of PDGFRβ --- p.51
Chapter 3.3.10 --- Summary of Image Analysis of Immunohistochemical staining --- p.51
Chapter 3.4 --- IN SITU ZYMOGRAPHY --- p.52
Chapter 4. --- DISCUSSION --- p.93
Chapter 4.1 --- DIAGNOSIS OF PATELLAR TENDINOSIS --- p.93
Chapter 4.2 --- HYPERCELLULARITY IN PATELLAR TENDINOSIS --- p.95
Chapter 4.3 --- COLLAGEN DISTURBANCE IN PATELLAR --- p.97
Chapter 4.4 --- INFLAMMATORY RESPONSE IN PATELLAR TENDINOSIS --- p.100
Chapter 4.5 --- THE EXPRESSION OF GROWTH FACTORS IN PATELLAR TENDINOSIS --- p.102
Chapter 4.6 --- PROPOSED PATHOGENESIS FOR PATELLAR TENDINOSIS --- p.105
Chapter 4.7 --- LIMITATION OF THIS STUDY --- p.108
Chapter 4.8 --- FUTURE STUDY --- p.109
Chapter 5. --- CONCLUSION --- p.111
BIBLIOGRAPHY --- p.113

Clinical immunohistochemistry (IHC) techniques are not yet fully standardized. In this project, a standardization method was developed and tested for proficiency testing (PT) in external quality assurance (EQA) and quality control (QC) in clinical IHC laboratories. The breast cancer markers estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor 2 (HER2) were used as a model system. Digital image analysis (IA) was used in conjunction with new calibrated and standardized cell line microarrays (CLMA). CLMAs built from nine formalin-fixed paraffin-embedded (FFPE) breast cancer cell lines were used for both QC controls and PT samples, instead of traditionally used FFPE tissues, in the standardization of breast cancer IHC. IA was used for measurement of IHC results, and compared to evaluation by the traditional expert-assessment method. Laboratory Score: Reference Score Ratio (LSRSR) was derived from Histo-Scores (HScores) determined by IA. HScores and LSRSRs were examined statistically and evaluated as histograms and boxplots to summarize and rank participant laboratory EQA results, in comparison to a reference sample or reference laboratories in two consecutive Canada-wide EQA runs. LSRSR-derived reference ranges were highly sensitive in evaluating laboratory EQA performance in PT as well as for monitoring of controls for QC. Laboratory on-slide tissue and cell-line IHC QA controls were assessed using IA and Levey Jennings QC charts. These charts were determined to be an excellent way to observe trending in laboratory IHC staining over time, particularly when cell line controls were used. This approach also reduced the time and labor costs for PT evaluation. Overall, cell line calibration controls were functionally equivalent or better than tissue-based controls in QC and PT mainly because of cell line biological homogeneity and sample availability. This study identified an optimal design for preparation of IHC cell line controls and PT samples for breast cancer markers. Optimal, intermediate staining cell line IHC controls were identified for all three breast cancer markers. Using IA with LSRSR and cell line samples is recommended for standardization of IHC methodology. This approach advances QA for diagnostic IHC and when implemented will improve patient care

碩士
國立臺灣大學
獸醫學研究所
93
Bartonella henselae is the causative agent of cat scratch diseases (CSD), which usually manifests as acute regional lymphadenopathy. From January 2001 through December 2004, 377 sera from 352 reportable CSD cases were examined by indirect fluorescence antibody assay (IFA). Ninety-nine patients were seropositive to B. henselae and which contributed to 28.1% seroprevalence in CSD suspected patients. Epidemiological features, like geographic and seasonal distribution or clinical presenting symptom were analyzed. There were 50.5% (50/99) of CSD seropositive patients were lived in north of Taiwan and 35.4% (35/99) of them were occurring in summer. The retrospective study of 152 CSD suspected cases demonstrated the seropositive results associated with cat contact, axillary and elbow lymphadenopathy. Immunohistologic studies for localization of B. henselae in collected 16 lymph nodes biopsies and 2 skin biopsies out of 352 patients were carried out by polyclonal antibody-based immunohistochemistry (IHC) and polymerase chain reaction (PCR) for comparing. IHC demonstrated the presence of the organism in intracellular location within granulomatous lesions in lymph node tissues. Ten lymph nodes from CSD suspected patients showed IHC positive and 3 of them were seronegative. The PCR results showed high sensitivity of 93.3% (14/15) cases positive. Immunohistochemistry can contribute to the etiologic diagnosis of Bartonella lymphadenopathy when serology and molecular techniques are not available.