Es wurde eine neue Färbemethode zur simultanen Darstellung von Estrogenrezeptoren (ER) und argyrophilen Nukleolus-organisierenden Regionen (AgNORs) entwickelt, um die Proliferationscharakteristika ER-positiver und ER-negativer Tumorzellen unabhängig voneinander zu bestimmen. Um eine mögliche gegenseitige Beeinflussung beider Färbemethoden auszuschließen wurden Serienschnitte 10 invasiv duktaler Mammakarzinome entweder mit einer der einzelnen Methoden oder mit der Simultanfärbemethode ER/AgNORs gefärbt und nachfolgend vergleichend untersucht. Durch Vermessung der histologischen Schnitte mit Hilfe der digitalen Bildanalyse konnten reziproke Effekte ausgeschlossen werden. Es konnte nachgewiesen werden, daß die simultane Färbung beider Marker zu einem reproduzierbaren und spezifischen Färberesultat führt. Es ist somit gerechtfertigt, AgNORs in immunhistochemisch gefärbten Zellen zu messen. Histologische Schnitte von 49 invasiven Mammakarzinomen wurden simultan für ER und AgNORs gefärbt. In jeweils 100 ER-positiven und ER-negativen Tumorzellkernen wurden die AgNORs mit Hilfe des Bildverarbeitungssystems AMBA vermessen. Zur Quantifizierung der AgNORs in immunhistochemisch gefärbten Zellkernen wurde das Programm AMBA\ orcolor angewendet. Die AgNOR-Konfiguration wird beschrieben durch Anzahl, Fläche und räumliche Verteilung innerhalb des Zellkerns. Zwischen ER-positiven und ER-negativen Zellen bestehen hochsignifikante Unterschiede. ER-negative Zellen des Mammakarzinoms besitzen eine größere Anzahl AgNORs (3,06 *0,67) verglichen mit den ER-positiven Zellen (1,65 *0,34). Nur die AgNOR-Parameter der ER-negativen Zellfraktion korrelierten mit anderen Malignitätsmarkern (Bloom-Richardson-Grading; Wachstumsfraktion (Ki-67)). Die ER-negativen Zellen lassen sich als Zellfraktion mit höherer Proliferationsaktivität charakterisieren. Die Ergebnisse zeigen, daß die ER-negativen Zellen des Mammakarzinoms einen entscheidenden Beitrag zur Tumorproliferation leisten und sehr wahrscheinlich die Progression der Tumorerkrankung bestimmen. / A new staining method for simultaneous demonstration of Estrogen receptors (ER) and argyrophilc Nucleolus-Organizer Regions (AgNORs) was developed to measure the proliferation characteristics of ER-positive and ER-negative tumour cell independently. To rule out possible reciprocal effects of the two staining procedures serial slides of 10 invasive ductale breast cancers were stained with either the single staining methods or the simultaneous ER/AgNOR-staining method and investigated comparatively. Measuring the slides by means of image analysis reciprocal effects could be excluded. It could be proved that a simultaneous staining of both markers leads to a reproducible and specific staining result. It is concluded that it is justified to measure AgNORs in immunohistochemically stained cells. Specimens of 49 invasive breast cancers were stained simultaneously for Estrogen receptors (ER) and argyrophilc Nucleolus-Organizer Regions (AgNORs). AgNORs in 100 ER-positive and 100 ER-negative tumour cell nuclei were measured by means of the image analysis system AMBA. For the quantification of AgNORs within immunohistochemically stained nuclei the measuring program AMBA\ orcolor was used. AgNOR number, area and distribution within the nucleus describe the AgNOR-configuration. Highly significant differences between ER-positive and ER-negative tumour cells were found. ER-negative breast cancer cells have a larger amount of AgNORs within their nuclei (3,06 *0,67) compared to ER-positive cells (1,65 *0,34). A special clustering phenomenon in ER-positive cells was found. Only the AgNOR-parameters of the ER-negative cell fraction were found to be associated with other indicators of malignancy (Bloom-Richardson-Grading; Growth-Fraction (Ki-67)). The ER-negative cells of breast cancer are characterised as the cell fraction with a higher proliferation activity. The results indicate that the ER-negative cells of breast cancer mainly contribute to the tumour proliferation characteristics and most likely to the progression of the tumour disease.

Submitted by PPG Odontologia (odontologia-pg@pucrs.br) on 2018-03-09T17:42:57Z No. of bitstreams: 1 RICARDO_LOSEKANN_PAIVA_TES.pdf: 1108231 bytes, checksum: a32ed38f5354b3e7c925afdd81c861e4 (MD5) / Approved for entry into archive by Tatiana Lopes (tatiana.lopes@pucrs.br) on 2018-03-15T13:00:03Z (GMT) No. of bitstreams: 1 RICARDO_LOSEKANN_PAIVA_TES.pdf: 1108231 bytes, checksum: a32ed38f5354b3e7c925afdd81c861e4 (MD5) / Made available in DSpace on 2018-03-15T13:37:24Z (GMT). No. of bitstreams: 1 RICARDO_LOSEKANN_PAIVA_TES.pdf: 1108231 bytes, checksum: a32ed38f5354b3e7c925afdd81c861e4 (MD5) Previous issue date: 2017-11-29 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Oral cytopathology may be used to monitor individuals exposed to risk factor for oral cancer. In this context, the study of field cancerization, a phenomenon involved in the initial stages of oral carcinogenesis, has gained relevance for the establishment of biomarkers that may identify individuals exposed to carcinogens with the greatest risk for developing oral cancer. The first article of this study aimed to compare cytopathological and histopathological characteristics of the clinically normal mucosa adjacent to oral squamous cell carcinoma. The nuclear area of cells obtained by cytological smear of this region was also analyzed and compared to that of individuals without lesions but exposed to smoking and/or alcohol and of patients not exposed to these risk factors. Ninety patients of both sexes over 40 years old were included. In patients with carcinoma, in addition to exfoliative cytology, tissue was obtained from the area adjacent to the tumor for histopathological examination. In smears stained with Papanicolaou, the nuclei of 50 intermediate cells were measured. Both histological sections and cytological smears were classified as low or high-risk. The sensitivity, specificity and accuracy of the cytopathological diagnosis in relation to the histopathological diagnosis, considered the gold standard, were 100, 75 and 75.86%, respectively. The mean nuclear area was significantly lower (p<0.05) in the patients not exposed to the risk factors in relation to the others. The cyto-histopathological comparison of the area adjacent to oral cancer showed good sensitivity, specificity and accuracy. In conclusion, this article demonstrated that nuclear area can be used to detect early cellular changes in the oral mucosa exposed to carcinogens and that mean percentage of nuclei larger than 100 ?m2 is the most indicated method for the assessment of these changes. The second article aimed to assess genetic damage and cell proliferation rate in the field of cancerization, i.e., the clinically normal mucosa adjacent to oral carcinoma. Cytologic smears from the same scrapes used in the first article were stained with silver and with the Feulgen reaction. The mean number of AgNORs/nucleus and micronuclei (MN) was significantly higher (p<0.05) in the Tobacco/Alcohol and Oral Cancer Groups than in the Control Group. Conversely, the mean number of NBUDs was higher in the Control Group compared with the other groups. The number of AgNORs/nucleus and MN/1,000 cells provide evidence of initial oral carcinogenesis at field cancerization areas. Cutoff values for inclusion of individuals exposed to carcinogens in longitudinal monitoring were ? 3.38 AgNORs/nucleus and/or ? 3 MN/1,000 cells. A prospective model including the biomarkers assessed in this study was proposed. / A citopatologia bucal pode ser aplicada como m?todo de monitoramento em indiv?duos expostos a fatores de risco ao c?ncer de boca. O campo de canceriza??o, representando as etapas iniciais da carcinog?nese bucal, torna-se uma ?rea atrativa ao estudo de biomarcadores que poder?o ser utilizados para identificar indiv?duos expostos a carcin?genos com maior risco ao desenvolvimento do c?ncer bucal. O primeiro artigo deste estudo visou comparar as caracter?sticas citopatol?gicas e histopatol?gicas da mucosa clinicamente normal adjacente ao carcinoma espinocelular bucal. Al?m disso, a ?rea nuclear das c?lulas obtidas desta regi?o, por meio de esfrega?o citol?gico, foi mensurada e comparada ? das c?lulas de indiv?duos sem les?o expostos ao fumo e/ou ?lcool e a de pacientes n?o expostos a estes fatores de risco. Foram inclu?dos 90 pacientes de ambos os sexos com idade superior a 40 anos. Nos pacientes com carcinoma, al?m da citologia esfoliativa, foi obtido material para exame histopatol?gico da ?rea adjacente ao tumor. Nos esfrega?os, corados com Papanicolaou, foram mensurados os n?cleos de 50 c?lulas intermedi?rias. Tanto os cortes histol?gicos, quanto os esfrega?os citol?gicos foram classificados em baixo ou alto risco. Ao associar as caracter?sticas citopatol?gicas e histopatol?gicas, verificou-se sensibilidade, especificidade e acur?cia de 100%, 75% e 75,86%, respectivamente. A m?dia da ?rea nuclear foi menor no grupo n?o exposto ao fumo e ao ?lcool, com diferen?a significativa (p<0,05) em rela??o aos demais. A associa??o cito-histopatol?gica da ?rea adjacente ao c?ncer bucal apresentou boa sensibilidade, especificidade e acur?cia. Al?m disso, constatou-se que a ?rea nuclear ? pass?vel de ser utilizada para detectar altera??es celulares precoces na mucosa bucal exposta a carcin?genos, sendo a m?dia percentual de n?cleos com mais de 100 ?m2 o m?todo de avalia??o mais indicado. O segundo artigo objetivou avaliar o dano gen?tico e a velocidade de prolifera??o celular no campo de canceriza??o, ou seja, na mucosa clinicamente normal adjacente ao c?ncer bucal. Os esfrega?os citol?gicos foram corados com a t?cnica das AgNORs e rea??o de Feulgen, utilizando o mesmo raspado citol?gico obtido no primeiro artigo. A m?dia das AgNORs/n?cleo e do micron?cleo (MN) dos grupos fumo/?lcool e carcinoma bucal foi superior, com diferen?a estatisticamente significativa (p<0,05) em rela??o ao grupo controle. Este, por sua vez, obteve m?dia superior de bot?es nucleares (NBUDs) em rela??o aos grupos fumo/?lcool e carcinoma bucal. Ambos os marcadores, n?mero das AgNORs/n?cleo e MN, evidenciaram a fase inicial da carcinog?nese bucal, representada no campo de canceriza??o. Valores iguais ou superiores a 3.38 AgNORs/n?cleo e/ou 3 MN/1000 c?lulas foram identificados como ponto de corte ideal para incluir um indiv?duo exposto a carcin?genos no monitoramento longitudinal. Um modelo prospectivo dos marcadores foi sugerido.

Made available in DSpace on 2017-07-10T14:38:24Z (GMT). No. of bitstreams: 1 Dissertacao Simone Cristina Girardi.pdf: 4377774 bytes, checksum: 366158b7c208a2a4a26392aebcbc096b (MD5) Previous issue date: 2015-02-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Pimelodidae is a family of fishes of South America, and although several taxonomic and molecular studies have been conducted, the phylogenetic relationships among the genera are not still fully understood. In order to provide data to assist in the understanding of the relationships within this family, cytogenetic studies were performed in two species of Iheringichthys and seven species of Pimelodus from three river systems. The specimens were collected in the Piquiri River, Upper Paraná River basin; in the Iguaçu River, downstream to the Iguaçu Falls in the Middle Paraná River basin; in the Iguaçu River, Lower Iguaçu River basin and in the Ijuí River, Upper Uruguay River basin. The analysis showed the presence of 2n=56 chromosomes for all species, corroborating the hypothesis of this basal diploid number for the family. The AgNORs, confirmed by 18S rDNA-FISH, were localized in the terminal position on long arm of a chromosome pair for all analyzed species, which has been reported for all species of Pimelodidae and may indicate a basal trait for the family. The heterochromatin distribution pattern found herein is similar to those described for other Pimelodidae, and allowed us to differentiate most of the species, becoming an important marker. The location of 5S rDNA sequences in Iheringichthys species allowed their differentiation, and can be used as a taxonomic marker. In Pimelodus species, it was verified a variation in the number and position of 5S rDNA sites. In P. britskii and P. maculates, sites of 5S rDNA and 18S were found in synteny, which may indicate a derived condition for these species, considering that they are the only for pimelodids species till now studied that have this feature. The results of this study provided data that contribute to the knowledge of the evolutionary history of the species for Pimelodidae; establishing phylogenetic relationships and assisting in the identification of these species. / Pimelodidae é uma família de peixes da região Neotropical, e embora vários estudos taxonômicos e moleculares tenham sido realizados, as relações filogenéticas entre seus gêneros ainda não são totalmente compreendidas. Com o intuito de fornecer dados para auxiliar no entendimento das relações dentro desta família, foram realizados estudos citogenéticos em duas espécies de Iheringichthys e em sete espécies de Pimelodus de três sistemas hidrográficos. Os exemplares foram coletados no rio Piquiri, Bacia do Alto rio Paraná; no rio Iguaçu, jusante às Cataratas do Iguaçu na Bacia do Médio rio Paraná; no rio Iguaçu, Bacia do Baixo rio Iguaçu e no rio Ijuí, Bacia do Alto rio Uruguai. As análises mostraram a presença de 2n=56 cromossomos em todas as espécies, reforçando a hipótese de número diplóide basal para a família. As AgRONs, confirmadas pela FISH-DNAr 18S, foram localizadas na região terminal do braço longo de um par de cromossomos em todas as espécies estudadas, sendo que posição terminal desta região é observada em todas as espécies de Pimelodidae e pode indicar um caracter basal da família. O padrão de distribuição de heterocromatina encontrado é semelhante ao observado em outros Pimelodidae, e permitiu diferenciar a maioria das espécies, sendo um importante marcador. A localização das sequências de DNAr 5S nas espécies de Iheringichthys permitiu diferenciá-las, podendo ser utilizado como marcador taxonômico. Em Pimelodus, variação quanto ao número e posição de sítios do DNAr 5S foi observada. Em P. britskii e P. maculatus os sítios de DNAr 5S e 18S foram localizados em sintenia, o que pode indicar uma condição derivada para estas espécies, visto que são as únicas espécies de Pimelodidae que apresentam esta característica até o momento. Os resultados do presente estudo fornecem dados que contribuem para o conhecimento da história evolutiva das espécies de Pimelodidae, permitem estabelecer relações filogenéticas e auxiliam na identificação destas espécies.