Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [105]-117).

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Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

The genome size of evolutionary polyploid, neopolyploid and hybrid sturgeons is well known for its high variability. Aim of this study was 1) to specify the genome size of polyploid and neopolyploid sturgeons using an analysis of 2-D and 3-D images of specifically stained cells nuclei, 2) to evaluate the samples of populations for cytogenetic analysis needs and thereafter, 3) to compare both methods and record the data either for next research or for negative selection from the broodstock. This test has been done at the laboratory of molecular, cellular and quantitative genetics, Faculty of Fisheries and Protection of Waters USB in Vodňany using all sturgeons spawners and using samples obtained from some foreign fish farms which cooperated with the faculty. The samples included A. ruthenus, A. baerii, A. stellatus, A. gueldenstaedtii and Huso huso, intentionally bred hybrids of A. gueldenstaedtii (8n) x A. baerii (12n), A. baerii (8n) x A. ruthenus (4n), A. gueldenstaedtii (8n) x A. baerii (10n) a A. gueldenstaedtii (8n) x A. ruthenus (4n). As methods have been chosen image cytometry and confocal microscopy which use image digitalization and subsequently its computer analysis. The genome size was measured from the size of specifically stained nuclei of erythrocytes in specimens sampled. Result of this study was measuring the genome size in sturgeons under study using different methods, recording the obtained data, description of spatial conformation changes of cell nucleus with increasing ploidy level and deduction of impact to their physiology and comparing the methods between each other. The conclusion is necessity of another sturgeons genome size determination , choice of the best methods for more effective search and research of non-standard individuals and subsequently an examination of their physiological differences.

Made available in DSpace on 2015-03-26T13:42:10Z (GMT). No. of bitstreams: 1 texto completo.pdf: 575171 bytes, checksum: 3cf08bba8bb6a93eab8dbf41ef32bd01 (MD5) Previous issue date: 2006-02-03 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Capsicum genus is one of the most important members of the Solanaceae family with high economic value. Out of 25 known species, five are extensively cultivated. Capsicum annuum is the most important commercially by its agronomic value as spice. In despite of this importance, the nuclear DNA content of C. annuum is still uncertain with values from the literature ranging from 5.52 to 10.80 pg. In face of the fast development of digital image technologies in cytometry, this study proposes the utilization of these resources to reevaluate the genomic size in C. annuum. The specific objectives were: (i) to determine the reliability parameters for image cytometry analysis by performing tests of equipment stability, linearity and uniformity of nuclei integrated optical density; (ii) to apply that parameters in analysis of erythrocytes nuclei from Gallus domesticus 'Leghorn' (male and female); and (iii) to develop image cytomery methodologies for determination of the nuclear DNA content of pepper (C. annuum L. 'Fortuna Super'). Hen and rooster blood samples were prepared by using the method of smear preparation. The slides were stained with Feulgen reaction and they were used to perform the quality control procedures and integrated optical density measurements. Root tips of C. annuum were fixed, stained with Feulgen reaction and squashed on a microscope slide. Once the parameters of the reliability for performing image cytometry were established, the application of those standardizations in the chicken red blood cells suggested that this technique allows identification of differences in the nuclear DNA content between rooster and hen. Analysis of variance demonstrated that the 4.32% difference obtained was significant and it was attributed to sexual chromosomes heteromorfism. C. annuum analyses allowed the calculation of integrated optical density values, and they were in iccordance with the international standards for DNA image cytometry. The 2C DNA value for C. annuum 'Fortuna Super', using Pisum sativum as the internal standard, was 8.10 pg. This data differs in 2% from the previously obtained by flow cytometry for the same plant population. Besides, the value obtained in this study differs in16% and 28% from those 2C values previously published by image cytometry and in 7% and 32% for flow cytometry. These results include information about C. annuum genome that can be useful in breeding programs, genome sequencing projects and for comparative studies of genome evolution. / Capsicum é um dos gêneros mais importantes economicamente da família Solanaceae. Das 25 espécies descritas, cinco são extensivamente cultivadas. Capsicum annuum é a mais importante comercialmente, pelo seu valor agronômico como especiaria. Apesar da sua importância, valores de conteúdo de DNA conflitantes, que variam de 5,52 a 10,80 pg, têm sido descritos para essa espécie. Diante dos avanços dos equipamentos digitais nas análises de citometria de imagem, o presente trabalho propôs o estabelecimento desses recursos para reavaliar o tamanho do genoma em C. annuum. Os objetivos específicos foram: (i) o estabelecimento de parâmetros de confiabilidade para análises em citometria de imagem, por meio da realização de testes de estabilidade do equipamento, linearidade e uniformidade da densidade óptica integrada dos núcleos; (ii) aplicação dessa calibração em núcleos de eritrócitos de Gallus domesticus Leghorn (macho e fêmea); e (iii) desenvolvimento de metodologias de citometria de imagem para determinação do conteúdo de DNA em pimentão (C. annuum L. Fortuna Super ). Lâminas de esfregaço de sangue de galinha e galo submetidas à reação de Feulgen foram utilizadas nos testes de calibração e para a determinação da densidade óptica integrada. As preparações citogenéticas de pimentão foram feitas pela técnica de esmagamento de meristemas radiculares submetidos à reação de Feulgen. Uma vez estabelecidos os parâmetros de confiabilidade para a realização da citometria de imagem, a aplicação dessas padronizações em análises de núcleos de eritrócitos de ave permitiu identificar diferenças de 4,32% no conteúdo de DNA nuclear entre machos e fêmeas de G. domesticus. Análise de variância demonstrou que a diferença encontrada era significativa, e essa foi atribuída ao heteromorfismo dos cromossomos sexuais. As análises de citometria de imagem em C. annuum permitiram o cálculo da densidade óptica integrada, atendendo aos parâmetros internacionalmente adotados para esse tipo de estudo. O conteúdo de DNA estimado para C. annuum Fortuna Super , utilizando Pisum sativum Ctirad com o padrão interno, foi de 8,10 pg. Esse valor difere em 2% do obtido previamente por citometria de fluxo, utilizando-se material nuclear foliar da mesma população de plantas. Além disso, o valor obtido difere em 16% e 28% dos valores 2C obtidos por citometria de imagem descritos na literatura e em 7% e 32% dos relatados por citometria de fluxo. Esses resultados abrangem informações acerca do genoma dessa espécie, que podem ser úteis em programas de melhoramento e seqüenciamento e em estudos evolutivos.

Background: The accuracy of DNA image cytometry as an investigation method for potentially malignant disorders of the oral cavity is currently still a subject of controversy, due to inconsistently applied definitions of DNA aneuploidy, small cohorts and different application techniques of the method. The aim of this study was to examine the accuracy of the method as a supplementary diagnostic tool in addition to the cytological examination using internationally consented definitions for DNA aneuploidy. Methods: A total of 602 samples from 467 patients with various oral lesions were included in this prospective study. Brush biopsies from each patient were first cytologically examined and categorized by a pathologist, second evaluated using DNA image cytometry, and finally compared to either histological biopsy result or clinical outcome. Results: Using the standard definition of DNA aneuploidy, we achieved a sensitivity of 93.5%, a positive predictive value for the detection of malignant cells of 98.0%, and an area under the curve of 0.96 of DNA ploidy analysis for the detection of severe oral epithelial dysplasia, carcinoma in situ or oral squamous cell carcinoma. Importantly, using logistic regression and a two-step model, we were able to describe the increased association between DNA-ICM and the detection of malignant cells (OR = 201.6) as a secondary predictor in addition to cytology (OR = 11.90). Conclusion: In summary, this study has shown that DNA ploidy analysis based on conventional specimens of oral brush biopsies is a highly sensitive, non-invasive, patient-friendly method that should be considered as an additional diagnostic tool for detecting malignant changes in the oral cavity.

<p>Receptor epidermalnog faktora rasta (EGFR) pripada porodici receptora protein-tirozin kinaze čija je aktivacija povezana sa proliferacijom malignih, invazijom, inhibicijom apoptoze, tumorskom angiogenezom i metastatskim &scaron;irenjem stoga ima važnu ulogu u karcinogenezi i tumorskoj progresiji. Aktivirane mutacije se odvijaju oko katalitičkog tirozin kinaza domena. Biopsijski, citolo&scaron;ki i hirur&scaron;ki uzorci se koriste u detekciji EGFR mutacija u momentu postavljanja dijagnoze adenokarcinoma ili karcinoma sa komponentom adenokarcinoma, najpouzdanije lančanom reakcijom polimeraze. Činjenica da primena ciljane molekularne terapije tirozin kinaza inhibitorima kod obolelih sa EGFR mutiranim adenokarcinomom pluća pobolj&scaron;ava prognozu bolesti, postoji rezistencija kod pojedinih tipova EGFR mutacija i povezanost histopatolo&scaron;kim i imunohistohemijskim karakteristikama tumora, da je bronholo&scaron;ki uzorak često jedini uzorak u kome je potrebno odrediti i molekularni profil tumora osnovni cilj ove disertacije bio je da se odredi učestalost i tip EGFR mutacija i povezanost sa karakteristikama adenokarcinoma. Da bi se taj cilj realizovao postavljeni su sekundarni ciljevi odnosno da se: izvr&scaron;i histopatolo&scaron;ka reklasifikacija adenokarcinoma pluća na osnovu kriterijuma koje je postavila internacionalna asocijacija za proučavanje carcinoma pluća, američko torakalno dru&scaron;tvo i evropsko respiratorno dru&scaron;tvo; odredi ekspresija TTF-1 u adenokarcinomu pluća i povezanost sa EGFR mutacionim statusom; odredi učestalost, tip i povezanost EGFR mutacija sa predominantnim tipom adenokarcinoma i utvrdi da li bronhoskopska biopsija može da bude reprezentativni uzorak za određivanje EGFR mutacionog statusa. Histopatolo&scaron;ka građa adenokarcinoma pluća u hirur&scaron;kim uzorcima je heterogenija u odnosu na biopsijske uzorke i ta razlika je statistički značajna (p&lt;0,001). Acinarno predominantni tip je najzastupljeniji u hirur&scaron;kim i biopsijskim uzorcima bez statistički značajne razlike u raspodeli predominantnih tipova u njima (p=0,65883). Predominantni tip u primarnom tumoru određuje predominantni tip u limfogenim metastazama. EGFR mutacije tipa insercija na egzonu 21 i L858R mutacija na egzonu 20 su detektovane kod tri od 60 (5%) bolesnika u pet od 120 uzoraka (tri hirur&scaron;ka i dva biopsijska uzorka), če&scaron;će kod žena, starijih od 60 godina, pu&scaron;ača i u solidno predominantnom tipu. Ne postoji statistički značajna razlika u koncentraciji izolovane DNK između EGFR mutiranih i wt EGFR adenokarcinoma u biopsijskim (p=0,132) i hirur&scaron;kim uzorcima (p=0,641). Procenat invalidnih rezultata prilikom određivanja EGFR mutacionog statusa u je veći u biopsijskim uzorcima u odnosu na hirur&scaron;ke uzorke. Postoji statistički značajna razlika izmeĐu broja TTF-1 pozitivnih i TTF-1 negativnih adenokarcinoma (p&lt;0,001), ali ne i u raspodeli ovih bolesnika prema polovima (p=0,1231), prosečnoj starosti, pu&scaron;ačkim navikama (p=0,6488) i prosečnoj veličini tumora (p=0,21). Postoji pozitivna korelacija između TTF-1 pozitivne ekspresije i EGFR mutacionog statusa stoga TTF-1 pozitivna ekspresija može da bude prediktor pozitivnog EGFR mutacionog statusa. Bronhoskopska biopsija je reprezentativni uzorak za određivanje EGFR mutacionog statusa zato &scaron;to: većina dijagnostičkih biopsijskih uzoraka ima vi&scaron;e od 100 očuvanih tumorskih ćelija, nema razlike u raspodeli predominantnih tipova u odnosu na hirur&scaron;ke uzorke, EGFR mutacije se detektuju u uzorcima sa manje od 100 tumorskih ćelija i manje od 20% volumenske gustine tumorskog tkiva, razlika između koncentracije izolovane DNK u EGFR mutiranim i wt EGFR adenokarcinomima u biopsijskim i hirur&scaron;kim uzorcima nije statistički značajna (p=0,132 i p=0,641).</p> / <p>Epidermal growth factor receptor (EGFR) belongs to the family of protein-tyrosin kinase family, whose activation is associated with the proliferation of malignant cells, invasion, inhibition of apoptosis, tumor angiogenesis and metastatic spread and thus plays an important role in carcinogenesis and tumor progression. Activated mutations take place around the catalytic tyrosine kinase domain. Biopsy, cytological and surgical specimens are used for the detection of EGFR mutations at the time of diagnosis of adenocarcinoma or carcinoma with an adenocarcinoma component, most reliably using a polymerase chain reaction. The fact that the application of molecular tyrosin kinase inhibitor therapy to patients with EGFR mutated lung adenocarcinoma improves the prognosis of the disease, there is resistance in certain types of EGFR mutations and connection with histopathological and immunohistochemical characteristics of tumor, that the bronchoscopic specimen is often the only specimen in which it is necessary to determine the molecular profile of the tumor, the primary objective of this thesis is to determine the frequency and type of EGFR mutations and their connection with the characteristics of adenocarcinoma. In order to realize this objective, the following secondary objectives have been set: to execute histopathological reclassification of lung adenocarcinoma based on the criteria set by the International Association for the Study of Lung Cancer, the American Thoracic Society and European Respiratory Society; determine the expression of TTF-1 in lung adenocarcinoma and connection with EGFR mutation status; determine the frequency, type and connection of EGFR mutations with predominant type of adenocarcinoma and confirm whether bronchoscopic biopsy may be a representative specimen for the determination of EGFR mutation status. Histopathological material of lung adenocarcinoma in surgical specimens is more heterogeneous in relation to biopsy specimens and such difference is statistically significant (p&lt;0,001). Acinar predominant type is the most common in surgical and biopsy specimens with no statistically significant differences in the distribution of predominant type among them (p=0,65883). The predominant type in the primary tumor determines the predominant type in lymphatic metastases. EGFR mutations in the type of insertions on exon 21 and L858R mutations on exon 20 have been detected in three out of 60 (5%) of patients in five out of 120 specimens (three surgical and two biopsy samples), more often in women older than 60, smokers and in a solid predominant type. There are no statistically significant differences in the concentration of isolated DNA between EGFR mutated and wt EGFR adenocarcinoma in biopsy (p=0,132) and surgical specimens (p=0,641). The percentage of invalid results in determining the EGFR mutation status is higher in biopsy specimens compared to the surgical specimens. There is a statistically significant difference between the number of TTF-1 positive and TTF-1 negative adenocarcinoma (p&lt;0,001), but not in the distribution of these patients according to gender (p=0,1231), average age, smoking habits (p=0,6488) and average tumor size (p-0,21). There is a positive correlation between TTF-1 positive expression and EGFR mutation status and therefore TTF-1 positive expression can be a predictor of positive EGFR mutation status. Bronchoscopic biopsy is a representative sample for the determination of EGFR mutation status because: most diagnostic biopsy specimens have more than 100 preserved tumor cells, there is no difference in the distribution of predominant types in relation to surgical specimens, EGFR mutations are detected in samples with less than 100 tumor cells and less than 20% of volume density of tumor tissue, the difference between the concentration of isolated DNA in EGFR mutated and wt EGFR adenocarcinoma in biopsy and surgical specimens is not statistically significant (p=0,132 and p=0,641).</p>