Orientador: Júlio César Teixeira / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T00:35:59Z (GMT). No. of bitstreams: 1 Paes_ElianaFerreira_M.pdf: 2469476 bytes, checksum: 7fdb355ac120c70d0bffe635016ba6af (MD5) Previous issue date: 2015 / Resumo: Introdução: O agente etiológico do câncer do colo do útero é um HPV de alto risco (hrHPV) e testes biomoleculares para detecção deste vírus em amostras do colo do útero tendem a ter um papel cada vez mais importante no rastreamento de lesões pré-câncer para o futuro. No momento, estes testes são de alto custo e de execução complexa. Objetivo: desenvolver e padronizar um teste de PCR multiplex marcado com fluorescência para detecção de DNA de hrHPV em amostras obtidas do colo do útero e comparar com um teste de referência. Metodologia: foi realizado um estudo piloto para descrição e padronização de uma metodologia de detecção e genotipagem de HPV, tipo PCR multiplex, com primers desenhados com base na região E7 de seis hrHPV (16, 18, 31, 33, 45 e 52), o teste `E7-HPV¿. Foi seguido um guia internacional para desenvolvimento de novos testes de HPV para rastreamento, que orienta uma avaliação inicial de 50 ou mais amostras de mulheres com NIC2+, comparação com um teste referência validado, e alcançar um índice de concordância kappa de 0,7, com sensibilidade de 90% da sensibilidade do teste referência. O teste referência adotado foi o cobas® HPV Test da Roche Diagnostics, chamado `cobas®¿, que identifica em grupo 12 hrHPV e genotipa os tipos 16 e 18, separadamente. Neste estudo foram utilizadas amostras de 60 pacientes, 55 com citologia ASCH/HSIL, coletadas entre Agosto e Setembro de 2013, que foram avaliadas por cada teste com cálculo da concordância dos resultados pelo índice de kappa e comparação da sensibilidade, com poder estatístico de 90%. Resultados: o teste teve sua descrição e padronização finalizada. Houve alta concordância entre os testes `E7-HPV¿ e cobas® na detecção do HPV 16 com kappa 0,972, com apenas um caso discordante, de paciente com NIC3 com cobas® negativo e com o teste `E7-HPV¿ positivo. Para o HPV18, os resultados dos testes demonstraram concordância total, com índice kappa=1,0. Quando comparadas as detecções dos tipos hrHPV não 16 e 18, seis casos foram positivos para o cobas® e negativos para o teste `E7-HPV¿, podendo ser decorrente da detecção do cobas® de tipos de HPV não contemplados no teste `E7-HPV¿. Ao contrário, o teste `E7-HPV¿ detectou dois hrHPV adicionais, com cobas® negativo (HPV31 e 52). A sensibilidade de detecção de NIC2+ foi igual para os dois testes avaliados. Conclusão: Foi possível desenvolver e padronizar uma técnica de PCR multiplex com metodologia para detecção de seis hrHPV. O desempenho do teste `E7-HPV¿ foi, pelo menos, equivalente ao teste referência (cobas® HPV Test) com kappa=0,972 e 100% da sensibilidade do teste referência na detecção de NIC2+ em pacientes sabidamente com lesões, superando os pré-requisitos necessários. Assim, os resultados satisfatórios do `E7-HPV¿ na primeira fase de avaliação possibilita a continuidade do desenvolvimento do mesmo, visando futura aplicação em maior escala / Abstract: Introduction: the etiologic agent of cervical cancer is a high-risk HPV (hrHPV) and biomolecular tests for detection of this virus in cervical samples tend to have an increasingly important role in screening for precancerous lesions for the future. At present, these tests are expensive and complex procedure. Purpose: to develop and standardize a multiplex PCR test, 'E7-HPV' test, to detect and genotyping of 6 hr-HPV DNA, tested in samples obtained from the cervix and compared with a reference test. Methods: A pilot study was performed to develop and standardize a methodology of the multiplex PCR with primers designed on the basis of six hrHPV E7 region (16, 18, 31, 33, 45 and 52) and labeled with fluorochrome 6-FAM. Viral detection was performed by capillary electrophoresis in automated sequencer. It was followed an international guide to development of new HPV testing for screening, which guides an initial assessment of 50 or more samples from women with CIN2+, and the results compared to a validated reference test to achieve a kappa index of 0.7, and 90% sensitivity of reference sensitivity test. The reference test adopted was the cobas® HPV Test from Roche Diagnostics, called 'cobas®', which identifies 12 hrHPV pooled and genotype HPV 16 and 18 separately. In this study, cervix samples were obtained from 60 women, 55 with ASCH/HSIL cytology, collected between August to September 2013. The samples were evaluated by each test, and a non-inferiority analysis was conducted in relation to the cobas® HPV Test, following international guidelines for the development of new tests with kappa index and sensitivity, with a statistical power of 90%. Results: The methodology of E7-HPV test was described and standardized. The 'E7-HPV' test and cobas® Test had a high concordance rate in HPV16 detection (kappa=0.972), with only one discordant case (CIN3 with negative cobas® and `E7-HPV¿ HPV16 positive) and total concordance in HPV18 detection (kappa=1.0). When comparing the detection of hrHPV not 16/18, six cases were positive for cobas® and negative for 'E7-HPV', maybe due to the detection for cobas® of HPV types not included in the 'E7- HPV ' test. Others three cases were `E7-HPV¿ positive (HPV16, 31 and 52) with negative cobas®. For diagnosis of CIN2 or worse, both tests had the same sensitivity. Conclusion: It was possible to develop and standardize a multiplex PCR methodology for detection and genotyping 6 hrHPV in cervical samples. The performance of 'E7-HPV' test was at least equivalent to the reference test for the detection of CIN2+, fulfilling the clinical validation criteria requirements. Thus, the satisfactory initial results of the 'E7-HPV' test evaluation indicate that the development can be continued, aiming future application on a larger scale / Mestrado / Oncologia Ginecológica e Mamária / Mestra em Ciências da Saúde

<p>Uprkos postojanju dijagnostičkih procedura za ranu dijagnostiku, sprovođenju preventivnog skrining programa, te dugom preinvazivnom periodu u kojem je moguće izvr&scaron;iti detekciju, karcinom grlića materice i dalje predstavlja jedan od vodećih zdravstvenih problema, imajući u vidu činjenicu da je uzrok obolevanja i umiranja velikog broja žena &scaron;irom sveta. Oko 85% karcinoma grlića materice dijagnostikuje se u zemljama u razvoju, čineći čak 15% svih malignih tumora kod žena, dok u razvijenim zemljama ovaj procenat iznosi 3,6% novootkrivenih karcinoma. Prema podacima Registra za rak Vojvodine, u AP Vojvodini u 2011. godini, od ukupnog broja novoregistrovanih malignih bolesti kod žena, karcinom grlića materice se nalazio na četvrtom mestu, sa procentualnim uče&scaron;ćem od 6,66%, dok je najveći broj novoobolelih slučajeva u starosnoj grupi 45&ndash;49 godina, pokazujući trend pomeranja prema mladjim starosnim grupama. Primena novih, savremenih dijagnostičkih metoda u inicijalnom zbrinjavanju obolelih od karcinoma grlića materice može doprineti pravilnijem tretmanu bolesti, utičući na tok bolesti, ishod i preživljavanje. PET/CT je hibridna imidžing metoda koja poslednjih godina zauzima značajno<br />mesto u određivanju stadijuma malignih bolesti i dijagnostikovanju recidiva, a kojom se stiče uvid u metaboličko/funkcionalni status ispitivanih tkiva i organa, superponiran na morfoanatomsku CT sliku. Kako je za cilj istraživanja postavljeno utvrđivanje značaja PET/CT pregleda u preoperativnoj evaluaciji karcinoma grlića materice, ispitanice koje su ispunjavale kriterijume za uključivanje u istraživanje su operisane nakon načinjenog PET/CT pregleda, a zatim su poređeni parametri dobijeni kliničkim pregledom, PET/CT pregledom, intraoperativnim pregledom i patohistolo&scaron;kim pregledom. Rezultati načinjenog istraživanja ukazuju da hibridni PET/CT imidžing ne demonstrira prednost u odnosu na dosada&scaron;nje preporuke vezane za preoperativnu dijagnostičku evaluciju ispitanica sa ranim stadijumom karcinoma grlića materice, naročito u detekciji postojanja primarnog tumora dimenzija manjih od 7mm i u detekciji prisustva mikrometastaza u lokoregionalnim limfnim čvorovima zbog jo&scaron; uvek ograničene prostorne rezolucije. PET/CT je pokazao izuzetno visoku specifičnost u utvrđivanju nepostojanja primarnog tumora i visoku komplementarnost sa patohistolo&scaron;kim nalazom. Uočeno je da PET/CT demonstrira statistički značajniju tačnost u proceni stepena pro&scaron;irenosti osnovne bolesti u odnosu na kliničko i intraoperativno utvrđivanje stepena pro&scaron;irenosti osnovne bolesti. Standardizovana vrednost preuzimanja radiofarmaka (SUV) zavisna od histolo&scaron;kog tipa karcinoma grlića materice, demonstrirajući najvi&scaron;e vrednosti u planocelularnom karcinomu i u lo&scaron;e diferentovanom tip G3 karcinomu grlića materice, pozitivno korelirajući sa promerom tumora. Pokazujući značajan potencijal u detekciji primarnog karcinoma grlića materice, kao i u detekciji metastaza u lokoregionalnim limfnim čvorovima, PET/CT nalazi svoje mesto u dijagnostičkoj obradi bolesnica sa karcinomom grlića materice.</p> / <p>Despite the existence of the diagnostic procedures for early diagnosis, implementing preventive screening programs and long preinvasive period in which it is possible to perform detection, cervical cancer remains one of the leading health problems, bearing in mind the fact that it is the cause of morbidity and mortality of a large number of women throughout the world. About 85% cases of cervical cancer are diagnosed in underdeveloped and developing countries. In economically underdeveloped countries, cervical cancer accounts for 15% of all malignant tumors in women, whereas in developed countries it is 3.6% of new cancers. According to data from the Cancer Registry of Vojvodina, in AP Vojvodina, in 2011, of the total number of newly registered malignant diseases in women, cervical cancer was in fourth place, with a percentage share of 6.66% , the largest number of new cases is in the 45-49 years age group, with the shifting the peak in the incidence of cervical cancer towards younger age groups. Applying of new diagnostic methods in the initial management of patients with cervical cancer, indirectly affect the course of the disease, its treatment, and survival. PET-CT hybrid imaging method in recent years occupies an important place in the staging of malignant disease and the diagnosis of recurrence, showing the functional state of certain tissues and organs (PET) with anatomical details (CT). As the aim of this study was to assess the importance of PET / CT scans in the preoperative evaluation of cervical cancer, the women who met the criteria were included in the study, and were operated after having PET / CT scans done, after which the parameters of the results of clinical examination, PET / CT examination, intraoperative examination and histopathological examination, were compared. It was observed that the hybrid PET / CT imaging does not demonstrate the advantage compared to the previous recommendations related to the preoperative diagnostic evaluation of patients with early-stage cervical cancer, especially in the detection of primary tumor measuring less than 7mm and the detection of the presence of micrometastases in the locoregional lymph nodes, due to the still limited spatial resolution. PET / CT showed extremely high specificity in determining the absence of primary tumor and high complementarity with histopathological findings. The study showed that PET / CT demonstrates statistically more significant accuracy in the assessment of the extent of the underlying disease, compared with the clinical and intraoperative determination of the extent of the underlying disease. It has been shown that the standardized uptake value of radiopharmaceuticals (SUV) depends on the histological type of cervical cancer, demonstrating the highest values in squamous-cell carcinoma and then in poorly differentiated G3 carcinoma of the cervix, positively correlating with the diameter of the tumor. Demonstrating significant potential in the detection of primary cervical cancer, as well as in the detection of metastases in locoregional lymph nodes, PET / CT has its place in diagnostic treatment of patients with cervical cancer.</p>

Die in der Bundesrepublik Deutschland übliche Therapie für das Zervixkarzinom der FIGO-Stadien IB-IIB ist die Wertheim-Meigs-Operation. Bei bestimmten Risikofaktoren wird häufig eine adjuvante Bestrahlung, gegebenenfalls eine postoperative Radioche-motherapie angeschlossen. Die Folge können zahlreiche Einschränkungen der gesund-heitsbezogenen Lebensqualität sein. Zur Verbesserung der postoperativen gesund-heitsbezogenen Lebensqualität wurde eine neue nervenschonende Operationsmetho-de, die totale mesometriale Resektion (TMMR), von Höckel und Kollegen der Universi-tätsfrauenklinik Leipzig entwickelt. Die vorliegende retrospektive Querschnittsstudie untersucht erstmals die Auswirkungen der TMMR im Vergleich zur Wertheim-Meigs-Operation in Bezug auf die postoperative gesundheitsbezogene Lebensqualität. Dazu wurden 110 Zervixkarzinompatientinnen mit der Hilfe der Fragebögen EORTC-QLQ-C30 und EORTC-QLQ-CX24 befragt. Die Ergebnisse dieser Pilotstudie deuten daraufhin, dass Patientinnen nach einer TMMR-Operation im Vergleich zu Frauen nach einer Wert-heim-Meigs-Operation in einigen Teilaspekten eine bessere Lebensqualität haben. So fanden sich bezüglich der postoperativen körperlichen Funktionsfähigkeit und der Rol-lenfunktion sowie der postoperative Ausprägung der Symptome Fatigue, Schmerzen, Diarrhö, Appetitlosigkeit und Dyspnoe in der vorliegenden Untersuchung signifikant bessere Werte in der Gruppe der mittels TMMR operierten Frauen im Vergleich zur Wertheim-Meigs-Gruppe. Dies kann zum Anlass genommen werden große multizentri-sche prospektive Studien durchzuführen.:BIBLIOGRAPHISCHE BESCHREIBUNG - 5 - ABKÜRZUNGSVERZEICHNIS - 6 - 1 EINLEITUNG - 7 - 2 KLINISCHE UND MEDIZINPSYCHOLOGISCHE GRUNDLAGEN - 9 - 2.1 DAS ZERVIXKARZINOM - 9 - 2.2 DIE WERTHEIM-MEIGS-OPERATION - 12 - 2.3 NERVENSCHONENDE OPERATIONSMETHODEN - 13 - 2.4 EINE NEUE OPERATIONSTECHNIK: DIE TOTALE MESOMETRIALE RESEKTION (TMMR) - 14 - 2.5 MORBIDITÄT UND LEBENSQUALITÄT - 16 - 2.5.1 MORBIDITÄT UND PATHOPHYSIOLOGIE DER ZERVIXKARZINOMTHERAPIE - 16 - 2.5.2 DER EINFLUSS DER THERAPIE AUF DIE LEBENSQUALITÄT BEI ZERVIXKARZINOMPATIENTINNEN - 18 - 2.5.3 WEITERE EINFLUSSFAKTOREN SOWIE INTERAKTIONEN DER LEBENSQUALITÄT - 26 - 2.6 FAZIT - 28 - 3 FRAGESTELLUNG - 30 - 4 METHODIK - 31 - 4.1 STUDIENBESCHREIBUNG - 31 - 4.2 PATIENTINNENKOLLEKTIV - 32 - 4.3 ERHEBUNGSINSTRUMENTE - 34 - 4.4 STATISTISCHE ANALYSE - 36 - 5 ERGEBNISSE - 37 - 5.1 STICHPROBENCHARAKTERISTIKA - 37 - 5.2 GESUNDHEITSBEZOGENE LEBENSQUALITÄT - 41 - 5.2.1 ERGEBNISSE DES GRUPPENVERGLEICHS IM ÜBERBLICK - 41 - 5.2.2 ERGEBNISSE DES GRUPPENVERGLEICHES IM DETAIL - 44 - 6 DISKUSSION - 52 - 6.1 ALLGEMEINE DISKUSSION - 52 - 6.2 DISKUSSION DER EINZELNEN LEBENSQUALITÄTSEBENEN - 53 - 6.3 METHODENKRITISCHE DISKUSSION - 65 - 6.4 FAZIT UND AUSBLICK - 67 - 7 ZUSAMMENFASSUNG - 69 - LITERATURVERZEICHNIS - 72 - TABELLEN- UND ABBILDUNGSVERZEICHNIS - 78 - ANLAGEN - 79 - SELBSTSTÄNDIGKEITSERKLÄRUNG - 86 - WISSENSCHAFTLICHE VERÖFFENTLICHUNG - 88 - DANKSAGUNG - 89 -

The thesis evaluates the results of experimental protocol involving the fertility sparing treatment procedure in early stage cervical carcinoma (LAP I protocol). Sentinel lymph node detection and experimental extirpation of afferent channels using laparoscopy and its technical aspects were analysed in prospective group of 85 women. The oncologic results and early/late morbidity show that established surgical procedures can be considered safe with minimal morbidity, provided that the indication criteria are met. The second part analyses the results of 148 women with no further pregnancy plans suffering from cervical tumors less than 2 cm in size with invasion less than half of the stroma (LAP II protocol). The oncological results in our defined group are very good and comparable to 'standard' procedure of modified radical hysterectomy type B or C with lower morbidity. In the separate section the thesis analyses the possibilities of laparoscopy in endometrial cancer treatment including the potentials of use of sentinel lymph node detection and technical aspects of laparoscopy in obese women. Currently the biggest controversy is the use of laparoscopy in malignant ovarian tumors. Our oncogynaecological study group at FN Motol prefers the laparotomic approach and we chose to include the set of advanced...

A model was developed in this study to understand women's cervical screening behaviour. It revealed that the interaction among institutional factors, risk appraisal, coping appraisal, and health beliefs and cultural factors contributed to the complex nature of screening behaviour among Chinese women. The institutional component provided the contextual factors within which women perceived the risk of cervical cancer, perceived the practice of cervical screening, and decided to take or not to take cervical screening. Risk appraisal provided the premise factor that induces women to seek coping strategies to reduce or remove the risk. During the process of coping appraisal, women's motivation to have cervical screening could be increased or decreased as the perceived benefits and costs of screening interacted with each other. The importance of the women's health beliefs and cultural factors was reflected in the way that they were affected by their notions of health behaviour and their cultural beliefs about cervical cancer risk and cervical screening participation. Commitment to participate in screening was a reinforcing factor inducing women to take up an offer of cervical screening. / Aim: To explore the knowledge and the perception of the risk of cervical cancer, identify the factors determining cervical screening behaviour, and develop a model to understand cervical screening behaviour among women in mainland China. / Background: Cervical cancer is the most common type of cancer, and is the second most common cause of cancer death in women in mainland China. Cervical screening is the most important intervention for the secondary prevention of cervical cancer. Theories of health behaviour and empirical research highlight risk perception as a significant factor motivating people to opt for cancer screening. However, little is known about the risk perception of cervical cancer and the factors influencing the screening participation of women in mainland China. / Conclusion: This study provides evidence of the complex factors influencing cervical screening behaviour and contributes new knowledge to the understanding of cervical screening behaviour within the Chinese cultural context. It further informs programmes for the promotion of cervical screening among this population. / Methods: A mixed method design consisting of two phases was used, employing both quantitative and qualitative methods of data collection. First, a cross-sectional survey was conducted to collect a baseline assessment of women's knowledge of cervical cancer and screening, their perceptions of the risk of cervical cancer, and the relationship between these factors and their cervical screening behaviour. Findings from this phase also guided the purposive sampling of participants in phase two. / Results: The findings from phase one demonstrated that the availability of an organized screening programme was a major motivator for women to opt for cervical screening. Multivariate analysis shows that having children (OR=2.57, p=0.026), a perception that visiting doctors regularly is important for health (OR-2.66, p=0.025), average (OR-4.84, 1)=0.006) and high levels of knowledge about cervical screening (OR-9.66, p=0.001) were significantly associated with having been screened in the previous three years. / Then in phase two, qualitative research was conducted using semi-structured interviews of 27 women, 16 of whom had been screened and 11 had not. The interview structure was based on an initial analysis of the data from phase one and from a review of the related literature. The data from the interviews were analyzed using latent content analysis, involving an interpretative reading of the symbolism underlying the surface structure in the text. The audio recordings of the interviews were transcribed verbatim in Chinese, and then the key phrases which were important for the objectives of the study were identified. The key phrases and words were grouped according to their commonality of meaning. Then, these groups of data were sorted and classified to create categories and sub-categories, which were mutually exclusive, explicit and accurate without overlapping. / Two themes emerged from the qualitative data from phase two. Theme I was that perceptions of cervical cancer and cervical screening included five categories: the perceived effects of suffering from cervical cancer; the perception of cervical screening; a lack of understanding about cervical cancer and screening; the perceived risk of cervical cancer; and factors related to the cultural beliefs system. Theme II was that the institutional and health care practitioner system included two categories: availability of an organised physical examination programme and the role of the health care practitioner in encouraging cervical screening utilization. / Gu, Can. / Adviser: Chan, Carmen. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 244-267). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

O câncer do colo do útero é o quarto tipo de câncer mais frequente em mulheres em todo mundo. No Brasil, estima-se que cerca de 16 mil novos casos ocorrem por ano. A redução deste tipo de câncer ao longo dos anos deve-se ao rastreamento das lesões intraepiteliais cervicais por meio do exame citológico de Papanicolaou. Em 2014, o Programa Nacional de Imunização (PNI) introduziu a vacina contra o papilomavírus humano (HPV) como prevenção primária deste câncer, uma vez que este vírus é uma causa necessária para o surgimento desta malignidade. A vacinação não substitui o rastreamento, visto que não há proteção contra todos os tipos de HPV de alto risco e nem imunização de toda a população. A incorporação do programa de vacinação interfere nos resultados do programa de rastreamento, pois leva a diminuição dos casos de câncer e lesões precursoras. Desta forma, existe a necessidade de explorar novas estratégias de rastreamento, considerando também outras tecnologias existentes. Objetivo: desenvolver um modelo do tipo Markov para realizar uma análise de custo-efetividade de estratégias de rastreamento do câncer do colo do útero para hipotéticas coortes imunizadas e não imunizadas contra o vírus do HPV no Brasil na perspectiva do Sistema Único de Saúde (SUS). Métodos: A primeira parte é a exploração e avaliação qualitativa de estudos de avaliação econômica sobre estratégias de rastreamento para prevenção do câncer do colo do útero que utilizaram um modelo do tipo Markov feita por meio de uma revisão sistemática. A reunião das várias abordagens utilizadas e das principais características destes modelos poderá auxiliar a construção de um modelo em cenários onde há poucos profissionais capacitados com esta técnica. Baseando-se nesta revisão e nas consultas a especialistas das áreas de ginecologia, virologia e epidemiologia, foi desenvolvido um modelo matemático de análise de decisão estático do tipo Markov que simula a história natural do câncer do colo do útero considerando a imunização contra o HPV. Este modelo simula o seguimento de uma coorte de mulheres, dos 10 anos até o óbito, cujos parâmetros foram estimados a partir de dados secundários (revisão da literatura, sistemas de informação em saúde e inquéritos populacionais) nacionais específicos do rastreamento e calibrados de forma a refletir as condições reais de rastreamento encontradas no Brasil. Resultados: A revisão dos modelos de Markov para avaliação econômica de estratégias de rastreamento do câncer do colo do útero mostrou que a declaração do problema e a descrição das estratégias a serem comparadas foram muito bem relatados. Em contrapartida, os itens de avaliação da incerteza e consistência do modelo e a consistência precisam melhorar o relato. Os resultados obtidos por meio da calibração do modelo se mostraram satisfatórios, pois alcançaram uma boa concordância com os dados empíricos. A análise do caso base sugeriu que a melhor estratégia foi o Teste HPV-DNA como triagem para o encaminhamento da citologia ou da colposcopia, com repetição a cada 5 anos, para mulheres entre 30 e 70 anos. Esta estratégia promove um ganho de 9,5 dias ao longo dos anos e detecta, a cada 100 mil mulheres, 6 casos a mais de câncer e 16 de NIC II/III. A razão de custo-efetividade incremental (RCEI) foi de R$16.056,94 por ano de vida ganho, na perspectiva do sistema de saúde. Conclusão: Estudos futuros devem considerar metodologias que levem em conta a incerteza, a heterogeneidade e a consistência no modelo de decisão e utilizar diretrizes validadas para o relato do estudo / Cervical cancer is the fourth most common cancer in women worldwide. In Brazil, it is estimated that around 16,000 new cases occur per year. The reduction of this type of cancer over the years owes to cervical intraepithelial lesions screening through pap smears. In 2014, the National Immunization Program (NIP) introduced a vaccine against human papillomavirus (HPV) as the primary prevention of this cancer, since this virus is a necessary cause for the onset of this malignancy. Vaccination does not replace screening because there is no protection against all types of high risk HPV nor immunization of the entire population. Incorporation of the vaccination program interferes with the results of the screening program, leading to a decreased number of cancer cases and precursor lesions. In this way, there is a need to explore new screening strategies, also considering other existing technologies. Objective: Determining a Markov based model to perform a cost-effectiveness analysis of cervical cancer screening strategies for hypothetical immunized and non-immunized cohorts against the HPV in Brazil from the perspective of the Unified Health System (UHS). Methods: The first part is a qualitative appraisal and assessment of economic evaluation studies on screening strategies for cervical cancer prevention using a Markov based model done through a systematic review. The combination of different approaches and of the main features of these models can be auxiliary in the construction of a model in scenarios where there are few professionals trained with this technique. Based on this review and consultations with specialists in the areas of gynecology, virology and epidemiology, a Markov model for decision analysis was developed, which simulates the natural history of cervical cancer considering immunization against HPV. This model simulates the follow-up of a cohort of women, from 10 years-old to death, whose parameters were estimated from secondary data, particular to screening and calibrated in order to reflect real screening conditions found in Brazil. Results: A review of Markov models for economic evaluation of cervical cancer screening strategies showed that the report of the problem statement and the description of the compared strategies were well conducted. In contrast, the uncertainties of the model and the consistency were the worst items. The results obtained by calibration of the model were satisfactory, since a good agreement with empirical data was achieved. The baseline case analysis suggested that the best strategy was the HPV-DNA Test as triage for cytology or colposcopy referral, repeated every 5 years, for women between 30 and 70 years-old. This strategy promotes a gain of 9.5 days over the years and detects, every 100,000 women, 6 cases of cancer and 16 of CIN 2/3. The incremental cost-effectiveness ratio (ICER) was R$16,056.94 per life years gained from the health system perspective. Conclusion: Future studies should consider methodologies that take into account uncertainty, heterogeneity and consistency in the decision model and use validated guidelines for the study report

Introdução: Na prática clínica se observa que há diferenças na incidência de efeitos colaterais entre pacientes submetidos ao mesmo esquema terapêutico de radioterapia. Tais diferenças podem ser entendidas como uma radiossensibilidade individual determinada geneticamente. Objetivos: Este estudo teve como objetivo avaliar os efeitos tardios na bexiga em pacientes com câncer do colo uterino tratadas com radioterapia, com ou sem cirurgia, e o valor prognóstico de três polimorfismos genéticos de base única com relação ao desenvolvimento de cistite actínica. Material e métodos: Foi realizada uma análise retrospectiva de 50 pacientes com carcinoma cervical tratadas entre 1999 e 2004, com um mínimo de 6,5 anos de seguimento. A dose de radioterapia na bexiga foi considerada como a soma da dose da radioterapia externa com a dose de braquiterapia no ponto de bexiga definido pelo ICRU 38 (Relato número 38 da Comissão Internacional de Unidades e Medidas em Radiação). Para as correlações entre dose e efeito, foi calculada a dose biológica efetiva (BED) para cada caso. Para a avaliação dos efeitos tardios em bexiga, além dos dados descritos em prontuário, foi feito um questionário específico dirigido aos sintomas urinários, foi realizada cistoscopia em todas as pacientes e a escala LENTSOMA (efeitos tardios no tecido normal/ subjetivo-objetivo tratamento e exames) foi aplicada, utilizando o pior grau do efeito encontrado nos diferentes métodos de avaliação. Variantes genéticas do códon 72 da p53 (Arginina / Prolina), MDM2 SNP309 T/G e ATMex39 5557 G>A foram identificadas usando o método de genotipagem de SNP ABI SNaPshot e os resultados foram correlacionados com a incidência e grau de cistite actínica. Resultados: Complicações clínicas tardias da bexiga foram registradas em 17 (34%) pacientes usando dados coletados dos prontuários e em 41 (82%) pacientes pelo questionário de existência e gravidade dos efeitos tardios da irradiação. Essas complicações foram diretamente correlacionadas com a BED. Vinte e oito pacientes (56%) desenvolveram cistite diagnosticada por cistoscopia (16% Grau 2-4). MDM2 SNP309 TT associado a TP53 (P72R) GG foram relacionados com o aumento da incidência de cistite. Conclusões: Cistite actínica, em grau 2 ou maior, foi elevada nessa população e apresentou uma maior incidência quando realizado um questionário específico para tal. Houve associação com maior dose de radioterapia (BED Gy3 > 100 Gy) e com MDM2 SNP309 TT associado a TP53 (P72R) GG. / Introduction: In clinical practice it is observed that there are differences in the incidence of side effects among patients undergoing the same regimen of radiotherapy. Such differences can be understood as a genetically determined individual radiosensitivity. Purposes: This study aimed to evaluate urinary bladder late effects in patients with uterine cervix cancer treated with radiotherapy with or without surgery and the prognostic value of three single nucleotide polymorphisms (SNPs) related to radiation cystitis. Material and methods: retrospective analysis of 50 patients with cervical carcinoma treated between 1999 and 2004 with a minimum of 6.5 years of follow-up was performed. The radiation dose in the bladder was considered as the dose delivered by external beam irradiation plus the brachytherapy dose in the ICRU Report 38 (International Commission of Radiation Units and Measurements report number 38) bladder point. For dose-effect correlations the biological effective dose (BED) was calculated for each case. For evaluation of bladder late effects, besides the data collected from the charts review, a specific query directed to urinary symptoms was applied to the patients and also a cystoscopy was performed in all of them. The LENTSOMA (late effects of normal tissues/subjective-objective management analytic) scale for bladder late effects was applied. Genetic variants of p53 codon72 (arginine/proline) polymorphism, MDM2 SNP309 T/G and ATMex39 5557G>A were identified by using ABI SNaPshot SNP genotyping method. And the results were correlated with the incidence and grade of radiation cystitis. Results: Clinical late bladder complications were recorded in 17 (34%) patients using data collected from the charts and in 41 (82%) patients by the questionnaire for the existence and severity of late irradiation effects. These complications were directly related with the BED. Twenty eight patients (56%) developed cystitis diagnosed by cystoscopy (16% Grade 2-4). MDM2 SNP309 TT associated with TP53 (P72R) GG was related with increased incidence of cystitis. Conclusions: Late radiation cystitis grade 2 or greater were high in this population and presented a higher incidence when a specific questionnaire was used. Higher radiation dose (BED Gy3 > 100 Gy) and MDM2 SNP309 TT associated with TP53 (P72R) GG were correlated with bladder late effects

Chan, Ho Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 263-283). / Abstracts in English and Chinese. / Examination Committee List --- p.i / Acknowledgements --- p.ii / Abstract --- p.iv / 摘要 --- p.vi / Table of Contents --- p.viii / List of Abbreviations --- p.xvii / List of Figures --- p.xx / List of Tables --- p.xxv / Chapter Chapter I --- Introduction --- p.1 / Chapter 1.1 --- SUMO (Small Ubiquitin-like Modifier) and SUMOylation --- p.1 / Chapter 1.1.1 --- "Ubiquitin, Ubiquitin-like proteins and SUMO isoforms" --- p.2 / Chapter 1.1.2 --- SUMO cycle --- p.5 / Chapter 1.1.2.1 --- SUMO conjugation consensus sequence --- p.5 / Chapter 1.1.2.2 --- SUMO maturation --- p.6 / Chapter 1.1.2.3 --- SUMO conjugation cascade --- p.7 / Chapter 1.1.2.4 --- SUMO deconjugation --- p.9 / Chapter 1.1.3 --- Mode of SUMO action --- p.12 / Chapter 1.1.4 --- Biological functions of SUMO --- p.13 / Chapter 1.1.4.1 --- SUMO in cancer --- p.14 / Chapter 1.2 --- Human cervical cancer and human papillomavirus (HPV) --- p.17 / Chapter 1.2.1 --- Infectious cycle of HPV-16 --- p.18 / Chapter 1.2.1.1 --- Viral entry --- p.18 / Chapter 1.2.1.2 --- Maintenance --- p.18 / Chapter 1.2.1.3 --- Deregulation of cell cycle --- p.19 / Chapter 1.2.1.4 --- Amplification and virion release --- p.20 / Chapter 1.2.2 --- Viral cancer induction --- p.22 / Chapter 1.2.2.1 --- Integration into the host genome --- p.22 / Chapter 1.2.2.2 --- Viral oncoproteins E6 and E7 --- p.23 / Chapter 1.2.3 --- SUMOylation and HPV --- p.24 / Chapter 1.2.3.1 --- Known examples of virus-host SUMOylation system interaction --- p.24 / Chapter 1.2.3.2 --- Other possible mode of virus-SUMO interaction --- p.26 / Chapter 1.3 --- A novel labeling method: biarsenical-tetracysteine labeling in SUMO study --- p.28 / Chapter 1.3.1 --- Potential use of 2As-4Cys system in SUMO studies --- p.31 / Chapter 1.3.2 --- Potential use of 2As-4Cys system in SUMO proteomics --- p.31 / Chapter 1.4 --- Objectives of the present study --- p.34 / Chapter Chapter II --- Proteomics investigation of SUMOylation in human cervical carcinoma cell line HeLa --- p.35 / INTRODUCTION --- p.35 / Chapter 2.1 --- MATERIALS --- p.37 / Chapter 2.1.1 --- Vectors for expression of SUMO and SUMOylation enzymes in E. coli --- p.37 / Chapter 2.1.2 --- E.coli cell strains --- p.38 / Chapter 2.1.3 --- Mammalian cell lines --- p.39 / Chapter 2.1.4 --- E.coli growth mediums --- p.40 / Chapter 2.1.5 --- Mammalian cell growth medium --- p.41 / Chapter 2.1.6 --- Reagents and buffers --- p.41 / Chapter 2.1.6.1 --- Reagents and buffers for molecular cloning --- p.41 / Chapter 2.1.6.2 --- Reagents and buffers for E.coli protein expression --- p.43 / Chapter 2.1.6.3 --- Reagents and buffers for mammalian cell culture --- p.44 / Chapter 2.1.6.4 --- Reagents and buffers for Western blot study --- p.45 / Chapter 2.1.7 --- Reagents and solutions for two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) sample preparation --- p.46 / Chapter 2.1.7.1 --- Reagents and solutions for 2-DE --- p.46 / Chapter i. --- 2-DE sample preparation --- p.46 / Chapter ii. --- First dimensional gel electrophoresis -isoelectric focusing (IEF) --- p.46 / Chapter iii. --- Second dimensional gel electrophoresis -SDS-PAGE --- p.47 / Chapter iv. --- Silver staining --- p.47 / Chapter 2.1.7.2 --- Reagents and solutions for mass spectrometry sample preparation --- p.48 / Chapter i. --- Destaining of silver stained gel spots --- p.48 / Chapter ii. --- Trypsin digestion --- p.48 / Chapter iii. --- Peptide extraction --- p.48 / Chapter iv. --- Desalting and concentration of peptide mixture --- p.49 / Chapter 2.2 --- METHODS --- p.50 / Chapter 2.2.1 --- Molecular cloning of SUMO-1 into pET-28m and pHM6 vectors --- p.50 / Chapter 2.2.1.1 --- Design of primers for the cloning of SUMO-1 --- p.50 / Chapter 2.2.1.2 --- DNA amplification by polymerase chain reaction (PCR) --- p.51 / Chapter 2.2.1.3 --- DNA extraction from agarose gels --- p.52 / Chapter 2.2.1.4 --- Restriction digestion of vectors and purified PCR products --- p.54 / Chapter 2.2.1.5 --- Ligation of SUMO cDNA into expression vector pET-28m and pHM6 --- p.55 / Chapter 2.2.1.6 --- Preparation of competent cells --- p.56 / Chapter 2.2.1.7 --- Transformation of ligated mixture into competent DH5a --- p.56 / Chapter 2.2.1.8 --- Preparation of plasmid DNA --- p.57 / Chapter 2.2.1.8.1 --- Mini-preparation of plasmid DNA --- p.57 / Chapter 2.2.1.8.2 --- Midi-preparation of plasmid DNA --- p.58 / Chapter 2.2.1.8.3 --- DNA quantification and quality measurement --- p.60 / Chapter 2.2.2 --- "Expression of His6-tagged SUMO, ubc9, TDG, GST-tagged El and MBP-tagged Prdx 1 with E.coli" --- p.60 / Chapter 2.2.3 --- "Purification of His6-tagged SUMO, ubc9, TDG, GST-tagged El and MBP-tagged Prdx 1" --- p.62 / Chapter 2.2.3.1 --- Affinity chromatography --- p.65 / Chapter 2.2.3.1.1 --- Ni-NTA affinity chromatography --- p.65 / Chapter 2.2.3.1.2 --- Heparin affinity chromatography --- p.66 / Chapter 2.2.3.1.3 --- Glutathione affinity chromatography --- p.66 / Chapter 2.2.3.1.4 --- Amylose affinity chromatography --- p.67 / Chapter 2.2.3.2 --- Ion exchange chromatography --- p.68 / Chapter 2.2.3.2.1 --- Anion exchange chromatography --- p.68 / Chapter 2.2.3.2.2 --- Cation exchange chromatography --- p.68 / Chapter 2.2.3.3 --- Size exclusion chromatography --- p.69 / Chapter 2.2.3.4 --- Purification strategies --- p.70 / Chapter 2.2.3.4.1 --- Purification of His6-tagged SUMO --- p.70 / Chapter 2.2.3.4.2 --- Purification of His6-tagged TDG --- p.71 / Chapter 2.2.3.4.3 --- Purification of His6-tagged ubc9 --- p.72 / Chapter 2.2.3.4.4 --- Purification of GST-tagged El --- p.73 / Chapter 2.2.3.4.5 --- Purification of MBP-tagged Prdx 1 --- p.74 / Chapter 2.2.4 --- HeLa and C-33A cell culturing and protein extraction --- p.75 / Chapter 2.2.4.1 --- HeLa and C-33A cell culturing --- p.75 / Chapter 2.2.4.2 --- Protein extraction for in vitro SUMOylation assay --- p.76 / Chapter 2.2.5 --- Protein quantification with Bradford assay --- p.76 / Chapter 2.2.6 --- In vitro SUMO conjugation assay --- p.77 / Chapter 2.2.6.1 --- In vitro SUMO conjugation system optimization --- p.77 / Chapter 2.2.6.2 --- In vitro SUMO conjugation of HeLa cell extract --- p.78 / Chapter 2.2.7 --- Transient transfection of pHM6-SUMO-l into HeLa cells and protein extraction from HeLa cells --- p.79 / Chapter 2.2.7.1 --- Transfection with lipofection method --- p.79 / Chapter 2.2.7.2 --- Determination of transfection efficiency --- p.80 / Chapter 2.2.7.3 --- Whole cell protein extraction of transfected cells --- p.81 / Chapter 2.2.8 --- Protein quantification with BCA assay --- p.81 / Chapter 2.2.9 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.83 / Chapter 2.2.10 --- Western blot analysis --- p.84 / Chapter 2.2.10.1 --- Electro-transfer blotting --- p.84 / Chapter 2.2.10.2 --- Immunoblotting with antibodies --- p.84 / Chapter 2.2.10.3 --- ECL detection --- p.85 / Chapter 2.2.10.4 --- Mild stripping for re-probing --- p.86 / Chapter 2.2.11 --- Two-dimensional gel electrophoresis (2-DE) --- p.86 / Chapter 2.2.11.1 --- Sample preparation --- p.86 / Chapter 2.2.11.2 --- First dimension gel electrophoresis -isoelectric focusing (IEF) --- p.87 / Chapter 2.2.11.3 --- Second dimension gel electrophoresis -SDS-PAGE --- p.88 / Chapter 2.2.11.3.1 --- Strip equilibration --- p.88 / Chapter 2.2.11.3.2 --- 16 x 18cm SDS-PAGE --- p.88 / Chapter 2.2.11.4 --- Visualization of proteins on SDS-polyacrylamide gel --- p.90 / Chapter 2.2.11.4.1 --- Silver staining --- p.90 / Chapter 2.2.11.4.2 --- Coomassie Blue® R250 staining --- p.91 / Chapter 2.2.12 --- Sample preparation for mass spectrometry analysis --- p.92 / Chapter 2.2.12.1 --- Destaining and trypsin digestion --- p.92 / Chapter 2.2.12.2 --- Extraction of peptide mixture --- p.93 / Chapter 2.2.12.3 --- Desalting and concentration of peptide mixture --- p.93 / Chapter 2.3 --- RESULTS --- p.95 / Chapter 2.3.1 --- Construction of recombinant pET-28m-SUMO-l and pHM6-SUMO-l --- p.95 / Chapter 2.3.2 --- "Purification of His6-tagged SUMO, ubc9, TDG and GST-tagged El" --- p.98 / Chapter 2.3.2.1 --- Purification of His6-SUMO --- p.98 / Chapter 2.3.2.2 --- Purification of His6-TDG --- p.101 / Chapter 2.3.2.3 --- Purification of His6-ubc9 --- p.104 / Chapter 2.3.2.4 --- Purification of GST-El --- p.106 / Chapter 2.3.3 --- In vitro SUMO conjugation assay --- p.108 / Chapter 2.3.3.1 --- Optimization of in vitro SUMO conjugation system --- p.108 / Chapter 2.3.3.2 --- In vitro SUMO conjugation of HeLa cell protein extract --- p.111 / Chapter 2.3.3.2.1 --- Protein extraction for in vitro sumoylation assay --- p.111 / Chapter 2.3.3.2.2 --- In vitro SUMOylation of HeLa cell lysate --- p.114 / Chapter 2.3.4 --- Differential proteomes of control and in vitro SUMOylated HeLa total cellular extract --- p.116 / Chapter 2.3.4.1 --- Mass spectrometric identification of differential protein candidates --- p.123 / Chapter 2.3.5 --- Overexpression of SUMO-1 in HeLa cells by transient transfection --- p.127 / Chapter 2.3.6 --- Differential proteomes of total cellular protein extract from control and SUMO-1 transfected HeLa cells --- p.128 / Chapter 2.3.6.1 --- Mass spectrometric identification of differential protein candidates --- p.132 / Chapter 2.4 --- Proteins identified in proteomic study with in vitro SUMOylation -Analysis of protein candidate --- p.133 / Chapter 2.4.1 --- Proteins identified from the in vitro investigation --- p.133 / Chapter 2.4.2 --- Verification of putative SUMO substrate Prdx 1 --- p.139 / Chapter 2.4.2.1 --- Purification of Prdx 1 --- p.139 / Chapter 2.4.2.2 --- In vitro SUMOylation of Prdx 1 --- p.142 / Chapter 2.4.3 --- Highlights of the proteins identified --- p.145 / Chapter 2.4.3.1 --- DJ-1 protein --- p.145 / Chapter 2.4.3.2 --- nm23A --- p.145 / Chapter 2.4.3.3 --- v-crk protein of CT10 --- p.146 / Chapter 2.4.3.4 --- Annexin I --- p.146 / Chapter 2.4.3.5 --- "Enolase 1, aldolase A, triosephosphate isomerase (TIM) and phosphoglycerate mutase 1" --- p.147 / Chapter 2.4.3.6 --- CyclophilinA(CypA) --- p.148 / Chapter 2.4.3.7 --- Stress induced phosphoprotein 1 (Stip 1) --- p.148 / Chapter 2.4.3.8 --- TSA and peroxiredoxin 1 (Prdx 1) --- p.149 / Chapter 2.5 --- Proteins identified in proteomic study with overexpression of SUMO-1 in HeLa cells -Analysis of protein candidate --- p.150 / Chapter 2.5.1 --- Proteins identified from the in vivo investigation --- p.150 / Chapter 2.5.2 --- Verification of upregulation of keratin 17 --- p.157 / Chapter 2.5.2.1 --- Immunoblotting against keratin 17 --- p.157 / Chapter 2.5.3 --- Highlights of the proteins identified --- p.159 / Chapter 2.5.3.1 --- "Heat shock proteins (Hsp 60, 70 and 27)" --- p.159 / Chapter 2.5.3.2 --- 14-3-3σ protein (SFN protein) --- p.161 / Chapter 2.5.3.3 --- PDZ-RGS3 --- p.162 / Chapter 2.5.3.4 --- "Keratins 8, 17" --- p.163 / Chapter 2.5.3.5 --- XIAP-1 --- p.164 / Chapter 2.5.3.6 --- ISG15 --- p.164 / Chapter 2.6 --- DISCUSSION --- p.166 / Chapter Chapter III --- Characterization of a novel fluorescent labeling method: Biarsencial-tetracysteine labeling in SUMO study --- p.182 / INTRODUCTION --- p.182 / Chapter 3.1 --- MATERIALS --- p.184 / Chapter 3.1.1 --- "Molecular cloning, protein expression and purification of pET-28m-4Cys 1 -SUMO-1 and pET-28m-4Cys2-SUMO-1" --- p.184 / Chapter 3.1.2 --- Mammalian cell culture and transient transfection of pHM6-4Cysl-SUMO-1 and pHM6-4Cys2-SUMO-l into HeLa cells --- p.184 / Chapter 3.1.3 --- Reagents and buffers --- p.184 / Chapter 3.1.3.1 --- Reagents and buffers for Lumio´ёØ in-gel labeling --- p.184 / Chapter 3.1.3.2 --- Reagents and buffers for Lumio´ёØ in cell labeling --- p.185 / Chapter 3.1.3.3 --- Reagents and buffers for immunostaining --- p.186 / Chapter 3.2 --- METHODS --- p.187 / Chapter 3.2.1 --- Molecular cloning of tetracysteine-tagged SUMO (4Cys-SUMO) into pET-28m and pHM6 vectors --- p.187 / Chapter 3.2.1.1 --- Design of primers and oligonucleotides encoding tetracysteine tag --- p.187 / Chapter 3.2.1.1.1 --- For 4Cysl-SUMO-1 --- p.187 / Chapter 3.2.1.1.2 --- For 4Cys2-SUMO-l --- p.188 / Chapter 3.2.1.2 --- DNA amplification of 4Cysl-SUMO-1 by Polymerase chain reaction (PCR) --- p.189 / Chapter 3.2.1.3 --- Restriction digestion of vectors and purified PCR products of 4Cysl-SUMO-1 --- p.191 / Chapter 3.2.1.4 --- Ligation of 4Cysl-SUMO into expression vector pET-28m and pHM6 --- p.191 / Chapter 3.2.1.5 --- Restriction digestion of pET-28m-SUMO and pHM6-SUMO for ligation with 4Cys2 oligos --- p.192 / Chapter 3.2.1.6 --- Ligation of 4Cys2 oligos to the digested pET-28m-SUMO and pHM6-SUMO plasmids --- p.193 / Chapter 3.2.1.6.1 --- Self-annealing of the 4Cys oligonucleotides --- p.193 / Chapter 3.2.1.6.2 --- Phosphorylation of ds 4Cys2 oligos and ligation to the plasmids --- p.193 / Chapter 3.2.2 --- Expression and purification of pET-28m-4Cys 1 -SUMO-1 and pET-28m-4Cys2-SUMO-1 in E.coli expression system --- p.195 / Chapter 3.2.3 --- Immunohistochemistry (IHC) staining of endogenous SUMO in HeLa cells --- p.196 / Chapter 3.2.4 --- In-cell labeling of 4Cysl/2-SUMO with Lumio´ёØ Reagent --- p.197 / Chapter 3.2.4.1 --- Preparation --- p.197 / Chapter 3.2.4.2 --- In-cell Lumio´ёØ labeling --- p.198 / Chapter 3.2.4.3 --- Detection and imaging of the labeled cells --- p.199 / Chapter 3.2.5 --- In-gel labeling of 4Cysl/2-SUMO with Lumio´ёØ Reagent --- p.199 / Chapter 3.2.5.1 --- Lumio´ёØ in-gel labeling --- p.199 / Chapter 3.2.5.2 --- Visualization and imaging of the labeled gel --- p.200 / Chapter a. --- UV illumination at 302 nm --- p.200 / Chapter b. --- Typhoon Trio TMLaser-scanning at 532 nm --- p.201 / Chapter 3.2.5.3 --- Detection limit of fluorescent 4Cys2-SUMO-l in SDS-PAGE --- p.201 / Chapter 3.2.5.4 --- In-gel labelling in two-dimensional electrophoresis (2-DE) --- p.202 / Chapter 3.2.5.4.1 --- Modification of equilibration buffer before SDS-PAGE --- p.202 / Chapter 3.3 --- RESULTS --- p.203 / Chapter 3.3.1 --- Adoption of old version of 4Cys-tag (4Cys 1) in SUMO study --- p.203 / Chapter 3.3.1.1 --- Construction of recombinant pET-28m-4Cys 1 -SUMO-1 and pHM6-4Cysl-SUMO-1 --- p.203 / Chapter 3.3.1.2 --- In vivo HA-4Cysl-SUMO-1 Lumio´ёØ labelling --- p.205 / Chapter 3.3.1.3 --- Immunohistochemistry (IHC) staining of endogenous SUMO in HeLa cells --- p.207 / Chapter 3.3.1.4 --- Expression and purification of His6-4Cysl-SUMO-1 --- p.208 / Chapter 3.3.1.5 --- Validation of 4Cys1-SUMO-1 conjugate by Lumio´ёØ in-gel labeling --- p.211 / Chapter 3.3.2 --- Adoption of a modified version of 4Cys-tag (4Cys2) in SUMO study --- p.213 / Chapter 3.3.2.1 --- Construction of recombinant pET-28m-4Cys2-SUMO-l and pHM6-4Cys2-SUMO-l --- p.213 / Chapter 3.3.2.2 --- In vivo HA-4Cys2-SUMO-l Lumio´ёØ labelling --- p.216 / Chapter 3.3.2.3 --- Expression and purification of His6-4Cys2-SUMO-1 --- p.219 / Chapter 3.3.2.4 --- Validation of 4Cys2-SUMO-l conjugate Lumio´ёØ in-gel labeling --- p.221 / Chapter 3.3.3 --- 2As-4Cys labeling in two-dimensional electrophoresis (2-DE) --- p.223 / Chapter 3.3.3.1 --- Detection limit of 4Cys2-SUMO-l in SDS-PAGE --- p.224 / Chapter 3.3.3.2 --- Lumio´ёØ labeling in 2-DE --- p.226 / Chapter 3.4 --- DISCUSSION --- p.232 / Chapter Chapter IV --- Conclusion and Future Perspectives --- p.242 / Chapter 4.1 --- Conclusion on proteomic study of SUMOylation --- p.242 / Chapter 4.2 --- Future perspectives of proteomic study of SUMOylation --- p.245 / Chapter 4.2.1 --- In vitro study --- p.245 / Chapter 4.2.2 --- In vivo study --- p.246 / Chapter 4.3 --- Conclusion of the investigation of biarsencial-tetracysteine (2As-4Cys) system application on SUMO study --- p.247 / Chapter 4.4 --- Future perspectives of the application of 2As-4Cys system application on SUMO study --- p.249 / Chapter 4.4.1 --- In cell study --- p.249 / Chapter 4.4.2 --- In gel study --- p.250 / Appendices --- p.251 / Chapter 1. --- Genotype of E.coli strains --- p.251 / Chapter 2. --- Vector maps --- p.252 / Chapter a. --- Vector map and MCS of pET-28a --- p.252 / Chapter b. --- Vector map and MCS of pHM6 --- p.253 / Chapter c. --- Vector information of pTwo-E --- p.254 / Chapter 3. --- Primers used in this study --- p.255 / Chapter 4. --- Nikon TE2000 filter sets spectrums --- p.257 / Chapter a. --- FITC/GFP filter set --- p.257 / Chapter b. --- RFP filter set --- p.257 / Chapter c. --- UV/DAPI/Hoechst filter set --- p.258 / Chapter 5. --- Akt signalling pathway diagram --- p.259 / Chapter 6. --- DNA sequence of SUMOs and 4Cys2 oligonucleotide --- p.260 / Chapter 7. --- Electrophoresis markers --- p.261 / References --- p.263

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Shading Accent 3"/> <w:LsdException Locked="false" Priority="61" Name="Light List Accent 3"/> <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 3"/> <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 3"/> <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 3"/> <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 3"/> <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 3"/> <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 3"/> <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 3"/> <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 3"/> <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 3"/> <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 3"/> <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 3"/> <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 3"/> <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 4"/> <w:LsdException Locked="false" Priority="61" Name="Light List Accent 4"/> <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 4"/> <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 4"/> <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 4"/> <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 4"/> <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 4"/ / <p>Free radicals are produced in our body under physiological conditions. Although in very low concentrations, they can show some toxic effects. While trying to bind electrons, in the chemical reaction of oxidation, they rapidly and unpredictably bind to adjacent molecules- proteins, lipids, carbohydrates and nucleic acids from which the structural elements of the cell are made, triggering the internal pathway of apoptosis. Antioxidants are substances that prevent or significantly reduce the oxidation of biomolecules. Oxidative stress is a condition that occurs when the production of free radicals exceeds the capacity of antioxidant enzymes to neutralize them. The antioxidant enzymes include: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S-transferase (GST). Lipid Peroxidation (LP) is the process of oxidation of polyunsaturated fatty acids by free radicals. Malondialdehyde is a biochemical marker by which it is possible to measure the degree of oxidative damage of cell membranes. The oxidative modification of DNA leads to a change in DNA structure that results in genetic damage. The most commonly used marker of oxidative stress is urinary 8-hydroxy-2-deoxiguanosine (8-OHdG). The damage to proteins, lipids and DNA is an important basis for many diseases such as atherosclerosis, neurodegenerative diseases, diabetes, obesity, aging, retinopathy, chronic inflammatory disease and cancer. Starting from the hypothesis that these biomolecules are different at different stages of the disease, they could represent a prognostic marker of the progression of the disease. The aim of the study was to examine whether there were differences between the control group (healthy women), the patients with precancerous lesions on the cervix (HSIL), the patients with early stage cervical cancer (FIGO Ia-Ib) and the patient with locally advanced cervical cancer (IIa - IV) in the indicators of DNA damage (determining the value of 8-OHdG), indicators of oxidative stress (by determining the lipid peroxidation intensity (TBARS)), indicators of antioxidant defense (by determining the activity of antioxidative enzymes of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase GPx), glutathione reductase (GR), and glutathione-S-transferase (GST)). In addition, the aim of the study was to compare the values of 8- OHdG, lipid peroxidation products (TBARS) and the activity of antioxidant enzymes (SOD, CAT, GST, GPx, GR) within the group of patients with early stage cervical cancer divided into two subgroups- with low and high risk in relation to the relapse of the disease. The research was performed at the Clinic for operative oncology, Department of Gynecology at the Institute of Oncology of Vojvodina, Medical Faculty in Novi Sad, Department of Pharmacy and the Institute for Health Care of Novi Sad in the period from 2013 to 2017. Samples of blood and urine of the patients were collected, prepared adequately and stored at -80 &deg; until the analysis. The activity of the antioxidant enzymes as well as the lipid peroxidation were determined by spectrophotometric methods, and the concentration of 8-OHdG was determined by gas chromatography with mass detection. The approval of the Ethical Committee of the Institute for Oncology of Vojvodina was obtained before conducting the research. It has been shown that there are statistically significant differences between the control group (healthy women), patient with precancerous cervical lesions (HSIL), the patients with early stage cervical cancer (FIGO Ia-Ib) compared to a group of patients with locally advanced cervical cancer (IIa-IV) in indicators of damage to DNA (concentration of 8-OHdG), indicators of oxidative stress (lipid peroxidation (TBARS)), indicators of antioxidant defense (activities of antioxidant enzymes SOD, CAT and GST). There was no difference between the groups in activity of glutathione peroxidase enzyme (GPx) and glutathione reductase (GR). There were no differences in the concentration of 8-OHdG, lipid peroxidation products (TBARS) and the activity of antioxidant enzymes (SOD, CAT, GST, GPx and GR) within the group of patients with locally restricted cervical cancer divided into two subgroups with low and high risk in relation on relapses of the disease. CAT and GST activities were the best predictors of disease recurrence among defined groups. Based on the activities of these two oxidative enzymes, the separation of the group of patients who did not experience disease recurrence after a follow-up period from the other two groups in which recurrence of the disease occurred was possible. Based on the obtained results it is concluded that it is possible to use the studied biomarkers as diagnostic markers in patients with cervical cancer. These biomolecules can help in the patient&#39;s classification into certain groups according to the stage of the disease, and consequently the more efficient choice of appropriate treatment. In addition, CAT and GST enzyme activity have been shown to be predictors of disease recurrence in defined patient groups.</p>

Source: Masters Abstracts International, Volume: 46-05, page: 2668. Adviser: Maria E. Fernandez. Includes bibliographical references