Ip Toi Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 81-105). / Abstracts in English and Chinese. / CONTENTS --- p.i-iii / ACKNOWLEDGEMENT --- p.iv / ABSTRACT --- p.v-vi / Chapter Chapter One --- INTRODUCTION --- p.1-2 / Chapter Chapter Two --- LITERATURE REVIEW --- p.3-37 / Chapter 2.1 --- Epidemiology and Etiology of Cervical and Endometrial Cancers --- p.3-4 / Chapter 2.1.1 --- Epidemiology and Etiology of Cervical cancer --- p.4 / Chapter 2.1.1.1 --- Incidence and Mortality --- p.4-6 / Chapter 2.1.1.2 --- Etiology --- p.6-8 / Chapter 2.1.2 --- Epidemiology and Etiology of Endometrial Cancer --- p.9 / Chapter 2.1.2.1 --- Incidence and Mortality --- p.9-11 / Chapter 2.1.2.2 --- Rick Factors --- p.11-14 / Chapter 2.2 --- Pathology of Cervical and Endometrial Cancers --- p.14 / Chapter 2.2.1 --- Pathology of Cervical Cancer --- p.14-15 / Chapter 2.2.1.1 --- Macroscopic Appearance --- p.15 / Chapter 2.2.1.2 --- Histology --- p.15-18 / Chapter 2.2.2 --- Staging of Cervical Cancer --- p.19-21 / Chapter 2.2.3 --- Pathology of Endometrial Cancer --- p.21 / Chapter 2.2.3.1 --- Macroscopic Appearance --- p.22 / Chapter 2.2.3.2 --- Histology --- p.22-24 / Chapter 2.2.4 --- Staging of Endometrial Cancer --- p.24-25 / Chapter 2.2 --- Introduction to Microsatellite Instability (MI) --- p.25 / Chapter 2.3.1 --- DNA structure --- p.25-27 / Chapter 2.3.2 --- Microsatellite --- p.27-28 / Chapter 2.3.3 --- Mismatch Repair (MMR) --- p.28-29 / Chapter 2.3.4 --- Microsatellite Instability (MI) --- p.30-33 / Chapter 2.3.5 --- Microsatellite Instability in various cancers --- p.33-37 / Chapter Chapter Three --- MATERIALS AND METHODS --- p.38-50 / Chapter 3.1 --- Materials --- p.38 / Chapter 3.1.1 --- Patients and Specimens --- p.38-39 / Chapter 3.1.2 --- Chemicals and Reagents --- p.39 / Chapter 3.1.2.1 --- Chemicals --- p.39-40 / Chapter 3.1.2.2 --- Solution --- p.40-41 / Chapter 3.1.2.3 --- Microsatellite Markers --- p.42 / Chapter 3.1.3 --- Major Equipment --- p.43 / Chapter 3.2 --- Methodology --- p.43 / Chapter 3.2.1 --- DNA Extraction --- p.43-45 / Chapter 3.2.2 --- DNA Amplification --- p.45 / Chapter 3.2.2.1 --- End-labeling of Primer --- p.45 / Chapter 3.2.2.2 --- Polymerase Chain Reaction (PCR) --- p.46 / Chapter 3.2.3 --- Electrophoresis of PCR Products and Autoradiography --- p.46-49 / Chapter 3.2.4 --- Determination Of Microsatellite Instability (MI) --- p.49 / Chapter 3.3 --- Statistical Analyses --- p.50 / Chapter Chapter Four --- Result --- p.51-66 / Chapter 4.1 --- Microsatellite Instability in Cervical Cancer --- p.51 / Chapter 4.1.1 --- Prevalence of MI in Cervical Cancer --- p.51 -54 / Chapter 4.1.2 --- MI and Age in Cervical Cancer --- p.55 / Chapter 4.1.3 --- MI and Histological Type in Cervical Cancer --- p.55-56 / Chapter 4.1.4 --- MI and Histologic Grades in Cervical Cancer --- p.56-57 / Chapter 4.1.5 --- MI and Clinical stage in Cervical Cancer --- p.57-58 / Chapter 4.1.6 --- MI and Clinical Status in Cervical Cancer --- p.58-59 / Chapter 4.2 --- Microsatellite Instability in Endometrial Cancer --- p.59 / Chapter 4.2.1 --- Prevalence of MI in Endometrial Cancer --- p.59-62 / Chapter 4.2.2 --- MI and Age Groups in Endometrial Cancer --- p.63 / Chapter 4.2.3 --- MI and Histological Type in Endometrial Cancer --- p.63-64 / Chapter 4.2.4 --- MI and Histologic Grades in Endometrial Cancer --- p.64-65 / Chapter 4.2.5 --- MI and Clinical stage of Endometrial Cancer --- p.65 / Chapter 4.2.6 --- MI and Clinical Status in Endometrial Cancer --- p.66 / Chapter Chapter Five --- Discussion --- p.67-77 / Chapter 5.1 --- MI detection --- p.67-71 / Chapter 5.2 --- MI of Cervical Cancer --- p.71 -74 / Chapter 5.3 --- MI of Endometrial Cancer --- p.74-77 / Chapter Chapter Six --- Conclusions --- p.78-80 / Reference --- p.81-112 / Appendix --- p.113-114

Poon Kin-yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 119-147). / Abstracts in English and Chinese. / ACKNOWLEDGMENT --- p.i / ABSTRACT --- p.iii / ABBREVIATIONS --- p.viii / TABLE OF CONTENTS --- p.x / Chapter CHAPTER I --- INTRODUCTION --- p.1 / Chapter 1.1 --- Cervical Intraepithelial Neoplasia (CIN) and Cervical Cancer --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.3 / Chapter 1.1.1.1 --- Descriptive Epidemiology --- p.4 / Chapter 1.1.1.2 --- Risk Factors --- p.7 / Chapter 1.1.2 --- Pathology --- p.22 / Chapter 1.1.2.1 --- Macroscopic Appearance --- p.22 / Chapter 1.1.2.2 --- Symptoms and Diagnosis --- p.23 / Chapter 1.1.2.3 --- Staging Classification --- p.25 / Chapter 1.1.2.4 --- Histopathology --- p.29 / Chapter 1.2 --- Microsatellite Instability (MSI) --- p.35 / Chapter 1.2.1 --- Microsatellite --- p.35 / Chapter 1.2.2 --- Mismatch Repair --- p.37 / Chapter 1.2.3 --- Microsatellite Instability (MSI) --- p.38 / Chapter 1.2.4 --- MSI in Various Cancers --- p.42 / Chapter 1.2.5 --- The Role of MSI in Carcinogenesis --- p.49 / Chapter 1.2.6 --- MSI as a Diagnostic / Prognostic Tool --- p.50 / Chapter CHAPTER II --- AIMS OF THE STUDY --- p.53 / Chapter CHAPTER III --- MATERIALS AND METHODS --- p.56 / Chapter 3.1 --- Materials --- p.56 / Chapter 3.1.1 --- Patients and Specimens --- p.56 / Chapter 3.1.2 --- Microsatellite Markers --- p.57 / Chapter 3.2 --- Methods --- p.59 / Chapter 3.2.1 --- Preparation of OCT-embedded Specimen Sections --- p.59 / Chapter 3.2.2 --- Microdissection of Epithelial Cells and Neoplastic Cells from Specimen Sections --- p.60 / Chapter 3.2.3 --- DNA Extraction --- p.60 / Chapter 3.2.3.1 --- Normal Blood --- p.61 / Chapter 3.2.3.2 --- Dissected Cells --- p.62 / Chapter 3.2.4 --- DNA Amplification --- p.64 / Chapter 3.2.4.1 --- End-labeling of Primers --- p.64 / Chapter 3.2.4.2 --- Polymerase Chain Reaction --- p.65 / Chapter 3.2.5 --- Denaturing Polyacrylamide Gel Electrophoresis --- p.66 / Chapter 3.2.6 --- Autoradiography --- p.67 / Chapter 3.2.7 --- Determination of MSI --- p.67 / Chapter 3.2.8 --- HPV Detection --- p.68 / Chapter 3.2.9 --- Statistical Analysis --- p.69 / Chapter CHAPTER IV --- RESULTS --- p.70 / Chapter 4.1 --- Incidence of MSI in Cervix --- p.70 / Chapter 4.1.1 --- Incidence of MSI in Normal Cervix --- p.70 / Chapter 4.1.2 --- Incidence of MSI in CIN --- p.70 / Chapter 4.1.3 --- Incidence of MSI in Cervical Carcinoma --- p.71 / Chapter 4.1.4 --- Correlation of MSI-positive with the Evolution of Cervical Neoplasm --- p.77 / Chapter 4.2 --- Correlation of MSI-positive with Clinicopathological Characteristics in Cervical Carcinoma --- p.77 / Chapter 4.2.1 --- MSI and Age --- p.80 / Chapter 4.2.2 --- MSI and Clinical Stage --- p.80 / Chapter 4.2.3 --- MSI and Histological Grade --- p.80 / Chapter 4.2.4 --- MSI and Clinical Status --- p.81 / Chapter 4.3 --- Comparison between Two Panels of Microsatellite Markers used in MSI Detection --- p.84 / Chapter 4.4 --- Human Papilloma Virus (HPV) Infection in Cervical Neoplasm --- p.89 / Chapter 4.4.1 --- HPV Infection and Typing in CIN and Cervical Carcinoma --- p.89 / Chapter 4.4.2 --- Correlation of MSI-positive with HPV Infection in Cervical Carcinoma --- p.94 / Chapter CHAPTER V --- DISCUSSION --- p.96 / Chapter 5.1 --- MSI Detection --- p.96 / Chapter 5.1.1 --- Techniques in MSI Assays --- p.98 / Chapter 5.1.2 --- Choice of Microsatellite Markers --- p.101 / Chapter 5.1.3 --- Diagnostic Criteria of MSI --- p.105 / Chapter 5.2 --- The Role of MSI in the Carcinogenesis of Cervical Neoplasm --- p.107 / Chapter 5.3 --- The Clinical Significant of MSI in Cervical Carcinoma --- p.111 / Chapter 5.4 --- The Interaction between HPV Infection and MSI in Cervical Carcinoma --- p.113 / Chapter CHAPTER VI --- CONCLUSION --- p.116 / REFERENCES --- p.119

Um dos maiores desafios na área forense é, sem dúvida, a identificação humana. O DNA tem sido o responsável por uma verdadeira revolução das técnicas de identificação nas últimas décadas a partir do estudo e da identificação de polimorfismos entre determinados marcadores moleculares existentes nos indivíduos. Os marcadores recomendados para a obtenção de perfis genéticos são loci de regiões microssatélite do DNA, também designados de Short Tandem Repeats (STR). O número de repetições dos marcadores STR é variável entre os indivíduos, criando polimorfismo e tornando-os, desta forma, ótimos marcadores para identificação humana. O objetivo desse trabalho foi estabelecer a distribuição da frequência alélica de 16 STRs preconizados no sistema CODIS, na capital e no Departamento Central de Paraguai. Foram estudadas 300 amostras de saliva coletadas com NUCLEIC-CARD(TM) Collection Device System, de indivíduos paraguaios dentre 20 e 70 anos de idade, morando em uma das 20 cidades estudadas. Para o processamento foi utilizado o kit AmpFLSTR Identifiler Direct PCR Amplification® seguindo as fases de genotipagem, amplificação e eletroforese capilar. Foi possível estabelecer o perfil genético de 259 amostras bem como os parâmetros forenses e, assim, calcular os loci mais polimórficos os quais foram FGA e D18S51 utilizando os softwares GenAlEx 6.5, Arlequin 3.5 e R 2.5. A distribuição das frequências alélicas de cada loci analisado permitiu estabelecer a caracterização genética da população estudada. Foi possível confirmar que a população do Paraguai se encontra em equilíbrio Hardy-Weinberg, com uma diversidade genética intrapopulacional de 0.794915 +/- 0.398307 e interpopulacional determinada pelo índice de fixação (FST) de 0.01112. A partir desse estudo, foi possível determinar as frequências alélicas de 15 STRs utilizados no sistema CODIS nacapital e no Departamento Central do Paraguai bem como a caracterização genética e os parâmetros forenses da população estudada. Todos os loci estudados na população paraguaia foram considerados muito informativos e úteis para a solução de problemas relacionados com identificação humana na amostra analisada / One of the biggest challenges in Forensic Sciences is undoubtedly human identification. DNA has been responsible for a true revolution in identification techniques in the last decades from the study and identification of polymorphisms between certain molecular markers in individuals. The recommended markers for obtaining genetic profiles are loci of DNA microsatellite regions, also called Short Tandem Repeats (STR). The number of repetitions of STR markers is variable among individuals, creating polymorphism and thus making them excellent markers for human identification. The aim of this study was to establish the distribution of the allelic frequency of 16 STRs recommended in the CODIS system, in the capital and in the Central Department of Paraguay. We studied 300 saliva samples collected from NUCLEIC-CARD (TM) Collection Device System of Paraguayan individuals between 20 and 70 years-old, living in one of the 20 cities studied. For the processing, the AmpFLSTR Identifiler Direct PCR Amplification® kit was used following the phases of genotyping, amplification and capillary electrophoresis. It was possible to establish the genetic profile of 259 samples as well as the forensic parameters and thus to calculate the most polymorphic loci which were FGA and D18S51 using the software GenAlEx 6.5, Arlequin 3.5 and R 2.5. The distribution of the allelic frequencies of each analyzed loci allowed establishing the genetic characterization of the studied population. It was possible to confirm that the population of Paraguay is in Hardy-Weinberg equilibrium, with an intrapopulational genetic diversity of 0.8046 +/- 0.0120 and interpopulational determined by the fixation index (FST) of 0.01112. From this study, it was possible to determine the allelic frequencies of 15 STRs used in the CODIS system in the capital and in the Central Department of Paraguay, as well as the genetic characterization and forensic parameters of the studied population. All the loci studied in the Paraguayan population were considered veryinformative and useful for the solution of problems related to human identification in the analyzed sample

by Wai-keung Leung. / Thesis (M.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 217-232). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

Um dos maiores desafios na área forense é, sem dúvida, a identificação humana. O DNA tem sido o responsável por uma verdadeira revolução das técnicas de identificação nas últimas décadas a partir do estudo e da identificação de polimorfismos entre determinados marcadores moleculares existentes nos indivíduos. Os marcadores recomendados para a obtenção de perfis genéticos são loci de regiões microssatélite do DNA, também designados de Short Tandem Repeats (STR). O número de repetições dos marcadores STR é variável entre os indivíduos, criando polimorfismo e tornando-os, desta forma, ótimos marcadores para identificação humana. O objetivo desse trabalho foi estabelecer a distribuição da frequência alélica de 16 STRs preconizados no sistema CODIS, na capital e no Departamento Central de Paraguai. Foram estudadas 300 amostras de saliva coletadas com NUCLEIC-CARD(TM) Collection Device System, de indivíduos paraguaios dentre 20 e 70 anos de idade, morando em uma das 20 cidades estudadas. Para o processamento foi utilizado o kit AmpFLSTR Identifiler Direct PCR Amplification® seguindo as fases de genotipagem, amplificação e eletroforese capilar. Foi possível estabelecer o perfil genético de 259 amostras bem como os parâmetros forenses e, assim, calcular os loci mais polimórficos os quais foram FGA e D18S51 utilizando os softwares GenAlEx 6.5, Arlequin 3.5 e R 2.5. A distribuição das frequências alélicas de cada loci analisado permitiu estabelecer a caracterização genética da população estudada. Foi possível confirmar que a população do Paraguai se encontra em equilíbrio Hardy-Weinberg, com uma diversidade genética intrapopulacional de 0.794915 +/- 0.398307 e interpopulacional determinada pelo índice de fixação (FST) de 0.01112. A partir desse estudo, foi possível determinar as frequências alélicas de 15 STRs utilizados no sistema CODIS nacapital e no Departamento Central do Paraguai bem como a caracterização genética e os parâmetros forenses da população estudada. Todos os loci estudados na população paraguaia foram considerados muito informativos e úteis para a solução de problemas relacionados com identificação humana na amostra analisada / One of the biggest challenges in Forensic Sciences is undoubtedly human identification. DNA has been responsible for a true revolution in identification techniques in the last decades from the study and identification of polymorphisms between certain molecular markers in individuals. The recommended markers for obtaining genetic profiles are loci of DNA microsatellite regions, also called Short Tandem Repeats (STR). The number of repetitions of STR markers is variable among individuals, creating polymorphism and thus making them excellent markers for human identification. The aim of this study was to establish the distribution of the allelic frequency of 16 STRs recommended in the CODIS system, in the capital and in the Central Department of Paraguay. We studied 300 saliva samples collected from NUCLEIC-CARD (TM) Collection Device System of Paraguayan individuals between 20 and 70 years-old, living in one of the 20 cities studied. For the processing, the AmpFLSTR Identifiler Direct PCR Amplification® kit was used following the phases of genotyping, amplification and capillary electrophoresis. It was possible to establish the genetic profile of 259 samples as well as the forensic parameters and thus to calculate the most polymorphic loci which were FGA and D18S51 using the software GenAlEx 6.5, Arlequin 3.5 and R 2.5. The distribution of the allelic frequencies of each analyzed loci allowed establishing the genetic characterization of the studied population. It was possible to confirm that the population of Paraguay is in Hardy-Weinberg equilibrium, with an intrapopulational genetic diversity of 0.8046 +/- 0.0120 and interpopulational determined by the fixation index (FST) of 0.01112. From this study, it was possible to determine the allelic frequencies of 15 STRs used in the CODIS system in the capital and in the Central Department of Paraguay, as well as the genetic characterization and forensic parameters of the studied population. All the loci studied in the Paraguayan population were considered veryinformative and useful for the solution of problems related to human identification in the analyzed sample

Wu, Feng. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 38-44). / Abstracts in English and Chinese. / Title Page --- p.i / Thesis Committee --- p.ii / Acknowledgment --- p.iv / Table of Contents --- p.v / List of Figures --- p.vii / List of Abbreviations and Symbols --- p.xi / Abstract (English version) --- p.xii / Abstract (Chinese version) --- p.xiii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Significance of DNA CCTG repeats --- p.1 / Chapter 1.2 --- Objectives of this work --- p.2 / Chapter 1.3 --- DNA structure --- p.3 / Chapter 2 --- Materials and Methods --- p.5 / Chapter 2.1 --- Sample design --- p.5 / Chapter 2.2 --- Sample preparation --- p.5 / Chapter 2.3 --- NMR spectroscopy --- p.6 / Chapter 2.4 --- Resonance assignment --- p.7 / Chapter 3 --- NMR Structural Studies of (CCTG)3 --- p.9 / Chapter 3.1 --- Overview --- p.9 / Chapter 3.2 --- NMR resonance assignments --- p.9 / Chapter 3.3 --- Formation of two-residue CT-loop in the middle repeat of (CCTG)3 --- p.12 / Chapter 3.4 --- C-bulge and T.T mispair in (CCTG)3 hairpin stem region --- p.13 / Chapter 3.5 --- Summary --- p.15 / Chapter 4 --- NMR Structural Studies of (CCTG)4 --- p.16 / Chapter 4.1 --- Overview --- p.16 / Chapter 4.2 --- Conformational exchange in (CCTG)4 --- p.16 / Chapter 4.3 --- Formation of two-residue CT-loops in different repeats of (CCTG)4 --- p.17 / Chapter 4.4 --- Mutational studies of (CCTG)4 --- p.19 / Chapter 4.4.1 --- Mutational studies on the 1st repeat of (CCTG)4: (CCTG)4-C2T --- p.19 / Chapter 4.4.2 --- Mutational studies on the 2nd repeat of (CCTG)4：(CCTG)4-C6T --- p.21 / Chapter 4.4.3 --- Mutational studies on the 3rd repeat of (CCTG)4：(CCTG)4-C 10T --- p.26 / Chapter 4.4.4 --- Mutational studies on the 4th repeat of (CCTG)4: (CCTG)4-C14T --- p.28 / Chapter 4.5 --- Summary --- p.33 / Chapter 5 --- Conclusions and Future Works --- p.35 / References --- p.38

Diss. (sammanfattning) Linköping : Linköpings universitet, 2008. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-07-19T13:40:55Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) tese final.pdf: 4052323 bytes, checksum: 74297c119131b87a26908be1d213027a (MD5) / Made available in DSpace on 2016-07-19T13:40:55Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) tese final.pdf: 4052323 bytes, checksum: 74297c119131b87a26908be1d213027a (MD5) Previous issue date: 2015-12-01 / CAPEs / O uso de insetos visando responder aos quesitos levantados em investigações criminais ganhou espaço nas últimas décadas entre os pesquisadores e profissionais desta área, assim como a combinação de técnicas de genética forense para a obtenção de DNA humano a partir destes organismos, em especial dos dípteros necrófagos. Desse modo, neste estudo objetivou-se obter e testar um protocolo de identificação de DNA humano extraído a partir de larvas de dípteros coletadas em cadáveres no Instituto de Medicina Legal de Pernambuco Antonio Persivo Cunha (IMLAPC/PE). Inicialmente, espécimes imaturos foram coletados no IMLAPC/PE e criados em dieta a base de carne moída bovina para possibilitar a identificação da espécie mais abundante e frequente que se cria neste substrato. A espécie Chrysomya albiceps (Diptera: Calliphoridae) foi selecionada como modelo experimental. Grupos de larvas dessa espécie foram submetidos a uma dieta baseada em carne moída e sangue humano por 48 horas, dissecadas e submetidas a extração de DNA, utilizandose duas metodologias comumente adotadas pelos laboratórios de genética forense: Kit DNA IQ™ e Método Fenol-Clorofórmio. O DNA extraído foi quantificado através de Nanodrop® e Real-Time PCR 7500 com uso do Quantifiler® Duo DNA Quantification. Para amplificação do DNA foram usados os kits para STR (short tandem repeats): AmpFℓSTR® Identifiler® Plus PCR Kit, Argus X-12® Kit e PowerPlex® Fusion System kit. As amostras amplificadas foram analisadas por eletroforese capilar em ABI PRISM 3500, permitindo observar que, para os kits utilizados houve perfis íntegros e compatíveis com a amostra referência, a partir da extração com kit DNA IQ™ e/ou método Fenol- Clorofórmio. Além disso, foram testados quatro meios de armazenagem comumente utilizados em zoologia: etanol 70%, etanol 95%, formol 4% e via seca. Após 24 horas de armazenagem, as amostras foram submetidas aos processos de análise de DNA e o formol 4% apresentou os melhores perfis de DNA. O fato de haver perfis passíveis de comparação confirma a utilidade das larvas de dípteros usadas para este fim, as quais podem futuramente ser usadas para correlacionar perfis genéticos com uma cena criminal. O aprimoramento destas técnicas é necessário para que o uso das larvas de dípteros muscóides com emprego para a entomogenética tenha mais difusão entre os meios acadêmico e forense. / The use of insects for investigations has gained ground in recent decades among researchers and criminal professionals. Recently, the use of these animals has been combined with forensic genetics techniques for obtaining human DNA from these. Among the main focus groups for this technique are the carrion flies that have the host DNA extracted from intestinal contents. Because the visibility of this branch of forensic biology, this study aimed to obtain and test a protocol for identifying human DNA extracted from larvae of Diptera at the Instituto de Medicina Legal de Pernambuco Antonio Persivo Cunha (IMLAPC/PE). The species Chrysomya albiceps (Diptera: Calliphoridae) was selected as an experimental model. Groups of larvae of this species were subjected to diet ground meat and human blood for 48 hours, dissected and subjected to DNA extraction using two methods commonly used by forensic genetics laboratories: DNA IQ™ Kit and Method phenol-chloroform. The extracted DNA was quantified by Nanodrop® and Real-Time PCR 7500 with use of Quantifiler® Duo DNA Quantification. For DNA amplification kits for STR (short tandem repeats) were used: AmpFℓSTR® Identifiler® Plus PCR Kit, Argus X-12® Kit and PowerPlex® Fusion System kit. The amplified samples were analyzed by capillary electrophoresis in ABI PRISM 3500, allowing to observe for kits used there have upright profiles and compatible with the reference sample, from the IQ™ DNA extraction kit and/or phenol-chloroform method. In addition, four storage means commonly used in zoology were tested: 70% ethanol, 95% ethanol, 4% formaldehyde and dry. After 24 hours of storage, the samples were submitted to DNA analysis processes and the 4% formaldehyde DNA showed the best profile. The fact that there be comparable profiles confirms the usefulness of dipteran larvae used for this purpose, which can further be used to correlate genetic profiles and a criminal scene. The improvement of these techniques is required for the use of larvae dipterae with employment for the entomogenetics have more diffusion among academic and forensic means.

Orientadores: Flavio Antonio Maës dos Santos, Maria Imaculada Zucchi / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T23:19:56Z (GMT). No. of bitstreams: 1 Silvestrini_Milene_D.pdf: 3891912 bytes, checksum: 6bb6bd65f788f261b8387e6c9daf17f8 (MD5) Previous issue date: 2014 / Resumo: A estrutura genética espacial de populações de plantas pode variar ao longo dos estádios ontogenéticos, através das gerações e entre diferentes condições ambientais. Estas mudanças são direcionadas por fatores ecológicos e evolutivos. As espécies pioneiras apresentam histórias de vida e estruturas populacionais características que são afetadas principalmente pelas mudanças ambientais geradas por distúrbios naturais ou antrópicos. O objetivo deste trabalho foi investigar como as características do ciclo de vida, os processos ecológicos e fatores genéticos associados aos distúrbios afetam a diversidade e estrutura genética de populações de uma espécie arbórea pioneira. Nós estudamos Croton floribundus Spreng. (Euphorbiaceae), uma espécie arbórea pioneira abundante em clareiras e em áreas secundárias da Floresta Estacional Semidecidual, em duas áreas com níveis contrastantes de distúrbios antrópicos: uma floresta primária e uma floresta secundária em estádio inicial de sucessão. A fim de abordar a principal questão deste estudo, nós avaliamos o padrão de distribuição da espécie sob as diferentes condições ambientais geradas por distúrbios naturais e antrópicos (Capítulo I); testamos e caracterizamos iniciadores universais cloroplastidiais (cpSSR) para C. floribundus (Capítulo II); desenvolvemos e caracterizamos marcadores microssatélites nucleares (SSR) para C. floribundus bem como examinamos algumas características citogenéticas da espécie com o objetivo de testar a ocorrência de poliploidia e avaliar sua implicação para o uso dos marcadores SSR (Capítulo III); avaliamos a diversidade e estrutura genética de C. floribundus entre duas classes de tamanho e entre populações em uma floresta primária e uma floresta secundária em estádio inicial de sucessão (Capítulo IV). C. floribundus foi frequente e igualmente distribuído em clareiras de todos os tamanhos na floresta primária, mas sua estrutura populacional variou entre áreas com níveis contrastantes de distúrbio antrópico. Seis locos cpSSR foram otimizados e caracterizados em C. floribundus. O estudo citogenético permitiu a caracterização mais precisa dos locos SSR, bem como forneceu novos dados sobre a origem e a evolução da espécie. O número de bivalentes observados na meiose, n = 56 (2n = 8x = 112), mostrou a ocorrência de poliploidia em todas as populações estudadas. Altos níveis de diversidade genética foram encontrados para C. floribundus. A dispersão de sementes e as colonizações (e extinções) foram determinantes para a estrutura genética em fina escala encontrada nas populações de C. floribundus em ambos os tipos de florestas. Além disso, os efeitos destes processos associados aos distúrbios antrópicos parecem aumentar fortemente a diferenciação genética entre as populações na floresta em estádio inicial de sucessão. As análises de marcadores moleculares nucleares e cloroplastidias sugeriram que o fluxo gênico por pólen é responsável por manter a diversidade genética dentro das populações de C. floribundus tanto na floresta primária quanto na floresta secundária em estádio inicial de sucessão. Nesta última, o fluxo gênico por sementes parece ser igualmente importante. Os resultados obtidos mostraram que a dinâmica de clareiras, o processo de colonização e a dispersão de pólen e sementes afetam a diversidade e estrutura genética da espécie arbórea pioneira, aumentando-os ou diminuindo-os conforme o número de colonizadores, número de populações-fonte, as taxas de fluxo gênico e o nível de perturbação antrópica da área / Abstract: The spatial genetic structure of plant populations may vary across life stages, across generations and among different environmental conditions. These changes are driven by evolutionary and ecological forces. Pioneer tree species exhibit particular life histories and population structures that are mainly affected by environmental changes generated by natural or human disturbances. Our aim was to investigate how the life-history traits, ecological processes, and the genetic factors associated to natural and human disturbances can affect the genetic diversity and structure of populations of a pioneer tree species. We studied Croton floribundus Spreng. (Euphorbiaceae), a pioneer tree species abundant in gaps and secondary areas of the semi-deciduous tropical forest, in two areas with contrasting levels of human disturbance: a primary forest and an early successional forest. In order to address the main question of this study, we examined the pattern of distribution of the species under the different environmental conditions generated by natural and human disturbances (Chapter I); tested and characterized universal chloroplast microsatellite (cpSSR) primers for C. floribundus (Chapter II); developed and characterized nuclear microsatellite (SSR) markers for C. floribundus as well as examined some cytogenetic traits of the species in order to test for polyploidy and to evaluate its implications for the appropriate use of the SSR markers (Chapter III); and evaluated the genetic diversity and structure of C. floribundus between two size classes and among populations in the primary forest and in the early successional forest (Chapter IV). C. floribundus was widespread and equally distributed along the gap size range in the primary forest, but its population structure varied between areas with contrasting levels of human disturbance. Six universal cpSSR loci were optimized and characterized for C. floribundus. The cytogenetic study allowed the accurate characterization of SSR loci as well as provided new data on the origin and evolution of the species. The number of bivalents observed in meiosis n=56 (2n=8x=112) showed the occurrence of polyploidy in all populations studied. High genetic diversity levels were found for C. floribundus. Seed dispersal and colonizations (and extinctions) were determinants of the fine-scale genetic structure of C. floribundus in both forest types. Also, their effects associated to the human disturbances seem to strongly increase the genetic differentiation among populations in the early successional forest. Analysis of nuclear and chloroplast markers suggested that gene flow by pollen is responsible for maintaining the genetic diversity within populations of C. floribundus in both primary and early successional forests. In the latter, gene flow by seeds seem to be equally important. The results showed that gap dynamics, colonization process, and pollen and seed dispersal affect the genetic diversity and structure of the pioneer tree species by increasing or decreasing them depending mainly on the number of colonizers, the number of source populations, the gene flow rates, and the level of human disturbance of the area / Doutorado / Ecologia / Doutora em Ecologia