La théorie de l’évolution repose sur la biologie moderne. Toutes les nouvelles espèces émergent d’une espèce existante. Il en résulte que différentes espèces partagent une ascendance commune, telle que représentée dans la classification phylogénétique. L’ascendance commune peut expliquer les similitudes entre tous les organismes vivants, tels que la chimie générale, la structure cellulaire, l’ADN comme matériau génétique et le code génétique. Les individus d’une espèce partagent les mêmes gènes mais (d’ordinaire) différentes séquences d’allèles de ces gènes. Un individu hérite des allèles de leur ascendance ou de leurs parents. Le but des études phylogénétiques est d’analyser les changements qui se produisent dans différents organismes pendant l’évolution en identifiant les relations entre les séquences génomiques et en déterminant les séquences ancestrales et leurs descendants. Une étude de phylogénie peut également estimer le temps de divergence entre les groupes d’organismes qui partagent un ancêtre commun. Les arbres phylogénétiques sont utiles dans les domaines de la biologie, comme la bio informatique, pour une phylogénétique systématique et comparative. L’arbre évolutif ou l’arbre phylogénétique est une exposition ramifiée les relations évolutives entre divers organismes biologiques ou autre existence en fonction des différences et des similitudes dans leurs caractéristiques génétiques. Les arbres phylogénétiques sont construits à partir de données moléculaires comme les séquences d’ADN et les séquences de protéines. Dans un arbre phylogénétique, les nœuds représentent des séquences génomiques et s’appellent des unités taxonomiques. Chaque branche relie deux nœuds adjacents. Chaque séquence similaire sera un voisin sur les branches extérieures, et une branche interne commune les reliera à un ancêtre commun. Les branches internes sont appelées unités taxonomiques hypothétiques. Ainsi, les unités taxonomiques réunies dans l’arbre impliquent d’être descendues d’un ancêtre commun. Notre recherche réalisée dans cette dissertation met l’accent sur l’amélioration des prototypes évolutifs appropriés et des algorithmes robustes pour résoudre les problèmes d’inférence phylogénétiques et ancestrales sur l’ordre des gènes et les données ADN dans l’évolution du génome complet, ainsi que leurs applications.[...] / The theory of evolution is based on modern biology. All new species emerge of an existing species. As a result, different species share common ancestry,as represented in the phylogenetic classification. Common ancestry may explainthe similarities between all living organisms, such as general chemistry, cell structure,DNA as genetic material and genetic code. Individuals of one species share the same genes but (usually) different allele sequences of these genes. An individual inheritsalleles of their ancestry or their parents. The goal of phylogenetic studies is to analyzethe changes that occur in different organisms during evolution by identifying therelationships between genomic sequences and determining the ancestral sequences and theirdescendants. A phylogeny study can also estimate the time of divergence betweengroups of organisms that share a common ancestor. Phylogenetic trees are usefulin the fields of biology, such as bioinformatics, for systematic phylogeneticsand comparative. The evolutionary tree or the phylogenetic tree is a branched exposure the relationsevolutionary between various biological organisms or other existence depending on the differences andsimilarities in their genetic characteristics. Phylogenetic trees are built infrom molecular data such as DNA sequences and protein sequences. Ina phylogenetic tree, the nodes represent genomic sequences and are calledtaxonomic units. Each branch connects two adjacent nodes. Each similar sequencewill be a neighbor on the outer branches, and a common internal branch will link them to acommon ancestor. Internal branches are called hypothetical taxonomic units. Thus,Taxonomic units gathered in the tree involve being descended from a common ancestor. Ourresearch conducted in this dissertation focuses on improving evolutionary prototypesappropriate and robust algorithms to solve phylogenetic inference problems andancestral information about the order of genes and DNA data in the evolution of the complete genome, as well astheir applications.

O câncer no colo do útero é o segundo tipo de neoplasia maligna mais prevalente nas mulheres no mundo todo e o seu rastreamento é realizado através do exame microscópico das células esfoliadas da mucosa cervical, o teste de Papanicolaou. Partindo-se da premissa que a infecção por Papilomavírus Humano oncogênico (HRHPV) é condição necessária para o desenvolvimento desta neoplasia, tem-se avaliado a possibilidade de empregar-se para a mesma finalidade, o rastreio, métodos que detectam o material genético viral. Estudos recentes demonstraram que estes testes moleculares apresentam maior sensibilidade e a substituição do teste citológico pelos moleculares vem ocorrendo em alguns países. O monitoramento da resposta ao tratamento das pacientes com câncer cervical é realizado por meio de testes inespecíficos como exames de imagem. Portanto, é desejável o desenvolvimento de um teste específico, não invasivo capaz de detectar precocemente a recidiva. Este estudo investigou a freqüência e os tipos de HPV presentes em tumores cervicais de pacientes brasileiras, e verificou a ocorrência de resultados HPV-negativos pelos testes moleculares, procurando investigar as causas de possíveis falhas dos testes. Além disso, padronizou e avaliou uma reação de PCR em tempo real capaz detectar o DNA do HPV-16 e 18 no plasma das pacientes. Foram incluídas no estudo 104 pacientes com diagnóstico confirmado de câncer cervical atendidas em três hospitais oncológicos do Estado de São Paulo, ICESP, IBCC e HC-Barretos, no período de novembro de 2009 a julho de 2011. Foram coletados um fragmento do tumor cervical e sangue total. O DNA extraído dos tumores foi submetido a genotipagem do HPV por meio da utilização dos kits Linear Array HPV Genotyping Test (LA) e PapilloCheck®, além de PCRs tipo específicas para HPV-16, 18 e 45. Amostras que apresentaram resultado HPV-negativo no PapilloCheck®, tiveram parte da região E1 seqüenciada para verificar potenciais causas do resultado falso-negativo. A amostra de plasma das pacientes que apresentavam HPV-16 e/ou 18 no tecido tumoral foi submetida à PCR em tempo real para avaliar a presença do DNA do HPV no plasma. Das 104 amostras de tecido tumoral, 103 foram positivas para HPV, sendo que os HPV-16 e 18, como infecção simples, foram os encontrados com maior freqüência (48,5%). Os testes LA e PapilloCheck® apresentaram 65,4% de concordância total. O PapilloCheck® apresentou 12,5% de resultados falso-negativos (N=13). A principal hipótese para explicar esses resultados é a presença de mutações na região de hibridização dos iniciadores e/ou das sondas. A reação de PCR em tempo real específica para HPV-16 e 18 padronizada no estudo apresentou baixa sensibilidade, mas alta especificidade e não deve ser utilizada como teste diagnóstico para o câncer cervical. A relação entre DNA de HPV no plasma e o prognóstico da paciente deve ser explorada no futuro / Invasive cervical cancer is the second most common cancer among women worldwide and screening programs are based on microscopical examination of cells exfoliated from the cervical mucosa, the Papanicolaou test. Since the infection by an oncogenic HPV (HR-HPV) has been shown to be necessary for the development of this neoplasia, molecular assays are being evaluated for the same application, primary screening. Indeed, recent studies showed these methods to be more sensitive and therefore are replacing the cytological test in many countries. Post treatment surveillance of invasive cervical cancer patients are made by unspecific tests as imaging exams. Hence, the development of a specific non invasive test able to detect premature recurrence is desired. This study investigated the HPV frequency and types on invasive cervical tumors among Brazilian patients observing the occurrence of HPV-negative results on molecular tests, trying to addrees the causes for the putative failures. Moreover, we standardized and evaluated a real time PCR method for HPV-16 and 18 DNA detection on patients plasma. One hundred and four women with invasive cervical cancer were recruited in three oncologic hospitals from São Paulo State, ICESP, IBCC and HCBarretos, in between November 2009 and July 2011. Tumor tissue and whole blood were collected. DNA extracted from the tumors were submitted to HPV detection and genotyping tests such as the Linear Array HPV Genotyping Test (LA) and PapilloCheck®, besides type specific PCRs for HPV-16, 18 e 45. Samples that showed an HPV-negative result on the PapilloCheck® were submitted to direct sequencing of E1 region to verify potential mismatches responsible for that. Plasma samples from patients with tumor tissue positive for HPV-16 and/or 18 were submitted to the real time PCR to evaluate the HPV DNA presence on plasma. Out of 104 cervical carcinomas, 103 were HPV positive, HPV-16 and 18, as single infection, were the most frequent types observed (48.5%). LA and PapilloCheck® showed 65.4% of total agreement. PapilloCheck® displaied 12.5% of false-negative results (N=13). The major hypothesis to explain these results is the presence of mismatches to the primers and/or probes annealing regions. Real time PCR specific for HPV-16 and 18 developed in this study showed low sensitivity, but a high specificity and cannot be used as an invasive cervical cancer diagnostic tool. The relationship between the presence of HPV DNA in the plasma and the patient prognostic shall be evaluated in the future

Among all existing alignment-free methods for comparing biological sequences, the sequence graphical representation provides a simple approach to view, sort, and compare gene structures. The aim of graphical representation is to display DNA or protein sequences graphically so that we can easily find out visually how similar or how different they are. Of course, only the visual comparison of sequences is not enough for the follow-up research work. We need more accurate comparison. This leads us to develop the application of the graphical representation for biological sequences. / In this thesis, we have two main contributions: (1) We construct a protein map with the help of our proposed new graphical representation for protein sequences. Each protein sequence can be represented as a point in this map, and cluster analysis of proteins can be performed for comparison between the points. This protein map can be used to mathematically specify the similarity of two proteins and predict properties of an unknown protein based on its amino acid sequence. (2) We construct a novel genome space with biological geometry, which is a subspace in RN . In this space each point corresponds to a genome. The natural distance between two points in the genome space reflects the biological distance between these two genomes. Our genome space will provide a new powerful tool for analyzing the classification of genomes and their phylogenetic relationships. / Yu, Chenglong. / Adviser: Luk Hing Sun. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 59-64). / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Yan, Changqing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 92-97). / Abstracts in English and Chinese. / ABSTRACT (ENGLISH) --- p.iii / ABSTRACT (CHINESE) --- p.iv / ACKNOWLEDGMENTS --- p.v / LIST OF ABBREVIATIONS --- p.vi / TABLE OF CONTENTS --- p.vii / LIST OF FIGURES --- p.ix / LIST OF TABLES --- p.x / Chapter CHAPTER ONE: --- BACKGROUND INFORMATION --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Deoxyribonucleic Acid --- p.2 / Chapter 1.3 --- DNA Studies --- p.9 / Chapter 1.4 --- Cisplatin Studies --- p.11 / Chapter 1.5 --- Scope of the Thesis --- p.13 / Chapter CHAPTER TWO: --- METHODOLOY AND COMPUTATION --- p.16 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.2 --- Molecular Dynamics Simulation --- p.16 / Chapter 2.3 --- Quantum Mechanics Calculation --- p.23 / Chapter 2.4 --- Verification of Methodology --- p.25 / Chapter 2.4.1 --- Backbone Torsion Angles --- p.25 / Chapter 2.4.2 --- N7-N7 Distance --- p.30 / Chapter 2.4.3 --- Location of HOMO --- p.33 / Chapter 2.5 --- Summary --- p.35 / Chapter CHAPTER THREE: --- UNDERSTANDING OF THE CISPLATIN-DNA CROSSLINKS --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- MO Analysis --- p.37 / Chapter 3.3 --- Potential Binding Products with the Ligand --- p.37 / Chapter 3.3.1 --- "1,2-d(GpG) Intrastrand Crosslink" --- p.43 / Chapter 3.3.2 --- "l,2-d(ApG) Intrastrand Crosslink" --- p.43 / Chapter 3.3.3 --- "l,3-d(GpXpG) Intrastrand Crosslink" --- p.44 / Chapter 3.3.4 --- d(GpC)d(GpC) Interstrand Crosslink --- p.44 / Chapter 3.3.5 --- d(GpXpC)d(GpXpC) Interstrand Crosslink --- p.44 / Chapter 3.3.6 --- Summary --- p.45 / Chapter 3.4 --- Potential Binding Products Analysis --- p.47 / Chapter 3.4.1 --- Site Identification Convention --- p.47 / Chapter 3.4.2 --- Potential Binding Products Analysis --- p.48 / Chapter 3.4.3 --- Applications --- p.53 / Chapter 3.5 --- Cisplatin-DNA Crosslink Products Analysis --- p.56 / Chapter 3.5.1 --- "1,2-d(GpG) and l,2-d(ApG) Intrastrand Crosslinks" --- p.61 / Chapter 3.5.2 --- "l,3-d(GpXpG) Intrastrand and d(GpXpC)d(GpXpC) Interstrand Crosslinks" --- p.62 / Chapter 3.5.3 --- d(GpC)d(GpC) Interstrand Crosslinks --- p.63 / Chapter 3.5.4 --- Platination at Terminal Positions --- p.65 / Chapter 3.6 --- Summary --- p.65 / Chapter CAHPTER FOUR: --- CONCLUDING REMARKS --- p.67 / APPENDIX I: BACKBONE TORSION ANGLES AND SUGAR RING CONFORMATIONS OF THE OPTIMIZED GEOMETRIES --- p.69 / APPENDIX II: BACKBONE TORSION ANGLES OF THE EXPERIMENTAL SEQUENCES FROM NUCLEIC ACID DATABASE (NDB) --- p.77 / REFERENCES --- p.92

Transcriptomics and gene expression profiling enables the elucidation of the genetic response of an organism to various environmental cues. Transcriptomics enables the deciphering of differences between two closely related organisms to the same environment and in contrast, enables the elucidation of genetic responses of the same organism to different environmental cues. Two major methods are utilized for the study of transcriptomes, high-throughput sequencing and microarray analysis. High-throughput sequencing technologies such as the Illumina platform are relatively new and protocols must be developed for the analyses of transcriptomes (RNA-sequencing). A RNA-seq protocol was developed and refined for the Illumina sequencing platform. This protocol was then utilized for the de novo sequencing of the steelhead salmon transcriptome. Hatchery steelhead exhibit a reduced fitness compared to wild steelhead that has been shown to be genetically based. Consequently, the steelhead transcriptome was assembled, annotated, and used to identify gene expression differences between hatchery and wild fish. We uncovered many differentially expressed genes involved in metabolic processes and growth and development. This work has created a better understanding of the genetic differences between hatchery and wild steelhead salmon. Brachypodium distachyon is a monocot grass important as a model for cereal crops and potential biofuels feedstocks. To better understand the genetic response of this plant to different environmental cues, a comprehensive assessment of the transcriptomic response was conducted under a variety of conditions including diurnal/circadian light/dark/temperature environments and different abiotic stress conditions. Using a whole-genome tiling DNA microarray, we identified that the majority of transcripts in Brachypodium exhibit a daily rhythm in their abundance that is conserved between rice and Brachypodium. We also identified numerous cis-regulatory elements dictating these rhythmic expression patterns. We also identified the genetic response to abiotic stresses such as salinity, drought, cold, heat, and high light. We uncovered a core set of genes which responds to all stresses, indicating a core stress response. A large number of transcription factors were uncovered as potential nodes for regulating the abiotic stress response in Brachypodium. Moreover, promoter elements that drive specific responses to discrete abiotic stresses were uncovered. Altogether, the transcriptome analyses in this work furthers our understandings of how particular organisms respond to environmental cues and better elucidates the relationship between genes and the environment. / Graduation date: 2012 / Access restricted to the OSU Community at author's request from Oct. 5, 2011 - April 5, 2012.

The advent of new technology enhance the speed and reduce the cost for sequencing biological data. Making biological sense of this genomic data is a big challenge to the algorithm design as well as the high performance computing society. There are many problems in Bioinformatics, such as how new functional genes arise, why genes are organized into chromosomes, how species are connected through the evolutionary tree of life, or why arrangements are subject to change. Phylogenetic analyses have become essential to research on the evolutionary tree of life. It can help us to track the history of species and the relationship between different genes or genomes through millions of years. One of the fundamentals for phylogenetic construction is the computation of distances between genomes. Since there are much more complicated combinatoric patterns in rearrangement events, the distance computation is still a hot topic as much belongs to mathematics as to biology. For the distance computation with input of two genomes containing unequal gene contents (with insertions/deletions and duplications) the problem is especially hard. In this thesis, we will discuss about our contributions to the distance estimation for unequal gene order data. The problem of finding the median of three genomes is the key process in building the most parsimonious phylogenetic trees from genome rearrangement data. For genomes with unequal contents, to the best of our knowledge, there is no algorithm that can help to find the median. In this thesis, we make our contributions to the median computation in two aspects. 1) Algorithm engineering aspect, we harness the power of streaming graph analytics methods to implement an exact DCJ median algorithm which run as fast as the heuristic algorithm and can help construct a better phylogenetic tree. 2) Algorithmic aspect, we theoretically formulate the problem of finding median with input of genomes having unequal gene content, which leads to the design and implementation of an efficient Lin-Kernighan heuristic based median algorithm. Inferring phylogenies (evolutionary history) of a set of given species is the ultimate goal when the distance and median model are chosen. For more than a decade, biologists and computer scientists have studied how to infer phylogenies by the measurement of genome rearrangement events using gene order data. While evolution is not an inherently parsimonious process, maximum parsimony (MP) phylogenetic analysis has been supported by widely applied to the phylogeny inference to study the evolutionary patterns of genome rearrangements. There are generally two problems with the MP phylogenetic arose by genome rearrangement: One is, given a set of modern genomes, how to compute the topologies of the according phylogenetic tree; Another is, given the topology of a model tree, how to infer the gene orders of the ancestor species. To assemble a MP phylogenetic tree constructor, there are multiple NP hard problems involved, unfortunately, they organized as one problem on top of other problems. Which means, to solve a NP hard problem, we need to solve multiple NP hard sub-problems. For phylogenetic tree construction with the input of unequal content genomes, there are three layers of NP hard problems. In this thesis, we will mainly discuss about our contributions to the design and implementation of the software package DCJUC (Phylogeny Inference using DCJ model to cope with Unequal Content Genomes), that can help to achieve both of these two goals. Aside from the biological problems, another issue we need to concern is about the use of the power of parallel computing to assist accelerating algorithms to handle huge data sets, such as the high resolution gene order data. For one thing, all of the method to tackle with phylogenetic problems are based on branch and bound algorithms, which are quite irregular and unfriendly to parallel computing. To parallelize these algorithms, we need to properly enhance the efficiency for localized memory access and load balance methods to make sure that each thread can put their potentials into full play. For the other, there is a revolution taking place in computing with the availability of commodity graphical processors such as Nvidia GPU and with many-core CPUs such as Cray-XMT, or Intel Xeon Phi Coprocessor with 60 cores. These architectures provide a new way for us to achieve high performance at much lower cost. However, code running on these machines are not so easily programmed, and scientific computing is hard to tune well on them. We try to explore the potentials of these architectures to help us accelerate branch and bound based phylogenetic algorithms.

The nucleotide sequence of a cloned RAPD DNA marker (OPI08) linked to a disease resistance gene in L. sativa (lettuce) revealed homology with the conserved domain of the reverse transcriptase of Ty3/gypsy retrotransposons. To further characterize the presence of Ty3/gypsy-like sequences in plants, sets of degenerate primers deduced from archetype retrotransposons were used for PCR amplification of a sequence domain characteristic of the reverse transcriptase and the integrase of Ty3/gypsy retrotransposons. The nucleotide sequence of two cloned DNA fragments of Z. mays (maize) and A. thaliana proved to be homologous with the conserved domains of the reverse transcriptase and the integrase of Ty3/gypsy retrotransposons. Southern blot analysis also demonstrated homology of the Z. mays clone to Lycopersicon (tomato) and L. sativa. This is the first report of Ty3/gypsy-like sequences in A. thaliana, and L. sativa. This research brings to six the number of plant species where this type of element has been reported, in contrast to the large number of plant Ty1/copia transposable elements described. It is not known whether these elements are actively transposing in plant genomes.

Background: Canine babesiosis is a tick-borne disease causing detrimental health effects on the domestic dogs with huge economic impact on the owners. The most complicated form of canine babesiosis is caused by a pathogenic Babesia rossi parasite. Canine babesiosis induced by B. rossi still remains the cause of mortality and morbidity in South African dogs, yet, the transcriptomic and genomic information of this parasite species is still not available. The transcriptomic and genomic information is essential in the disease development and processes for the design of effective disease control strategies. Consequently, our understanding of the mechanisms underlying the pathogenesis of the different genotypes of B. rossi remains limited. A previous study suggested a relationship between the parasite genotype and the disease phenotype. To date, thirteen B. rossi genotypes have been identified and associated with diverse clinical signs in their hosts. Hence the aim of this study was to sequence RNA from samples representing B. rossi genotypes, 19, 29 and 31, in order to have insight on the overall transcriptome of this parasite and to establish if there would be significant differences among the genotypes. Methodology: To screen for B. rossi positive samples, total DNA was extracted from 20 blood samples collected from sick domestic dogs presented at the Onderstepoort Veterinary Academic Hospital (OVAH). Babesia rossi infections were confirmed using the PCR-Reverse Line Blot (RLB) hybridization assay. Further confirmation of infection status was done by amplification of the B. rossi Erythrocyte Membrane Antigen 1 (BrEMA1) gene in all the DNA samples using qualitative PCR (qPCR), followed by sequencing of PCR products. Subsequently, total RNA was extracted from the 20 B. rossi-infected blood samples collected from the same dogs in which DNA was extracted. Three samples representing B. rossi genotypes 19, 29 and 31 were selected for transcriptome analysis. RNA sequencing was performed using the Illumina HiSeq 2000 to allow transcriptome analysis. De novo assembly was performed independently for all three transcriptomes using the Trinity software. The unigenes generated from specific transcriptome assemblies were subjected to global functional annotation using Blast2GO version 2.8.0 software, followed by KEGG database for annotation of biological pathways, and DAVID version 6.7, for COG classification to predict and classify their functions. Results: The sample representing B. rossi genotype 31 was excluded in the transcriptome analysis due to low RNA mass, which usually compromises the quality of the library used in RNA sequencing.Thus, a total of 26 747 238 and 25 709 627 paired-end reads were obtained from B. rossi genotypes 19 and 29, respectively. De novo transcriptome assembly produced a total of 3019 unigenes, with an average length of 419 bp and N50 of 362 bp in B. rossi genotype 19, and 2727 unigenes with an average length of 441 bp and N50 of 362 in B. rossi genotype 29. A total of 1193 unigenes were common between B. rossi genotype 19 and 29, while 1828 unigenes were exclusively detected in B. rossi genotype 19; and 1534 were specific to B. rossi genotype 29. Between the two B. rossi genotypes, a total of 4553 unigenes were obtained, representing the overall B. rossi transcriptome. From the overall transcriptome, 12.3% (n=558) of the unigenes could be annotated with 53 different gene ontology (GO) functional categories. About 34% (n=1550) of the unigenes represented in the overall transcriptome mapped to 237 KEGG pathways and only 2.5% (114) could be annotated in the COG database. Conclusion: Although, there were no striking differences in the transcriptomes of B. rossi genotypes 19 and 29, this study presents the first transcriptomic resource for B. rossi, which will highly contribute to our genetic understanding of B. rossi and provide a platform for future gene expression studies. Hypothetical proteins identified in this study will require further characterization as they may have a critical role in the biology and pathogenicity of B. rossi parasite. / Life and Consumer Sciences / M. Sc. (Life Sciences)

O câncer no colo do útero é o segundo tipo de neoplasia maligna mais prevalente nas mulheres no mundo todo e o seu rastreamento é realizado através do exame microscópico das células esfoliadas da mucosa cervical, o teste de Papanicolaou. Partindo-se da premissa que a infecção por Papilomavírus Humano oncogênico (HRHPV) é condição necessária para o desenvolvimento desta neoplasia, tem-se avaliado a possibilidade de empregar-se para a mesma finalidade, o rastreio, métodos que detectam o material genético viral. Estudos recentes demonstraram que estes testes moleculares apresentam maior sensibilidade e a substituição do teste citológico pelos moleculares vem ocorrendo em alguns países. O monitoramento da resposta ao tratamento das pacientes com câncer cervical é realizado por meio de testes inespecíficos como exames de imagem. Portanto, é desejável o desenvolvimento de um teste específico, não invasivo capaz de detectar precocemente a recidiva. Este estudo investigou a freqüência e os tipos de HPV presentes em tumores cervicais de pacientes brasileiras, e verificou a ocorrência de resultados HPV-negativos pelos testes moleculares, procurando investigar as causas de possíveis falhas dos testes. Além disso, padronizou e avaliou uma reação de PCR em tempo real capaz detectar o DNA do HPV-16 e 18 no plasma das pacientes. Foram incluídas no estudo 104 pacientes com diagnóstico confirmado de câncer cervical atendidas em três hospitais oncológicos do Estado de São Paulo, ICESP, IBCC e HC-Barretos, no período de novembro de 2009 a julho de 2011. Foram coletados um fragmento do tumor cervical e sangue total. O DNA extraído dos tumores foi submetido a genotipagem do HPV por meio da utilização dos kits Linear Array HPV Genotyping Test (LA) e PapilloCheck®, além de PCRs tipo específicas para HPV-16, 18 e 45. Amostras que apresentaram resultado HPV-negativo no PapilloCheck®, tiveram parte da região E1 seqüenciada para verificar potenciais causas do resultado falso-negativo. A amostra de plasma das pacientes que apresentavam HPV-16 e/ou 18 no tecido tumoral foi submetida à PCR em tempo real para avaliar a presença do DNA do HPV no plasma. Das 104 amostras de tecido tumoral, 103 foram positivas para HPV, sendo que os HPV-16 e 18, como infecção simples, foram os encontrados com maior freqüência (48,5%). Os testes LA e PapilloCheck® apresentaram 65,4% de concordância total. O PapilloCheck® apresentou 12,5% de resultados falso-negativos (N=13). A principal hipótese para explicar esses resultados é a presença de mutações na região de hibridização dos iniciadores e/ou das sondas. A reação de PCR em tempo real específica para HPV-16 e 18 padronizada no estudo apresentou baixa sensibilidade, mas alta especificidade e não deve ser utilizada como teste diagnóstico para o câncer cervical. A relação entre DNA de HPV no plasma e o prognóstico da paciente deve ser explorada no futuro / Invasive cervical cancer is the second most common cancer among women worldwide and screening programs are based on microscopical examination of cells exfoliated from the cervical mucosa, the Papanicolaou test. Since the infection by an oncogenic HPV (HR-HPV) has been shown to be necessary for the development of this neoplasia, molecular assays are being evaluated for the same application, primary screening. Indeed, recent studies showed these methods to be more sensitive and therefore are replacing the cytological test in many countries. Post treatment surveillance of invasive cervical cancer patients are made by unspecific tests as imaging exams. Hence, the development of a specific non invasive test able to detect premature recurrence is desired. This study investigated the HPV frequency and types on invasive cervical tumors among Brazilian patients observing the occurrence of HPV-negative results on molecular tests, trying to addrees the causes for the putative failures. Moreover, we standardized and evaluated a real time PCR method for HPV-16 and 18 DNA detection on patients plasma. One hundred and four women with invasive cervical cancer were recruited in three oncologic hospitals from São Paulo State, ICESP, IBCC and HCBarretos, in between November 2009 and July 2011. Tumor tissue and whole blood were collected. DNA extracted from the tumors were submitted to HPV detection and genotyping tests such as the Linear Array HPV Genotyping Test (LA) and PapilloCheck®, besides type specific PCRs for HPV-16, 18 e 45. Samples that showed an HPV-negative result on the PapilloCheck® were submitted to direct sequencing of E1 region to verify potential mismatches responsible for that. Plasma samples from patients with tumor tissue positive for HPV-16 and/or 18 were submitted to the real time PCR to evaluate the HPV DNA presence on plasma. Out of 104 cervical carcinomas, 103 were HPV positive, HPV-16 and 18, as single infection, were the most frequent types observed (48.5%). LA and PapilloCheck® showed 65.4% of total agreement. PapilloCheck® displaied 12.5% of false-negative results (N=13). The major hypothesis to explain these results is the presence of mismatches to the primers and/or probes annealing regions. Real time PCR specific for HPV-16 and 18 developed in this study showed low sensitivity, but a high specificity and cannot be used as an invasive cervical cancer diagnostic tool. The relationship between the presence of HPV DNA in the plasma and the patient prognostic shall be evaluated in the future

CAPES / A produção de carne suína no Brasil tem dado um grande salto nos últimos anos, no seu aspecto quantitativo e qualitativo. Isso permitiu posicionar o Brasil dentre os principais atuantes mundiais no setor da suinocultura. A garantia da inocuidade de alimentos depende da capacidade das indústrias em minimizar a contaminação de alimentos por microrganismos patogênicos, principalmente durante o processamento e estocagem, e controlar para que a garantia continue sendo mantida. Dentre os patógenos existentes, destaca-se Listeria monocytogenes que é um importante patógeno de origem alimentar emergente e causadora de infecções localizadas e generalizadas, levando o individuo ao óbito. Neste sentido, o objetivo deste estudo foi avaliar a qualidade sanitária do processo produtivo de um abatedouro-frigorifico de suínos, quanto à presença de L. monocytogenes. Inicialmente foram padronizados os métodos para aplicação da PCR e análise de sequenciamento posteriormente aplicado nas amostras coletadas. Duas coletas foram realizadas em um frigorífico que abate suínos localizado na região dos Campos Gerais – PR. Também verificou-se os prazos e custos apresentados por laboratórios particulares brasileiros, fazendo uma comparação com a técnica molecular. A quantidade de amostras positivas da seunda coleta foi superior em relação a primeira, 10 e 3 respectivamente. Com o sequenciamento foi possível observar que a origem da contaminação encontra-se no setor de triparia. Também foi possível verificar que poucos laboratórios possuem a PCR como método de diagnóstico, e provou-se ainda que a técnica pode ser uma ótima alternativa por se apresentar econômica e viável para indústria de carnes. / The production of pork in Brazil has taken a great leap in recent years, in quantitative and qualitative. This allowed position Brazil among the main active worldwide in the field of pig farming. The guarantee of the safety of food depends on the ability of industries to minimise the contamination of food by pathogenic micro-organisms, especially during processing and storage, and to ensure that the guarantee will continue to be maintained. Among the existing pathogens, stands out Listeria monocytogenes, which is an important pathogen of food origin emerging and causing localized infections and generalized, leading the individual to death. In this sense, the objective of this study was to evaluate the health quality of the production process from an abattoir-fridge of pigs, as the presence of L. monocytogenes. Initially were standardized methods for the application of PCR analysis and sequencing of subsequently applied in the samples collected. Two samples were collected in a fridge that slaughter pigs located in the region of Campos Gerais - PR. Also it was found that the deadlines and costs presented by private laboratories Brazilians, making a comparison with the molecular technique. The number of positive samples of seunda collection was superior to that of the first, 10 and 3 respectively. With the sequencing was possible to observe that the source of contamination is in the sector of triparia. It was also possible to observe that few laboratories have the PCR as a method of diagnosis, and it has been proved that the technique can be a great alternative to present economic and feasible for meat industry.