Rearranjos genômicos são alterações estruturais na molécula de DNA e podem ser a causa de inúmeras doenças genéticas. O mecanismo gerador dessas alterações é bem variável. Ele pode ser recorrente, por intermédio de low copy repeats (LCRs), resultando num rearranjo causado por recombinação homóloga não-alélica (NAHR), ou não recorrente, ou seja, sem intermédio de um hotspot. Dentre os mecanismo não recorrentes temos: a junção das extremidades não-homólogas (NHEJ - non-homologous end joining) e a junção mediada por micro-homologia (MMEJ - microhomology-mediated end joining), a replicação em série por deslizamento (SRS), a SRS induzida por quebra (BISRS), a replicação induzida pela quebra de DNA por homologia (MMBIR - microhomology-mediated break induced replication), o enrolamento da forquilha de replicação e mudança de molde de DNA (FoSTeS - fork stalling and template switching). A análise dos pontos de quebra dos rearranjos genômicos pode fornecer informações importantes para uma maior compreensão da arquitetura genômica e seu papel na geração das anormalidades estruturais. O objetivo deste trabalho foi sequenciar os pontos de quebra genômicos a fim de identificar o mecanismo formador das alterações encontradas. Para isso, investigamos o panorama estrutural de 10 pacientes por sequenciamento por meio de linked reads (10X Genomics) e sequenciamos os pontos de quebra previamente identificados por array CytoSNP-12 (Illumina) de 12 pacientes com rearranjos genômicos estruturais por utilizando a captura por Nextera Rapid Capture (Illumina). A investigação por linked reads revelou rearranjos estruturais em 5 pacientes, destacando translocações encontradas em dois pacientes, impossíveis de serem detectadas por metodologias de sequenciamento que não envolva long reads. Foi possível sugerir os mecanismos causadores dessas alterações como NHEJ. O sequenciamento após a captura por Nextera foi capaz de identificar elementos que permitiram definir o mecanismo em três pacientes (NAHR E FoSTeS/MMBIR) e sugerir em mais dois pacientes (NHEJ). Com a estratégia utilizada foi possível sequenciar pontos de quebra por meio do flanqueamento das regiões identificadas por array, identificar os elementos genômicos presentes nos pontos de quebra e os mecanismos formadores dessas alterações / Genomic rearrangements are structural changes in the DNA molecule and can be the cause of numerous genetic diseases. The mechanisms that generate these alterations can occur in different ways. It can be recurrent, mediated by low copy repeats (LCRs), resulting in a rearrangement cause by non-alellic homologue recombination (NAHR), or non-recurrent, without a hotspot. Among non-recurrent mechanisms there are: non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), serial replication slippage (SRS), break-induced serial replication slippage (BISRS), microhomology-mediated break induced replication (MMBIR) and fork stalling and template switching (FoSTeS). Analysis of the breakpoints of genomic rearrangements may provide important information for a better understanding of genomic architecture and its role in generating structural abnormalities. The aim of this work was to sequence the genomic breakpoints in order to identify the mechanism that formed the alterations found. To do this, we investigated the genomic structure of 10 patients by linked reads (10X Genomics) sequencing and sequenced the breakpoints previously identified by Illumina CytoSNP-12 array of 12 patients with structural genomic imbalances by using Nextera Rapid Capture (Illumina). The research by linked reads revealed structural rearrangements in 5 patients, highlighting translocations found in two patients, impossible to be detected by sequencing methodologies that did not involve long reads. It was possible to suggest the mechanisms causing these changes as NHEJ. The sequencing after capture by Nextera was able to identify elements that allowed us to determine the formation mechanism in three patients (NAHR and FoSTeS / MMBIR) and to suggest in two patients (NHEJ). With the approach employed here, it was possible to sequencing breakpoints by flanking the regions identified by array, identifying the genomic elements present at breakpoints and the formation mechanisms of the alterations

Shum Ka Chiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 174-182). / Abstracts in English and Chinese. / Acknowledgement --- p.ii / Abstract --- p.iii / 摘要 --- p.vi / Table of content --- p.viii / List of figures --- p.xvi / List of tables --- p.xxii / Abbreviations --- p.xxv / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Importance of authentication of Traditional Chinese Medicines --- p.1 / Chapter 1.1.1 --- Confusing nomenclatures --- p.1 / Chapter 1.1.2 --- Similar morphologies of different medicinal materials --- p.2 / Chapter 1.1.3 --- Toxicities of medicinal materials --- p.2 / Chapter 1.1.4 --- Conservation of natural products --- p.2 / Chapter 1.2 --- TCM listed in the Pharmacopoeia of People's Republic of China --- p.3 / Chapter 1.3 --- Overview of mis-use and intoxication of TCM --- p.4 / Chapter 1.4 --- Ordinances regulating Chinese medicines as natural products --- p.7 / Chapter 1.4.1 --- Laws governing Chinese medicine --- p.7 / Chapter 1.4.2 --- Laws governing endangered species --- p.8 / Chapter 1.5 --- Current technologies in the authentication of Traditional Chinese Medicines and their limitations --- p.9 / Chapter 1.6 --- Historical applications of Radix Aconiti --- p.12 / Chapter 1.7 --- Modern applications of Radix Aconiti --- p.16 / Chapter 1.8 --- Research on Radix Aconiti and its chemical components --- p.17 / Chapter 1.8.1 --- Chemistry --- p.17 / Chapter 1.8.2 --- Pharmacology --- p.19 / Chapter 1.8.3 --- Molecular interaction --- p.22 / Chapter 1.9 --- Brief review on the systematics and phylogeny of Aconitum --- p.23 / Chapter 1.10 --- Historical applications of Radix Aucklandiae and related materials --- p.25 / Chapter 1.11 --- Modern applications of Radix Aucklandiae and related material --- p.27 / Chapter 1.12 --- Research on Aucklandiae and related material and their chemical components --- p.28 / Chapter 1.12.1 --- Chemistry --- p.28 / Chapter 1.12.2 --- Pharmacology --- p.29 / Chapter 1.13 --- Brief review on the systematics and phylogeny of Aucklandia and related medicinal species --- p.31 / Chapter 1.14 --- Authentication by DNA sequencing --- p.33 / Chapter 1.14.1 --- Introduction --- p.33 / Chapter 1.14.2 --- Criteria of sequence markers --- p.36 / Chapter 1.14.3 --- Model used to process polymorphism in DNA sequences --- p.37 / Chapter 1.15 --- Screening for novel markers --- p.38 / Chapter 1.15.1 --- Reason for screening novel markers --- p.38 / Chapter 1.15.2 --- Basic principle --- p.39 / Chapter 1.16 --- Introduction to gas chromatography- mass spectrometry --- p.40 / Chapter 1.16.1 --- Basic principles and components of GC-MS --- p.41 / Chapter 1.16.2 --- Advantages and limitations of GC-MS --- p.42 / Chapter 1.16.3 --- Usage of GC-MS on natural product analysis --- p.43 / Chapter 1.16.4 --- Chemometric analysis --- p.44 / Chapter 1.17 --- Objectives --- p.46 / Chapter Chapter 2. --- Materials and Methods --- p.47 / Chapter 2.1 --- Plant samples --- p.47 / Chapter 2.1.1 --- Samples of Aconitum --- p.47 / Chapter 2.1.2 --- Samples of Aucklandia and related species --- p.51 / Chapter 2.2 --- DNA extraction method --- p.58 / Chapter 2.2.1 --- Reagents --- p.58 / Chapter 2.2.2 --- Methods --- p.59 / Chapter 2.3 --- Chemical extraction methods --- p.61 / Chapter 2.4 --- Chemical standard extraction and purification method --- p.62 / Chapter 2.5 --- DNA sequencing --- p.63 / Chapter 2.5.1 --- Reagents --- p.63 / Chapter 2.5.2 --- Methods --- p.65 / Chapter 2.6 --- Genomic subtraction --- p.70 / Chapter 2.7 --- Search for species-specific markers from the subtraction library --- p.74 / Chapter 2.8 --- Gas chromatography- mass spectrometry --- p.74 / Chapter 2.9 --- GC-MS chemometric analysis --- p.75 / Chapter Chapter 3. --- Authentication of Aconitum by DNA Sequencing --- p.76 / Chapter 3.1 --- Introduction --- p.76 / Chapter 3.2 --- Methods --- p.77 / Chapter 3.3 --- Results - 5S spacer --- p.77 / Chapter 3.3.1 --- Sequence information --- p.77 / Chapter 3.3.2 --- Sequence similarity --- p.78 / Chapter 3.3.3 --- Phylogram study --- p.81 / Chapter 3.4 --- Results -psbA-trnH --- p.85 / Chapter 3.4.1 --- Sequence information --- p.85 / Chapter 3.4.2 --- Sequence similarity --- p.85 / Chapter 3.4.3 --- Phylogram study --- p.87 / Chapter 3.5 --- Discussion --- p.91 / Chapter 3.5.1 --- Overview of nuclear ribosomal 5S spacer --- p.91 / Chapter 3.5.2 --- Extensive polymorphism of 5S spacer --- p.91 / Chapter 3.5.3 --- Distribution of samples in the phylograms constructed by 5S spacer --- p.93 / Chapter 3.5.4 --- Utility of 5S spacer for authentication --- p.94 / Chapter 3.5.5 --- Overview of psbA-trnH spacer --- p.94 / Chapter 3.5.6 --- Distribution of samples in the phylograms constructed by psbA-trnH spacer --- p.95 / Chapter 3.5.7 --- A distinctive region of inversion --- p.96 / Chapter 3.5.8 --- Utility of psbA-trnH for authentication --- p.97 / Chapter Chapter 4. --- Screening for Novel Markers for Authentication of Aconitum --- p.98 / Chapter 4.1 --- Introduction --- p.98 / Chapter 4.2 --- Methods --- p.99 / Chapter 4.3 --- Results - subtracted clones --- p.99 / Chapter 4.4 --- Results - SSH6 --- p.104 / Chapter 4.4.1 --- Sequence information --- p.104 / Chapter 4.4.2 --- Sequence similarity --- p.105 / Chapter 4.5 --- Results-SSH15 --- p.107 / Chapter 4.5.1 --- Sequence information --- p.107 / Chapter 4.5.2 --- Sequence similarity --- p.107 / Chapter 4.5.3 --- Phylogram study --- p.109 / Chapter 4.6 --- Results-SSH45 --- p.113 / Chapter 4.6.1 --- Sequence information --- p.113 / Chapter 4.6.2 --- Sequence similarity --- p.113 / Chapter 4.6.3 --- Phylogram study --- p.115 / Chapter 4.7 --- Discussion --- p.119 / Chapter 4.7.1 --- Utility of subtraction in screening markers --- p.119 / Chapter 4.7.2 --- SSH6 --- p.121 / Chapter 4.7.3 --- SSH15 --- p.122 / Chapter 4.7.4 --- SSH45 --- p.123 / Chapter 4.7.5 --- Hybridization in Aconitum --- p.124 / Chapter 4.7.6 --- Inferring species identities of samples from the market --- p.126 / Chapter 4.8 --- Conclusion --- p.128 / Chapter Chapter 5. --- Assessment of Aucklandia lappa and Related Species by GC-MS --- p.129 / Chapter 5.1 --- Introduction --- p.129 / Chapter 5.2 --- Methods --- p.130 / Chapter 5.3 --- Results --- p.130 / Chapter 5.3.1 --- Extraction of essential oil --- p.130 / Chapter 5.3.2 --- GC-MS analysis --- p.131 / Chapter 5.3.3 --- Peak alignment and hierarchical cluster analysis --- p.133 / Chapter 5.3.4 --- Purification of chemical markers from Aucklandia lappa --- p.148 / Chapter 5.3.5 --- Standardization of the purified chemical markers --- p.148 / Chapter 5.3.6 --- Quantitative analysis of chemical markers --- p.152 / Chapter 5.4 --- Discussion --- p.154 / Chapter 5.4.1 --- Analysis of chemical composition --- p.154 / Chapter 5.4.2 --- A comparison on chemometric methods --- p.154 / Chapter 5.4.3 --- Similarity of chemical profiles --- p.156 / Chapter 5.4.4 --- Dendrogram analysis --- p.157 / Chapter 5.4.5 --- Utility of GC-MS in authentication of A. lappa and related species --- p.159 / Chapter 5.4.6 --- Limitations --- p.159 / Chapter 5.4.7 --- Comparison with molecular data --- p.161 / Chapter 5.4.8 --- Contents of dehydrocostuslactone and costunolide --- p.163 / Chapter 5.4.9 --- Locality study --- p.164 / Chapter 5.5 --- Conclusion --- p.165 / Chapter Chapter 6. --- General Discussion --- p.167 / Chapter 6.1 --- DNA sequencing --- p.168 / Chapter 6.2 --- Genomic subtraction --- p.169 / Chapter 6.3 --- Future work on molecular authentication --- p.170 / Chapter 6.4 --- Future work on authentication of Aconitum --- p.170 / Chapter 6.5 --- Gas chromatography- mass spectrometry --- p.171 / Chapter 6.6 --- Future work on authentication by GC-MS --- p.172 / Chapter 6.7 --- Future work on authentication of Aucklandia lappa and related species … --- p.173 / References --- p.174 / Appendix A. Sequence Alignment of 5S Spacer from Aconitum Species --- p.183 / Appendix B. Sequence Alignment of psbA- trnH Spacer from Aconitum Species --- p.188 / Appendix C. Sequences of Subtracted Clones from Aconitum --- p.191 / Appendix D. Sequence Alignment of SSH6 from Aconitum Species --- p.194 / Appendix E. Sequence Alignment of SSH15 from Aconitum Species --- p.195 / Appendix F. Sequence Alignment of SSH45 from Aconitum Species --- p.200 / Appendix G. Gas Chromatograms of Essential Oil Extracts of Aucklandia lappa and Related Species --- p.202

Tse, Sui Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 95-104). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgement --- p.v / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Motivation --- p.1 / Chapter 1.1.1 --- Basic Concepts and Definitions --- p.2 / Chapter 1.2 --- Evolutionary Algorithms --- p.5 / Chapter 1.2.1 --- Chromosome Encoding --- p.6 / Chapter 1.2.2 --- Selection --- p.7 / Chapter 1.2.3 --- Crossover --- p.9 / Chapter 1.2.4 --- Mutation --- p.10 / Chapter 1.2.5 --- Elitism --- p.11 / Chapter 1.2.6 --- Niching --- p.11 / Chapter 1.2.7 --- Population Manipulation --- p.13 / Chapter 1.2.8 --- Building Blocks --- p.13 / Chapter 1.2.9 --- Termination Conditions --- p.14 / Chapter 1.2.10 --- Co-evolution --- p.14 / Chapter 1.3 --- Local Search --- p.15 / Chapter 1.4 --- Memetic Algorithms --- p.16 / Chapter 1.5 --- Objective --- p.17 / Chapter 1.6 --- Summary --- p.17 / Chapter 2 --- Background --- p.18 / Chapter 2.1 --- Multiple Drugs Tumor Chemotherapy --- p.18 / Chapter 2.2 --- Bioinformatics --- p.22 / Chapter 2.2.1 --- Basics of Bioinformatics --- p.24 / Chapter 2.2.2 --- Applications on Biomedical Systems --- p.26 / Chapter 3 --- A New Drug Administration Dynamic Model --- p.29 / Chapter 3.1 --- Three Drugs Mathematical Model --- p.31 / Chapter 3.1.1 --- Rate of Change of Different Subpopulations --- p.32 / Chapter 3.1.2 --- Rate of Change of Different Drug Concen- trations --- p.35 / Chapter 3.1.3 --- Toxicity Effects --- p.35 / Chapter 3.1.4 --- Summary --- p.36 / Chapter 4 --- Memetic Algorithm - Iterative Dynamic Program- ming (MA-IDP) --- p.38 / Chapter 4.1 --- Problem Formulation: Optimal Control Problem (OCP) for Mutlidrug Optimization --- p.38 / Chapter 4.2 --- Proposed Memetic Optimization Algorithm --- p.40 / Chapter 4.2.1 --- Iterative Dynamic Programming (IDP) . . --- p.40 / Chapter 4.2.2 --- Adaptive Elitist-population-based Genetic Algorithm (AEGA) --- p.44 / Chapter 4.2.3 --- Memetic Algorithm 一 Iterative Dynamic Programming (MA-IDP) --- p.50 / Chapter 4.3 --- Summary --- p.56 / Chapter 5 --- MA-IDP: Experiments and Results --- p.57 / Chapter 5.1 --- Experiment Settings --- p.57 / Chapter 5.2 --- Optimization Results --- p.61 / Chapter 5.3 --- Extension to Other Mutlidrug Scheduling Model . --- p.62 / Chapter 5.4 --- Summary --- p.65 / Chapter 6 --- DNA Sequencing by Hybridization (SBH) --- p.66 / Chapter 6.1 --- Problem Formulation: Reconstructing a DNA Sequence from Hybridization Data --- p.70 / Chapter 6.2 --- Proposed Memetic Optimization Algorithm --- p.71 / Chapter 6.2.1 --- Chromosome Encoding --- p.71 / Chapter 6.2.2 --- Fitness Function --- p.73 / Chapter 6.2.3 --- Crossover --- p.74 / Chapter 6.2.4 --- Hill Climbing Local Search for Sequencing by Hybridization --- p.76 / Chapter 6.2.5 --- Elitism and Diversity --- p.79 / Chapter 6.2.6 --- Outline of Algorithm: MA-HC-SBH --- p.81 / Chapter 6.3 --- Summary --- p.82 / Chapter 7 --- DNA Sequencing by Hybridization (SBH): Experiments and Results --- p.83 / Chapter 7.1 --- Experiment Settings --- p.83 / Chapter 7.2 --- Experiment Results --- p.85 / Chapter 7.3 --- Summary --- p.89 / Chapter 8 --- Conclusion --- p.90 / Chapter 8.1 --- Multiple Drugs Cancer Chemotherapy Schedule Optimization --- p.90 / Chapter 8.2 --- Use of the MA-IDP --- p.91 / Chapter 8.3 --- DNA Sequencing by Hybridization (SBH) --- p.92 / Chapter 8.4 --- Use of the MA-HC-SBH --- p.92 / Chapter 8.5 --- Future Work --- p.93 / Chapter 8.6 --- Item Learned --- p.93 / Chapter 8.7 --- Papers Published --- p.94 / Bibliography --- p.95

Chen, Zhang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (p. 109-114). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 摘要 --- p.III / ACKNOWLEDGEMENTS --- p.IV / PUBLICATIONS --- p.VI / CONTRIBUTORS --- p.VII / TABLE OF CONTENTS --- p.VIII / LIST OF TABLES --- p.XIII / LIST OF FIGURES --- p.XIV / LIST OF ABBREVIATIONS --- p.XVI / Chapter SECTION I : --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- PRENATAL DIAGNOSIS OF FETAL TRISOMY BY NEXT GENERATION SEQUENCING TECHNOLOGY --- p.2 / Chapter 1.1 --- FETAL TRISOMY --- p.2 / Chapter 1.2 --- CONVENTIONAL PRENATAL DIAGNOSIS OF FETAL TRISOMIES --- p.3 / Chapter 1.3 --- CELL FREE FETAL D N A AND ITS APPLICATION IN PRENATAL DIAGNOSIS --- p.5 / Chapter 1.4 --- NEXT GENERATION SEQUENCING TECHNOLOGY --- p.5 / Chapter 1.5 --- SUBSTANTIAL BIAS IN THE NEXT GENERATION SEQUENCING PLATFORM --- p.9 / Chapter 1.6 --- PRENATAL DIAGNOSIS OF TRISOMY BY NEXT GENERATION SEQUENCING --- p.10 / Chapter 1.7 --- AIMS OF THIS THESIS --- p.11 / Chapter SECTION I I : --- MATERIALS AND METHODS --- p.13 / Chapter CHAPTER 2: --- METHODS FOR NONINVASIVE PRENATAL DIAGNOSIS OF FETAL TRISOMY MATERNAL PLASMA DNA SEQUENCING --- p.14 / Chapter 2.1 --- STUDY DESIGN AND PARTICIPANTS --- p.14 / Chapter 2.1.1 --- Ethics Statement --- p.14 / Chapter 2.1.2 --- "Study design, setting and participants" --- p.14 / Chapter 2.2 --- MATERNAL PLASMA D N A SEQUENCING --- p.17 / Chapter 2.3 --- SEQUENCING DATA ANALYSIS --- p.18 / Chapter SECTION I I I : --- TRISOMY 13 AND 18 DETECTION BY THE T21 BIOINFORMATICS ANALYSIS PIPELINE --- p.21 / Chapter CHAPTER 3: --- THE T21 BIOINFORMATICS ANALYSIS PIPELINE FOR TRISOMY 13 AND 18 DETECTION --- p.22 / Chapter 3.1 --- INTRODUCTION --- p.22 / Chapter 3.2 --- METHODS --- p.23 / Chapter 3.2.1 --- Bioinformatics analysis pipeline for trisomy 13 and 18 detection --- p.23 / Chapter 3.3 --- RESULTS --- p.23 / Chapter 3.3.1 --- Performance of the T21 bioinformatics analysis pipeline for trisomy 13 and 18 detection --- p.23 / Chapter 3.3.2 --- The precision of quantifying chrl 3 and chrl 8 --- p.27 / Chapter 3.4 --- DISCUSSION --- p.29 / Chapter SECTION IV : --- IMPROVING THE T21 BIOINFORMATICS ANALYSIS PIPELINE FOR TRISOMY 13 AND 18 DETECTION --- p.30 / Chapter CHAPTER 4: --- IMPROVING THE ALIGNMENT --- p.31 / Chapter 4.1 --- INTRODUCTION --- p.31 / Chapter 4.2 --- METHODS --- p.32 / Chapter 4.2.1 --- Allowing mismatches in the index sequences --- p.32 / Chapter 4.2.2 --- Calculating the mappability of the human reference genome --- p.33 / Chapter 4.2.3 --- Aligning reads to the non-repeat masked human reference genome --- p.34 / Chapter 4.2.4 --- Trisomy 13 and 18 detection --- p.34 / Chapter 4.3 --- RESULTS --- p.34 / Chapter 4.3.1 --- Increasing read numbers by allowing mismatches in the index sequences --- p.34 / Chapter 4.3.2 --- Increasing read numbers by using the non-masked reference genome for alignment . --- p.38 / Chapter 4.3.3 --- Allowing mismatches in the read alignment --- p.42 / Chapter 4.3.4 --- The performance of trisomy 13 and 18 detection after improving the alignment --- p.47 / Chapter 4.4 --- DISCUSSION --- p.50 / Chapter CHAPTER 5: --- REDUCING THE GC BIAS BY CORRECTION OF READ COUNTS --- p.53 / Chapter 5.1 --- INTRODUCTION --- p.53 / Chapter 5.2 --- METHODS --- p.54 / Chapter 5.2.1 --- Read alignment --- p.54 / Chapter 5.2.2 --- Calculating the correlation between GC content and read counts --- p.55 / Chapter 5.2.3 --- GC correction in read counts --- p.55 / Chapter 5.2.4 --- Trisomy 13 and 18 detection --- p.56 / Chapter 5.3 --- RESULTS --- p.56 / Chapter 5.3.1 --- GC bias in plasma DNA sequencing --- p.56 / Chapter 5.3.2 --- Correcting the GC bias in read counts by linear regression --- p.59 / Chapter 5.3.3 --- Correcting the GC bias in read counts by LOESS regression --- p.65 / Chapter 5.3.4 --- Bin size --- p.72 / Chapter 5.4 --- DISCUSSION --- p.75 / Chapter CHAPTER 6: --- REDUCING THE GC BIAS BY MODIFYING THE GENOMIC REPRESENTATION CALCULATION --- p.77 / Chapter 6.1 --- INTRODUCTION --- p.77 / Chapter 6.2 --- METHODS --- p.78 / Chapter 6.2.1 --- Modifying the genomic representation calculation --- p.78 / Chapter 6.2.2 --- Trisomy 13 and 18 detection --- p.78 / Chapter 6.2.3 --- Combining GC correction and modified genomic representation --- p.78 / Chapter 6.3 --- RESULTS --- p.79 / Chapter 6.3.1 --- Reducing the GC bias by modifying genomic representation calculation --- p.79 / Chapter 6.3.2 --- Combining GC correction and modified genomic representation --- p.86 / Chapter 6.4 --- DISCUSSION --- p.89 / Chapter CHAPTER 7: --- IMPROVING THE STATISTICS FOR TRISOMY 13 AND 18 DETECTION --- p.91 / Chapter 7.1 --- INTRODUCTION --- p.91 / Chapter 7.2 --- METHODS --- p.92 / Chapter 7.2.1 --- Comparing chrl 3 or chrl8 with other chromosomes within the sample --- p.92 / Chapter 7.2.2 --- Comparing chrl 3 or chrl 8 with the artificial chromosomes --- p.92 / Chapter 7.3 --- RESULTS --- p.93 / Chapter 7.3.1 --- Determining the trisomy 13 and 18 status by comparing chromosomes within the samples --- p.93 / Chapter 7.3.2 --- Determining the trisomy 13 and 18 status by comparing chrl3 or chrl 8 with artificial chromosomes --- p.97 / Chapter 7.4 --- DISCUSSION --- p.100 / Chapter SECTION V : --- CONCLUDING REMARKS --- p.102 / Chapter CHAPTER 8: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.103 / Chapter 8.1 --- THE PERFORMANCE OF THE T21 BIOINFORMATICS ANALYSIS PIPELINE DEVELOPED FOR TRISOMY 21 DETECTION IS SUBOPTIMAL FOR TRISOMY 13 AND 18 DETECTION --- p.103 / Chapter 8.2 --- THE ALIGNMENT COULD BE IMPROVED BY ALLOWING ONE MISMATCH IN THE INDEX AND USING THE NON-REPEAT MASKED HUMAN REFERENCE GENOME AS THE ALIGNMENT REFERENCE --- p.104 / Chapter 8.3 --- THE PRECISION OF QUANTIFYING CHR13 AND CHR18 COULD BE IMPROVED BY THE G C CORRECTION OR THE MODIFIED GENOMIC REPRESENTATION --- p.104 / Chapter 8.4 --- THE STATISTICS FOR TRISOMY 13 AND 18 DETECTION COULD BE IMPROVED BY COMPARING CHR13 OR CHR18 WITH ARTIFICIAL CHROMOSOMES WITHIN THE SAMPLE --- p.105 / Chapter 8.5 --- PROSPECTS FOR FUTURE WORK --- p.106 / REFERENCE --- p.109

O Hespesvírus 8 humano (HHV-8) é hiperendêmico na população indígena, mas os seus mecanismos de transmissão ainda são desconhecidos. Método: Os anticorpos contra o antígeno LANA e lítico do HHV-8 foram detectados por imunofluorescência, em 339 indígenas e 181 não-indígenas da Amazônia brasileira. Marcadores sorológicos de transmissão oro-fecal (hepatite A), parenteral (hepatites B e C) e sexual (herpes simples 2 e sífilis) foram detectados por Elisa específicos. O DNA do HHV-8, extraído da saliva, foi detectado por nested-PCR e sequenciado. Resultados: Os anticorpos contra o antígeno LANA ou lítico foram detectados em 79,1% nos indígenas e 6,1% nos não-indígenas. A soroprevalência do HHV-8 aumentou com a idade entre os indígenas sendo que as crianças já apresentavam alta prevalência, mas não houve diferença com relação ao sexo em nenhuma das populações. As populações indígenas e nãoindígenas não apresentaram diferenças na soroprevalência para os marcadores de transmissão oro-fecal e parenteral, entretanto a soroprevalência de marcadores de transmissão sexual foi menor entre os indígenas. O DNA do HHV-8 na saliva foi detectado em 23 % dos indígenas soropositivos. A detecção de DNA do HHV-8 diminuiu com a idade e foi mais comum em homens. As amostras positivas foram sequenciadas e agrupadas como subtipo E. Conclusão: Os dados sugerem a hipótese de transmissão horizontal e precoce, via saliva, do subtipo E do HHV- 8 na população indígena. / Human herpes virus type 8 (HHV-8) is hyperendemic in Amerindian populations, but its modes of transmission are unknown. Objectives: 1. Study the Human Herpesvirus-8 Infection and Oral Shedding in Amerindian and Non-Amerindian Populations in the Brazilian Amazon Region. 2. Methods: Antibodies against either HHV-8 latency-associated nuclear antigen (LANA) or HHV-8 lytic antigen were detected, by immunofluorescence assays, in 339 Amerindians and 181 non-Amerindians from the Brazilian Amazon. Serological markers of oro-fecal (hepatitis A), parenteral (hepatitis B and C), and sexual (herpes simplex virus type 2 and syphilis) transmission were measured by specific ELISA. Salivary HHV-8 DNA was detected by use of a nested polymerase chain reaction assay and was sequenced. Results: Antibodies against either LANA antigen or lytic were detected in 79.1% of Amerindians and in 6.1% of non-Amerindians. HHV-8 seroprevalence increased with age among Amerindians and already had high prevalence in childhood but was not sex specific in either population. The 2 populations did not differ in seroprevalence of oro-fecal or parenteral markers, but seroprevalence of markers of sexual transmission was lower among Amerindians. HHV-8 DNA in saliva was detected in 23 % of HHV-8 seropositive Amerindians. Detection of HHV-8 decreased with age and was more common in men. HHV-8 DNA samples were sequenced, and all clustered as subtype E. Conclusion: The data support the hypothesis of early acquisition and horizontal transmission, via saliva, of HHV-8 subtype E in Amerindian populations.

INTRODUÇÃO: Na última década surgiu uma nova técnica, o sequenciamento de nova geração, que, contrário ao método tradicional de Sanger, permite o sequenciamento em paralelo e em larga escala de múltiplos genes, ou até mesmo todos os genes humanos, a menor custo e com uma análise mais acelerada. Essa técnica possibilitou a descoberta de novos genes responsáveis por diversas doenças mendelianas, sendo rapidamente incorporada no contexto clínico. OBJETIVOS: comparar os resultados das técnicas de Sanger e sequenciamento de nova geração em amostras controle; introduzir a técnica de sequenciamento de nova geração no contexto clínico nas casuísticas de doenças ósseas genéticas e RASopatias; avaliar a sensibilidade diagnóstica desta técnica em amostras sem dados clínicos fornecidos. MÉTODOS: o sequenciamento de nova geração (sob a forma de um painel de genes customizado ou do exoma) foi realizado em amostras com mutações identificadas previamente por Sanger e em dois grupos de doenças mendelianas, 144 pacientes com doenças ósseas e 79 com RASopatias, além de 90 amostras sem dados clínicos conhecidos (45 casos e 45 controles). A técnica de Sanger foi aplicada em 29 amostras de doenças ósseas e em 81 amostras para confirmação de variantes identificadas pelo sequenciamento de nova geração. RESULTADOS: A sensibilidade da técnica de sequenciamento de nova geração foi estimada em 95,92% e a especificidade em 98,77%. Na casuística de doenças ósseas, a sensibilidade diagnóstica das amostras sequenciadas por Sanger foi de 69% (20/29), por painel customizado, 60% (75/125) e por exoma, 63% (12/19). Na casuística de RASopatias, a sensibilidade diagnóstica através do exoma foi de 46% (36/79). Como resultado deste trabalho, dois genes novos associados a RASopatias (LZTR1 e SOS2) e um associado a uma displasia esquelética (PCYT1A) foram identificados. Na análise das amostras sem conhecimento prévio da hipótese clínica foi obtida uma sensibilidade diagnóstica de 46,67% (21/45), mas que chegou a 73,08% (14/26) para as hipóteses de erros inatos do metabolismo. CONCLUSÕES: Foi demonstrado que a sensibilidade e a especificidade da técnica do sequenciamento de nova geração são altas e correspondentes a valores encontrados por outros grupos na literatura. Essa técnica foi considerada apropriada não apenas no contexto de pesquisa, demonstrado aqui pela descoberta de três novos genes associados a doenças mendelianas, como também para análises clínicas. Neste estudo a técnica foi aplicada com sucesso no contexto clínico, seja pelo painel customizado, seja pelo exoma, com uma positividade semelhante à encontrada pela técnica de Sanger. Mesmo na análise de amostras sem história clínica prévia foi possível identificar variantes patogênicas em quase metade dos casos, e numa porcentagem ainda maior quando a doença era um erro inato do metabolismo. Essa sensibilidade é comparável à obtida pela espectroscopia de massas em Tandem aplicada à triagem de múltiplas condições simultaneamente, o que sugere que a técnica do sequenciamento de nova geração poderá ser incorporada ao programa de triagem neonatal no futuro, ampliando o emprego de testes genéticos em complementaridade aos testes bioquímicos tradicionais / INTRODUCTION: In the last decade a new technique, the next generation sequencing, has emerged, which, contrary to the traditional Sanger method, performs parallel and high-throughput sequencing of multiple genes, or even all human genes, at a lower cost and with a faster analysis. This technique allowed the discovery of new genes responsible for several Mendelian diseases and has been quickly incorporated into the clinical context. OBJECTIVES: to compare the results of Sanger technique and next generation sequencing in control samples; to apply the next generation sequencing technique in the clinical practice to the cases of genetic skeletal disorders and RASopathies; to evaluate the diagnostic yield of this technique in samples without clinical data provided. METHODS: Next generation sequencing (in the form of a customized gene panel or exome) was performed in samples with mutations previously identified by Sanger sequencing and in two groups of Mendelian diseases, 144 patients with skeletal disorders and 79 patients with RASopathies, besides 90 samples with unknown clinical data (45 cases and 45 controls). The Sanger technique was applied in 29 samples of skeletal disorders and in 81 samples for confirmation of variants identified by next generation sequencing. RESULTS: The sensitivity of the next generation sequencing technique was estimated at 95.92% and the specificity at 98.77%. In the case of skeletal disorders, the diagnostic yield of the samples sequenced by Sanger was 69% (20/29), by customized panel, 60% (75/125), and by exome, 63% (12/19). In the individuals with RASopathies, the diagnostic yield through exome sequencing was 46% (36/79). As a result of this study, two new genes associated with RASopathies (LZTR1 and SOS2) and one associated with a skeletal dysplasia (PCYT1A) were identified. In the analysis of the samples without previous knowledge of the clinical hypothesis, a total diagnostic yield of 46.67% (21/45) was obtained, but it was up to 73.08% (14/26) in the group with hypothesis of inborn errors of metabolism. CONCLUSIONS: It was demonstrated that the sensitivity and specificity of the next generation sequencing technique are high and are similar to values found by other groups in the literature. The technique was considered appropriate not only for research, as demonstrated here by the identification of three new genes associated to Mendelian diseases, but also for clinical analysis. In this study the technique was successfully applied in the clinical context, both by customized panel and by exome, with positivity similar to that obtained by the Sanger technique. Even in the analysis of samples with no previous clinical history, it was possible to identify pathogenic variants in almost half of the cases, and an even greater percentage was obtained when the disease was an inborn error of metabolism. This sensitivity is comparable to that obtained by Tandem mass spectroscopy applied to multi-condition screening, suggesting that the next generation sequencing technique may be incorporated in the future into the neonatal screening program, increasing the use of genetic testing in complementarity to the biochemical tests

Infecções invasivas por Trichosporon spp. ocorrem com maior frequência em pacientes neutropênicos, principalmente portadores de doenças hematológicas malignas, e estão associadas a elevados índices de mortalidade devido às dificuldades na identificação do patógeno e à resistência aos fármacos mais empregados na terapêutica antifúngica. A identificação das espécies de Trichosporon é importante tanto para estudos epidemiológicos, como para associar aspectos clínicos com as espécies causadoras das infecções. Além disso, auxilia no tratamento da enfermidade, uma vez que a suscetibilidades aos fármacos antifúngicos pode variar de acordo com a espécie. Além disso, as leveduras do gênero Trichosporon sintetizam a glucuronoxilomanana (GXM) em sua parede celular, que pode estar envolvida no mecanismo de virulência do patógeno. Este estudo teve como objetivo determinar, por identificação fenotípica e molecular, espécies isoladas de pacientes internados em unidades hospitalares, comparando os resultados obtidos por ambos os métodos; avaliar diferenças na distribuição dessas espécies em relação às formas invasivas e não invasivas da infecção; determinar o perfil de suscetibilidade dessas leveduras aos antifúngicos, empregando um método de micro-diluição de referência e um método comercial; e avaliar a presença de GXM na parede celular dos isolados. Foram avaliados 74 isolados obtidos de amostras clínicas de pacientes do Hospital das Clinicas da FMUSP e de outras unidades hospitalares do Estado de São Paulo, no período de 2003 a 2011. Dezenove amostras foram isoladas de sítios estéreis do organismo (infecções invasivas) e 55 foram isoladas de urina e cateter (isolados não invasivos). Para a identificação das espécies, os isolados foram submetidos a análises fenotípicas, que incluíram estudo macro e micromorfológico, provas fisiológicas e avaliação do perfil bioquímico por sistema automatizado VITEK 2. A identificação molecular foi realizada pelo sequenciamento das regiões IGS e D1/D2 do DNA ribossomal. O perfil de suscetibilidade dos 74 isolados foi analisado pelo método de micro-diluição EUCAST (referência) com os fármacos fluconazol (FCZ), itraconazol (ITZ), voriconazol (VCZ), cetoconazol (CTZ), anfotericina B (AMB) e 5-fluocitosina (5FC); e pelo método de micro-diluição comercial Sensititre YeastOne, com os mesmos fármacos empregados no EUCAST, acrescidos do posaconazol (POS) e caspofungina (CAS). Os valores das concentrações inibitórias mínimas (CIM), erros categórico e essencial, bem como outros parâmetros foram comparados entre os dois métodos. A presença de GXM na parede celular dos 74 isolados foi determinada por citometria de fluxo, empregando anticorpo monoclonal anti-GXM. Os resultados dos estudos morfológicos e fisiológicos foram insuficientes para definir as espécies dos 74 isolados. Pela assimilação de carboidratos analisada pelo sistema VITEK 2, verificou-se que 71 isolados foram identificados como T. asahii (17 de infecção invasiva e 54 não invasivos), um isolado como T. mucoides (invasivo), e para dois isolados (um invasivo e um não invasivo), a identificação não foi conclusiva. Para estes últimos foi realizado o auxanograma (método manual), e a identificação permaneceu inconclusiva, pois pelo perfil de assimilação, os isolados poderiam ser identificados como T. asahii ou T. faecale. Pela técnica de sequenciamento, 62 dos 74 isolados foram identificados como T. asahii, demonstrando 82,4% de concordância com o sistema VITEK 2. Onze isolados com identificações discordantes pertenciam às espécies T. inkin (8), T. faecale (2) e T. dermatis (1), como determinado por sequenciamento. Dos dois isolados com identificação inconclusiva pelo VITEK 2, um foi identificado pela técnica molecular como T. asahii, enquanto para o outro isolado não foi possível definir a espécie. Portanto, dos 74 isolados do estudo, 62 foram identificados como T. asahii, 8 como T. inkin, 2 como T. faecale e 1 T. dermatis; dois isolados permaneceram sem identificação conclusiva. Os resultados dos testes de suscetibilidade in vitro mostraram que, em ambos os métodos, VCZ apresentou a melhor atividade antifúngica. Pelo método EUCAST, foram obtidos valores elevados de CIM para AMB, enquanto o mesmo não foi observado no teste comercial. Neste último, foram observados valores elevados de CIM para FCZ, POS e CAS. Em relação à 5FC, os valores de CIM 90% por ambos os testes foram elevados (16mg/L). Diferenças significantes foram observadas entre os valores de CIM obtidas pelos dois métodos, e percentuais relativamente elevados de erros categóricos graves quando o método comercial foi comparado ao de referência. Não houve diferença estatística significante de valores de CIM entre isolados de infecção invasiva e não invasiva, exceto para ITZ e 5FC. Cerca de 30% dos isolados obtidos de casos de infecção invasiva e não invasivos apresentaram resistência cruzada entre os azóis FCZ e VCZ, e uma pequena porcentagem apresentou multirresistência. Para a análise de GXM na parede celular dos 74 isolados do estudo, foi avaliada a intensidade de fluorescência emitida pela citometria de fluxo, não tendo sido observada diferença estatística significante entre isolados invasivos e não invasivos. O estudo permitiu concluir que T. asahii foi a espécie mais isolada das amostras clínicas obtidas de sítios estéreis e não estéreis. A metodologia clássica de identificação fenotípica não foi suficiente para definir as espécies do gênero Trichosporon, e o sistema VITEK 2 apresentou discordância quando comparado à técnica molecular para as espécies não T. asahii. Em relação aos testes de suscetibilidade in vitro, VCZ apresentou-se mais adequado para a inibição das leveduras, enquanto os fármacos AMB, FCZ e POS não foram eficazes para a maior parte dos isolados. As discordâncias encontradas entre o método de referência e o comercial sugerem que, para o segundo, são necessárias mais avaliações para seu emprego em rotina laboratorial para o gênero Trichosporon. A detecção de GXM não resultou em diferenças entre os isolados de ambos os grupos; no entanto, para se determinar o efeito protetor do polissacarídeo contra a ação de macrófagos, ensaios de fagocitose devem ser realizados / Invasive Trichosporon spp. infections occur more frequently in neutropenic patients, especially those with hematologic malignancies, and are associated with high mortality rates due to difficulties in identifying the pathogen and treating patients with drugs most currently employed in antifungal therapy. Trichosporon species identification is important for epidemiological studies and to better define eventual species-specific clinical association. Additionally, antifungal susceptibility may vary according to the species. Furthermore, glucuronoxylomannan (GXM) is a cell wall-associated polysaccharide produced by genus Trichosporon, which may be involved in virulence mechanisms of this pathogen. This study aimed (i) to identify Trichosporon species isolated from hospitalized patients by both phenotypic and molecular methods, comparing results; (ii) to verify the distribution of these species in invasive and non-invasive infection episodes; (iii) to determine the in vitro activities of various antifungals agents against the Trichosporon spp. isolates, employing a reference micro-dilution method and a commercial system; (iv) and to analyze the surface expression of GXM. Seventy-four Trichosporon spp. isolates obtained from clinical specimens of patients admitted to the Hospital das Clínicas-FMUSP and to other hospitals in the state of São Paulo, from 2003 to 2011, were included in the study. Nineteen samples were isolated from sterile deep sites (invasive infections) and 55 were isolated from catheter and urine samples (non-invasive isolates). All isolates were submitted to phenotypic analysis, which consisted in morphological features observation, physiological tests and determination of the biochemical profile by VITEK 2 system. Molecular identification was performed by sequencing of IGS1 and D1/D2 regions from the ribosomal DNA. The susceptibility antifungal profiles of the 74 isolates were analyzed by both the EUCAST micro-dilution method (reference) employing fluconazole (FCZ), itraconazole (ITZ), voriconazole (VCZ), ketoconazole (CTZ), amphotericin B (AMB) and 5 - flucytosine (5FC), and the commercial micro-dilution test Sensititre YeastOne, with the same drugs employed in EUCAST plus posaconazole (POS) and caspofungin (CAS). The minimum inhibitory concentration values (MIC), categorical and essential errors as well as other susceptibility parameters were compared between both methods. The cell wall expression of GXM of all isolates was measured by flow cytometry employing an anti-GXM monoclonal antibody. The morphological and physiological features of the Trichosporon spp. isolates were insufficientto define species. The carbohydrate assimilation analysis, performed by VITEK 2 system, has resulted in 71 isolates identified as T. asahii (17 from invasive infections and 54 non-invasive isolates) and one isolate as T. mucoides (invasive). The species identification for the two remaining isolates (one invasive and one non-invasive) was inconclusive. For this reason, a manual auxanogram was performed with these isolates, resulting again in non-conclusive species identification. By the automated sequencing method, 62 of the 74 isolates were identified as T. asahii, showing 82.4% of agreement with the VITEK 2 identification. Eleven isolates were identified by sequencing as T. inkin (8), T. faecale (2) and T. dermatis (1), showing disagreement identification with the VITEK 2 system. Regarding the two isolates with inconclusive results by the carbohydrate assimilation, the molecular technique identified one as T. asahii, whereas for the other isolate the sequencing was also unable to define species. Therefore, among the 74 studied isolates, 62 were identified as T. asahii, eight as T. inkin, two as T. faecale and one as T. dermatis; and two isolates remained with unconclusive identification. Almost all Trichosporon spp. isolates displayed susceptibility to VCZ with both methods. By the EUCAST method, high values of MIC were observed for AMB, while by the commercial test especially the invasive isolates showed susceptibility to this drug. Additionally, the Sensititre kit provided elevated MIC values for FCZ, POS and CAS. In regards to 5FC, the MIC 90% values were consistently high (16 mg/L) in both methodologies. The MIC values obtained by both EUCAST and commercial methods were compared, resulting in significant differences of MIC values for all tested antifungal drugs; major categorical errors occurred at relatively high percentage with the commercial method. No statistically significant differences in MIC values were verified when invasive and non-invasive isolates were compared. Around 30% of both invasive and non-invasive isolates showed cross-resistance to FCZ and VCZ, while a small number of isolates was multiresistant. The GXM analysis by cytometry demonstrated no significant differences between invasive and non- invasive isolates. This study demonstrated that T. asahii was the most frequently isolated species from both deep and non-sterile sites of the patients. The classical phenotypic methodology was not able to define Trichosporon species, and the VITEK 2 system identification showed disagreement with the sequencing technique for the non-T. asahii species. Regarding the in vitro susceptibility tests, VCZ was the most effective drug against the isolates, whereas most of them appear to be less susceptible to AMB, FCZ and POS. The discrepancies in the Trichosporon spp. susceptibility results between the reference and commercial methods suggest that the latter requires further evaluation tests before it can be used in routine laboratory. Although the GXM expression seemed to be equal in both invasive and non-invasive Trichosporon spp. isolates, phagocytic assays should be performed in order to determine the protective effect of the polysaccharide against phagocytosis

Analysis of gene expression has undergone a technological revolution. What was impossible 6 years ago is now routine. High-throughput DNA sequencing machines capable of generating hundreds of millions of reads allow, indeed force, a major revision toward the study of the genome’s functional output—the transcriptome. This thesis examines the history of DNA sequencing, measurement of gene expression by sequencing, isoform complexity driven by alternative splicing and mammalian piRNA precursor biogenesis. Examination of these topics is framed around development of a novel RNA-templated DNA-DNA ligation assay (SeqZip) that allows for efficient analysis of abundant, complex, and functional long RNAs. The discussion focuses on the future of transcriptome analysis, development and applications of SeqZip, and challenges presented to biomedical researchers by extremely large and rich datasets.

INTRODUÇÃO: A principal causa de mortalidade em pacientes com fibrose cística é o declínio da função pulmonar, relacionada à infecção respiratória de repetição. A rinossinusite crônica pode contribuir na deterioração da função pulmonar, porque o nariz e seios paranasais podem representar um reservatório de potenciais patógenos que causam as infecções pulmonares recorrentes ou crônicas. Métodos como o sequenciamento de nova geração, na identificação do microbioma, mostraram a natureza polimicrobiana das infecções respiratórias em fibrose cística, com a caracterização de agentes infecciosos não detectados nos métodos convencionais de cultura. Ainda muito pouco se sabe a respeito da composição e diversidade do microbioma desses pacientes. OBJETIVO: Descrever a composição do microbioma bacteriano do meato médio e do escarro de pacientes adultos com fibrose cística. Comparar riqueza, diversidade e dominância do microbioma dos pacientes com doença pulmonar discreta ou moderada com pacientes com doença pulmonar grave. PACIENTES E MÉTODOS: Foi avaliado o microbioma do meato médio e escarro de 31 adultos com fibrose cística, utilizado a análise do gene 16S rRNA por meio do sequenciamento de nova geração. RESULTADOS: Staphylococcus, Streptococcus e Corynebacterium foram os gêneros mais abundantes no meato médio e Pseudomonas, Haemophilus e Prevotella, no escarro. Nos pacientes com doença grave, observamos um aumento na prevalência de Pseudomonas nos dois sítios estudados isoladamente. Na análise pareada de escarro e meato médio, obtivemos concordância na composição do microbioma apenas em pacientes com doença discreta a moderada, o mesmo não foi observado no grupo com doença grave. CONCLUSÃO: O avanço nos conhecimentos da composição e diversidade do microbioma nas vias aéreas dos pacientes com fibrose cística é fundamental para o entendimento da fisiopatologia da doença, além de seu papel na criação novas perspectivas e possibilidades de tratamentos. Esse é o primeiro trabalho brasileiro a estudar o microbioma de vias aéreas em pacientes com fibrose cística. Nossos achados estão em concordância com a literatura internacional, ao apontar a Pseudomonas como importante elemento na fisiopatologia da doença, presente tanto no escarro como no meato médio dos pacientes com doença pulmonar grave / INTRODUCTION: The main cause of mortality in patients with cystic fibrosis is the decline in lung function, related to recurrent respiratory infection. Chronic rhinosinusitis leads to significant morbidity and contributes to the pathophysiology of lung disease. In cystic fibrosis, the nose and paranasal sinuses may represent a reservoir of potential respiratory pathogens and contribute to recurrent or chronic lung infections. Culture independent molecular detection methods of microbiome have shown the polymicrobial nature of respiratory infections in cystic fibrosis, with the characterization of undetectable pathogenic agents in conventional culture methods. Composition and diversity of the airway microbiome is still poor explored. METHODS: This study evaluated the airway microbiome of 31 adult cystic fibrosis patients, with the analysis of the 16S rRNA by the next generation sequencing. RESULTS: Staphylococcus, Streptococcus e Corynebacterium were the most abundant genera in middle meatus and Pseudomonas, Haemophilus e Prevotella, in sputum. In patients with advanced disease, we noticed an increase in Pseudomonas prevalence in both sample types studied separately. In paired analysis, sputum and middle meatus have shown a similarity in microbiome composition in patients with mild or moderate disease. This was not observed in patients with advanced disease. CONCLUSION: Advances in the knowledge of the composition and diversity of the airway microbiome of cystic fibrosis patients are essential for understanding the pathophysiology of the disease. It has an important role in creating new perspectives and possibilities of treatments. There is a lack in the literature of studies with evaluation of the airway microbiome of these patients in Brazil. This is the first Brazilian study to evaluate the airway microbiome of cystic fibrosis patients. Our finds agreed with international literature, when point at Pseudomonas role in disease pathophysiology, present in sputum and middle meatus of patients with advanced disease

Introdução: Pearlman e colaboradores relacionou a presença de mutações ativadoras no gene MAP3K1 com o desenvolvimento testicular anormal em pacientes com disgenesia gonadal 46,XY familial, embora os estudos em camundongos tenham demonstrado que o gene Map3k1 não é essencial para a determinação testicular. No desenvolvimento gonadal masculino, a ligação do MAP3K1 à proteína RHOA promove uma fosforilação normal de p38 e ERK1/2, o que determina um bloqueio da via da beta-catenina pela MAP3K4. Já no desenvolvimento feminino, ocorre uma hiper fosforilação de p38 e ERK1/2, o que determina a ativação da via da beta-catenina e o bloqueio da via de retroalimentação positiva do SOX9 e o desenvolvimento testicular. Objetivos: Pesquisar a presença de variantes alélicas do gene MAP3K1 em pacientes portadores de distúrbios do desenvolvimento sexual (1) 46,XY por anormalidades do desenvolvimento gonadal e avaliar a repercussão funcional das variantes identificadas. Casuística e Métodos: Quarenta e sete pacientes com disgenesia gonadal 46,XY (17 com a forma completa e 29 com a forma parcial) e uma paciente com DDS 46,XY de causa etiológica não conhecida foram estudados. As regiões codificadoras do gene MAP3K1 foram amplificadas e sequenciadas pelo método de Sanger ou painel customizado de genes-alvo associados ao DDS. Estudo in vitro utilizando o método de detecção colorimétrica In-Cell ELISA com anticorpos específicos para detecção de ERK1/2 e AKT, fosforilado e não fosforilado foi realizado em fibroblastos obtidos por biópsia de pele e mantidos em cultura celular de 3 indivíduos portadores de variantes no MAP3K1. A quantificação da fosforilação de p38 e ERK por ensaio de citometria em células linfoblastóides mutadas foram realizados em amostras de 4 indivíduos portadores de variantes no MAP3K1 em estudo realizado em colaboração. Imunohistoquímica com anticorpos anti Caspase-3 foram realizadas em tecidos gonadais parafinados das pacientes portadoras de variantes alélicas nos genes MAP3K1 e FGFR2. Resultados: Vinte e uma variantes alélicas, sete das quais ainda não descritas na literatura, foram identificadas no gene MAP3K1. Quatro novas variantes alélicas exônicas e não sinônimas (p.Leu639Pro, p.Leu447Trp, p.Thr657Arg e p.Cys691Arg) foram identificadas em heterozigose; todas foram classificadas como deletérias para a proteína nos estudos de predição \"in silico\", não foram identificadas em indivíduos controles brasileiros estudados e não estão descritas nos bancos de dados populacionais. A variante p.Leu639Pro foi identificada em duas irmãs com disgenesia gonadal 46,XY portadoras da variante p.Ser453Leu no gene FGFR2 identificada previamente. A variante intrônica c.834+1G >T identificada em heterozigose foi classificada como deletéria à proteína na análise no site de predição para alteração de \"splicing\". Os ensaios colorimétricos para detecção de ERK1/2 e AKT, fosforilado e não fosforilado foram inconclusivos. Os estudos in vitro de avaliação dos níveis de fosforilação de p38 e ERK evidenciaram uma maior fosforilação nas culturas celulares mutantes para o MAP3K1 quando comparado com a linhagem celular selvagem, resultado estatisticamente significativo ( p < 0,001) e que corrobora com os dados publicados previamente. A imunohistoquímica com anticorpos anti Caspase-3 mostrou uma maior marcação em células germinativas nos tecidos gonadais das pacientes portadoras das variantes no MAP3K1 e FGFR2 do que no tecido testicular normal, porém marcações foram identificadas também em células germinativas de tecidos testiculares de indivíduos com DDS 46,XY de outras etiologias. Conclusões: Os achados sugerem fortemente a participação das mutações identificadas no MAP3K1 na etiologia dos distúrbios do desenvolvimento sexual dos pacientes estudados. Porém, uma melhor compreensão dos mecanismos de participação da via MAPK nas redes gênicas de regulação do processo de determinação testicular humano ainda é necessário / Introduction: Pearlman et al. associated the presence of activating mutations in MAP3K1 gene with abnormal testicular development in patients with familial 46,XY gonadal dysgenesis, although studies in mice have shown that the Map3k1 gene is not essential for testicular determination. In male gonadal development, the binding of MAP3K1 to the RHOA protein promotes a normal phosphorylation of p38 and ERK1/2, and a blockade of the beta- catenin pathway is determined by MAP3K4. In the female development, hyperphosphorylation of p38 and ERK1/2 occurs. p38 and ERK1/2 hyperphosphorylated determine the activation of the beta-catenin pathway, the blockade of the positive feedback pathway of SOX9 and the testicular development. Objectives: To investigate the presence of allelic variants of the MAP3K1 gene in patients with 46,XY disorders of sex development (DSD) due to abnormalities of gonadal development and to evaluate the functional repercussion of the identified variants. Patients and Methods: Forty-seven patients with 46,XY gonadal dysgenesis (17 patients with complete form and 29 with partial form) and one patient with 46,XY DSD of unknown cause were studied. The MAP3K1 coding regions were amplified and sequenced by Sanger method or by custom panel of target genes associated with DSD. In-Cell ELISA assay with specific antibodies for the detection of phosphorylated and non-phosphorylated ERK1/2 and AKT was performed on fibroblasts obtained by skin biopsy and kept in cell culture of 3 individuals with MAP3K1 variants. Quantification of p38 and ERK phosphorylation by cytometric assay on mutated lymphoblastoid cells were performed on samples from 4 subjects with MAP3K1 variants in a collaborative study. Immunohistochemistry with anti-Caspase-3 antibodies were performed on paraffinembedded gonadal tissues of patients with MAP3K1 and FGFR2 allelic variants. Results: Twenty-one allelic variants, seven of them have not yet been described in the literature, were identified in the MAP3K1. Four novel exonic and non-synonymous allelic variants (p.Leu639Pro, p.Leu447Trp, p.Thr657Arg and p.Cys691Arg) were identified in heterozygous state; all of them were classified as deleterious in silico prediction sites; they were not identified in Brazilian control subjects and they were not described in the human genetic variation databases. The p.Leu639Pro variant was identified in two sisters with 46,XY gonadal dysgenesis carrying the previously identified FGFR2 variant (p Ser453Leu). The intronic c.834+1G > T variant identified in heterozygous state was classified as deleterious in the prediction sites. Colorimetric assays for the detection of phosphorylated and nonphosphorylated ERK1/2 and AKT were not significant. In vitro studies to evaluate p38 and ERK phosphorylation levels evidenced increased phosphorylation in the MAP3K1 mutant cells when compared to the wild type cells line; a statistically significant result (p < 0.001) that confirmed previously published data. The immunohistochemistry study with anti-Caspase-3 antibodies showed that the gonadal tissues of patients with MAP3K1 and FGFR2 variants exhibited more apoptotic germ ceIls than normal testicular tissue, but stained germ cells were also identified in the testicular tissues of the 46,XY DSD controls.Conclusions: These findings strongly suggest the participation of MAP3K1 mutations in the etiology of the testicular abnormalities of the 46,XY DSD patients of this study. However, a better understanding of the mechanisms of MAPK pathway in the gene regulatory networks of the human testicular determination process is still necessary