Chaperone proteins are essential components in the maintenance and turnover of the proteome. Many chaperones play integral functions in the folding and unfolding of cellular substrates under many conditions, including heat stress. Most chaperones can be characterized into two categories; the typical ATP dependent chaperones and the ATP independent chaperones. One ATP independent chaperone class it the Small Heat Shock Proteins (sHSPs), which as molecular life vests and are thought to protect misfolding proteins from irreversible aggregation. One such organism, the cyanobacterium Synechocystis sp. PCC 6803, is an excellent model for the study and understanding of these proteins and their functions in vivo. The genome of Synechocystis encodes only one sHSP, Hsp16.6, and it has be shown to be essential for acquired thermotolerance. Two mutant derivatives of Hsp16.6 with single amino acid substitutions in the N-terminal arm (L9P and E25K) have loss-of-function phenotypes similar to knock out strains, but each has very different biochemical properties. The mutant L9P has an inability to interact with putative substrates during heat stress in vivo, while the mutant E25K appears unable to release substrates. Using a directed evolution approach, suppressors have been isolated that recover the lost thermotolerance of their respective parent strains, either L9P (16 suppressors) or E25K (10 suppressors). Illumina sequencing and comparative genomics have been used to identify alterations in the genomes of the suppressor strains in order to define genetic circuits involved in thermotolerance.

The epigenome carries dynamic information that controls gene expression and maintains cell identity during both disease and normal development. The inherent plasticity of the epigenome paves new avenues for developing diagnostic and therapeutic tools for human diseases. Microfluidic technology has improved the sensitivity and resolution of epigenomic analysis due to its outstanding ability to manipulate nanoliter-scale liquid volumes. In this thesis, I report three projects focusing on low-input, cell-type-specific and spatially resolved histone modification profiling on microfluidic platforms. First, I applied Microfluidic Oscillatory Washing-based Chromatin Immunoprecipitation followed by sequencing (MOWChIP-seq) to study the effect of culture dimensionality, hypoxia stress and bacterium infection on histone modification landscapes of brain tumor cells. I identified differentially marked regions between different culture conditions. Second, I adapted indexed ChIPmentation and introduced mu-CM, a low-input microfluidic device capable of performing 8 assays in parallel on different histone marks using as few as 20 cells in less than 7 hours. Last, I investigated the spatially resolved epigenome and transcriptome of neuronal and glial cells from coronal sections of adult mouse neocortex. I applied unsupervised clustering to identify distinct spatial patterns in neocortex epigenome and transcriptome that were associated with central nervous system development. I demonstrated that our method is well suited for scarce samples, such as biopsy samples from patients in the context of precision medicine. / Doctor of Philosophy / Epigenetic is the study of alternations in organisms not caused by alternation of the genetic codes. Epigenetic information plays pivotal role during growth, aging and disease. Epigenetic information is dynamic and modifiable, and thus serves as an ideal target for various diagnostic and therapeutic strategies of human diseases. Microfluidics is a technology that manipulates liquids with extremely small volumes in miniaturized devices. Microfluidics has improved the sensitivity and resolution of epigenetic analysis. In this thesis, I report three projects focusing on low-input, cell-type-specific and spatially resolved histone modification profiling on microfluidic platforms. Histone modification is one type of epigenetic information and regulates gene expression. First, we studied the influence of culture condition and bacterium infection on histone modification profile of brain tumor cells. Second, we introduced mu-CM, combining a low-input microfluidic device with indexed ChIPmentation and is capable of performing 8 assays in parallel using as few as 20 cells. Last, we investigated spatial variations in the epigenome and transcriptome across adult mouse neocortex, the outer layer of brain involving in higher-order function, such as cognition. I identified distinct spatial patterns responsible for central nervous system development using machine learning algorithm. Our method is well suited for studying scarce samples, such as cells populations isolated from patients in the context of precision medicine.

Insertion and deletion (indel), a common form of genetic variation, has been shown to cause or contribute to human genetic diseases and cancer. Despite this importance and being the second most abundant variant type in the human genome, indels have not been studied as much as the single nucleotide polymorphism (SNP). With the advance of next-generation sequencing technology, many indel calling tools have been developed. However, performance comparison of commonly used tools has shown that (1) the tools have limited power in identifying indels and there are significant number of indels undetected, and (2) there is significant disagreement among the indel sets produced by the tools. These findings indicate the necessity of improving the existing tools or developing new algorithms to achieve reliable and consistent indel calling results. Two indels are biologically equivalent if the resulting sequences are the same. Storing biologically equivalent indels as distinct entries in databases causes data redundancy and misleads downstream analysis. It is thus desirable to have a unified system for identifying and representing equivalent indels. This dissertation describes UPS-indel, a utility tool that creates a universal positioning system for indels so that equivalent indels can be uniquely determined by their coordinates in the new system. Results show that UPS-indel identifies more redundant indels than existing algorithms. While mapping short reads to the reference genome, a significant number of short reads are unmapped and excluded from downstream analyses, thereby causing information loss in the subsequent variant calling. This dissertation describes Genesis-indel, a computational pipeline that explores the unmapped reads to identify missing novel indels. Results analyzing sequence alignment of 30 breast cancer patients show that Genesis-indel identifies many novel indels that also show significant enrichment in oncogenes and tumor suppressor genes, demonstrating the importance of rescuing indels hidden in the unmapped reads in cancer and disease studies. Somatic mutations play a vital role in transforming healthy cells into cancer cells. Therefore, accurate identification of somatic mutations is essential. Many somatic mutations callers are available with different strengths and weaknesses. An ensemble approach integrating the power of the callers is warranted. This dissertation describes SomaticHunter, an ensemble of two callers, namely Platypus and VarDict. Results on synthetic tumor data show that for both SNPs and indels, SomaticHunter achieves recall comparable to the state-of-the-art somatic mutation callers and the highest precision, resulting in the highest F1 score. / Doctor of Philosophy / Insertion and deletion (indel), a common form of genetic variation in the human genome, is associated with genetic diseases and cancer. However, indels are heavily understudied due to experimental and computational challenges. This dissertation addresses the computational challenges in three aspects. First, the current approach of representing indels is ambiguous and causes significant database redundancy. A universal positioning system, UPS-indel, is proposed to represent equivalent indels unambiguously and the UPS-indel algorithm is theoretically proven to find all equivalent indels and is thus exhaustive. Second, a significant number of indels are hidden in DNA reads not mapped to the reference genome. Genesis-indel, a computational pipeline that explores the unmapped reads to identify novel indels that are initially missed, is developed. Genesis-indel has been shown to uncover indels that can be important genetic markers for breast cancer. Finally, mutations occurring in somatic cells play a vital role in transforming healthy cells into cancer cells. Therefore, accurate identification of somatic mutation is essential for a better understanding of cancer genomes. SomaticHunter, an ensemble of two sensitive variant callers, is developed. Simulated studies using whole genome and whole exome sequences have shown that SomaticHunter achieves recall comparable to state-of-the-art somatic mutation callers while delivering the highest precision and therefore resulting in the highest F1 score among all the callers compared.

Next-generation sequencing (NGS) technologies have made it possible to rapidly sequence the human genome, heralding a new era of health-care innovations based on personalized genetic information. However, these NGS technologies generate data at a rate that far outstrips Moore\'s Law. As a consequence, analyzing this exponentially increasing data deluge requires enormous computational and storage resources, resources that many life science institutions do not have access to. As such, cloud computing has emerged as an obvious, but still nascent, solution. This thesis intends to investigate and design an efficient framework for running and managing large-scale data-intensive scientific applications in the cloud. Based on the learning from our parallel implementation of a genome analysis pipeline in the cloud, we aim to provide a framework for users to run such data-intensive scientific workflows using a hybrid setup of client and cloud resources. We first present SeqInCloud, our highly scalable parallel implementation of a popular genetic variant pipeline called genome analysis toolkit (GATK), on the Windows Azure HDInsight cloud platform. Together with a parallel implementation of GATK on Hadoop, we evaluate the potential of using cloud computing for large-scale DNA analysis and present a detailed study on efficiently utilizing cloud resources for running data-intensive, life-science applications. Based on our experience from running SeqInCloud on Azure, we present CloudFlow, a feature rich workflow manager for running MapReduce-based bioinformatic pipelines utilizing both client and cloud resources. CloudFlow, built on the top of an existing MapReduce-based workflow manager called Cloudgene, provides unique features that are not offered by existing MapReduce-based workflow managers, such as enabling simultaneous use of client and cloud resources, automatic data-dependency handling between client and cloud resources, and the flexibility of implementing user-defined plugins for data transformations. In-general, we believe that our work attempts to increase the adoption of cloud resources for running data-intensive scientific workloads. / Master of Science

[ES] Las distrofias hereditarias de la retina (DHR) son un conjunto de trastornos caracterizados por la muerte, generalmente, progresiva de fotorreceptores y células del epitelio pigmentario retiniano. Las DHR afectan a más de 2 millones de personas en el mundo, produciendo una pérdida parcial y, en muchos casos, total de la visión, para la cual no existe ningún tratamiento que revierta o frene la progresión de la enfermedad. Se caracterizan por una elevada heterogeneidad clínica y genética, identificándose hasta la fecha mutaciones en más de 250 genes responsable de algún tipo de DHR. Esta tesis doctoral se ha centrado en el diagnóstico genético de 208 pacientes afectados de DHR no sindrómica (DHR-NS), mediante secuenciación masiva dirigida a un panel de 117 genes y cinco regiones intrónicas profundas en las que previamente se habían descrito mutaciones. Las variantes identificadas en los pacientes se priorizaron de acuerdo a la mínima frecuencia alélica y se clasificaron según los criterios de la guía del American College of Medical Genetics and Genomics. Las variantes de número de copias identificadas mediante la secuenciación del panel se validaron utilizando multiplex ligation-dependent probe amplification o array genómico. A todos los pacientes se les realizó un estudio oftalmológico, más o menos exhaustivo, incluyendo pruebas electrofisiológicas. Además, se realizaron ensayos in vitro con minigenes para comprobar la repercusión de ciertas variantes sobre el procesamiento del ARNm. Para ello, se empleó el plásmido pSPL3 en el que se introdujo la mutación a estudio, la transformación se realizó en bacterias E. coli electrocompetentes y la transfección en células HEK-293. Mediante la secuenciación del panel de genes, se obtuvo una tasa diagnóstica del 60%. Las mutaciones en los pacientes resueltos se distribuyeron en 30 genes diferentes, de las cuales 38 se describen por primera vez en este trabajo. Los genes mutados con mayor frecuencia en estos pacientes fueron ABCA4, USH2A, RPGR y PRPH2. Adicionalmente, se identificaron ocho variantes de número de copias en los genes EYS, ABCA4, PRPF31, MERTK, CRB1 y ROM1, demostrando el enorme potencial de estas tecnologías. En total, se reclasificaron el 10% de las familias estudiadas y se observó variabilidad fenotípica intra e inter familiar en pacientes con mutaciones en C1QTNF5, CERKL y PROM1. Además, destacamos que el 50% de las mujeres heterocigotas para RPGR y sintomáticas presentaron retinosis pigmentaria (RP) con asimetría interocular. Asimismo, se identificó un caso con posible digenismo entre los genes CNGA1 y CNGB1, y la duplicación completa de ROM1 en dos familias no relacionadas diagnosticadas de RP. Por otra parte, se han identificado seis familias con mutaciones en dos genes que potencialmente ambos podrían ser la causa de la DHR. En el ensayo funcional mediante minigenes, se observó que seis de las variantes analizadas presentaron un splicing aberrante. Los resultados de esta tesis destacan la utilidad clínica de la secuenciación masiva dirigida para las DHR-NS y la importancia de un examen oftalmológico completo que nos permita establecer nuevas asociaciones genotipo-fenotipo y ampliar el conocimiento de este grupo de enfermedades. Identificar la causa de la enfermedad es esencial para mejorar el manejo del paciente, realizar un asesoramiento genético preciso y beneficiarse de los futuros tratamientos y ensayosclínicos basados en terapia génica. / [CA] Les distròfies hereditàries de la retina (DHR) són un conjunt de trastorns caracteritzats per la mort, generalment, progressiva de fotoreceptors i cèl¿lules de l'epiteli pigmentari retiniano. Les DHR afecten més de 2 milions de persones al món, produint una pèrdua parcial i, en molts casos, total de la visió, per la qual no hi ha cap tractament que revertisca o frene la progressió de la malaltia. Es caracteritzen per una elevada heterogeneïtat clínica i genètica, identificant-se fins a la data mutacions en més de 250 gens responsable d'algun tipus de DHR. Esta tesi doctoral s'ha centrat en el diagnòstic genètic de 208 pacients afectats de DHR no sindròmica (DHR-NS), per mitjà de seqüenciació massiva dirigida a un panell de 117 gens i cinc regions intròniques profundes en les que prèviament s'havien descrit mutacions. Les variants identificades en els pacients es van prioritzar d'acord a la mínima freqüència allèlica i es van classificar segons els criteris de la guia de l'American College of Medical Genetics and Genomics. Les variants de nombre de còpies identificades per mitjà de la seqüenciació del panell es van validar utilitzant multiplex ligation-dependent probe amplification o array genòmic. A tots els pacients se'ls va realitzar un estudi oftalmològic, més o menys exhaustiu, incloent proves electrofisiològiques. A més, es van realitzar assajos in vitro amb minigens per comprovar la repercussió de certes variants sobre el processament de l'ARNm. Per a això, es va emprar el plasmidi pSPL3 en què es va introduir la mutació a estudi, la transformació es va realitzar en bacteris E. coli electrocompetentes i la transfecció en cèl-lules HEK-293. Per mitjà de la seqüenciació del panell de gens, es va obtindre una taxa diagnòstica del 60%. Les mutacions en els pacients resolts es van distribuir en 30 gens diferents, de les quals 38 es descriuen per primera vegada en este treball. Els gens mutats amb major freqüència en estos pacients van ser ABCA4, USH2A, RPGR i PRPH2. Addicionalment, es van identificar huit variants de nombre de còpies en els gens EYS, ABCA4, PRPF31, MERTK, CRB1 i ROM1, demostrant l'enorme potencial d'estes tecnologies. En total, es van reclassificar el 10% de les famílies estudiades i es va observar variabilitat fenotípica intra i inter familiar en pacients amb mutacions en C1QTNF5, CERKL i PROM1. A més, destaquem que el 50% de les dones heterozigotes per RPGR i simptomàtiques van presentar retinosi pigmentària (RP) amb asimetria interocular. Així mateix, es va identificar un cas amb possible digenismo entre els gens CNGA1 i CNGB1, i la duplicació completa de ROM1 en dues famílies no relacionades diagnosticades de RP. D'altra banda, s'han identificat sis famílies amb mutacions en dos gens que potencialment ambdós podrien ser la causa de la DHR. En l'assaig funcional per mitjà de minigens, es va observar que sis de les variants analitzades van presentar un splicing aberrant. Els resultats d'esta tesi destaquen la utilitat clínica de la seqüenciació massiva dirigida per a les DHR-NS i la importància d'un examen oftalmològic complet que ens permeta establir noves associacions genotip-fenotip i ampliar el coneixement d'este grup de malalties. Identificar la causa de la malaltia és essencial per millorar el maneig del pacient, realitzar un assessorament genètic precís i beneficiar-se dels tractaments basats en teràpia gènica. / [EN] Inherited retinal dystrophies (IRD) are a group of disorders generally characterized by the progressive death of photoreceptors and retinal pigment epithelial cells. IRD affect more than 2 million people in the world, producing a partial and, in many cases, total loss of vision, for which there is no treatment that reverses or slows the progression of the disease. They are characterized by a high clinical and genetic heterogeneity, identifying mutations in more than 250 genes responsible for some type of IRD to date. This doctoral thesis has focused on the genetic diagnosis of 208 patients affected by non-syndromic IRD (NS-IRD), through targeted genomic sequencing focuses on a panel of 117 genes and five deep intronic regions in which mutations have previously been identified. The identified variants in patients are prioritized according to the minimum allelic frequency and are classified according to the criteria of the American College of Medical Genetics and Genomics guide. The copy number variants identified by panel sequencing were validated using multiplex ligation-dependent probe amplification or genomic array. All patients underwent an ophthalmological study, more or less comprehensive, including electrophysiological tests. In addition, in vitro assays with minigenes were performed to verify the impact of certain variants on mRNA processing. To do this, pSPL3 plasmid in which the mutation was introduced was used, the transformation was performed in electrocompetent E. coli bacteria and transfection in HEK-293 cells. By the panel sequencing, a diagnostic rate of 60% was obtained. The mutations in the resolved patients were distributed in 30 different genes, of which 38 were described for the first time in this work. The most frequently mutated genes in these patients were ABCA4, USH2A, RPGR and PRPH2. In addition, eight copy number variants were identified in the genes EYS, ABCA4, PRPF31, MERTK, CRB1 and ROM1, that demonstrated the enormous potential of these technologies. In total, 10% of the families studied were reclassified and intra and inter-family phenotypic variability was detected in patients with mutations in C1QTNF5, CERKL and PROM1. In addition, we highlight that 50% of the symptomatic and heterozygous RPGR women showed retinitis pigmentosa (RP) with interocular asymmetry. Likewise, we identified a case with possible digenism between the CNGA1 and CNGB1 genes, and the complete duplication of ROM1 in two unrelated families diagnosed with RP. On the other hand, six families have been identified with mutations in two genes that could potentially be the cause of IRD. Functional assays by minigenes showed that six of the studied variants affected splicing. The results of this thesis highlight the clinical utility of targeted genomic sequencing in IRD and the importance of a complete ophthalmological examination that allows us to establish new genotype-phenotype associations and expand the knowledge of this group of diseases. Identifying the cause of the disease is essential to improve patient management, to perform an accurate genetic counseling and to take advantage of treatments based on gene therapy. / Rodríguez Muñoz, A. (2020). High-throughput strategies for molecular diagnosis of nonsyndromic inherited retinal dystrophies [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/139095

Forest trees represent a unique group of organisms combined with ecological and economic importance. Owing to their random mating system and widespread geographical distribution, they harbor abundance genetic variation both within and among populations. Despite their importance, research in forest trees has been underrepresented majorly due to their large and complex genome and scarce funding. However, recent climate change and other associated problems such as insect outbreaks, diseases and stress related damages have urged scientists to focus more on trees. Furthermore, the advent in high-throughput sequencing technologies have allowed trees to be sequenced and used as reference genome, which provided deeper understanding between genotype and environment. Whole genome sequencing is still not possible for organisms having large genomes including most tree species, and it is still not feasible economically for population genomic studies which require sequencing hundreds of samples. To get around this problem, genomic reduction is required. Sequence capture has been one of the genomic reduction techniques enabled studying the subset of the DNA of interest. In this paper, our primary goal is to outline challenges, provide guidance about the utility of sequence capture in trees, and to leverage such data in genome-wide association analyses to find the genetic variants that underlie complex, adaptive traits in spruce and pine, as well as poplar. Results of this research will facilitate bridging the genomic information gap between trees and other organisms. Moreover, it will provide better understanding how genetic variation governs phenotype in trees, which will facilitate both marker assisted selection for improved traits as well as provide guidance to determine forest management strategies for reforestation to mitigate the effects of climate change. / Ph. D. / Forests are under extensive threat including increased demand in wood consumption, climate changes and associated diseases and stress related damages. Up until very recently, researches in trees have been relatively slower owing to their large and complex genomes. However, this has dramatically changed mainly due to the advancement in sequencing technology. There have been more and more studies performed identifying novel genes that are responsible for improved characteristics. In this study, we provided guidance about how to better utilize sequencing technology and identified genes that are potentially related with adaptation in trees.

Yes / Background. Increased availability of Next Generation Sequencing (NGS) techniques allows, for the first time, to distinguish relapses from reinfections in patients with multiple Buruli ulcer (BU) episodes. Methodology. We compared the number and location of single nucleotide polymorphisms (SNPs) identified by genomic screening between four pairs of Mycobacterium ulcerans isolates collected at the time of first diagnosis and at recurrence, derived from a collection of almost 5000 well characterized clinical samples from one BU treatment center in Benin. Principal Findings. The findings suggest that after surgical treatment—without antibiotics—the second episodes were due to relapse rather than reinfection. Since specific antibiotics were introduced for the treatment of BU, the one patient with a culture available from both disease episodes had M. ulcerans isolates with a genomic distance of 20 SNPs, suggesting the patient was most likely reinfected rather than having a relapse. Conclusions. To our knowledge, this study is the first to study recurrences in M. ulcerans using NGS, and to identify exogenous reinfection as causing a recurrence of BU. The occurrence of reinfection highlights the contribution of ongoing exposure to M. ulcerans to disease recurrence, and has implications for vaccine development. / This work was supported by the UBS Optimus Foundation (Zurich, Switzerland) and the Department of Economy, Science and Innovation of the Flemish Government (Belgium). KV was supported by a VLADOC PhD scholarship of VLIRUOS (Belgium).

Acredita-se que o Brasil é o centro de diversidade de vírus transmitidos por ácaros do gênero Brevipalpus (VTB). Alguns desses VTB infectam culturas fundamentais para o agronegócio brasileiro como citros e café, além de maracujá e de várias plantas ornamentais. Na última década os genomas de dois deles, Citrus leprosis virus C (CiLV-C) e Coffee ringspot virus (CoRSV) foram sequenciados, mas ainda é escasso o conhecimento sobre a diversidade genética e processos evolutivos envolvidos na população dessas espécies. Neste contexto, o objetivo deste trabalho foi caracterizar molecularmente novas estirpes de CiLV-C e CoRSV que infectam citros e café, respectivamente. E revelar as relações filogenéticas com espécies de VTB conhecidas, assim como avaliar a variabilidade genética da população de CiLVC no Brasil. Para o estudo de CiLV-C, 47 amostras de Citrus sinensis apresentando sintomas típicos da leprose dos citros foram coletadas em diferentes regiões do Brasil no período de 2011-2015. A presença de CiLV-C foi detectada por RT-PCR em todas as amostras coletadas e, posteriormente, foi realizado o sequenciamento de quatro regiões do genoma viral (p29, p15, RI e MP) de cada isolado. As sequências obtidas foram utilizadas no estudo de filogenia e variabilidade da população de CiLV-C no Brasil. Foi demonstrado que a população de CiLV-C apresenta uma variabilidade relativamente baixa; entretanto, foi identificada a existência de duas linhagens dentro da espécie, nomeadas Cor e SJRP. Os genomas completos de CiLV-C SJRP e também do dicorhavirus tentativo CoRSV identificado em Limeira, SP, foram obtidos mediante o sequenciamento de RNA de pequeno tamanho (siRNA). Cada sequência foi validada mediante o sequenciamento de fragmentos gerados por RT-PCR ao longo do genoma. CiLV-C SJRP apresenta cerca de 85% de identidade de nucleotídeo com o membro-tipo do gênero Cilevirus e exibe evidências de recombinação com isolados da linhagem Cor, a prevalente no território brasileiro. Globalmente, o genoma de CoRSV Limeira apresenta mais de 90% de identidade de nucleotídeo com isolado CoRSV Lavras, o que indica que ambos os isolados são membros da mesma espécie tentativa de dichorhavirus. / South America is most likely the center of diversity of Brevipalpus transmitted viruses (BTV). Some of these BTV infect major crops of the Brazilian agribusiness such as citrus and coffee. Passion fruit and several other ornamental plants are affected as well. The genome of two of these viruses, Citrus leprosis virus C (CiLV-C) and Coffee ringspot virus (CoRSV) were sequenced, but the knowledge about several molecular characteristics and processes involved in the evolution of their populations are still scarce. Thus, the objective of this study was to molecularly characterize new isolates of BTV infecting citrus and coffee, reveal the phylogenetic relationships with known species of BTV, and assess the genetic variability of the population of CiLV-C in Brazil. For CiLV-C studies, 47 samples of Citrus sinensis showing typical symptoms of leprosis were collected in different Brazilian regions during 2011-2015. The presence of CiLV-C was detected by RT-PCR in all the collected samples and four regions of the viral genome (p29, p15, IR and MP) of each isample were sequenced. It has been shown that the CiLV-C population has relatively low variability; although the existence of two lineages named Cor and SJRP were identified in this work. The complete genomes of one isolate of the lineage SJRP (CiLV-C SJRP) and that of the tentative dicorhavirus CoRSV found in Limeira, SP, were obtained by small RNA (siRNA) Sequencing. Validation was performed by sequencing fragments generated by RT-PCR using specific primers throughout the genome. CiLV-C SJRP has about 85% nucleotide identity with the genome of the type-member of the Cilevirus genus and shows evidence of recombination with isolates of the lineage Cor, which are prevalent in Brazil. CoRSV isolate Limeira has more than 90% of nucleotide identity with CoRSV Lavras, indicating that both isolates are members of the same tentative species of dichorhavirus.

Acredita-se que o Brasil é o centro de diversidade de vírus transmitidos por ácaros do gênero Brevipalpus (VTB). Alguns desses VTB infectam culturas fundamentais para o agronegócio brasileiro como citros e café, além de maracujá e de várias plantas ornamentais. Na última década os genomas de dois deles, Citrus leprosis virus C (CiLV-C) e Coffee ringspot virus (CoRSV) foram sequenciados, mas ainda é escasso o conhecimento sobre a diversidade genética e processos evolutivos envolvidos na população dessas espécies. Neste contexto, o objetivo deste trabalho foi caracterizar molecularmente novas estirpes de CiLV-C e CoRSV que infectam citros e café, respectivamente. E revelar as relações filogenéticas com espécies de VTB conhecidas, assim como avaliar a variabilidade genética da população de CiLVC no Brasil. Para o estudo de CiLV-C, 47 amostras de Citrus sinensis apresentando sintomas típicos da leprose dos citros foram coletadas em diferentes regiões do Brasil no período de 2011-2015. A presença de CiLV-C foi detectada por RT-PCR em todas as amostras coletadas e, posteriormente, foi realizado o sequenciamento de quatro regiões do genoma viral (p29, p15, RI e MP) de cada isolado. As sequências obtidas foram utilizadas no estudo de filogenia e variabilidade da população de CiLV-C no Brasil. Foi demonstrado que a população de CiLV-C apresenta uma variabilidade relativamente baixa; entretanto, foi identificada a existência de duas linhagens dentro da espécie, nomeadas Cor e SJRP. Os genomas completos de CiLV-C SJRP e também do dicorhavirus tentativo CoRSV identificado em Limeira, SP, foram obtidos mediante o sequenciamento de RNA de pequeno tamanho (siRNA). Cada sequência foi validada mediante o sequenciamento de fragmentos gerados por RT-PCR ao longo do genoma. CiLV-C SJRP apresenta cerca de 85% de identidade de nucleotídeo com o membro-tipo do gênero Cilevirus e exibe evidências de recombinação com isolados da linhagem Cor, a prevalente no território brasileiro. Globalmente, o genoma de CoRSV Limeira apresenta mais de 90% de identidade de nucleotídeo com isolado CoRSV Lavras, o que indica que ambos os isolados são membros da mesma espécie tentativa de dichorhavirus. / South America is most likely the center of diversity of Brevipalpus transmitted viruses (BTV). Some of these BTV infect major crops of the Brazilian agribusiness such as citrus and coffee. Passion fruit and several other ornamental plants are affected as well. The genome of two of these viruses, Citrus leprosis virus C (CiLV-C) and Coffee ringspot virus (CoRSV) were sequenced, but the knowledge about several molecular characteristics and processes involved in the evolution of their populations are still scarce. Thus, the objective of this study was to molecularly characterize new isolates of BTV infecting citrus and coffee, reveal the phylogenetic relationships with known species of BTV, and assess the genetic variability of the population of CiLV-C in Brazil. For CiLV-C studies, 47 samples of Citrus sinensis showing typical symptoms of leprosis were collected in different Brazilian regions during 2011-2015. The presence of CiLV-C was detected by RT-PCR in all the collected samples and four regions of the viral genome (p29, p15, IR and MP) of each isample were sequenced. It has been shown that the CiLV-C population has relatively low variability; although the existence of two lineages named Cor and SJRP were identified in this work. The complete genomes of one isolate of the lineage SJRP (CiLV-C SJRP) and that of the tentative dicorhavirus CoRSV found in Limeira, SP, were obtained by small RNA (siRNA) Sequencing. Validation was performed by sequencing fragments generated by RT-PCR using specific primers throughout the genome. CiLV-C SJRP has about 85% nucleotide identity with the genome of the type-member of the Cilevirus genus and shows evidence of recombination with isolates of the lineage Cor, which are prevalent in Brazil. CoRSV isolate Limeira has more than 90% of nucleotide identity with CoRSV Lavras, indicating that both isolates are members of the same tentative species of dichorhavirus.

With the development of the internet protocol (IP) and digitization for the global telecommunication industry, the convergence rate of communications and broadcasting has been improved. According to these motives, the domestic telecommunication industry modify present commercial operation managements and combine with the communication networks, the fixed-mobile communication (FMC) networks, and the mobile communication networks into the all-internet protocol (all-IP) communication network structure based on the extended upgrade communication network system. The domestic telecommunication industry expect that the integrated heterogeneous network, including the speech data, the video data, and the communication services, which can provide the omnipresent customizable mobile communication network services and obtain the advance business opportunities in terms of the future development of digital convergence. Hence, the domestic telecommunication industry not only build the next generation network structure to satisfy their demands, but also develop the management information system (MIS) to monitor the operation of telecommunication networks for ensure the quality of communication services and achieve the development of next generation networks. A primary consideration is to assure the usability of MIS for the telecommunication industry and the customers based on the profit rates and the omnipresent mobile network services, respectively¡C However, the current status of the telecommunication industry that meet the many difficult challenges and problems to construct the next generation MIS. For examples, the all-IP-based open network structure will be used instead of the close network structure, the different generation telecommunication systems combine with the operation and maintenance information system, the information security incident, and so on. It is must to consider highly of these situations between the major links above. Otherwise, the service usability of MIS will be destroyed. In this thesis, we adopt the case study approach to analyze the MIS construction process of the domestic telecommunication operator. During the build process, the MIS construction of next generation telecommunication networks must to suffice for the flexibility, the safety, and the stabilization and need to make sure the critical mission on stable operation condition, the lower service interruption, the higher usability. The implementation of the next generation MIS, which will help support the crucial operation procedure of the conglomerate and cope with the fast variation of the market demands.