Os isolados de nematóides entomopatogênicos provenientes do solo de Monte Negro (RO) receberam o prefixo LPP seguido de um número sequencial. As linhagens de bactérias simbiontes receberam o prefixo MN como referência ao local de isolamento. As culturas dos isolados de nematóides entomopatogênicos LPP foram estabelecidas em nosso laboratório, sendo necessário cultivar também o lepidóptero Galleria mellonella para as infeções visando manter os nematóides em cultura. Os isolados, tanto de nematóides como das bactérias, foram identificados através do sequenciamento de genes ribossômicos (ITS 1 e 2 além do 5.8S para os nematóides e 16S para as bactérias). Análises integradas, abrangendo morfologia (em colaboração com a Dra. Claudia Dolinski e Inês Machado) e análises filogenéticas permitiram a identificação dos nematóides. Essas análises morfomoleculares mostram que as linhagens não podem ser descritas como novas espécies, baseadas apenas no local de isolamento, mas como pertencentes à espécies já descritas e bem caracterizadas. As linhagens de nematóides foram identificadas como pertencentes a duas espécies do gênero Heterorhabditis (Rhabditida): Heterorhabditis baujardi (LPP5, 7, 8, 10 e 11) Heterorhabditis indica (LPP1, 2, 3, 4, 9). A bactéria entomopatogênica simbionte isolada a partir do nematoide Heterorhabditis baujardi linhagem LPP7 foi identificada como pertencente à espécie Photorhabdus luminescens subsp. luminescens linhagem MN7. Análises filogenéticas mostram que há similaridade entre o isolado bacteriano e as outras linhagens já descritas de Photorhabdus luminescens subsp. luminescens. Ao mesmo tempo em que eram isoladas as bactérias simbiontes, ocorreu o isolamento de outra espécie de bactéria forética dos nematóides entomopatogênicos, identificada após sequenciamento parcial do 16S, como pertencentes ao gênero Ochrobactrum. As linhagens isoladas foram denominadas OMN2, OMN3, OMN4. / The entomopathogenic nematodes isolates from Monte Negro (RO) soils received the prefix LPP followed by a sequential number. The strains of bacterial symbionts received the prefix MN as a reference to its isolation site. Cultures of LPP entomopathogenic nematodes were established in our laboratory and the need for suitable hosts were supplied by cultivating the lepidopteran Galleria mellonella. Nematode and bacterial isolates were identified through ribosomal genes (ITS 1 and 2, 5.8S for nematodes and 16S for bacteria) sequencing. Integrated analysis, including morphology (in collaboration with Dr. Claudia Dolinski and Inês Machado) and a phylogenetic approach allowed the identification of nematodes. These morpho-molecular analysis clearly showed that our strains can not be described as new species, solely based on the isolation site critheria. It should be described as belonging to previously described and well characterized species. The nematode strains were identified as belonging to two species of the genus Heterorhabditis (Rhabditida): Heterorhabditis baujardi (LPP5, 7, 8, 10 and 11) Heterorhabditis indica (LPP1, 2, 3, 4, 9). The entomopathogenic bacterial symbiont isolated from the nematode Heterorhabditis baujardi strain LPP7 was identified as belonging to the species Photorhabdus luminescens subsp. luminescens strain MN7. Phylogenetic analysis showed similarities between isolated bacterial strains and other previously described Photorhabdus luminescens subsp. luminescens strains. In paralel work, along with bacterial symbionts isolation, other entomopathogenic nematodes foretic bacteria were isolated and identified after partial sequencing of 16S, as belonging to the genus Ochrobactrum. The isolated strains were named OMN2, OMN3, OMN4.

Gene expression heterogeneity underlies critical bacterial phenotypes including antibiotic tolerance, pathogenesis, and communication. Though microbial population heterogeneity has been appreciated for decades, we still lack a complete view of single-cell gene expression and phenotypic states. Various tools, including bulk RNA-seq and proteomics, are available for probing all genes on a population-level. Conversely, fluorescent protein reporters and in situ hybridization can capture single-cell states but only for a limited number of genes. Single-cell RNA-sequencing (scRNA-seq), which can quantify expression of all genes with resolution for individual cells, has revolutionized studies of heterogeneous eukaryotic populations. However, adaptation to bacteria has been hindered by technical barriers. This thesis will describe the development of high-throughput scRNA-seq for bacteria and its application to uncover a distinct transcriptional state of rare antibiotic-tolerant cells called persisters. Chapter 2 presents prokaryotic expression profiling by tagging RNA in situ and sequencing (PETRI-seq), our novel scRNA-seq technology. I will detail how PETRI-seq was optimized to overcome bacteria-specific challenges, including lack of mRNA polyadenylation, thick cell walls, and extremely low mRNA abundance. Using combinatorial indexing, PETRI-seq uniquely barcodes tens of thousands of gram-negative and/or gram-positive cells in a single experiment at low cost. In proof-of-concept experiments, we show robust discrimination of E. coli growth phases and identification of rare prophage activation in S. aureus. PETRI-seq will be broadly useful for characterizing bacterial heterogeneity in many contexts. Chapter 3 describes an expansive investigation into antibiotic persistence in E. coli. When a population is treated with lethal antibiotics, persisters are rare cells that can survive the exposure by assuming a relatively dormant state. Understanding the gene expression state and molecular drivers of persistence has been a longstanding goal with major potential to inform drug development and clinical practice. We have applied PETRI-seq to multiple models of E. coli persistence and discovered a distinct transcriptional state underlying this phenotype. In parallel, we used genome-wide CRISPR-interference to probe the functional contribution of every gene to the persistence phenotype. We discovered multiple driver genes and pathways. Comprehensive validation established Lon protease and YqgE as key gene products modulating translation rate, post-starvation dormancy, and persistence. Our work is a major step in defining the physiological state of persistence and the molecular processes leading cells into this state. In all, this thesis demonstrates how a new generation of systems approaches, including scRNA-seq and CRISPR-interference, enable new discoveries about long-studied phenomena. The overarching approach is broadly applicable with potential to inspire a wide range of microbiology studies.

Solano-violeta (Solanum violaefolium) é uma ornamental rasteira usada pra cobrir solos de áreas sombreadas. Um vírus que induz manchas anelares nas folhas, tentativamente designado de mancha anular do S. violaefoliumm (S. violaefolium ringspot vírus - SvRSV), transmitido pelo ácaro Brevipalpus phoenicis foi encontrado nesta planta em jardins de Piracicaba, SP. Trata-se de um vírus baciliforme que se acumula no lúmen do retículo endoplasmático induzindo viroplasma citoplasmático, assemelhando-se a outros vírus do tipo citoplasmático, dos transmitidos por ácaros Brevipalpus (Acari: Tenuipalpidea). Este trabalho relata algumas de suas propriedades biológicas e a caracterização molecular parcial. SvRSV foi ser transmitido mecanicamente e pelo B. phoenicis a várias outras espécies botânicas, sempre causando lesões localizadas. Destas, Datura stramonium mostrou-se a melhor como hospedeira experimental. As propriedades físicas do SvRSV in vitro foram: temperatura de inativação - 40-45 ºC; ponto final de diluição - 10-3-10-4; longevidade in vitro- 12 dias. Posteriormente, observou-se também infestação destas plantas por B. obovatus que em ensaios preliminares transmitiu o SvRSV. Em secções ultrafinas, as partículas do SvRSV mostraram-se ligeiramente mais delgadas que as de outros vírus do tipo citoplasmático, transmitidos por Brevipalpus, e por outro lado formavam eventualmente partículas mais longas, às vezes de ca. 1 µm. Como os demais vírus, do tipo citoplasmático, transmitido por Brevipalpus, induz a formação de um viroplasma denso e vacuolado no citoplasma. Em casos favoráveis foram observadas fases do processo de morfogênese por "brotação" a partir do material do viroplasma. Dada sua labilidade não foi possível conseguir sua purificação apesar das inúmeras tentativas, usando diferentes protocolos. Logrou-se a extração de dsRNA a partir de D. stramonium e a partir dele, obter-se dois fragmentos do genoma viral, identificados como parte da proteína de movimento e da replicase, após seu sequenciamento. Foram produzidos pares de "primer" baseado nestas seqüências que amplificaram especificamente, por RT-PCR, fragmentos de DNA de tamanho esperado, a partir do RNA total extraído de lesões foliares de S. violaefolium e D. stramonium infetados. Sondas baseadas nas seqüências obtidas hibridizaram com ss- e dsRNA de lesões de D. stramonium. Ensaios preliminares de RT-PCR e hibridização não resultaram em reação com alguns outros vírus transmitidos por Brevipalpus, do tipo citoplasmático, inclusive o da leprose dos citros (CiLV-C). / Solanum violaefolium is an ornamental Solanaceae, with prostrate, trailing growth cultivated in shaded areas. Plants exhibiting necrotic ringlike spots on the leaves have been found in several gardens and parks at Piracicaba - SP. The ringspot symptoms on the leaves is caused by a vírus, named S. violaefolium ringspot virus (SvRSV), and is transmisible by mite Brevipalpus phoenicis (Acari: Tenuipalpidae). Short bacilliform particles are present within the cisternae of endoplasmic reticulum (ER) and often electron dense viroplasm is present in the cytoplasm, characterist of the cytoplasmatic type of Brevipalpus-borne virus. The present study reports some of its biological properties and partial molecular caracterization. SvRSV is easily transmitted to many plant species either by viruliferous B. phoenicis or mechanically, always causing local lesions. Datura stramonium was proved to be better as experimental host. Its physical properties in vitro were: inactivation thermal point - 40-45 ºC; final diluition point - 10-3-10-4; longevity in vitro - 12 days. Afterwards, its was observed that S. violaefolium plants were infested by B. obovatus that transmitted SvRSV in preliminary assays. Thin sections revealed that SvRSV particles are slightly thinner and sometimes appear very long. In some favorable sections intermediate steps of viral particle morphogenesis by a budding process of the dense material of the viroplasm toward the lumen of ER could be seen. Due to the fragility of the particles, several attempts to purify the virus have failed, despite many protocols tried. It was possible, however, to extract dsRNA from infected tissue of D. stramonium, and two segments of viral genome, respectively with homology to movement protein (mp) and replicase (rep) of some known viruses were obtained. Primers were designed based in these sequences, which amplified by RT-PCR, fragments of DNA of expected size from total RNA extracts from leaves lesions of infected S. violaefolium and D. stramonium. Probes based on obtained sequences hibridizated with ss- and dsRNA from lesions of S. violaefolium and D. stramonium. Preliminary assays of RT-PCR and hybridization did not result in positive reaction with other cytoplasmatic type of Brevipalpus-borne viruses, including citrus leprosis (CiLVC).

Solano-violeta (Solanum violaefolium) é uma ornamental rasteira usada pra cobrir solos de áreas sombreadas. Um vírus que induz manchas anelares nas folhas, tentativamente designado de mancha anular do S. violaefoliumm (S. violaefolium ringspot vírus - SvRSV), transmitido pelo ácaro Brevipalpus phoenicis foi encontrado nesta planta em jardins de Piracicaba, SP. Trata-se de um vírus baciliforme que se acumula no lúmen do retículo endoplasmático induzindo viroplasma citoplasmático, assemelhando-se a outros vírus do tipo citoplasmático, dos transmitidos por ácaros Brevipalpus (Acari: Tenuipalpidea). Este trabalho relata algumas de suas propriedades biológicas e a caracterização molecular parcial. SvRSV foi ser transmitido mecanicamente e pelo B. phoenicis a várias outras espécies botânicas, sempre causando lesões localizadas. Destas, Datura stramonium mostrou-se a melhor como hospedeira experimental. As propriedades físicas do SvRSV in vitro foram: temperatura de inativação - 40-45 ºC; ponto final de diluição - 10-3-10-4; longevidade in vitro- 12 dias. Posteriormente, observou-se também infestação destas plantas por B. obovatus que em ensaios preliminares transmitiu o SvRSV. Em secções ultrafinas, as partículas do SvRSV mostraram-se ligeiramente mais delgadas que as de outros vírus do tipo citoplasmático, transmitidos por Brevipalpus, e por outro lado formavam eventualmente partículas mais longas, às vezes de ca. 1 µm. Como os demais vírus, do tipo citoplasmático, transmitido por Brevipalpus, induz a formação de um viroplasma denso e vacuolado no citoplasma. Em casos favoráveis foram observadas fases do processo de morfogênese por “brotação” a partir do material do viroplasma. Dada sua labilidade não foi possível conseguir sua purificação apesar das inúmeras tentativas, usando diferentes protocolos. Logrou-se a extração de dsRNA a partir de D. stramonium e a partir dele, obter-se dois fragmentos do genoma viral, identificados como parte da proteína de movimento e da replicase, após seu sequenciamento. Foram produzidos pares de “primer” baseado nestas seqüências que amplificaram especificamente, por RT-PCR, fragmentos de DNA de tamanho esperado, a partir do RNA total extraído de lesões foliares de S. violaefolium e D. stramonium infetados. Sondas baseadas nas seqüências obtidas hibridizaram com ss- e dsRNA de lesões de D. stramonium. Ensaios preliminares de RT-PCR e hibridização não resultaram em reação com alguns outros vírus transmitidos por Brevipalpus, do tipo citoplasmático, inclusive o da leprose dos citros (CiLV-C). / Solanum violaefolium is an ornamental Solanaceae, with prostrate, trailing growth cultivated in shaded areas. Plants exhibiting necrotic ringlike spots on the leaves have been found in several gardens and parks at Piracicaba – SP. The ringspot symptoms on the leaves is caused by a vírus, named S. violaefolium ringspot virus (SvRSV), and is transmisible by mite Brevipalpus phoenicis (Acari: Tenuipalpidae). Short bacilliform particles are present within the cisternae of endoplasmic reticulum (ER) and often electron dense viroplasm is present in the cytoplasm, characterist of the cytoplasmatic type of Brevipalpus-borne virus. The present study reports some of its biological properties and partial molecular caracterization. SvRSV is easily transmitted to many plant species either by viruliferous B. phoenicis or mechanically, always causing local lesions. Datura stramonium was proved to be better as experimental host. Its physical properties in vitro were: inactivation thermal point – 40-45 ºC; final diluition point - 10-3-10-4; longevity in vitro – 12 days. Afterwards, its was observed that S. violaefolium plants were infested by B. obovatus that transmitted SvRSV in preliminary assays. Thin sections revealed that SvRSV particles are slightly thinner and sometimes appear very long. In some favorable sections intermediate steps of viral particle morphogenesis by a budding process of the dense material of the viroplasm toward the lumen of ER could be seen. Due to the fragility of the particles, several attempts to purify the virus have failed, despite many protocols tried. It was possible, however, to extract dsRNA from infected tissue of D. stramonium, and two segments of viral genome, respectively with homology to movement protein (mp) and replicase (rep) of some known viruses were obtained. Primers were designed based in these sequences, which amplified by RT-PCR, fragments of DNA of expected size from total RNA extracts from leaves lesions of infected S. violaefolium and D. stramonium. Probes based on obtained sequences hibridizated with ss- and dsRNA from lesions of S. violaefolium and D. stramonium. Preliminary assays of RT-PCR and hybridization did not result in positive reaction with other cytoplasmatic type of Brevipalpus-borne viruses, including citrus leprosis (CiLVC).

Orientador: Aricio Xavier Linhares / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T02:54:55Z (GMT). No. of bitstreams: 1 Thyssen_PatriciaJacqueline_D.pdf: 2658711 bytes, checksum: 6ae5d4aeb98843b0a14d74606d3f57c1 (MD5) Previous issue date: 2005 / Resumo: A correta identificação e avaliação da idade de insetos envolvidos com a decomposição de cadáveres é de suma importância para a estimativa do intervalo pós-morte (IPM) na área das ciências forenses, particularmente quando o IPM é baseado em informações sobre o ciclo de vida de insetos necrófagos. Entretanto, a análise destes parâmetros em insetos, especialmente quando se encontram em seus estágios imaturos, é difícil mesmo para taxonomistas bem treinados. Além das minúsculas diferenças morfológicas que há entre várias espécies, algumas variáveis tais como temperatura e substâncias tóxicas podem afetar o seu tempo de desenvolvimento gerando um erro no cálculo do IPM. Entre os insetos envolvidos neste processo, as larvas de dípteros da família Calliphoridae são freqüentemente as mais predominantes consumidoras de carcaça e estão presentes em todos os estágios de decomposição. Assim, este estudo teve como objetivo caracterizar morfologicamente e avaliar o tempo de desenvolvimento e as exigências térmicas das formas imaturas de duas espécies de dípteros em diferentes temperaturas: Hemilucilia segmentaria (Fabricius) e Hemilucilia semidiaphana (Rondani) (Calliphoridae). Todos os experimentos foram realizados em câmaras climáticas com temperaturas controladas em 10, 15, 20, 25, 30 e 35ºC, com fotoperíodo de 12 horas e umidade relativa de 70%. Dieta artificial própria para larvas foi oferecida para que estas completassem seu desenvolvimento. Neste estudo, além da descrição e caracterização morfológica tradicional, também foram utilizadas as técnicas da reação em cadeia da polimerase, associada ao polimorfismo baseado no comprimento do fragmento de restrição (PCR-RFLP), para a identificação das duas espécies / Abstract: The correct identification and age determination of insect species involved in cadaver decomposition is of particular importance in estimating the post-mortem interval (PMI) in forensic sciences, particularly since the PMI is based on information on the life cycle of necrophagous insects. However, the correct identification of several insects species, especially in their immature stages, is difficult even for experienced taxonomists. In addition to the minuscule morphological differences between several species, there are some variables such as temperature and toxic substances that may affect the developmental time of insects, generating errors in the estimate of the PMI. Among the insects that are involved in cadaver decomposition, maggots of blowflies (Calliphoridae) are often the most important consumers of carrion and are present in all stages of decomposition. Thus, this study aimed to characterize morphologically and to evaluate the developmental time and the thermal requirements of the immature stages of two species of blowflies reared in different temperatures: Hemilucilia segmentaria (Fabricius) e Hemilucilia semidiaphana (Rondani) (Calliphoridae). All experiments were done in growth chambers with temperatures set at 10, 15, 20, 25, 30 and 35ºC, photophase of 12 hours and relative humidity at 70%. The maggots were reared using an artificial diet for their complete development. In addition to traditional morphological description and characterization of the immatures, the usefulness of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to identify the two species mentioned above was also assessed in this study / Doutorado / Parasitologia / Doutor em Parasitologia

Human genetics is one of the strongest risk factors for complex diseases. Understandingthe effects of genetic variations not only serves as a fundamental approach to studying disease mechanisms but also offers unprecedented opportunities for improved clinical screening, disease diagnosis and therapeutic discoveries. Despite decades of extensive DNA sequencing and genetic research involving large cohorts, two major challenges remain. First, the majority of disease risk genes remain unidentified due to limited statistical power. Second, the functional effects of rare variants, especially missense variants, in disease risk genes are understudied. In this thesis, I describe new computational approaches to address those challenges using statistical genetics and machine learning methods implementing intuition of biological mechanisms. First, I worked on a statistical framework that can identify disease related pathways from de novo coding variants data. I applied this framework to study the genetics of esophageal atresia / tracheoesophageal fistula (EA/TEF) and identified several potential disease causal pathways that involved in endosome trafficking. Next, I developed a new method to identifying disease risk genes by integrating genetic (rare de novo variants) and functional genomics data. Identifying risk genes using rare variants typically has low statistical power due to the rarity of genotype data. Using functional genomics data has the potential to address this challenge as it serves as informative priors of disease risk. Therefore, I developed a statistical method called VBASS. VBASS is a semi-supervised algorithm that uses a neural network to encode biological priors, such as cell type-specific expression values, into a rigorous Bayesian statistical model to increase statistical power. On simulated data, VBASS demonstrated proper error rate control and better power than current state-of-the-art methods. We applied VBASS to congenital heart disease (CHD) and autism spectrum disorder (ASD), identifying several novel disease risk genes along with their associated cell types. Finally, I focused on predicting the functional mechanisms of missense variants that cause diseases. Pathogenic missense variants may act through different modes of action (e.g., gain-of-function or loss-of-function) by affecting various aspects of protein function. These variants may result in distinct clinical conditions requiring different treatments, yet current computational tools cannot distinguish between them because their predictions heavily relied on evolutional conservation data. The recent breakthrough of AI-powered protein structure prediction tools provides an opportunity to address this challenge because the functional mechanisms of variants is intrinsically embedded in its structural properties. Therefore, I developed a deep learning method called PreMode. PreMode is a pretrained SE(3)-equivariant graph neural network model designed to capture the effects of missense variants from their structural contexts and evolutionary information. I pretrained PreMode using labeled pathogenicity data to enable the model to learn a general representation of variant effects, followed by protein-specific transfer learning to predict mode-of-action effects. I applied PreMode to the mode-of-action predictions of 17 genes and demonstrated that PreMode achieved state-of-the-art performance compared to existing models. PreMode has various applications, including identifying novel gain/loss-of-function variants, improving the study design of deep mutational scans and optimization in protein engineering.