Orientadores: Zanoni Dias, Guilherme Pimentel Telles / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Computação / Made available in DSpace on 2018-08-23T01:03:41Z (GMT). No. of bitstreams: 1 Peixoto_BrunoMalveira_M.pdf: 1497265 bytes, checksum: 9127980450af9b4a7d8fe679c48f2310 (MD5) Previous issue date: 2013 / Resumo: Metagenômica é o estudo genético de uma amostra ambiental, e permite a análise de organismos incultiváveis em laboratório. É uma área de estudo nova e possui muitos desafios computacionais, com poucas ferramentas dedicadas disponíveis. O objetivo deste trabalho é realizar um estudo metagenômico da diversidade de organismos microbianos de amostras da unidade de compostagem da Fundação Parque Zoológico de São Paulo, analisando e comparando os programas e métodos existentes. O estudo das comunidades microbianas que ali vivem é de grande importância para um melhor entendimento do papel biológico desses organismos. Dados simulados foram testados para validar os métodos utilizados com os dados reais e os resultados mostram que mesmo programas conhecidos são sensíveis aos bancos de dados de referência que utilizam / Abstract: Metagenomics is the genetic study of an environmental sample, and allows the analysis of non-culturable organisms. It is a new area of genetic study and has many computational challenges, with few dedicated tools available. The objective of this work is to realize a metagenomic study of the microbial diversity of samples from the composting unit of Fundação Parque Zoológico de São Paulo, analyzing and comparing existing programs and methods. The study of the microbial communities that live there is of great importance to a better understanding of the biological role of these organisms. Simulated data were tested to validate the methods we used with the real data and the results show that even well-known tools are biased by the reference databases it uses / Mestrado / Ciência da Computação / Mestre em Ciência da Computação

Orientador: Fabio Marcio Squina / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T12:41:11Z (GMT). No. of bitstreams: 1 Alvarez_ThabataMaria_D.pdf: 22982311 bytes, checksum: 1bc2a260df2bfab4948ffb299f13a63f (MD5) Previous issue date: 2013 / Resumo: Devido à necessidade do desenvolvimento de fontes de energias renováveis é de grande interesse a descoberta de novas enzimas envolvidas na desconstrução da parede celular vegetal para a produção de bicombustíveis. A metagenômica é uma poderosa ferramenta para a descoberta de novos genes em comunidades microbianas que não são passíveis de cultivo pelas técnicas tradicionais. Neste contexto, o objetivo desta tese foi o desenvolvimento de estratégias metagenômicas para prospecção de novas enzimas atuantes na degradação da biomassa vegetal no metagenoma de solo de canavial bem como a caracterização funcional das mesmas. A biblioteca metagenômica construída com DNA extraído de um consórcio microbiano especializado na degradação de bagaço de cana-de-açúcar explodido a vapor e deslignificado foi empregada nos experimentos de triagem funcional de alto desempenho. Como resultado, foram identificados três clones positivos com atividade celulolítica e dois clones com atividade xilanolítica. A análise dos insertos de cada um dos clones resultou na localização de ORFs cujas sequências de aminoácidos apresentaram identidade com domínios conservados de glicosil hidrolases da família 5 (celulases E-1 e E-2), família 6 (celulase E-3), família 10 (xilanase X-1) e família 16 (glicosil hidrolase X-2). A celulase E-1 apresentou em sua estrutura além do domínio catalítico, E-1 Cat, um domínio de ligação a carboidratos, denominado E-1 CBM, que não apresentou identidade de sequência com domínios conservados conhecidos. A análise funcional do E-1 CBM revelou tratar-se de um CBM específico para cadeias de glucano com grau de polimerização mínimo de cinco unidades de glicose. Ensaios de atividade enzimática em diferentes substratos mostraram que E-1 Cat atuou especificamente na hidrólise das ligações glicosídicas do tipo ß(1,4) entre resíduos de glicose. Os maiores valores de atividade enzimática foram obtidos em pH 7,0 e temperatura de 50ºC. Os parâmetros cinéticos calculados em CMC foram Km igual a 6,05 ± 0,37 mg/mL, Vmax de 42,51 ± 1,2 ?mol/min/mg e eficiência catalítica kcat/Km de 4,06 mL/mg/s. A enzima apresentou termoestabilidade a 40ºC por cinco horas. A atividade enzimática de E-1 Cat em celulose cristalina e bagaço de cana-de-açúcar explodido a vapor resultou na liberação de açúcares solúveis, evidenciando sua potencial aplicação em processos de conversão da biomassa vegetal. Ensaios de atividade em diferentes substratos mostraram que X-1 apresentou maior atividade enzimática em xilana não ramificada, nas condições de pH e temperatura de 6,0 e 45ºC, respectivamente. Os parâmetros cinéticos calculados utilizando como substrato xilana de madeira de faia foram Km de 2,18 ± 0,13 mg/mL, Vmax de 1.435 ± 30,4 ?mol/min/mg e kcat/Km de 496,32 mL/mg/s. Em relação à termoestabilidade, a enzima se manteve estável a 40ºC e 50ºC por seis horas. A hidrólise de substratos complexos com X-1 resultou na liberação de xilooligossacarídeos, xilobiose e xilose, que são compostos que apresentam potencial aplicação nas indústrias alimentícias e de biocombustíveis. Os resultados obtidos neste estudo validaram a abordagem metagenômica desenvolvida para a descoberta de novos genes codificantes para glicosil hidrolases. Além disso, a estratégia descrita nesta tese pode ser estendida para a descoberta de uma miríade de bioprodutos de interesse biotecnológico / Abstract: Due to the necessity of development of renewable sources of energy, it is of great interest the discovery of novel enzymes involved in plant cell wall deconstruction for biofuels production. Metagenomics is a powerful tool for the discovery of novel genes in microbial communities that are not liable to cultivation by traditional techniques. In this context, the aim of this thesis was the development of metagenomic strategies for prospection of novel enzymes involved in plant biomass degradation in sugarcane field soil metagenome and functional characterization of the identified enzymes. The metagenomic library constructed with DNA extracted from a microbial consortium specialized in degradation of steam exploded delignified sugarcane bagasse was used in the experiments of high-performance functional screening. As a result, we identified three positive clones with cellulolytic activity and two clones with xylanolytic activity. The analysis of the inserts from each clone resulted in the location of ORFs whose amino acid sequences showed identity to conserved domains of glycoside hydrolase family 5 (cellulases E-1 and E-2), family 6 (cellulase E-3), family 10 (xylanase X-1) and family 16 (glycoside hydrolase X-2). Cellulase E-1 exhibited in addition to the catalytic domain, E-1 Cat, a carbohydrate binding module, called E-1 CBM, which showed no sequence identity with known conserved domains. Functional analysis of E-1 CBM showed that it is a CBM specific for glucan chains with a degree of polymerization of at least five units of glucose. Assays with a set of different substrates revealed that E-1 Cat hydrolyzed specifically ß(1,4) glycoside bonds between glucose residues. The highest value of enzymatic activity was obtained at pH 7.0 and temperature of 50°C. The kinetic parameters Km, Vmax and catalytic efficiency kcat/Km calculated using CMC were 6.05 ± 0.37 mg/mL, 42.51 ± 1.2 ?mol/min/mg and 4.06 mL/mg/s, respectively. The enzyme showed thermal stability at 40°C for five hours. The enzymatic activity of E-1 Cat in crystalline cellulose and steam exploded sugarcane bagasse resulted in the release of soluble sugars, demonstrating its potential application in processes of biomass conversion. The xylanase X-1 showed higher enzyme activity in debranched xylan, in reactions conducted in pH 6.0 and temperature of 45°C. The kinetic parameters Km, Vmax and catalytic efficiency kcat/Km calculated using beechwood xylan were 2.18 ± 0.13 mg/mL, 1,435 ± 30.4 ?mol/min/mg and 496.32 mL/mg/s, respectively. In relation to thermal stability, the enzyme was stable at 40°C and 50°C for six hours. The hydrolysis of complex substrates resulted in the release of xylo-oligosaccharides, xylobiose and xylose, which are compounds that have potential application in food and biofuels industries. The results of this study validated the metagenomic approach developed for the discovery of novel genes coding for glycoside hydrolases. Moreover, the strategy described in this work can be extended to the discovery of a myriad of byproducts of biotechnological interest / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular

Human microbial research has become increasingly popular in biomedical areas due to the importance of role of human microbiome in human health. One purpose of studying human microbiome is to detect differentially abundant features from a limited group of subjects across biological conditions. Metagenomic analyses of the human microbial communities are extensively used for biomedical applications due to its reliable and evident comparative discoveries across more than one metagenomes when multiple communities are taken into consideration. Next-generation sequencing technology helps to detect taxonomic compositions of specific features/species contained in human microbial communities. Statistical analysis often starts by generating the Operational Taxonomic Units (OTUs) using taxonomic compositions to classify groups of closely associated human microbiomes. Oftentimes, the counts of features are observed as matched count data with excess zeros. Such data lead some differential abundance analysis methods to apply Zero-Inflated Poisson (ZIP) or Zero-Inflated Negative Binomial (ZINB) regression for modeling the microbial abundance. However, over-dispersion as well as within-subject variation and correlation of matched count data render the standard ZIP and ZINB regression inadequate. To account for the inherent within-subject variation and correlation, independent random effect terms are commonly included in the regressions. Therefore, a robust method that accounts the effect of matched samples and correlated random effects while considering over-dispersion and excess zeros of count data is need for statistical analysis. In this paper, a statistical method, the two-part correlated ZINB model with correlated random effects (cZINB), is proposed for testing the matched samples with repeated measurements.

>Magister Scientiae - MSc / Pharmaceutical research and development strategies rely on the constant discovery of novel natural products as potential drugs. Recent studies have shown that the microorganisms associated with sponges are the true producers of some previously isolated compounds. This study created a large collection of bacterial symbionts associated with the South African marine sponge, Hamacantha esperioides. The bioactivity assays performed, showed that 44 isolates produced compounds with antimicrobial or anti-inflammatory activity. The successful identification of novel species that produce potential natural products highlights the importance of cultivation-dependent methods. To further screen for natural products, a cultivation-independent approach was used. A sequenced-based method, based on the biosynthetic genes of polytheonamide, was developed to screen for proteusins in sponge metagenomic DNA and the genomes of bacterial symbionts. The degenerate primers could amplify the targeted genes from DNA known to contain homologues. Evaluation of the primers' specificity showed non-specific amplification of genes, some containing similar conserved domains as the target genes. This study demonstrated that the use and development of cultivation-dependent and -independent screens are important for the discovery of novel natural products from the symbiotic bacteria of South African sponges. / National Research Foundation (NRF)

This dissertation documents the development of an environmental framework for monitoring antimicrobial resistance gene (ARG) dissemination in the aquatic environment. The work opens with a review of the relevant literature and outlines the importance of an environmental framework for monitoring ARG dissemination as part of antimicrobial resistance risk assessments. The ability to interrogate sequencing data quickly and easily for the presence of ARGs is crucial in order to facilitate their monitoring in the environment. As current laboratory methods for the detection and surveillance of antimicrobial resistant bacteria in the environment were limited in their effectiveness and scope, the dissertation begins by describing the design and implementation of a Search Engine for Antimicrobial Resistance (SEAR), a pipeline and web interface for detection of horizontally-acquired ARGs in raw sequencing data. The suitability of metagenomic methods for monitoring the ARG content of effluents from faecal sources was then assessed via a pilot study of a river catchment. Novel metagenomes generated from effluents entering the catchment were interrogated for ARGs. The relative abundance of ARGs in effluents were determined to be higher relative to the background environment, as were sequences relating to human and animal pathogens and mobile genetic elements. Thus, effluents were implicated in the dissemination of ARGs throughout the aquatic environment. To determine if ARGs were potentially in use in the environment, the expression of ARGs within effluents was then evaluated across a series of longitudinal samples through the use of metatranscriptomics, and the presence of potential environmental antimicrobial selection pressures was examined. This demonstrated that the abundance of ARGs, as well as antimicrobial usage at the effluent source, was correlated with the transcription of ARGs in aquatic environments. The work described in this dissertation has also found that horizontally transmitted ARGs were present in pathogenic endospore-forming bacteria commonly found across the aquatic environment, potentially providing a mechanism for ARG persistence in the environment. Finally, these findings were integrated into a universal framework for monitoring ARG dissemination in aquatic environments and used to highlight the developments required to incorporate this framework into future environmental ARG research and to facilitate antimicrobial resistance risk assessments.

Prokaryotes are the main actors in biogeochemical cycles that are fundamental in global nutrient cycling. The characterization of microbial communities and isolates can enhance the comprehension of such cycles. Potentially novel biochemical processes can be discovered in particular environments with unique characteristics. The Red Sea can be considered as a unique natural laboratory due to its peculiar hydrology and physical features including temperature, salinity and water circulation. Moreover the Red Sea is subjected to hydrocarbon pollution by both anthropogenic and natural sources that select hydrocarbon degrading prokaryotes. Due to its unique features the Red Sea has the potential to host uncharacterized novel microorganisms with hydrocarbondegrading pathways. The focus of this thesis is on the characterization at the metagenomic level of the water column of the Red Sea and on the isolation and characterization of novel hydrocarbon-degrading species and genomes adapted to the unique environmental characteristics of the basin. The presence of metabolic genes responsible of both linear and aromatic hydrocarbon degradation has been evaluated from a metagenomic survey and a meta-analysis of already available datasets. In parallel, water column-based microcosms have been established with crude oil as the sole carbon source, with aim to isolate potential novel bacterial species and provide new genome-based insights on the hydrocarbon degradation potential available in the Red Sea.

The demand for novel and robust microbial biocatalysts for industrial and pharmaceutical applications continue to grow at a fast pace.This warrants a continuous need for advanced tools and technologies to exploit the vast metabolic potential of microorganisms in different environments. Unlike culture-based studies that can only reveal the metabolic potential of cultivable microorganisms, functional metagenomics charts the enzymatic potential of the entire microbial communities in a given environment. This method has substantially contributed to the effective discovery of unique microbial genes for industrial and medical applications. Functional metagenomics involves the extraction of microbial DNA directly from environmental samples,construction of an expression library containing the entire microbial genome, and screening the libraries for the presence of desired phenotypes. Therefore, development of a pipeline for analyzing and screening metagenomic libraries is essential for rapid detection of the desired features from thousands of clones of a single library. Here, we developed a pipeline for high-throughput screening of the lipolytic genes from the Red Sea.Further, a high-throughput single cell microfluidics platform combined with a laser-based fluorescent screening bioassay was deployed to discover new lipolytic genes. Our analysis led to the identification of 24 microbial genes for lipases and esterase from a metagenomic library of the Red Sea water. The results further showed that the constructed pipeline is robust in conducting functional metagenomics and for the discovery of new genes. It also implies that the Red Sea is a rich under- investigated source of natural resources of new genes and gene products.

Coral reefs are among the most diverse marine ecosystems, but they are threatened by climate change. The foundation of reef ecosystems is the coral holobiont or metaorganism that consists of the coral animal host, photosynthetic microalgae, bacteria, and viruses (among other organisms). While microalgae provide the energy for corals to build the massive three-dimensional skeletons, bacteria support functions related to metabolism, immunity, and environmental adaptation. Conversely, the function of viruses is less well understood. Although viruses were previously associated with coral disease and bleaching, we are missing an overall understanding of the diversity and identity of viruses associated with corals, in particular for understudied areas such as the Red Sea. Here we characterized coral-associated viral community composition using a large metagenomic and metatransciptomic dataset covering > 1 billion sequences across > 100 coral samples collected from 14 different coral species in the central Red Sea. The viral sequence portion shows that coral species significantly differ from each other, but the most abundant viral families were consistently present. Notably, we found a pervasive abundance of Baculoviridae in metagenomes. In contrast, Polydnaviridae were the most abundant viruses in metatranscriptomes, highlighting that the combined approach of metagenomics and metatranscriptomics is informative with regard to deciphering viral diversity and activity. Our study provides a first comprehensive description of viruses associated with Red Sea corals. In line with previous studies, we confirm the presence of Baculoviridae, Polydnaviridae, Phycodnaviridae, Mimiviridae, and Herpesviridae, which may be considered viral families that are globally and commonly associated with corals. The reason for the pervasive abundance of Baculoviridae in Red Sea corals at present remains unknown, but it is tempting to speculate that the association is related to the uniquely warm and salty environment of the Red Sea.

Water reuse is increasingly pursued to alleviate global water scarcity. However, wastewater treatment process does not achieve full removal of biological contaminants from wastewater, and hence some microorganisms and their genetic elements can be disseminated into the reclaimed water distribution systems (RWDS). A systematic identification and characterization of these biological contaminants is required. However, a broad characterization for large-scale data are limited. In this study, reclaimed water samples are investigated through metagenomic analyses to assess their bacterial and functional (metal resistance genes (MRGs); virulence factors (VFs)) communities at the entry and exit points of the RWDS. Furthermore, water quality data are investigated to evaluate the potential relationship with these metagenomic annotations. This study found that the organic carbon content was likely relevant to the increase of bacteria and functional genes in RWDS. It was also found that the variation of functional genes was not associated with their host, inferring the role of horizontal gene transfers or promiscuity of hosts for various functional genes. Furthermore, Pseudomonas was identified in one RWDS with significant increase at both bacterial and functional levels.

The human microbiome, the complex and dynamic ecosystem that populates our body, performs essential functions such as aiding digestion and protecting us from harmful pathogens. An increasing number of diseases are found to be associated with a shift of balance - or dysbiosis - of the microbiome. However, we still know little about this delicate balance and how it depends on different microbial functions. In this thesis project, I used metagenomic sequencing data to study the variability of microbes and their functions in different areas of the human body. First, in an attempt to characterize the dysbiosis associated with periodontitis, I examined the microbial community of the oral cavity in presence and absence of this chronic inflammatory disease. Specifically, I catalogued the phylogenetic signatures composed of tetramer nucleotide frequencies and observed that the disease state occupies a much narrower region than the healthy one. This result suggests that upon onset of the disease, through host cell invasion, pathogenic bacteria may find a more consistent environment for their parasitic lifestyle. Motivated by these findings, I sought further evidence of an environment-specific use of metabolic functions in the oral and gut communities. Rather than focusing on the abundance of individual metabolic functions, I evaluated their diversity, i.e., the extent to which these functions are performed by different classes of organisms. My hypothesis was that such diversity may confer increased robustness to taxonomic variability. Using metagenomic sequencing data and NCBI's Protein Clusters database, I characterized the multiplicity of gene families associated with a given metabolic function. I found that different human body sites display different degrees of metabolic functional diversity, as assessed by Shannon entropy. For some well-studied gene functions, such as those involved in glycolytic pathways, I found entropy signatures consistent with the known degree of oxygen availability of different environmental niches. Conversely, in an unsupervised analysis, I identified functions with nontrivial entropy signatures. These results pave the way for a new way to inspect human microbiome activity, and could help understand its functional resilience and suggest ways to shift its balance towards healthy configurations.