Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response system and can occur when the immune system over- or under- reacts to an infection. Klebsiella spp. has been found to be one of the leading causes of sepsis, and the increasing occurrence of antibiotic resistance observed has become a major concern in clinical care. To study the genome and increase knowledge of the biodiversity of K. pneumoniae, K. variicola, and K. oxytoca, bacterial isolates were collected from blood, urine, nasopharynx, and wounds of patients with suspected sepsis. Next-generation sequencing was performed, and the presence of antibiotic resistance genes and plasmids were studied. Furthermore, a prediction of traits for each phylogroup was performed and the results from whole-genome sequencing were compared to phenotypic results. Among the K. pneumoniae isolates obtained, almost half had been misidentified by standard phenotypic methods and were found to be K. variicola, K. quasipneumoniae, and K. quasivariicola. A significant difference in the number of antibiotic resistance genes were observed between K. pneumoniae and K. variicola compared to K. oxytoca, however no significant difference was observed between K. pneumoniae and K. variicola, suggesting the underestimated pathogenicity of K. variicola. A genetic agreement was observed between the type of beta-lactamase harboured and presence or absence of nitrogen-fixation genes to the phylogroup, providing a way of species identification. Further studies should be conducted on the pathogenicity and virulence of K. variicola and K. quasipneumoniae to avoid misidentification, find organism-specific treatments, and narrow down the antibiotic prescription. / Biodiversitet vid Sepsis

Falhas reprodutivas são importante causa de prejuízos econômicos na suinocultura. Elas implicam na diminuição do número de leitões nascidos vivos e aumentam o descarte de animais e as taxas de reposição de matrizes, levando à redução da produtividade do rebanho. Embora a maioria dos casos de natimortalidade sejam associados a fatores não infecciosos, os agentes infecciosos possuem um papel importante e ainda pouco conhecido na etiologia deste quadro. Até o presente, nenhum trabalho foi realizado visando o estudo do conjunto de vírus que possam estar presentes em matrizes com eventos de natimortalidade por ocasião do parto. Em função disso, o presente trabalho teve por objetivo examinar o viroma do soro de matrizes suínas com e sem casos de natimortalidade. Foram coletadas 94 amostras de soro de matrizes de seis granjas distribuídas em cinco municípios do Rio Grande do Sul. Em cada granja foram formados dois pools de soros: um composto por matrizes que pariram (um ou mais) natimortos e outro por matrizes que pariram leitegadas sem natimortos. Os pools foram submetidos à extração de ácido nucleico viral, enriquecimento e sequenciamento de alto desempenho, buscando a identificação de agentes que possam representar um fator de risco à natimortalidade em suínos Não foi possível identificar diferenças significativas nos viromas de matrizes correlacionadas à ocorrência de natimortalidade. Não obstante, foi possível identificar uma ampla variedade de genomas virais, a maioria deles correspondendo a vírus das famílias Anelloviridae. Este estudo permitiu ainda identificar 20 genomas completos de três espécies de vírus: torque teno vírus suíno 1a e 1b, circovírus suíno tipo 3 (PCV3) e vírus circulares DNA fita simples codificantes de replicase (CRESS), seis dos quais até o presente ainda não reportados em suínos. Em duas granjas, em matrizes que apresentaram natimortalidade, foram identificados genomas de PCV3, cuja participação como potencial causador de problemas reprodutivos precisa ser futuramente investigada. Não foram identificados vírus com genoma de RNA. Este estudo traz uma contribuição ao conhecimento do viroma em soros de matrizes suínas e, paralelamente, busca contribuir para o esclarecimento das possíveis causas de natimortalidade de origem infecciosa em suínos. / Reproductive failure in swine herds is an important cause of economic losses. It leads to a decrease in the number of piglets reared per sow and may imply in the need for replacement of sows, reducing the productivity in a herd. Although the majority of cases of stillbirths have been attributed to non-infectious causes, several infectious agents have been implicated in the etiology of such condition. Nevertheless, other as yet unknown agents may be involved in the pathogenesis of stillbirths. The aim of this work was to investigate the virome in sera of sows without and with one or more cases of stillbirth in the litter. Sera were collected from 94 sows of six commercial farms in five municipalities in the state of Rio Grande do Sul, Brazil. Two pools of sera were collected from each farm: one representative of sows that had at least on stillbirth in the last litter and another composed by sera of sows that had litters with no stillbirths. The pools were subjected to nucleic acid extraction, enrichment and high throughput sequencing. No significant differences were detected in the serum viromes of sows with or without stillbirth Nevertheless, it was possible to identify a wide variety of viral genomes, most of these representing viruses of Anelloviridae family. In addition, the present work allowed the identification of 20 complete genome sequences including torque teno sus virus 1a and 1b, porcine circovirus 3 (PCV3) and circular rep-encoding ssDNA viruses (CRESS), including six species not previously reported in swine. In two farms, PCV3 genomes were identified in the serum pools of sows which had cases of stillbirth. The role for this virus as a potential cause of reproductive failure needs additional investigations. No genomes of viruses with RNA genomes were identified. This study provides a contribution to the knowledge on the serum virome of pregnant sows. In addition, it is expected to aid in the identification of possible causes of stillbirth in swine.

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/ Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Coal gasification stripped gas liquor (CGSGL) wastewater contains large quantities of complex organic and inorganic pollutants which include phenols, ammonia, hydantoins, furans, indoles, pyridines, phthalates and other monocyclic and polycyclic nitrogen-containing aromatics, as well as oxygen- and sulphur-containing heterocyclic compounds. The performance of most conventional aerobic systems for CGSGL wastewater is inadequate in reducing pollutants contributing to chemical oxygen demand (COD), phenols and ammonia due to the presence of toxic and inhibitory organic compounds. There is an ever-increasing scarcity of freshwater in South Africa, thus reclamation of wastewater for recycling is growing rapidly and the demand for higher effluent quality before being discharged or reused is also increasing. The selection of hybrid fixed-film bioreactor (HFFBR) systems in the detoxification of a complex mixture of compounds such as those found in CGSGL has not been investigated. Thus, the objective of this study was to investigate the detoxification of the CGSGL in a H-FFBR bioaugmented with a mixed-culture inoculum containing Pseudomonas putida, Pseudomonas plecoglossicida, Rhodococcus erythropolis, Rhodococcus qingshengii, Enterobacter cloacae, Enterobacter asburiae strains of bacteria, as well as the seaweed (Silvetia siliquosa) and diatoms. The results indicated a 45% and 79% reduction in COD and phenols, respectively, without bioaugmentation. The reduction in COD increased by 8% with inoculum PA1, 13% with inoculum PA2 and 7% with inoculum PA3. Inoculum PA1 was a blend of Pseudomonas, Enterobacter and Rhodococcus strains, inoculum PA2 was a blend of Pseudomonas putida iistrains and inoculum PA3 was a blend of Pseudomonas putida and Pseudomonas plecoglossicida strains. The results also indicated that a 70% carrier fill formed a dense biofilm, a 50% carrier fill formed a rippling biofilm and a 30% carrier fill formed a porous biofilm. The autotrophic nitrifying bacteria were out-competed by the heterotrophic bacteria of the genera Thauera, Pseudaminobacter, Pseudomonas and Diaphorobacter. Metagenomic sequencing data also indicated significant dissimilarities between the biofilm, suspended biomass, effluent and feed microbial populations. A large population (20% to 30%) of unclassified bacteria were also present, indicating the presence of novel bacteria that may play an important role in the treatment of the CGSGL wastewater. The artificial neural network (ANN) model developed in this study is a novel virtual tool for the prediction of COD and phenol removal from CGSGL wastewater treated in a bioaugmented H-FFBR. Knowledge extraction from the trained ANN model showed that significant nonlinearities exist between the H-FFBR operational parameters and the removal of COD and phenol. The predictive model thus increases knowledge of the process inputs and outputs and thus facilitates process control and optimisation to meet more stringent effluent discharge requirements. / Thesis (PhD)--University of Pretoria, 2017. / Chemical Engineering / PhD / Unrestricted

Orientador: Erick da Cruz Castelli / Resumo: O gene HLA-C está localizado dentro do Complexo Principal de Histocompatibilidade (MHC), a região mais variável do genoma humano. HLA-C codifica moléculas que participam principalmente do processo de apresentação de antígenos intracelulares aos linfócitos T citotóxicos e interage com receptores presentes nas células NK, modulando sua atividade. A variabilidade das moléculas HLA permite a apresentação de diferentes peptídeos por um mesmo indivíduo e aumenta o repertório de peptídeos apresentados por uma população. Além disso, HLA-C é expresso na interface materno-fetal pelo trofoblasto, onde possui uma importante função imunomodulatória por meio da interação com receptores KIR das células NK maternas. Devido a sua dupla função, alguns alelos de HLA-C ou combinações de alelos HLA-C/KIR têm sido associados a diversos contextos fisiológicos e patológicos. Contudo, a variabilidade desse gene tem sido explorada principalmente para os éxons, em especial os éxons 2 e 3, que codificam os domínios de ligação ao peptídeo, enquanto a variabilidade de outros segmentos gênicos importantes (como outros éxons, íntrons e regiões regulatórias) foram pouco estudadas. Este estudo avaliou a variabilidade genética e assinaturas de seleção natural ao longo do gene HLA-C em uma amostra de 418 indivíduos do Brasil e 108 indivíduos do Benin. Considerando as duas populações, detectamos 359 sítios de variação ao longo da região analisada. Os haplótipos promotores, codificadores e de 3’NT apresentaram um... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Human Leucocyte antigen-C (HLA-C) is a classical HLA class I molecule that binds and presents peptides to cytotoxic T lymphocytes in the cell surface. HLA-C has a dual function since it also interacts with KIR receptors expressed in NK and T cells, modulating their activity. The structure and diversity of the HLA-C regulatory regions, as well as the relationship among variants along the HLA-C locus, is poorly addressed, and no population-based study explored the complete HLA-C variability in different population samples. Here we first present a new molecular and bioinformatics method to evaluate the full HLA-C segment, including regulatory sequences. Then, we applied this method to survey the HLA-C diversity in two geographically distinct population samples, one admixed from Brazil (São Paulo State) and one less admixed from Benin. Our results indicate that the HLA-C promoter and 3’UTR are very polymorphic, with the presence of few but highly divergent haplotypes. However, both these regulatory regions present conserved segments that are shared among different primates. Nucleotide diversity was higher in other exonic segments rather than exons 2 and 3, and also higher in the second half of the 3’UTR region. We detected evidence of balancing selection on the entire HLA-C locus and positive selection in exon 1, for both populations. HLA-C motifs previously associated with KIR interaction and expression regulation are similar between both populations, but the frequency of amino ... (Complete abstract click electronic access below) / Doutor

No description available.

During the last decade, the use of Next-Generation Sequencing(NGS) technologies has sky-rocketed. The vast amount of data produced by these platforms require processing and analysis. This is usually performed at locations remote from the sequencing facilities thereby introducing the need for data-transportation to the place of analysis. The use of internet transfer would greatly facilitate the process, however since NGS data is considered to be personal sensitive information the handling of the data is highly regulated by the General Data Protection Regulation(GDPR). During this project, a web-based application was developed for the privacy-protecting transfer of personal sensitive data, implementing an in-motion encryption scheme which ensures data integrity and authenticity. The application consists of three scripts: the HTML web page with JavaScript functionality, a PHP script responsible for connection establishment and integrity verification, and a Python script executing the majority of the server-side operations. The resulting application uses the symmetric encryption algorithm AES in GCM mode, using a key size of 128 bits and transfers 60 Kibibytes of the file at a time. The key is established by using the asymmetric RSA encryption scheme with a 4096 bit key pair. SHA-256 is used for verifying the integrity of the transferred files. The JavaScript encryption speed is 584 MB/s and the Python decryption speed 251 MB/s. While the focus of the project was to optimize the application for NGS data, it is not limited to this type of file and can transfer different formats, enabling the use in multiple different fields.

Hepatitis B Virus (HBV) is a DNA virus and belongs to the genus Orthohepadnavirus of the Hepadnaviridae family which represents one of two animal viruses with a DNA genome which replicates by reverse transcription of a viral RNA intermediate. Nucleotide variation led to further sub-classification into 8 genotypes (A to H). The reverse transcription step within its life cycle is prone to the introduction of errors and recombination when dually infected. This leads to a viral quasispecies which forms during the course of infection with many minor population variants; such variants can however only be detected by means of ultra-deep sequencing. A recent study in the Department of Medical Virology (UP) by Mayaphi et al. identified a number of the specimens that partitioned away from the typical subgenotype A1 clades with high bootstrap values and longer branch lengths. Thus, the main objective of the current study was to characterize the full genome of all variants for the outliers observed in the aforementioned study, inclusive of potential recombination, dual infection and minor populations. Twenty samples were selected from a previous cohort for purposes of the present study. The viral DNA was extracted and amplified by PCR according to the methods described by Günther et al. with modified primer sets. Nineteen of the samples were successfully amplified and 15 of these were sequenced. Specimens were sequenced by NGS on the Illumina MiSeq™ sequencer and sequence data used to reconstruct the viral quasispecies of each specimen. Further analyses of the reconstructed variants included molecular characterization as well as phylogenetic analysis and screening for recombination and drug resistance mutations. Full genome coverage was obtained for twelve of the fifteen samples and full genome variants reconstructed, generating nearly 40 full genomes. Phylogenetic analysis showed that the majority of the samples are of genotype A, more specifically of subgenotype A1, differing by less than 4% from known sequences. The phylogenetic analysis revealed a similar clade of outliers, where four samples clustered together with significant bootstrap support (75%) and a fifth sample partitioned separate from, yet close to, this clade, away from the typical African A1 clade. This clade was assigned to genogroup III. Three samples were of the Asian A1 clade (genogroup I) with remaining specimens grouping within genotype D and E. The variants showed low diversity within each specimen with some differing at but a few positions across the genome while even the most diverse quasispecies differed by less than a percentage (32 positions). Several unique and atypical positional variations were observed amongst study samples of which some were present in but one of the variants for that sample. Twenty-six lead to shared amino acid changes. Some observed changes, such as A1762T/G1764A and G1896A, could explain the serological patterns such as HBeAg negativity while others, such as C2002T, were previously implicated in disease progression and severity. Sample N199 presented a longer branch length and revealed short regions within the genome that display evidence of recombination between HBV/A1 and HBV/A2. The results illustrate the utility of NGS technology in characterizing viral variants. / Dissertation (MSc)--University of Pretoria, 2014. / lk2014 / Medical Virology / MSc / Unrestricted

In a cell, the DNA undergoes transcription to form mature transcripts, some of which in turn undergo translation to form proteins. Although over 85% of the human genome is transcribed, it comprises only about 2% protein-coding genes, the rest being noncoding. One of the non-coding gene elements, called long non-coding RNAs (lncRNAs), are emerging as key players in various regulatory roles in the human genome. The generally accepted theory posits lncRNAs to be over 200 nucleotides long and to be able to grow over 10 kilobases, bearing a similarity with mRNAs. The majority of lncRNAs undergo alternative splicing and are weakly polyadenylated in combination with complex secondary structures. Among the annotated lncRNAs, so far it has been only a meagre portion for which functional roles have been detected, while functions of the vast majority remain to be discovered. Observed functional roles include thus far gene expression regulation through various mechanisms at transcriptional and post-transcriptional levels. With the advent of next-generation sequencing (NGS) and advances in RNA sequencing technology (RNA-Seq), it is easier to reconstruct the transcriptome by extracting information about the splicing machinery. RNA-Seq has helped consortia like GENCODE, ENCODE, and others to curate their annotation catalogues. In this PhD thesis, certain aspects of the human lncRNA transcriptome will be explored, such as the challenges in lncRNA annotation. Those challenges stem from the lack of signals that are common in mRNAs and make them easier to detect, for instance signals of ORFs and transcription start sites. Concurrently, owing to a lack of understanding of the connection between sequence and function, lncRNAs have been typically annotated based upon their location in relation to mRNAs and their functions have been predicted through a guilt-by-association approach. In the first part of the PhD research work, the splice junctions in the lncRNA transcriptome were mapped in an attempt to explore the isoform diversity of lncRNAs by using sequencing data from B-cell lymphoma. In this phase of the research work, multiple junction-spanning reads from the sequencing data with a very large read depth were found to represent the splice junctions. Using GENCODE v19 as a reference it was found that the human transcriptome harbours a large number of rare exons and introns that have remained unannotated. Concomitantly, it can be inferred that the current human transcriptome annotation is confined to a very well-defined set of splice variants. However, although the isoforms are well-defined, the same cannot be said about their biological functions and it remains to be explored why the processing machinery of lncRNAs is restricted to a set of very few splice sites. In the human genome, small regulatory RNAs like miRNAs and small nucleolar RNAs (snoRNAs) overlap with lncRNAs in their genomic loci. To further understand the human transcriptome, in the second part of the PhD research work, a study was undertaken in an attempt to distinguish the miRNA and snoRNA hosting lncRNAs from the lncRNAs that did not have any overlaps with the smaller RNAs. To this end, machine learning techniques were implemented on curated datasets employing features inspired by a few of the prevalent features used in published lncRNA detection tools encompassing not just sequence information, but also secondary structure and conservation information. Classification was attempted through supervised as well as unsupervised learning approaches; random forests for the former, PCA and k-means for the latter. In the end, the three RNA classes could not be separated with certitude, especially when the hosted RNA was not supplied to the classifier, however, this lack of detectable association can be confirmed to be of biological interest. It suggests that the function of host genes is not closely tied to the function of the hosted genes at least in this case. Nevertheless, understanding the dynamics of snoRNA and miRNA host genes can improve the knowledge of functional evolution of lncRNAs, as the fact that the smaller RNA genes are conserved makes it comparably easier to trace the host lncRNAs over much larger evolutionary timescales than most other lncRNAs. With the accelerated availability of sequencing techniques it can be expected that expanded investigation into conservation patterns and host gene functions will be possible in the near future.

Aujourd’hui, le marché des services est devenu de plus en plus concurrentiel. Les exigences des clients pour des offres de service en adéquation avec leurs usages et leurs préférences conduisent les fournisseurs à proposer de nouveaux services qui répondent à ce nouveau besoin pour se démarquer des concurrents et attirer de nouveaux clients. Avec la convergence des réseaux et celle des services de nouvelle génération (NGN/NGS), de nouveaux services sont apparus. Les utilisateurs sont nomades et veulent utiliser leurs services de différentes manières n’importe où, n’importe quand et par n’importe quel type de terminal, et cela avec une continuité de service et une qualité de service de bout en bout. Ainsi, fournir des services personnalisés aux clients dans un environnement hétérogène et mobile devient un challenge pour les opérateurs et les fournisseurs de service pour améliorer le retour sur investissement (ROI) et le délai de mise sur le marché (TTM). Nos réflexions à propos de la fourniture des services personnalisés selon les besoins fonctionnels et non-fonctionnels (QoS) des usagers, nous ont conduits à identifier les besoins du nouveau contexte NGN/NGS défini par l’intersection de ces trois éléments «user-centric, mobilité et QoS». Comment piloter dynamiquement la QoS de bout en bout pour une session unique «user-centric»? Comment assurer le « service Delivery» dans un contexte de mobilité et d’ubiquité? Ces nouveaux besoins, nous ont motivé à proposer des solutions à travers trois contributions principales qui prennent en considération la vision utilisateur et opérateur. Notre première contribution porte sur le modèle organisationnel. Nous proposons une nouvelle organisation avec un maximum de flexibilité, d’adaptabilité et d’autogestion, qui permet de piloter la QoS à chaque niveau de l’architecture (équipement, réseau et service). Dans cette organisation nous avons défini des acteurs et le rôle que joue chacun d’eux par rapport à la prise de décision au cours de la session de l’utilisateur, et cela pour maintenir la QoS de bout en bout dans un environnement qui est totalement hétérogène et mobile.Notre deuxième contribution traite du composant de service autonomique. Avec la complexité de la personnalisation des services dans un contexte hétérogène et mobile et le besoin de satisfaire la QoS de bout en bout, les ressources services doivent être prises en compte au même titre que les ressources réseaux. Donc, un degré élevé d’autosuffisance, d’autogestion et d’automatisation est demandé dans la ressource service (composant de service) pour améliorer le service delivery. Pour cela, nous proposons un composant de service autonomique «ASC: Autonomic Service Component» basé sur un agent de QoS intégré qui s’autocontrôle et s’autogère pour adapter dynamiquement ses ressources en réponse à un changement de situations au cours de la session de l’utilisateur. Notre troisième proposition couvre le modèle protocolaire. La session de services personnalisés nécessite des interactions plus flexibles au niveau service pour avoir une session unique avec une continuité de service. Nous proposons un protocole de signalisation SIP+ qui permet la négociation de la QOS des services personnalisés dès la phase d’initialisation de la session et de la renégociation de la QoS pendant l’usage, pour maintenir le service avec la QoS requise à travers une session unique.De façon plus concrète, nous présentons nos expérimentations à travers un scenario et une plate-forme de démonstration qui nous permet de tester la faisabilité et la performance de nos contributions. Les apports et les perspectives de cette thèse sont consignés en conclusion. / Nowadays, the services market has become increasingly competitive. Customer requirements for service offerings in line with their uses and preferences led providers to offer new services to meet this new need and to stand out from competitors and attract new customers. With the success of the network and service convergence (NGN / NGS), new services have emerged. A mobile user desires to access his services anywhere, anytime and on any type of terminal.Thus, providing customized services to clients while ensuring the service continuity and the end-to-end quality of service in a heterogeneous and mobile environment became a challenge for mobile operators and service providers to improve the return on investment (ROI) and time-to-market (TTM). Our thinking about the provision of customized services according to the functional and non-functional (QoS) needs of the users has led us to identify the needs of the new context NGN / NGS defined by the intersection of these three elements "user-centric, mobility and QoS". How to dynamically control the end-to-end QoS for a single "user-centric" session? How to ensure the "Service Delivery" in the context of mobility and ubiquity? These new needs have led us to propose solutions through three main contributions that take into account the user and the operator vision. Our first contribution concerns the organizational model. We have proposed a new organization with a maximum of flexibility, adaptability and self-management which allows the control of the QoS at each level of the architecture (equipment, network and service). In this organization, we have defined actors and the role of each one in relation to the decision-making process during the user session in order to maintain the end-to-end QoS in an environment that is totally heterogeneous and mobile. Our second contribution addresses the autonomic service component. With the complexity of services personalization in a heterogeneous and mobile context and the need to satisfy the end to end QoS, services and network resources must be taken into account. Therefore, a high degree of self-sufficiency, self-management and automation is required in the resource service to improve the service delivery. We have therefore proposed an autonomic service component based on an integrated QoS-agent which is self-controlled and self-managed to dynamically adapt its resources in response to changing situations during the user’s session. Our third proposal covers the model protocol. The personalized services session requires more flexible interactions at the service level in order to obtain a single session with service continuity. We have proposed a signalling protocol SIP + that allows the negotiation of the QoS of personalized services at the session initialization phase and the renegotiation of the QoS during the utilization to maintain the service with the required QoS through a unique session. More concretely, we have presented our experiments through a scenario and demonstration platform that allows us to test the feasibility and the performance of our contributions. The contributions and perspectives of this thesis are stated in the conclusion.