<p>Environmental stress affects plant development and productivity. Sulfur deficiency is a key nutrient deficiency that adversely affects crop yield. The model plant Arabidopsis thaliana has played an informative role in deciphering the mechanisms involved in sulfur assimilation, as well as, the response to limited conditions. Using Arabidopsis thaliana as a model to investigate gene expression in the root, microarray data sets have been generated. These data sets consist of whole root sections for 6 time points across 72 hours, and enriched populations of 5 radial cell-types and 4 sections of 3 developmental zones of the root at 3 hrs on sulfur limited conditions. With these data it was determined which cellular tissues and developmental zones were affected most by sulfur limited conditions. Furthermore, a novel phenotype was characterized that occurs in roots after growth on low sulfur conditions. Cellular inclusions build up within the cytoplasm of mature cortical root cells. These inclusions have been termed "sulfur pox" and their composition remains to be determined.</p> / Dissertation

No description available.

Lo sviluppo delle tecnologie di sequenziamento ha portato alla nascita di strumenti in grado di produrre gigabasi di dati di sequenziamento in una singola corsa. Queste tecnologie, comunemente indicate come Next Generation Sequencing o NGS, producono grandi e complessi dataset la cui analisi comporta diversi problemi a livello bioinformatico. L'analisi di questo tipo di dati richiede la messa a punto di pipelines computazionali il cui sviluppo richiede un lavoro di scripting necessario per concatenare i softwares già esistenti. Questa tesi tratta l'aspetto metodologico dell'analisi di dati NGS ottenuti con tecnologia Illumina. In particolare in essa sono state sviluppate tre pipelines bioinformatiche applicate ai seguenti casi studio: 1) uno studio di espressione genica mediante RNA-seq in "Olea europaea" finalizzato all’indagine dei meccanismi molecolari alla base dell’acclimatazione al freddo in questa specie; 2) uno studio mediante RNA-seq finalizzato all’identificazione dei polimorfismi di sequenza nel trascrittoma di due razze bovine mirato a produrre un ampio catalogo di marcatori di tipo SNPs; 3) il sequenziamento, l’assemblaggio e l’annotazione del genoma di un ceppo di Lactobacillus plantarum che mostrava potenziali proprietà probiotiche. / The advance in sequencing technologies has led to the birth of sequencing platforms able to produce gigabases of sequencing data in a single run. These technologies commonly referred to as Next Generation Sequencing or NGS produce millions of short sequences called “reads” generating large and complex datasets that pose several challenges for Bioinformatics. The analysis of large omics dataset require the development of bioinformatics pipelines that are the organization of the bioinformatics tools in computational chains in which the output of one analysis is the input of the subsequent analysis. A work of scripting is needed to chain together a group of existing software tools.This thesis deals with the methodological aspect of the data analysis in NGS sequencing performed with the Illumina technology. In this thesis three bioinformatics pipelines were developed.to the following cases of study: 1) a global transcriptome profiling of “Oleaeuropeae” during cold acclimation, aimed to unravel the molecular mechanisms of cold acclimation in this species; 2) a SNPs profiling in the transcriptome of two cattle breeds aimed to produce an extensive catalogue of SNPs; 3) the genome sequencing, the assembly and annotation of the genome of a Lactobacillus plantarum strain showing probiotic properties.

It is well known that cells in tissues display a large heterogeneity in gene expression due to differences in cell lineage origin and variation in the local environment at different sites in the tissue, a heterogeneity that is difficult to study by analyzing bulk RNA extracts from tissue. Recently, genome-wide transcriptome analysis technologies have enabled the analysis of this variation with single-cell resolution. In order to link the heterogeneity observed at molecular level with the morphological context of tissues, new methods are needed which achieve an additional level of information, such as spatial resolution. In this thesis I describe the development and application of padlock probes and rolling circle amplification (RCA) as molecular tools for spatially-resolved transcriptome analysis. Padlock probes allow in situ detection of individual mRNA molecules with single nucleotide resolution, visualizing the molecular information directly in the cell and tissue context. Detection of clinically relevant point mutations in tumor samples is achieved by using padlock probes in situ, allowing visualization of intra-tumor heterogeneity. To resolve more complex gene expression patterns, we developed in situ sequencing of RCA products combining padlock probes and next-generation sequencing methods. We demonstrated the use of this new method by, for the first time, sequencing short stretches of transcript molecules directly in cells and tissue. By using in situ sequencing as read-out for multiplexed padlock probe assays, we measured the expression of tens of genes in hundreds of thousands of cells, including point mutations, fusions transcripts and gene expression level. These molecular tools can complement genome-wide transcriptome analyses adding spatial resolution to the molecular information. This level of resolution is important for the understanding of many biological processes and potentially relevant for the clinical management of cancer patients. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Screening of a deeply sequenced transcriptome using Illumina sequencing as well as the genome of the tardigrade Hypsibius dujardini revealed a set of five opsin genes. To clarify the phylogenetic position of these genes and to elucidate the evolutionary history of opsins in Panarthropoda (Onychophora + Tardigrada + Arthropoda), we reconstructed the phylogeny of broadly sampled metazoan opsin genes using maximum likelihood and Bayesian inference methods in conjunction with carefully selected substitution models. According to our findings, the opsin repertoire of H. dujardini comprises representatives of all three major bilaterian opsin clades, including one r-opsin, three c-opsins, and a Group 4 opsin (neuropsin/opsin-5). The identification of the tardigrade ortholog of neuropsin/opsin-5 is the first record of this opsin type in a protostome, but our screening of available metazoan genomes revealed that it is also present in other protostomes. Our opsin phylogeny further suggests that two r-opsins, including an "arthropsin", were present in the last common ancestor of Panarthropoda. While both r-opsin lineages were retained in Onychophora and Arthropoda, the "arthropsin" was lost in Tardigrada. The single (most likely visual) r-opsin found in H. dujardini supports the hypothesis of monochromatic vision in the panarthropod ancestor, whereas two duplications of the ancestral panarthropod c-opsin have led to three c-opsins in tardigrades. Although the early-branching nodes are unstable within the metazoans, our findings suggest that the last common ancestor of Bilateria possessed six opsins: two r-opsins, one c-opsin, and three Group 4 opsins, one of which (Go opsin) was lost in the ecdysozoan lineage.

Die bodenbürtigen, pflanzenpathogenen Pilze der Verticillium Familie sind über die ganze Welt verteilt. Bislang gibt es keine Fungiszide, die einen Befall der Wirtspflanzen verhindern können. Auf infizierten Feldern ist dem Pilz für viele Jahre ein Überleben im Boden in sogenannten Dauerformen (Mikrosklerotien, melanisierte Hyphen) möglich. Auch nach langer Zeit können diese auskeimen und der Pilz dann die Wirtspflanzen befallen, was ihn zu einem wirtschaftlichen Problem macht. Die Infektion der Wirtspflanzen erfolgt durch die Wurzeln; und nachdem der Pilz in die Xylemgefäße der Pflanze eingedrungen ist, wächst er innerhalb dieser vasculären Leitungsbahnen. Die infizierten Pflanzen zeigen Infektionssymptome wie frühzeitige Chlorose der Blätter, absterbendes Gewebe sowie verfärbte Gefäße in Stamm und Wurzel. Für die Landwirtschaft ist die Erforschung der Interaktionen zwischen Pflanzen und dem pathogenene Pliz von großer Bedeutung um mögliche Angriffspunkte zur Pilzbekämpfung zu finden. Diese Arbeit fokussiert sich auf zwei spezifische Gene in Verticillium dahliae, die für die Pathogenität des Pilzes eine Rolle spielen. Des Weiteren wurde die Gen­Regulation in Verticillium longisporum durch Analyse eines Transkriptoms untersucht. Die Studie hat zum Ziel die hohe Anpassung des Pilzes an die Bedingungen innerhalb des vaskulären Systems der Wirtspflanze zu zeigen, in dem dem saprophytischen Pilz nur eine limitierten Menge an Nährstoffe zugänglich ist. Das untersuchte V. dahliae Protein VdThi4 ist Teil der Thiamin Biosynthese (Vitamin B1). Dieses Mitochondriell lokalisierte Protein ist mit seinen weiteren Funtktionen in den DNA Reparaturmechanismus und die zelluläre Antwort auf oxidativen Stress (induziert durch ROS) eingebunden. Das Fehlen des Proteins führt im Pilz zum Verlust der pathogenen Eigenschaften auf der Wirtspflanze Solanum lycopersicum. Neben Proteinen wichtiger Stoffwechselwege spielen sekretierte Proteine eine große Rolle bei der Wirtsinfektion. Solche Proteine sind der Erste Kontakt zwischen Pathogen und Wirt und werden von beiden Seiten abgesondert. Infektionsversuche an S. lycopersicum zeigten, dass der Pilz das membrangebundene V. dahliae Protein VdPa14_2 für die Wirtspflanzeninfektion benötigt. Dieses Protein scheint die Resistenz gegen oxidativen Stress in der Zelle zu verringern in der Synthese schwarzen Melanins involviert zu sein. Für die Infektion von Wirtspflanzen und die Pathogenität benötigt V. dahliae viele verschiedene Proteine aus verschiedenen Stoffwechelwegen, wie die analysierten Proteine VdThi4 und VdPa14_2, die in der Zelle verschiedene Funktionen haben, aber beide für die Pathogenität des Pilzes benötigt werden. Um ein umfassenderes Bild der Interaktion zwischen Pilz und Pflanze zu bekommen, wurde die Genregulation in V. longisporum untersucht. Hierfür wurde ein Transkriptom erstellt. Es wurden sowohl die spezifisch- als auch die gleich-regulierten Transkripte während der in situ Kultivierung des Pilzes in Xylem­Saft aus Brassica napus, sowie der in vitro Kultivierung in simuliertem Xylem medium (SXM) untersucht. Beide Medien stellten sich während der Analyse als grundverschieden heraus. Das SXM stellt hingegen früherer Annahmen keine Simmulation der in vivo Konditionen innerhalb des vaskulären Systems einer Pflanze dar.

Malaria is a pernicious disease that has greatly impacted and continues to affect the human population. While much research has been performed to understand the underlying nature of this disease, gaps in the knowledge-base persist. In order to address these deficiencies, a multi-disciplinary, multi-institutional project has been funded to study the systems biology of the host pathogen interaction during malaria infection in both humans and non-human primates. In the course of investigating the transcriptome during two 100-day experiments in Macaca mulatta, this work elucidated many of the underlying molecular pathways of the host and parasite that are affected by antimalarial drugs, as well as through host-pathogen interactions. The malaria-disease-related host pathways are related to, not surprisingly, immune-associated signalling and hematopoesis, and the altered parasite pathways demonstrate an association between disease severity and parasite life stage abundance. Continuing integration of this research with other data-types collected during the course of these experiments will improve our understanding of malaria systems biology and improve targeted malaria therapies.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2010. / Includes bibliography. / Title page: Dept. of Genetics, Faculty of Science / ENGLISH ABSTRACT: Recent advances in medical pharmacology have identified the immune-potentiating effects of β-1,3-glucans on mammalian immune systems. Extensive research has identified and described the mechanisms of action and receptor binding specificity of different β-1,3-glucans as well as their structural and functional relationships. Molecular mass, solubility, structural order, degree of branching as well as chemical modification all determine the effectiveness of the β-1,3-glucan immune-modulating activities, which typically include; macrophage activation, antibody adjuvant activities, reduction of LDL-cholesterol, leukocyte mitogenic activities, cytokine and chemokine production as well as antiviral and antitumor activities. Currently β-1,3-glucans have been sold commercially under the name β-glucan, mostly in the form of Betafectin, a genetically modified yeast derived β-1,3-glucan. Recent studies of different β-1,3-glucans have identified the pharmacological activities of paramylon, a Euglena derived β-1,3-glucan. Although paramylon has relatively low immune-stimulating activities, chemical modification of the paramylon granule increased immune-potentiation with specific antimicrobial and anti-HIV activities. Due to these specific immune-potentiating activities, paramylon is novel in terms of both structure as well as functional activity. In terms of biotechnological application, paramylon is greatly favoured as it is synthesized as an insoluble membrane bound granule in the cytosol of Euglena where most plant and fungal β-1,3-glucan synthases are cell membrane bound highly regulated multifunctional complexes, synthesizing β-1,3-glucan as cell wall components. Due to the novel granular nature of paramylon, expression in other systems with genetic modification could potentially further increase immuno-potentiating activities. In this study, different approaches were attempted in order to identify the genes involved in paramylon synthesis including; constructing and screening a Euglena gracilis cDNA library, sequence analysis of the purified proteins as well as transcription analysis of the sequenced transcriptome and genome of E. gracilis. Putative candidates that encode subunits of the paramylon synthase complex have been identified. / AFRIKAANSE OPSOMMING: Onlangse vordering in mediese farmakologie het die immuun-stimulerende effek van β-1,3-glukane op die soogdier immuunsisteem geïdentifiseer. Intensiewe navorsing het die meganisme van die werking en reseptor bindingspesifisiteit van verskillende β-1,3-glukane, asook hulle struktuur en funksionele verhoudings, geïdentifiseer. Die molekulêre massa, oplosbaarheid, strukturele orientasie, mate van vertakking asook chemiese modifikasies bepaal almal die effektiwiteit van die β-1,3-glukaan immuun-modulerende aktiwiteite. Tipiese immuno-moduleringsaktiwiteite sluit makrofaag aktivering, teenliggaampie adjuvant aktiwiteite, verlaging van LDL-cholesterol, leukosiet mitogeniese aktiwiteite, sitokien en chemokien produksie asook anti-virale en antitumor aktiwiteite in. Huidiglik word β-1,3-glukane onder die naam β-glukaan verkoop meestal in die vorm van Betafectin, ‘n geneties gemodifiseerde gis wat van β-1,3-glukaan afkomstig is. Onlangse studies van verskillende β-1,3-glukane het die farmakologiese aktiwiteit van paramylon, ‘n Euglena afkomstige β-1,3-glukaan geïdentifiseer. Alhoewel paramylon relatiewe lae immuun-stimulerende aktiweite toon, verhoog chemiese modifikasies van die paramylon granules immuun-stimulering, spesifiek die anti-mikrobiese en anti-MIV aktiwiteite. Weens hierdie spesifieke immuun-stimulerende aktiweite, word paramylon as nuut beskou veral in terme van beide struktuur asook funksionele aktiwiteit. In terme van biotegnologiese toepassing, verkry paramylon voorkeur aangesien dit as ‘n onoplosbare membraangebonde granule in die sitosol van Euglena gesintetiseer word terwyl meeste plant en fungus β-1,3-glukaan sintases hoogs gereguleerde multifunksionele selmembraan gebonde komplekse is wat β-1,3-glukaan asook ander selwand komponente sintetiseer. Weens die unieke granulêre natuur van paramylon, sal uitdrukking in ander sisteme ‘n moontlike industrie skep waar deur die transgeniese uitdrukking van granulêre paramylon verdere verbetering van die immuun-stimulerings aktiwiteite kan lei. In hierdie studie is verskillende benaderings aangewend om die gene wat by paramylon sintese betrokke is te identifiseer, dit sluit in die konstruksie en sifting van ‘n E. gracilis cDNS biblioteek, aminosuur volgorde analise van gesuiwerde proteiene asook die transkripsionele analise van die volgorde van die transkriptoom en genoom van E. gracilis. Moontelike kandidate wat vir die subeenhede van die paramylon syntase kompleks kodeer is geïdentifiseer.

More than a decade after the completion of the human genome project, the function of a large number of genes remains to be elucidated. Forward and reverse genetic approaches have proven to be powerful tools to study gene function and have provided insights into fundamental biological processes. Furthermore, functional genetic screening can lead to a better understanding of the action of endogenous and exogenous stimuli such as hormones or drugs on biological systems. Thus far, systematic and unbiased studies have largely been limited to model organisms. However, complex disease-relevant genotypes and phenotypes cannot be studied in entirety in lower organisms creating a need for systematic approaches in human cells. This thesis describes a series of studies using forward and reverse genetic approaches combined with state-of-the-art technology in haploid human cells. The first chapter describes the development of a quantitative phenotypic read-out using a novel application of RNA-sequencing that allows the functional annotation of genes in signalling pathways. The presented data demonstrate that the employed shallow RNA-sequencing method is scalable and suitable as a read-out for reverse genetic screening. The second chapter focuses on the implementation of this method in a large reverse genetic study in human cells to functionally annotate tyrosine kinases in signalling pathways upon stimulation with a set of ten polypeptides and small molecules. The screens revealed known and unexpected interactions between different signalling molecules and pathways, validating the technical approach in a biological context. The third chapter presents a pilot study describing the set-up of a forward genetic technique for compound mode-of-action screening using a pooled human mutant cell line collection. The chemical genetic approach displayed sufficient sensitivity and allowed to monitor thousands of gene-drug interactions simultaneously. Together, this thesis combines elements to advance technological and biological aspects of functional genomics and chemical genetics.

Mycoplasma hyopneumoniae, Mycoplasma flocculare e Mycoplasma hyorhinis são capazes de aderir e colonizar o trato respiratório de suínos. Enquanto a presença de M. flocculare é considerada assintomática, M. hyopneumoniae e M. hyorhynis são relacionados ao desenvolvimento de patologias. M. hyopneumoniae é o agente etiológico da pneumonia enzoótica suína e M. hyorhynis além dos pulmões pode atingir outros sítios e hospedeiros, estando relacionado a artrites, poliserosites e desenvolvimento de vários tipos de câncer em humanos. Apesar dos avanços tecnológicos na área de genômica, raros são os dados quanto ao papel de M. flocculare no trato respiratório suíno. Além do mais, informações relativas à transcrição gênica nessas espécies são escassas, apesar da importância desses microrganismos. Neste estudo são apresentados os dados da sequência do genoma de uma linhagem de M. flocculare, bem como do genoma de um novo isolado de M. hyopneumoniae. Com estas novas sequências foram realizadas análises de genômica comparativa visando a identificação de características que pudessem explicar os diferentes comportamentos quanto à patogenicidade dessas espécies. Além disso, a análise global dos transcritomas de cada uma das espécies foi realizada e o perfil transcricional entre M. hyopneumoniae, M. flocculare e M. hyorhynis foi analisado comparativamente objetivando identificar características peculiares para cada um dos mapas transcricionais, além de compreender a coordenação do modo de transcrição gênica em Mycoplasma. De um modo geral, as três espécies de Mycoplasma que habitam o trato respiratório suíno possuem grandes semelhanças na composição gênica, assim como na abundância de transcritos. A análise do repertório transcricional, mostra que os genomas são transcritos quase que em sua totalidade, incluindo as regiões intergênicas, nas três espécies. M. hyopneumoniae e M. flocculare apresentam conteúdo gênico e perfil transcricional muito semelhantes. Uma importante diferença encontrada entre estas duas espécies refere-se à presença exclusiva de genes e transcritos de adesinas específicas. M. hyorhynis possui genes e transcritos exclusivos, os quais sabidamente estão relacionados à sua capacidade mutacional, de invasividade e infecção de diferentes sítios. Por fim, a análise comparativa dos genomas, e a obtenção dos mapas transcricionais para M. hyopneumoniae, M. flocculare e M. hyorhynis, foram abordagens que resultaram em um grande número de informações, as quais são importantes para embasamento de futuros estudos de caracterização dos mecanismos moleculares, como os eventos de regulação da transcrição gênica, no gênero Mycoplasma. / Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and Mycoplasma flocculare are able to adhere and to colonize the swine respiratory tract. While M. flocculare presence is virtually assymptomatic, M. hyopneumoniae and M. hyorhynis infections may cause respiratory disease. M. hyopneumoniae is the causative agent of swine enzootic pneumonia and M. hyorhynis may affect the lungs and other sites in a diversity of hosts and has been related to arthritis, poliserosites and to the development of several types of human cancer. Despite genomics technological advances, there are very few data about the possible role of M. flocculare in the swine respiratory tract. Moreover, little information about gene transcription is available in these species, despite the importance of these microorganisms. In this work the genome sequences of M. flocculare and a new isolate of M. hyopneumoniae are presented. A comparative genomic analyzes was performed to identify possible characteristics that may help to explain the different behaviors of these species in the swine respiratory tracts. Furthermore, a transcriptome map of each species was performed and a comparative transcriptional profile analysis between M. hyopneumoniae, M. flocculare and M. hyorhynis was undertaken to identify the exclusive features for each of the transcriptional maps, in addition to understanding the coordination mode of gene transcription in Mycoplasma. In general, the three Mycoplasma species that inhabit the swine respiratory tract have a similar gene composition as well as the abundance of transcripts. The transcriptome maps showed that most of the predicted genes are transcribed from these Mycoplasma genomes, as well as some intergenic regions. M. hyopneumoniae and M. flocculare present very similar gene content and transcriptional profile. However, an important difference between these two species is related to the exclusive presence of genes and transcripts of some specific adhesins. M. hyorhynis presents exclusive genes and transcripts that have been related to its invasiveness, mutation rate and infection of different sites. Finally, the comparative analysis of the genomes and transcriptional maps between M. hyopneumoniae, M. flocculare and M. hyorhynis have resulted in a large amount of information, which are important for future studies of the molecular characterization, as transcriptional regulation in the Mycoplasma spp.