Parasitoid wasps are hyperdiverse, with current estimates suggesting that they may account for up to 20% of all insect species. Though their ecological significance and their importance in integrated pest management cannot be denied, these taxa remain understudied and, due to their small size, are often overlooked. However, recent advances in molecular techniques are helping to reverse this trend by providing tools which scientists can use to better understand species limits and host interactions. Parasitoid wasps are often morphologically cryptic and their accurate delimitation requires the analysis of DNA sequence data from fast-evolving genes in addition to morphological characters. The research presented here demonstrates the utility of a new molecular locus in species delimitation. Also, a morphological key to the species of a genus occurring in America, north of Mexico is presented. The interactions between parasitoid wasps and their hosts are highly complex. On the wasp side, it involves the production venom, which likely contains bountiful natural resources. In this study, the venom proteins of wasps of the genus Megarhyssa (Hymenoptera: Ichneumonidae) are identified. Putative functions are assigned to these proteins and possible applications are discussed. One of the proteins identified is the enzyme, laccase, which is associated with the degradation and digestion of wood. The sequence of the gene coding for this laccase was analyzed and used to create recombinant proteins in a baculovirus-insect cell expression system. Future work investigating this enzyme is necessary to determine its activity against the plant cell wall. The research presented here provides insight into the identification and venom composition of ichneumonid wasps. The results contribute to our knowledge of this understudied taxon and indicate that there is much to be gained from further research in this field which will become increasingly practicable as molecular techniques advance and become more affordable.

Thesis (PhD (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2007. / One of the aims of direct observed therapy strategy implemented by the World Health Organization was to prevent the development of drug resistant tuberculosis. However, in recent years a dramatic increase and spread in multidrug resistant tuberculosis has been observed. In this study, a molecular epidemiological approach was used to understand and rapidly detect drug resistance in high incidence tuberculosis communities of the Western Cape, South Africa. Previous studies showed that, drug resistant tuberculosis occurs as a result of spontaneous mutations in particular genes. Using molecular techniques, we developed an algorithm to rapidly detect isoniazid, rifampicin and ethambutol drug resistance in tuberculosis patients from a short term mini culture. Rapid detection of drug resistance is important to prevent future transmission events. In addition, accurate ethambutol resistance testing is of particular importance, since treatment of patients infected with multidrug resistant strains with second line anti-tuberculosis drugs depend on the ethambutol test results. In a comprehensive study, we found that the algorithm performs well when compared to the traditional culture method currently used by the routine laboratories. However, the results showed that more then 90 % of ethambutol resistance is missed by the routine laboratories. This has important implications for the tuberculosis control program, since patients infected with the drug resistant strain may be on inappropriate treatment. In this study, we found that certain strains have a selective advantage to become drug resistant and transmit and this implies that they are more virulent and fit than other strains. This observation has also been made for strains within the same genotype family. The more transmissible drug resistant strains cause large drug resistant outbreaks. This study highlights the complexity of the drug resistant epidemic, and confirms that it is a major problem in local communities. Application of molecular methods has provided us with tools to study how resistance might develop. We have demonstrated how we made use of a newly developed method to detect a multidrug resistant outbreak in the study community. The applications of transcriptomics identified several genes that might play a role in isoniazid resistance. Using this data a model was proposed whereby isoniazid resistant strains can compensate for the toxic effect of the drug. Application of comparative genomics by whole genome sequencing will be used to assist us in the further understanding of the mechanisms of drug resistance. This study also conclude that we should continue in our attempts to develop faster diagnostics for both first and second line drugs and that we must not loose site that all of this research must in the end benefit the patients.

Social stress is the most common stressor experienced by humans and exposure to social stress is thought to cause or exacerbate neuropsychiatric illness. Social stress also leads to behavioral and physiological responses in many animal models that closely mirror the symptoms of fear and anxiety in humans. Our laboratory uses Syrian hamsters to study behavioral responses to social stress. Hamsters are highly territorial, but after losing an agonistic encounter, hamsters exhibit a striking behavioral change, abandoning all territorial aggression and instead becoming highly submissive. This behavioral shift is termed conditioned defeat. Epigenetic modifications, such as changes in histone acetylation, are a possible molecular mechanism underlying such behavioral shifts. Histone deacetylase (HDAC) inhibitors have been shown to enhance fear learning and conditioned place preference for drugs of abuse, while suppressing histone acetylation with histone acetyltransferase (HAT) inhibitors impairs long-term memory formation. The first goal of this study was to test the hypothesis that histone acetylation is a molecular mechanism underlying conditioned defeat. We found that animals given an HDAC inhibitor systemically before social defeat later exhibited increased conditioned defeat. This treatment also suppressed defeat-induced immediate-early gene activity in the infralimbic cortex but not the basolateral amygdala. Next, we demonstrated that administration of an HDAC inhibitor in the infralimbic cortex before defeat enhanced stress-induced behavioral responses while HAT inhibition blocked these behavioral changes. Although both males and females exhibit conditioned defeat, the behavioral expression is more pronounced in males. We next used transcriptomic analysis to investigate potential genetic mechanisms leading to this sexually dimorphic expression and to further delineate the role of acetylation in stress-induced behavioral changes. We sequenced the whole brain transcriptome of male and female hamsters as well as the transcriptome of basolateral amygdala, a nucleus necessary for the acquisition and expression of conditioned defeat, of dominant, subordinate, and control animals. Our analysis revealed that numerous genes relating to histone acetylation, including several HDACs, were differentially expressed in animals of different social status and between sexes. Together, these data support the hypotheses that histone modifications underlie behavioral responses to social stress and that some of these modifications are sexually dimorphic.

Cellular life is highly complex. In order to expand our understanding of the workings of human cells, in particular in the context of health and disease, detailed knowledge about the underlying molecular systems is needed. The unifying theme of this thesis concerns the use of data derived from sequencing of RNA, both within the field of transcriptomics itself and as a guide for further studies at the level of protein expression. In paper I, we showed that publicly available RNA-seq datasets are consistent across different studies, requiring only light processing for the data to cluster according to biological, rather than technical characteristics. This suggests that RNA-seq has developed into a reliable and highly reproducible technology, and that the increasing amount of publicly available RNA-seq data constitutes a valuable resource for meta-analyses. In paper II, we explored the ability to extrapolate protein concentrations by the use of RNA expression levels. We showed that mRNA and corresponding steady-state protein concentrations correlate well by introducing a gene-specific RNA-to-protein conversion factor that is stable across various cell types and tissues. The results from this study indicate the utility of RNA-seq also within the field of proteomics. The second part of the thesis starts with a paper in which we used transcriptomics to guide subsequent protein studies of the molecular mechanisms underlying malignant transformation. In paper III, we applied a transcriptomics approach to a cell model for defined steps of malignant transformation, and identified several genes with interesting expression patterns whose corresponding proteins were further analyzed with subcellular spatial resolution. Several of these proteins were further studied in clinical tumor samples, confirming that this cell model provides a relevant system for studying cancer mechanisms. In paper IV, we continued to explore the transcriptional landscape in the same cell model under moderate hypoxic conditions. To conclude, this thesis demonstrates the usefulness of RNA-seq data, from a transcriptomics perspective and beyond; to guide in analyses of protein expression, with the ultimate goal to unravel the complexity of the human cell, from a holistic point of view. / <p>QC 20161121</p>

No description available.

High-throughput sequencing has greatly influenced the amount of data produced and biological questions asked and answered. Sequencing approaches have also enabled rapid development of related technological fields such as single-cell and spatially resolved expression profiling. The introductory parts of this thesis give an overview of the basic molecular and technological apparatus needed to analyse the transcriptome in cells and tissues. This is succeeded by a summary of present investigations that report recent advancements in RNA profiling. RNA integrity needs to be preserved for accurate gene expression analysis. A method providing a low-cost alternative for RNA preservation was reported. Namely, a low concentration of buffered formaldehyde was used for fixation of human cell lines and peripheral blood cells (Paper I). The results from bulk RNA sequencing confirmed gene expression was not negatively impacted with the preservation procedure (r2&gt;0.88) and that long-term storage of such samples was possible (r2=0.95). However, it is important to note that a small population of cells overexpressing a limited amount of genes can skew bulk gene expression analyses making them sufficient only in carefully designed studies. Therefore, gene expression should be investigated at the single cell resolution when possible. A method for high-throughput single cell expression profiling termed microarrayed single-cell sequencing was developed (Paper II). The method incorporated fluorescence-activated cell sorting, sample deposition and profiling of thousands of barcoded single cells in one reaction. After sample attachment to a barcoded array, a high-resolution image was taken which linked the position of each array barcode sequence to each individual deposited cell. The cDNA synthesis efficiency was estimated at 17.3% while detecting 27,427 transcripts per cell on average. Additionally, spatially resolved analysis is important in cell differentiation, organ development and pathological changes. Current methods are limited in terms of throughput, cost and time. For that reason, the spatial transcriptomics method was developed (Paper III). Here, the barcoded microarray was used to obtain spatially resolved expression profiles from tissue sections using the same imaging principle. The mouse olfactory bulb was profiled on a whole-transcriptome scale and the results showed that the expression correlated well (r2=0.94-0.97) as compared to bulk RNA sequencing. The method was 6.9% efficient, reported signal diffusion at ~2 μm and accurately deconvoluted layer-specific transcripts in an unbiased manner. Lastly, the spatial transcriptomics concept was applied to profile human breast tumours in three dimensions (Paper IV). Unbiased clustering revealed previously un-annotated regions and classified them as parts of the immune system, providing a detailed view into complex interactions and crosstalk in the whole tissue volume. Spatial tumour classification divulged that certain parts of the tumour clearly classified as other subtypes as compared to bulk analysis providing useful data for current practice diagnostics. The last part of the thesis discusses a look towards the future, how the presented methods could be used, improved upon or combined in translational research. / <p>QC 20170109</p>

<p>Lymphomas are a heterogeneous group of hematological malignancies composed of diseases with diverse molecular origins and clinical outcomes. Derived from immune cells of lymphoid origin, lymphoma can arise from lymphoid cells present anywhere in the body, from the spleen and lymph nodes to peripheral sites like the liver and intestines. Current strategies for lymphoma diagnosis involve primarily histopathological examinations of the tumor biopsy, including cytogenetics and immunophenotyping. As more data becomes available, diagnoses may increasingly depend on genomic features that define each disease. Classification of lymphoid neoplasms is generally based on the cell of origin, or the lineage of the normal cell that the cancer is thought to arise from. Lymphomas can be classified into dozens of distinct diagnostic entities, though any two patients with the same diagnosis may have very different outcomes and molecular underpinnings, so we need to understand both the commonalities of patients with the same disease and the unique features that may require personalized treatment strategies. Patient prognosis in lymphoma depends greatly on the type of lymphoma, ranging from nearly curable diseases with over 90% five-year survival rates, to most patients dying in the first year in the worse entities. Greater clarity is needed in the role of the underlying genomics that contribute to these variable treatment responses and clinical outcomes. </p><p>Next-generation sequencing approaches allow us to delve into the molecular underpinnings of lymphomas, in order to gain insight about the origin and evolution of these diseases. High-throughput sequencing protocols allow us to examine the whole genome, exome, epigenome, or transcriptome of cancer cells in tens to hundreds of patients for each disease. As cost of sequencing is reduced, and the ability to generate more data increases, we face increasing computational challenges to both process and interpret the wealth of data available in cancer genomics. Developing efficient and effective bioinformatics tools is necessary to transform billions of sequencing reads into actionable hypotheses on the role of certain genes or biological pathways in a specific cancer type or patient. </p><p>In this dissertation, I present several strategies and applications of integrative computational genomics in lymphoma, with contributions throughout the research process, from development of initial assays and quality control strategies for the sequencing data, to joint analysis of clinical and genomic data, and finally through follow-up experimental models for lymphoma. </p><p>First, I focus on two rare T cell lymphomas, hepatosplenic T cell lymphoma (HSTL) and enteropathy associated T cell lymphoma (EATL), which are both diseases with very poor clinical outcomes and a previous dearth of knowledge on the genetic basis of the diseases. We define the somatic mutation landscape of HSTL, through application of exome sequencing and find SETD2 to be the most highly mutated gene. We further utilize the exome sequencing data to investigate copy number alterations and show a significant survival difference between cases with and without certain arm-level copy number alterations. Knockdown of SETD2 in an HSTL cell line, followed by RNA sequencing, demonstrates the role of SETD2 loss in proliferation and cell cycle changes, linking the SETD2 mutations to a potential oncogenic mechanism. Furthermore, we investigate the potentially targetable mutations in the JAK-STAT pathway and demonstrate oncogenic downstream molecular phenotypes and potential druggability of these mutations. In the enteropathy associated T cell lymphoma study, we apply exome and RNA sequencing to a large EATL cohort. Our findings show a significant role for loss of function mutations in chromatin modifiers and JAK-STAT signaling genes. EATL can be separated into two subtypes, Type I and Type II, which we show to have convergent genomic features, in the face of divergent gene expression. RNA sequencing data defines a distinct separation between the two subtypes. Delving further into the role of SETD2 in these T cell lymphomas, we generate a mouse model with a conditional knockout of SETD2 in T cells and demonstrate a role for SETD2 in altering the lineage development of T cells. </p><p>To understand more about why certain genetic abnormalities are recurrent in some disease entities and not others, we turn to the cell of origin for clues. We pair two different lymphomas, Burkitt lymphoma and mantle cell lymphoma, with their associated cells of origin, germinal center B cells and naive B cells. These closely related cell types have much in common as B cells, but from studies of their transcriptomes, we know that there are many molecular differences that distinguish the two. In this work, after looking more closely at mantle cell lymphoma genomics, we look at the underlying chromatin markers that define the epigenomes of these B cells. We test the association between chromatin markers and mutation rates of genes between these two cell types and lymphomas, and find that genes with more open chromatin may have a higher mutation rate, when comparing closely related cells and lymphomas. Finally, I present my work on developing an RNA sequencing based strategy for defining the complete transcriptome of diffuse large B cell lymphoma (DLBCL). Gene expression profiling with microarray has shown the existence of two subtypes in DLBCL, activated B cell like (ABC) and germinal center B cell like (GCB). However, the role for non-coding RNAs, alternative splicing, and mutations, in these two subtypes and the larger group is previously not well understood. We develop a strand-specific RNA sequencing strategy that will allow the investigation of the total RNA transcriptome in DLBCL, including microRNAs, lncRNAs, and other important non-coding RNAs. Furthermore, we show that RNA sequencing can be used to distinguish the two subtypes, including through RNA sequencing based mutation calls, as well as through differentially expressed lncRNAs that we define for the first time in DLBCL.</p><p>Broadly, this dissertation contributes novel findings in the field of lymphoma genomics, as well as presenting a framework for computational integrative genomics that can guide future studies. The heterogeneity of lymphoma across cases requires us to dive deep into individual diseases, even rare ones, as well as appreciate the similarities and differences across lymphomas. To improve diagnoses, prognoses, and treatment options, we need to understand the molecular origins of lymphoma. Using a range of molecular and computational approaches, we can move closer to true personalized medicine at the genomic level.</p> / Dissertation

Rice blast disease is caused by the filamentous ascomycete fungus Magnaporthe oryzae and is the most destructive disease of cultivated rice. The pathogen elaborates a specialized infection structure called the appressorium. The morphological and physiological transitions that lead to appressorium formation of M. oryzae are stimulated through perception of environmental signals and are tightly regulated by cell cycle checkpoints. External stimuli are internalized by a variety of intracellular MAP kinase signaling pathways, and the major pathway regulating appressorium morphogenesis and plant infection is the Pmk1 MAP kinase signaling pathway. The central kinase, Pmk1, is required for appressorium morphogenesis and the homeobox and C2/H2 Zn-finger domain transcription factor, called Mst12, is required for appressorium formation and tissue invasion. The Mst12 null mutant is able to form melanised appressoria, but it is non-pathogenic. To understand the mechanism of appressorium morphogenesis and penetration peg formation, genome-wide comparative transcriptional profiling analysis was performed for the Δpmk1 and Δmst12 mutant using RNA-seq and HiSeq 2000 sequencing. This thesis reports the identification of gene sets regulated by the Pmk1 signalling pathway and defines the sub-set of these genes regulated by Mst12. I show that a hierarchy of transcription factors is likely to operate downstream of Pmk1 to regulate the main processes required for appressorium morphogenesis and plant infection. I also report the role of Mst12 in cytoskeletal re-organisation and show that it is necessary for septin-dependent F-actin polymerisation at the base on the appressorium prior to plant infection. This is consistent with the major transcriptional changes observed by RNA-seq. The thesis also reports experiments that strongly suggest that appressorium mediated plant penetration is regulated by an S-phase checkpoint which operates independently of the conventional DNA damage and repair response, and the Cds1 and Chk1 checkpoint kinases. Transcriptional profiling results are consistent with the S-phase checkpoint operating downstream of the Pmk1 MAP kinase signalling pathway. An integrated model for the operation of the Pmk1/Mst12 signalling pathways and the hierarchical control of appressorium morphogenesis in the rice blast fungus is presented.

La salinité du sol affecte la croissance des plantes glycophytes telle que Arabidopsis thaliana et le riz. Chez les plantes vasculaires, les racines sont composées de divers types de cellules organisées en cercles concentriques. Chaque type de cellules racinaires possède une fonction biologique spécifique et coordonnée avec les autres cellules qui composent cette même racine. Il est probable que la réponse des gènes au stress salin soit spécifique du type cellulaire, ce qui ne peut être révélé par des études à l'échelle de l'organe entier. Afin d'étudier les réponses spécifiques, notre approche a été de générer des profils de transcriptome pour deux types de cellules racinaires chez les plantes modèles, Arabidopsis et riz. Les deux types de cellules étudiées ont été choisis en raison de leur rôle possible soit dans le stockage du sodium dans les cellules corticales, soit dans son transport dans les cellules du péricycle chez Arabidopsis ou du cylindre central chez le riz. Des plantes exprimant la protéine fluorescente verte (GF) spécifiquement dans un type de cellule racinaire furent utilisées pour cette analyse. Les cellules ont donc pu être isolées chez le riz et Arabidopsis grâce à la technique de cytométrie en flux.L'analyse du transcriptome des cellules du péricycle et du cylindre central montrent que les cellules corticales sont plus réactives au stress salin et qu'une large majorité des gènes est sous–exprimée chez les deux plantes modèles. D'après les analyses d'expression des cellules du cortex d'Arabidopsis, trois voies métaboliques sont significativement sous-exprimées en réponse au stress salin: la voie de biosynthèse des phénylpropanoïdes, le transport de l'eau and le métabolisme secondaire. La régulation de gènes impliqués dans le transport de l'eau et des nutriments démontre l'importance des cellules corticales dans le mouvement des solutés. Chez le riz, les profils des deux types cellulaires étudiés révèlent une forte réaction de défense ; en effet le métabolisme protéique et la régulation de la transcription sont fortement sous-exprimés dans les cellules corticales alors que les cellules du cylindre central modifient et activent les gènes correspondant à divers catégories fonctionnelles telles que la réplication de l'ADN et le transport. Des gènes candidats ont été sélectionnés dans les deux types cellulaires d'Arabidopsis. Des lignées mutantes n'exprimant pas ces gènes ont été testées en stress salin dans des conditions hydroponiques. Les résultats ont révélé un phénotype accumulant moins de sodium dans les parties aériennes (20% par rapport au génotype sauvage) pour certaines de ces lignées mutantes. Ce travail est la première étude de transcriptome utilisant des types spécifiques de cellules racinaires chez le riz. L'identification de gènes et voies métaboliques répondant au stress salin dans le cortex et le cylindre central de la racine ouvre de nouveaux axes de recherche et va permettre d'élucider la complexité des processus biologiques d'adaptation au stress salin. / Soil salinity reduces the growth of glycophytic plants such as Arabidopsis thaliana and rice. In vascular plants, roots are organized into concentric layers of cells and each layer has a specific biological function coordinated with other cell types in the root. Therefore, genes differentially expressed in response to a salt stress are also likely to be changing only in specific cell types, and thus may not be revealed at the organ level. In order to identify novel salt-responsive genes, cell-type specific transcriptomic approaches were undertaken in Arabidopsis thaliana and rice, with application of physiologically reasonable salt stress (50mM) over 48 hours. Two cell-types from the root were chosen in both species for their potential role in salt storage and transport: cortical and pericycle/stelar cells respectively. Cell-types of interest expressing specifically Green Fluorescent Protein (GFP) were isolated from the rest of the root using fluorescence-activated cell sorting (FACS).The outer layer of the root was found to be responding more than the inner part of the root after 48 hours of salt stress, with an overall down-regulation in both rice and Arabidopsis. Arabidopsis cortical cells responding to salt seem to regulate the cell wall biosynthesis, which may modulate the shape of the cells or alter the apoplastic movements of solutes in response to salt. Genes related to transport were affected by salt in Arabidopsis, with the crucial role of cortical cells in the movement of solutes being evident. Rice cortical cells respond to salt by showing a more extreme defence reaction in changing the protein metabolism and the regulation of transcription. The response of the inner part of the rice root to 48 hours of mild salt stress showed up-regulation of genes implicated in broader functional categories. The biological relevance of genes revealed using cell-type specific transcriptomics was demonstrated in a salt assay using knock-out (KO) lines of candidate genes from both cell-types in Arabidopsis thaliana. Three KO mutant lines showed 20% reduction in shoot sodium after 5 weeks of salt stress and were also able to maintain a higher shoot dry weight. These transcriptomic studies of isolated stelar and cortical cells in response to a mild salt stress have revealed salt responsive genes and pathways, indicating new areas for further study, and contributing to our understanding of the complex responses of plants to their environment at the cellular level.

Doctor of Philosophy / Entomology / Yoonseong Park / Tick salivary secretion is crucial for survival and for successful feeding. Tick saliva includes excretory water/ions and bioactive components for compromising the hosts' immune responses, and provides a direct route for pathogen transmission. Control of the tick salivation involves autocrine/paracrine dopamine, the most potent stimulator of tick salivation. Our research group reported the presence of two dopamine receptors in the salivary glands of the blacklegged tick (Ixodes scapularis): dopamine receptor (D1) and invertebrate specific D1-like dopamine receptor (InvD1L). Dopamine-induced salivary secretion was orchestrated by two distinct physiological roles via activation of the two dopamine receptors (Chapter 2). Low concentration of dopamine activated D1 receptor on epithelial cells of salivary gland acini leading inward fluid transport. High concentration of dopamine activated InvD1L receptors on axonal projections innervating myoepithelial cells modulating pumping/gating actions for emptying luminal saliva into the main duct. Thus, ticks coordinated salivary secretion with duo dopamine receptors. Dopamine-mediated saliva production involves an important downstream component, Na/K-ATPase (Chapter 3). Na/K-ATPase was found in the epithelial cells of all types of acini. However, Na/K-ATPase had two different functions in salivary secretion in different acini: 1) dopamine-mediated production of primary saliva in distally located salivary gland acini type-2/- 3, and 2) dopamine-independent resorption in proximally located salivary gland acini type-1. Type-1 acini were also found to function in direct water absorption of off-host ticks, which could be a potential route for delivery of acaricides. Chapter 4 investigated the comparative transcriptomics of the lone star tick underlying the processes of pathogen acquisition. Differential expression analyses in pathogen-exposed ticks revealed a number of transcripts that are important in the tick-pathogen interaction. These included genes for tick immunity against pathogen and for modulation of tick physiology facilitating a pathogen’s invasion and proliferation. My study expanded the understanding of physiological mechanisms controlling tick salivation. In addition, transcriptomics of ticks in interaction with pathogen identified several genes that are relevant in vector/pathogen interactions. The knowledge obtained in my study will facilitate to the development of novel methods for the disruption of tick feeding and pathogen transmission.