La protéine DnaA, ou un homologue, est présente dans la plupart des organismes vivants parce qu'elle joue un rôle clé pour la réplication de l'ADN. Dans Escherichia coli, l'expression de DnaA, l'initiateur central de la réplication de l'ADN, est donc étroitement régulée. Des études antérieures ont montré qu'une forte surexpression de cette protéine conduit à une diminution de la viabilité cellulaire, alors que son absence induit l'arrêt de la division cellulaire. Même si ces conditions extrêmes sont bien étudiées, la transition entre l'arrêt de la division et la croissance normale n'a pas été analysée quantitativement.Nous avons modifié génétiquement Escherichia coli pour mettre l'expression de l'ARN polymérase et de DnaA sous le contrôle deux systèmes inductibles distincts. Pour contrôler l’expression de DnaA, nous avons utilisé un système d’induction se trouvant déjà dans la cellule, le système uhp. Le promoteur du gène uhpT est induit par le glucose-6-phosphate extracellulaire. Nous avons tout d’abord étudié les caractéristiques d’induction de ce système et ensuite caractérisé les phénomènes biologiques déclenchées par les variations de la concentration de DnaA. Les méthodes utilisées combinent des mesures sur une population des bactéries, avec celles en cellule unique, en utilisant la microscopie in vivo en temps réel et des systèmes microfluidiques. Les expériences de microscopie révèlent des phénomènes stochastiques en raison du faible nombre de molécules des composants du système d'induction uhp. En corrélant les observations de population et de cellules uniques, nous donnons une interprétation quantitative du comportement observé. Comme une application potentielle de notre système de contrôle, nous envisageons la possibilité d’arrêter la division cellulaire afin de transformer la cellule en un «sac d'enzymes» pour la production biotechnologique de métabolites. / The DnaA protein, or a homologue, is present in most living organisms because it plays a key role for DNA replication. In Escherichia coli, the expression of DnaA, the central initiator of DNA replication, is therefore tightly regulated. Previous studies have shown that a large overexpression of this protein leads to a decrease in cell viability, while its absence induces the arrest of cell division. Even though these extreme conditions are well studied, the transition from division arrest to normal growth has not been quantitatively analyzed.We genetically engineered Escherichia coli to put the expression of RNA polymerase and the expression of DnaA under the control of two distinct, inducible systems. For the control of DnaA expression, we used a regulatory system already present in the cell, the uhp system. The promoter of the uhpT gene is induced via extracellular glucose-6-phosphate. We characterized the induction characteristics of this system and studied the biological phenomena triggered by varying concentrations of DnaA, using population measurements and single cell, time lapse microscopy of microcolonies or cells grown in a microfluidics device. The microscopy experiments reveal stochastic phenomena due to the low number of molecules of components of the induction system and of DnaA. Confronting population and single cell observations we are able to give a quantitative interpretation of the observed behavior. As a potential application of our control system, we explored the possibility of freezing cell division in order to turn the cell into a “bag of enzymes” for the biotechnological production of metabolites.

Chlamydomonas reinhardtii possesses a single-cell-based CO2-concentrating mechanism (CCM). The CCM is an important element of algal photosynthesis, metabolism, growth and biomass production, which works by increasing the concentration of inorganic carbon (Ci) in the pyrenoid, a dense RuBisCO-packed structure within the chloroplast. This suppresses RuBisCO oxygenase activity and associated photorespiration. The enhanced efficiency of CO2 assimilation in the pyrenoid via CCM had been modelled theoretically as a requirement for successful CCM in higher plant systems. The ultimate aim of my research is to understand the biogenesis of the pyrenoid using a set of CCM mutants with pyrenoidal defects. Immunofluorescence methods and spot growth tests under different CO2 concentrations were performed on mutants with CCM defects generated by an insertional mutagenesis screen. Morphological and physiological characterisation of these mutants revealed differences in the pyrenoid morphology, the ability for RuBisCO to aggregate into the pyrenoid and the formation of thylakoidal tubule network associated with the pyrenoid. The thylakoid tubule network may be linked to the transport of inorganic carbon into the pyrenoid as part of the CCM. Further characterisation of one of the mutants gave rise to the hypothesis that the gene of interest, Cre11.g467712 (SAGA), is a multi-functional anchor protein related to the structural formation of the pyrenoid and may be another essential component of the pyrenoid.

Recent advances in sequencing technology have given access to information extracted on a single cell level. Single cell RNA sequencing enables for transcriptomes to be sequenced, allowing for studies within and between cell types. A recently developed protocol, based on Smart-seq2, and the Proximity ligation essay, allows for the detection of protein data from single cells, in parallel with RNA. The combination of the transcriptomic and proteomic data will enhance researchers’ ability to explore cell states. In this study, we are comparing a new pulldown protocol with the widely-used Smart-seq2, as well as against FACS sorted cells. Our results show differences in the RNA sequenced between the two protocols, as well the prediction of cell cycle state based on their data. Using RNA extracted from the pulldown protocol in different time points, we also calculate the direction of development for the cells. We expect that the incorporation of proteomic data will shed light to relevant biological questions related to the cell function.

Vascular smooth muscle cell (VSMC) accumulation is a hallmark of atherosclerosis and vascular injury. However, fundamental aspects of proliferation and the phenotypic changes within individual VSMCs, which underlie vascular disease remain unresolved. In particular, it is not known if all VSMCs proliferate and display plasticity, or whether individual cells can switch to multiple phenotypes. To assess whether proliferation and plasticity in disease is a general characteristic of VSMCs or a feature of a subset of cells, multi-colour lineage labelling is used to demonstrate that VSMCs in injury-induced neointimal lesions and in atherosclerotic plaques are oligo-clonal, derived from few expanding cells, within mice. Lineage tracing also revealed that the progeny of individual VSMCs contribute to both alpha Smooth muscle actin (aSma)-positive fibrous cap and Mac-3-expressing macrophage-like plaque core cells. Co-staining for phenotypic markers further identified a double-positive aSma+ Mac3+ cell population, which is specific to VSMC-derived plaque cells. In contrast, VSMC-derived cells generating the neointima after vascular injury generally retained expression of VSMC markers and upregulation of Mac3 was less pronounced. Monochromatic regions in atherosclerotic plaques and injury-induced neointima did not contain VSMC-derived cells expressing a different fluorescent reporter protein, suggesting that proliferation-independent VSMC migration does not make a major contribution to VSMC accumulation in vascular disease. Similarly, VSMC proliferation was examined in an Angiotensin II perfusion model of aortic aneurysm in mice, oligo-clonal proliferation was observed in remodelling regions of the vasculature, however phenotypic changes were observed in a large proportion of VSMCs, suggesting that the majority of VSMCs have some potential to modulate their phenotype. To understand the mechanisms behind the inherent VSMC heterogeneity and observed functionality, the single cell transcriptomic techniques Smart-seq2 and the Chromium 10X system were optimized for use on VSMCs. The work within this thesis suggests that extensive proliferation of a low proportion of highly plastic VSMCs results in the observed VSMC accumulation after injury, and the atherosclerotic and aortic aneurysm models of cardiovascular disease.

DNA methylation is an important epigenetic mark spanning all of life's kingdoms. In humans, DNA methylation has been associated with a wide range of age-related pathologies, including type II diabetes and cancer. More recently, in humans, changes in DNA methylation at specific positions in the genome have been found to be predictive of chronological age. Interestingly, DNA methylation age is also predictive of health status and time-to-death. A better understanding of what these DNA methylation changes represent and whether they might be causative in the ageing process will be important to ascertain. However, at present there is no animal model system with which this process can be studied at a mechanistic level. Furthermore, it is becoming increasingly apparent that many disease states that increase in prevalence with age are not caused by all cells within the individual, but are often the result of changes to a subset of cells. This underscores the importance of studying these processes at the single cell level. The recent advances in single cell sequencing approaches now mean that we can study multiple layers of biology within the same single cell, such as the epigenome and the transcriptome (scM&T-Seq). Unfortunately, we are still only able to probe these important aspects of single cell biology in a static sense. This is a major limitation in the study of ageing because ageing and age-related disease processes are inherently dynamic. As such, it is incumbent upon us to develop approaches to assay single cell biology in a dynamic manner. 
In this thesis, I describe an epigenetic age predictor in the mouse. This predictor is tissue-independent and can accurately predict age (with an error of 3.33 weeks) and can record deviations in biological age upon interventions including ovariectomy and high fat diet both of which are known to reduce lifespan. Next, I describe the analysis of a homogeneous population of muscle satellite cells (MuSCs) that I have interrogated at the single cell level, using single cell combined transcriptome and methylome sequencing (scM&T-seq). I found that with age there was increased global transcriptional variability and increased feature-specific methylome variability. These findings explain the loss of functionality of these cells with age. Lastly, I describe two imaging approaches to study DNA methylation dynamically in single cells. Using these methods, I demonstrate that it is possible to accurately determine methylation status across a wide spectrum of global methylation levels and that by using such approaches novel information about dynamic methylation processes can be obtained. These methods represent the first to study DNA methylation dynamically.

Cryptosporidium is a protozoan parasite infecting the intestines of its hosts, leading to acute diarrheal disease. Out of 26 recognized species, 14 are known to infect humans. Of most importance, from a human perspective are Cryptosporidium parvum and Cryptosporidium hominis, of which the former is known to have zoonotic potential. Globally, cryptosporidiosis affect people with lowered immune status particularly hard; among children under five it is the most important parasitic cause of gastroenteritis. In the region of KwaZulu-Natal, on the east coast of South Africa, Cryptosporidium is considered endemic. Drinking water is frequently collected from river systems and as Cryptosporidium spp. can be transmitted via contaminated water, this may be one source of infection. Research on the species distribution is important for outbreak investigations and prevention efforts. In water and wastewater such speciation is commonly performed using immunomagnetic separation, an antibody dependent method. There is however a suspicion that these antibodies have less affinity to some species and hence contorts the detected species distribution. An alternative approach is therefore of interest.   In the present study, Cryptosporidium diversity in wastewater collected from four different wastewater treatment plants in KwaZulu-Natal, is evaluated with an optimized antibody-free workflow and a single cell platform. It was shown that the workflow is suitable for complex samples, such as wastewater. Furthermore, diversity was assessed with amplicon sequencing, revealing four different species and genotypes. Further modifications of the methods used could benefit the field of Cryptosporidium research, along with improving global health and preventing disease outbreaks.

There is a growing appreciation that eukaryotic genomes are transcribed into numerous, previously undetected - and thus uncharacterized regulatory long non-coding RNAs (lncRNAs). Recent studies are primarily focused on lncRNAs transcribed from intergenic regions and enhancers, leaving antisense lncRNAs the least studied group of lncRNAs. At the same time, antisense transcription occurs in up to 74 % of human gene loci, frequently - from the opposite strand of genes encoding proteins involved in regulation of transcription. Here, we identified HIPSTAR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), a novel conserved lncRNA that is transcribed antisense to the TFAP2A gene. Unlike previously reported antisense lncRNAs, HIPSTR expression does not correlate with the expression of its antisense counterpart. Although HIPSTAR and TFAP2A are co-expressed in in vitro derived neural crest and trophoblast cells, only HIPSTAR and not TFAP2A is specifically expressed in a subset of cells within 8-cell- and morula-stage human embryos. We show that, similar to HIPSTAR, in the individual cells of developing human embryos or of stable cell lines the expression of lncRNAs is more highly heterogeneous than the expression of mRNAs. Finally, we demonstrate that HIPSTAR depletion in HEK293 and H1BP, a human embryonic stem cell line, predominantly affects the expression levels of genes involved in early organismal development and cell differentiation. Together, we show that expression of HIPSTAR and hundreds other lncRNAs is highly heterogeneous in human embryos and cell lines. We use HIPSTAR to exemplify the functional relevance of lncRNAs with heterogeneous and developmental stage-specific expression patterns. / Tem sido cada vez mais reconhecido que a transcrição dos genomas eucarióticos produz múltiplos transcritos novos, anteriormente não detectados e ainda não caracterizados, sendo que a maioria é constituida de RNAs não-codificantes longos (lncRNAs) regulatórios. Estudos recentes estão focados principalmente nos lncRNAs transcritos de regiões intergênicas e enhancers; assim, o grupo dos lncRNAs antisenso permanece o menos estudado de todos. Ao mesmo tempo, a transcrição antisenso ocorre em até 74% dos loci de genes humanos, frequentemente - a partir da fita oposta de genes que codificam proteínas envolvidas na regulação da transcrição. No presente trabalho, nós identificamos HIPSTR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), um lncRNA novo conservado que é transcrito a partir da fita antisenso do gene TFAP2A. Ao contrário do anteriormente relatado para os lncRNAs antisenso, a expressão de HIPSTR não está correlacionada com a expressão do gene da fita oposta. HIPSTR e TFAP2A são co-expressos em células da crista neural e em trofoblastos derivadas in vitro, mas somente HIPSTR e não TFAP2A está especificamente expresso num subconjunto de células de embriões humanos nos estágios de 8-células e mórula. Mostramos que, semelhante a HIPSTR, a expressão de lncRNAs é mais altamente heterogênea que a expressão de mRNAs em células individuais de embriões humanos em desenvolvimento ou em linhagens estáveis de células. Finalmente, nós demonstramos que a depleção de HIPSTAR em células HEK293 e H1BP, uma linhagem de células tronco embrionárias humanas, afeta predominantemente os níveis de genes envolvidos no início do desenvolvimento do organismo e na diferenciação de células. No conjunto, nós mostramos que a expressão de HIPSTR e de centenas de outros lncRNAs é altamente heterogênea em embriões humanos e linhagens celulares. Usamos HIPSTR para exemplificar a relevância funcional de lncRNAs com padrões de expressão heterogêneos e estágio-de-desenvolvimento específicos.

Miniaturization of biological analysis is a trend in the field of biotechnology aiming to increase resolution and sensitivity in biological assays. Decreasing the reaction volumes to analyze fewer analytes in each reaction vessel enables the detection of rare analytes in a vast background of more common variants. Droplet microfluidics is a high throughput technology for the generation, manipulation and analysis of picoliter scale water droplets an in immiscible oil. The capacity for high throughput processing of discrete reaction vessels makes droplet microfluidics a valuable tool for miniaturization of biological analysis. In the first paper, detection methods compatible with droplet microfluidics was expanded to include SiNR FET sensors. An integrated droplet microfluidics SiNR FET sensor device capable of extracting droplet contents, transferring a train of droplets to the SiNR to measure pH was implemented and tested. In paper II, a workflow was developed for scalable and target flexible multiplex droplet PCR using fluorescently color-coded beads for target detection. The workflow was verified for concurrent detection of two microorganisms infecting poultry. The detection panel was increased to multiple targets in one assay by the use of target specific capture probes on color-coded detection beads.   In paper III, droplet microfluidics has been successfully applied to single cell processing, demonstrated in paper III, where reverse transcription was performed on 65000 individually encapsulated mammalian cells. cDNA yield was approximately equivalent for reactions performed in droplets and in microliter scale. This workflow was further developed in paper IV to perform reverse transcription PCR in microfluidic droplets for detection of exosomes based on 18S RNA content. The identification of single exosomes based on RNA content can be further developed to detect specific RNA biomarkers for disease diagnostics. Droplet microfluidics has great potential for increasing resolution in biological analysis and to become a standard tool in disease diagnostics and clinical research. / <p>QC 20171024</p>

The Central Dogma of molecular biology describes a framework for how genetic information is transferred in cells, placing RNA as a messenger between DNA and translated proteins. During the last years, interest in RNA research has grown tremendously due to the increasing understanding and recognition of the importance of RNA in regulation of gene expression, biochemical catalysis, and genome integrity surveillance. Most importantly, RNA content, unlike DNA, changes constantly, fine-tuning the cellular response to match the environmental conditions. There is a clear potential for RNA biomarkers, reflecting both the natural and pathological conditions in vivo. Various methods have been developed to study RNA, of which the most common tools and techniques are described in this thesis. Since many of these gold standard methods are based on detecting RNA derivative (cDNA), there is a wide scope for efficient alternative tools directly targeting RNA. In Paper I, the spatiotemporal expression of human adenovirus-5 mRNA in epithelial and blood cells infected with the virus has been studied. For this, padlock probes and rolling circle amplification (RCA) were used to visualize, quantify and analyse both viral and host cell cDNAs in different infection scenarios, at single cell level. In Paper II, direct RNA detection fidelity has been evaluated using padlock probes. A novel type of probe (iLock) that is activated on RNA via invasive cleavage mechanism, prior to RCA was developed in this approach. Using iLocks, a substantial improvement of direct RNA sensing fidelity has been observed. In Paper III, RNA modifications were introduced in otherwise DNA iLock probes to enhance the probes’ efficiency on miRNAs. Using chimeric iLock probes, multiplexed differentiation of conserved miRNA family members were performed with next- generation sequencing-by-ligation readout. Efficient replication of chimeric probes used in Paper III implies that the Phi29 DNA polymerase readily accepts RNA-containing circles as amplification substrates. In Paper IV, real-time RCA monitoring for measurement of amplification rates and analysis of amplification patterns of various RNA-containing circles was achieved. Moreover, the RCA products were sequenced as a proof for the reverse-transcriptase activity of the Phi29 DNA polymerase. This thesis effectively contributes to a better understanding of mechanisms influencing RNA detection with, but not limited to, padlock probes. It expands the available RNA analyses toolkit with novel strategies and solutions, which can be potentially adapted for RNA-focused research, in general and molecular diagnostics, in particular. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

Malgré les progrès réalisés dans le séquençage de l’ADN, nous n’avons pas encore compris comment le phénotype d’un organisme se rapporte au contenu de son génome. Cependant, il est devenu clair que l'impact des gènes dépend du contexte. La simple présence d'un gène dans un génome ne nous informe pas du moment où il est exprimé et des autres gènes qui y sont exprimés. Comprendre comment l'expression des gènes est régulée est un élément nécessaire pour comprendre comment les phénotypes émergent d'un génotype donné. Les facteurs de transcription, qui peuvent activer ou réprimer l'expression d'un gène, forment un réseau complexe d'interactions entre eux et leurs gènes ciblés. Ce réseau consiste en une hiérarchie de groupes de facteurs de transcription fortement liés, chacun lié à des processus cellulaires distincts. La structure de ce réseau de régulation transcriptionnelle est-elle significative pour la réponse transcriptionnelle d'une cellule? Ici, nous utilisons une protéine de liaison à l'ADN programmable appelée CRISPR (répétitions courtes palindromiques groupées régulièrement) pour perturber l'expression génique des régulateurs globaux au sein du réseau de régulation transcriptionnelle. Ces régulateurs mondiaux régulent de nombreux processus cellulaires distincts et ont de nombreuses cibles génétiques. Le système CRISPR nous permet de perturber ces régulateurs dans toutes les combinaisons possibles, y compris les perturbations d'ordre supérieur avec tous les régulateurs mondiaux potentiellement ciblés perturbés en même temps. Nous enregistrons ensuite à la fois le modèle d'expression du transciptome en utilisant le séquençage de l'ARN et l'adéquation de chaque souche. Nous trouvons que la structure du réseau de régulation augmente la dimensionnalité de la réponse transcriptionnelle plutôt que de la réduire. Cela se traduit par une épistasie importante au-delà des interactions par paires. Cela a des implications sur la façon dont ces réseaux évoluent. L'épistasie par paires que nous trouvons entre les facteurs de transcription globaux repose sur la présence ou l'absence d'autres perturbations. Cela implique que d'autres perturbations pourraient agir comme des mutations de potentialisation. Le nombre de voies d'évolution potentielles augmente avec les épistasies d'ordre élevé, même si cela ne nous dit rien sur la qualité de ces voies. Fait important, les répliques de cette thèse sont toujours en cours et les données présentées ici n’ont pas encore exclu les artefacts expérimentaux. / Despite advances in DNA sequencing, we have yet to understand how an organism’s phenotype relates to the contents of their genome. However it has become clear that the impact of genes are context dependant. The mere presence of a gene within a genome does not inform us of when it is expressed, and which other genes are expressed along with it. Understanding how gene expression is regulated is a necessary piece of understanding how phenotypes emerge from a given genotype. Transcription factors, which can activate or repress the expression of a gene, form a complex network of interactions between themselves and their targeted genes. This network consists of a hierarchy of groups of strongly connected transcription factors, each relating to distinct cellular processes. Is the structure of this transcriptional regulatory network significant to the transcriptional response of a cell? Here we use a programmable DNA binding protein called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) to perturb gene expression of global regulators within the transcriptional regulatory network. These global regulators are regulating many distinct cellular processes and have many genetic targets. The CRISPR system allows us to perturb these regulators in all possible combinations, including higher order perturbations with potentially all targeted global regulators perturbed at the same time. We then record both the expression pattern of the transciptome using RNA sequencing, and the fitness of each strain. We find that the structure of the regulatory network increases the dimensionality of the transcriptional response rather than reducing it. This results in significant high order epistasis beyond pair-wise interactions. This has implications for how these networks evolve. The pair-wise epistasis we find between global transcription factors rely on the presence or absence of other perturbations. This implies that other perturbations could act as potentiating mutations. The number of potential evolutionary paths increases with high order epistasis, although this alone tells us nothing about the quality of those paths. Importantly, the replicates for this thesis are still on-going and the data presented here has not yet excluded experimental artefacts.