Introduction. La prééclampsie (PE), l’accouchement prématurée (AP) et le retard de croissance intra-utérin (RCIU) sont des grands syndromes obstétricaux qui affectent 5 à 12 % des grossesses. Ces complications sont des facteurs de risque majeurs pour la santé à la fois de la mère et du fœtus, à court et à long terme, impactant ainsi les générations actuelles et à venir. L’inflammation joue un rôle central durant la grossesse, et sa modulation doit être minutieusement régulée afin de garantir le bon développement du placenta et du fœtus. Par conséquent, ces complications obstétricales sont indépendamment liées à l’inflammation et au dysfonctionnement du placenta. Malheureusement, les études qui investiguent ces pathologies ont concentré leurs efforts sur des compartiments individuels (maternels ou placentaires) au sein de conditions isolées, sans parvenir à les intégrer entre elles, empêchant ainsi leur comparaison et une compréhension globale. De plus, les complications associées à la grossesse dans la période post-partum, telles que la prééclampsie du post-partum (PPPE), ont été largement négligées en raison de la complexité des modèles d’étude et d’un manque de sensibilisation quant aux changements dynamiques post-partum. Ainsi, j’ai émis l’hypothèse qu’en intégrant différents compartiments au sein de pathologie distincte et en comparant systématiquement celles-ci, je serais en mesure de découvrir de nouvelles signatures spécifiques à chaque pathologie, tant dans les complications obstétricales bien étudiées que dans celles moins étudiées. Pour ce faire, mes objectifs étaient d’abord (1) d’obtenir une vision intégrée des contributions immunitaires des compartiments maternel et placentaire dans l’AP par rapport aux grossesses à terme sans complication. De plus, j’ai procédé à la (2) comparaison des placentas issus de pathologies cliniquement distinctes, mais se chevauchant afin d’étudier leurs profils moléculaires. Enfin, j’ai investigué (3) le profil immunitaire maternel dans la pathologie peu étudiée de la PPPE afin d’identifier d’éventuels mécanismes sous-jacents et de nouvelles cibles thérapeutiques. Méthodes. J’ai utilisé des techniques de séquençage d’ARN total (bulk RNAseq) et à cellules uniques (scRNAseq) pour étudier les profils moléculaires des tissus placentaires et des cellules immunitaires en circulation respectivement. Ceci a été effectué à partir de trois cohortes totalisant 174 femmes ayant accouché avec ou sans complications de la grossesse (CTL, PE, AP, RCIU, et PPPE). J’ai également analysé les profils inflammatoires dans la circulation maternelle et à l’interface materno-fœtale de ces patientes en utilisant des techniques de cytométrie de flux, d’ELISA, de multiplex, d’immunohistologie et de déconvolution cellulaire. Résultats. Les compartiments maternels et placentaires dans l’AP ont révélé une augmentation partagée de l’inflammation. L’analyse des transcriptomes placentaires révélait l’expression différentielle de gènes, à la fois connus et nouveaux, enrichis pour les voies inflammatoires, principalement en raison de l’AP spontanée. Cette inflammation était soutenue par les niveaux élevés de médiateurs inflammatoires dans le plasma, le placenta et les membranes fœtales des personnes atteintes de l’AP. Les modifications systémiques dans les médiateurs pourraient être liées aux altérations dans les proportions de cellules immunitaires, tandis que les changements dans les médiateurs locaux pourraient être associés aux lésions structurelles et inflammatoires accrues. L’analyse des placentas issus de complications cliniquement distinctes a confirmé l’existence d’une inflammation locale dans l’AP ainsi qu’un profil similaire dans le RCIU. En effet, nous avons observé que les signatures moléculaires étaient distinctes d’une pathologie à l’autre, mettant en évidence des différences entre la PE précoce et tardive, tout en montrant que la présence de co-pathologies, en particulier pour l’AP avec ou sans RCIU, impactait les transcriptomes placentaires. Nous avons également découvert de nouveaux changements dans la circulation maternelle qui suggèrent un affaiblissement des fonctions immunitaires dans la PPPE. Par ailleurs, nous avons constaté que la présence de cellules CD163+ à l’interface materno-fœtale avait une forte capacité à prédire quelles patientes développeraient une PPPE, ce qui offre des perspectives prometteuses en tant que biomarqueur non invasif et prédictif, malgré l’absence de placenta. Conclusions. L’inflammation est associée à la PE, à l’AP, et au RCIU, mais avec des profils placentaires distincts qui dépendent du caractère spontané ou iatrogène de la PTB ainsi que de la présence de co-pathologies, ce qui met en lumière leurs complexités sous-jacentes. Il existe une distinction claire entre les compartiments inflammatoires maternels et placentaires dans l’AP, ce qui suggère que le placenta pourrait jouer un rôle plus important. Nous apportons la preuve que ces pathologies ne sont pas seulement distinctes dans leurs manifestations cliniques, mais aussi dans leurs profils placentaires. Même lorsque le placenta n’est plus présent, nous pouvons l’exploiter pour comprendre les mécanismes sous-jacents, ainsi que de développer des stratégies pour identifier, diagnostiquer et guider les patientes pendant et dans les semaines qui suivent leur grossesse. / Introduction. Preeclampsia (PE), preterm birth (PTB), and intrauterine growth restriction (IUGR) are some of the Great Obstetrical Syndromes plaguing 5-12% of all human pregnancies. These pathologies are major risk factors for short- and long-term maternal and neonatal health outcomes, affecting current and future generations. A central component to pregnancy is inflammation, whose fluctuations must be properly orchestrated for the proper development of the placenta and the fetus. Consequently, these pregnancy complications have been independently linked to inflammation and placental dysfunction. Previous studies investigating these pathologies have focused on individual compartments (maternal or placental) within singular conditions and have failed to integrate them, preventing their comparison and a more complete understanding. Additionally, pregnancy-associated conditions in the postpartum period, such as postpartum preeclampsia (PPPE) have been largely neglected due to the complexity of study designs and a lack of awareness regarding postpartum dynamics. Thus, I hypothesized that by integrating different compartments within an individual pathology and by comparing between pathologies in a systematic manner, I would be able to uncover novel pathology-specific signatures in both well-studied and under-studied pregnancy complications. To accomplish this, my objectives were first (1) to obtain an integrated view of the immune contributions of both the maternal and placental compartments in PTB compared to uncomplicated term pregnancies, followed by (2) the comparison of placentas from clinically distinct but overlapping pathologies to address their molecular profiles. Lastly, I aimed to (3) investigate the maternal immune profile in the under-studied pathology of PPPE to identify possible underlying mechanisms and novel therapeutic targets. Methods. I leveraged bulk-RNA and single-cell sequencing techniques to investigate molecular profiles of placental tissues and circulating immune cells, respectively, on samples from 174 subjects delivering either with or without pregnancy complications (CTL, PE, PTB, IUGR, and PPPE). This was complimented by the analysis of the inflammatory profiles in maternal circulation and at the maternal-fetal interface of these patients using flow cytometry, ELISA, multiplex, immunohistology, and cellular deconvolution techniques. Results. The integration of maternal and placental compartments in PTB revealed a number of known and novel differentially expressed genes enriched for inflammatory pathways - driven largely by spontaneous PTB. This inflammation was supported by the increase in inflammatory mediators in the plasma, placenta, and fetal membranes of women with PTB. Systemic changes in mediators may be related to the dysregulated proportions of immune cells, whereas the changes in local mediators may be related to the increased structural and inflammatory lesions. Analysis of placentas from clinically distinct complications supported similar inflammatory profiles in PTB and IUGR. I observed distinct molecular signatures between the pathologies, highlighting distinctions between EOPE and LOPE while showing that co-occurring conditions, especially PTB with or without IUGR, significantly affected placental transcriptomes. Lastly, I discovered novel changes in maternal circulation suggestive of dampened immune function in PPPE, and found that although the placenta was no longer present, the presence of CD163+ cells at the interface had a strong ability to predict which patients would go on to develop PPPE and should be further investigated as a potential non-invasive and predictive biomarker. Conclusions. Inflammation is associated with PE, PTB, and IUGR, but with distinct placental profiles that are dependent on PTB etiology as well as the presence of co-pathologies, which sheds light on their underlying complexities. There is clear distinction between the maternal and placental inflammatory compartments in PTB suggesting the placenta may be a stronger contributor. We provide evidence that these pathologies are not only distinct in their clinical manifestations but also in their placental profiles, and that even once the placenta is no longer present, we can leverage it to understand underlying mechanisms, develop strategies to identify, diagnose and guide patients during and in the weeks following pregnancy.

<p dir="ltr">The genetic perturbations caused by spaceflight on biological systems tend to have a system-wide effect which is often difficult to deconvolute into individual signals with specific points of origin. Single cell multi-omic data can provide a profile of the perturbational effects but does not necessarily indicate the initial point of interference within a network. The objective of this project is to take advantage of large scale and genome-wide perturbational or Perturb-Seq datasets by using them to pre-train a generalist machine learning model that is capable of predicting the effects of unseen perturbations in new data. Perturb-Seq datasets are large libraries of single cell RNA sequencing data collected from CRISPR knock out screens in cell culture. The advent of generative machine learning algorithms, particularly transformers, make it an ideal time to re-assess large scale data libraries in order to grasp cell and even organism-wide genomic expression motifs. By tailoring an algorithm to learn the downstream effects of the genetic perturbations, we present a pre-trained generalist model capable of predicting the effects of multiple perturbations in combination, locating points of origin for perturbation in new datasets, predicting the effects of known perturbations in new datasets, and annotation of large-scale network motifs. We demonstrate the utility of this model by identifying key perturbational signatures in RNA sequencing data from spaceflown biological samples from the NASA Open Science Data Repository.</p>

Στην παρούσα διδακτορική διατριβή προτείνονται μεθοδολογίες υπολογιστικής νοημοσύνης για την επεξεργασία και ανάλυση δεδομένων γονιδιακής έκφρασης μικροσυστοιχιών cDNA. Πιο συγκεκριμένα, στο πρώτο σκέλος αναπτύσσονται δύο νέες προσεγγίσεις για την εύρωστη εκτίμηση και διόρθωση του θορύβου υποβάθρου: η διόρθωση υποβάθρου βάσει εκατοστημορίων και η διόρθωση υποβάθρου βάσει παλινδρόμησης loess. Οι προσεγγίσεις αυτές καινοτομούν κυρίως στο ότι χρησιμοποιούν μία εύρωστη εκτίμηση του θορύβου υποβάθρου, γεγονός που τις καθιστά ιδανικές σε περιπτώσεις, όπου τα δεδομένα είναι θορυβώδη. Επιπροσθέτως, αναπτύσσεται ένα νέο, γενικής χρήσεως, πλαίσιο για τη συστηματική αξιολόγηση του βαθμού επίδρασης των μεθόδων διόρθωσης υποβάθρου. Μέσω του πλαισίου αυτού, οι δύο προτεινόμενες προσεγγίσεις, καθώς και άλλες ευρέως χρησιμοποιούμενες μέθοδοι, αξιολογούνται βάσει εφαρμογής τους σε διαφορετικά σύνολα δεδομένων αυτο-υβριδοποίησης, με τις πρώτες να εμφανίζουν ιδιαιτέρως καλή απόδοση. Το πλαίσιο αυτό καινοτομεί στο ότι ενσωματώνει νέα κριτήρια και τρόπους γραφικής απεικόνισης. Τόσο οι προτεινόμενες μέθοδοι εκτίμησης και διόρθωσης θορύβου υποβάθρου, όσο και το πλαίσιο συστηματικής αξιολόγησής τους, συνιστούν μία νέα, ενδελεχή μελέτη που προσανατολίζει στην εφαρμογή ή απόρριψη μίας συγκεκριμένης προσέγγισης, συνεισφέροντας εν τέλει στην κατάκτηση καλλίτερης ποιότητας δεδομένων μικροσυστοιχιών. Επίσης, στο δεύτερο σκέλος της διατριβής αναπτύσσεται ένα νέο, ολοκληρωμένο και γενικής χρήσεως πλαίσιο ανάλυσης δεδομένων μικροσυστοιχιών ούτως, ώστε να διερευνηθεί το ζήτημα εάν στην T-λευχαιμική κυτταρική σειρά CCRF-CEM επικρατούν εγγενείς ή επίκτητοι μηχανισμοί αντοχής στην πρεδνιζολόνη. Συγκεκριμένα, καταλλήλως επιλεχθέντα δεδομένα μικροσυστοιχιών cDNA – που διευκολύνουν την εξέταση τόσο της εξαρτώμενης από τη συγκέντρωση δράσης, όσο και της δυναμικής της ανταπόκρισης στην πρεδνιζολόνη (πρώιμη και όψιμη δράση) – γίνονται αντικείμενο επεξεργασίας και ενδελεχούς ανάλυσης, και βάσει συγκεκριμένων, προ-διατυπωμένων συλλογισμών, προσεγγίζεται το εν λόγω ερώτημα. Το πλαίσιο αυτό είναι καινοτόμο, εφόσον, πέραν του ότι ενσωματώνει μία πρωτότυπη ακολουθία μεθόδων, προσεγγίζει συστηματικά το πρόβλημα της εγγενούς ή επίκτητης αντοχής, συνεισφέροντας, έτσι, στην ευρύτερη προσπάθεια διερεύνησης των επακριβών μηχανισμών αντοχής των λευχαιμικών κυττάρων στα γλυκοκορτικοειδή. Τα αποτελέσματα από την εφαρμογή του στα δεδομένα της εν λόγω κυτταρικής σειράς συνηγορούν υπέρ της ύπαρξης μίας σύνθετης ανταπόκρισης του υπό μελέτη συστήματος στα γλυκοκορτικοειδή, η οποία όμως τείνει περισσότερο προς έναν εγγενή μηχανισμό αντοχής. / In the present Ph.D. thesis, computational intelligence methods for processing and analyzing cDNA microarray gene expression data are designed and developed. More specifically, in the first part of this thesis, the problem of background estimation and correction of two-channel microarray data is addressed and two novel algorithms are proposed, namely the percentiles-based and the loess-based background correction methods. Both approaches are based on the multiplicative model of background, while utilizing robust background noise estimators, thus making them ideal for noisy datasets. Furthermore, a new, generic framework for the systematic evaluation of the impact of the background estimating methodologies is suggested, whereupon the aforementioned methods as well as other approaches are evaluated by application to various publicly available self-self hybridization datasets. As suggested by this thorough, comparative evaluation our algorithms perform very well regarding noise reduction. The evaluation framework, which is based mainly on different and widely used statistical measures, incorporates new criteria and visualization methods. Moreover, it represents a novel, detailed contribution to the examination of the impact of background correction methods to the final interpretation of microarray experiments, conferring explicit guidance on the pros and cons of them and when they should be applied. Additionally, in the second part of this thesis, a new, generic, computational microarray data analysis framework is described, in order to examine the hypothesis of whether the resistant T-cell leukemia cell line CCRF-CEM posses an intrinsic or exert an acquired mechanism of resistance and to investigate the molecular imprint of this, upon prednisolone treatment. More analytically, using the above explained computational analysis workflow, microarray data that enable the examination of both the dose effect of prednisolone exposure and the dynamics (early and late) of the molecular response of the cells at the transcriptomic layer, are systematically analyzed based on specific, predefined formulations. The analysis of the results supports a complex mechanism of action for the cells which seems to favor though more the intrinsic mechanism of resistance.

Acidophilic iron oxidizing bacteria of the betaproteobacterial genus “Ferrovum” are ubiquitously distributed in acid mine drainage (AMD) habitats worldwide. Since their isolation and maintenance in the laboratory has proved to be extremely difficult, members of this genus are not accessible to a “classical” microbiological characterization with exception of the designated type strain “Ferrovum myxofaciens” P3G. The present study reports the characterization of “Ferrovum” strains at genome and transcriptome level. “Ferrovum” sp. JA12, “Ferrovum” sp. PN-J185 and “F. myxofaciens” Z-31 represent the iron oxidizers of the mixed cultures JA12, PN-J185 and Z-31. The mixed cultures were derived from the mine water treatment plant Tzschelln close to the lignite mining site in Nochten (Lusatia, Germany). The mixed cultures also contain a heterotrophic strain of the genus Acidiphilium. The genome analysis of Acidiphilium sp. JA12-A1, the heterotrophic contamination of the mixed culture JA12, indicates an interspecies carbon and phosphate transfer between Acidiphilium and “Ferrovum” in the mixed culture, and possibly also in their natural habitat. The comparison of the inferred metabolic potentials of four “Ferrovum” strains and the analysis of their phylogenetic relationships suggest the existence of two subgroups within the genus “Ferrovum” (i.e. the operational taxonomic units OTU-1 and OUT-2) harboring characteristic metabolic profiles. OTU-1 includes the “F. myxofaciens” strains P3G and Z-31, which are predicted to be motile and diazotrophic, and to have a higher acid tolerance than OTU-2. The latter includes two closely related proposed species represented by the strains JA12 and PN-J185, which appear to lack the abilities of motility, chemotaxis and molecular nitrogen fixation. Instead, both OTU-2 strains harbor the potential to use urea as alternative nitrogen source to ammonium, and even nitrate in case of the JA12-like species. The analysis of the genome architectures of the four “Ferrovum” strains suggests that horizontal gene transfer and loss of metabolic genes, accompanied by genome reduction, have contributed to the evolution of the OTUs. A trial transcriptome study of “Ferrovum” sp. JA12 supports the ferrous iron oxidation model inferred from its genome sequence, and reveals the potential relevance of several hypothetical proteins in ferrous iron oxidation. Although the inferred models in “Ferrovum” spp. share common features with the acidophilic iron oxidizers of the Acidithiobacillia, it appears to be more similar to the neutrophilic iron oxidizers Mariprofundus ferrooxydans (“Zetaproteobacteria”) and Sideroxydans lithotrophicus (Betaproteobacteria). These findings suggest a common origin of ferrous iron oxidation in the Beta- and “Zetaproteobacteria”, while the acidophilic lifestyle of “Ferrovum” spp. may have been acquired later, allowing them to also colonize acid mine drainage habitats.

Text in English / Corridor disease is an economically important tick-borne disease of cattle in southern Africa. The disease is caused by Theileria parva and transmitted by the vectors, Rhipicephalus appendiculatus and R. zambeziensis. There is currently no vaccine to protect cattle against T. parva that is permitted in South Africa. To develop recombinant anti-tick vaccines against Corridor disease, comprehensive databases of genes expressed in the tick’s salivary glands are required. Therefore, in Chapters 2 and 3, mRNA from the salivary glands of R. appendiculatus and R. zambeziensis was sequenced and assembled using next generation sequencing technologies. Respectively, 12 761 and 13 584 non-redundant protein sequences were predicted from the sialotranscriptomes of R. appendiculatus and R. zambeziensis and uploaded to public sequence domains. This greatly expanded the number of sequences available for the two vectors, which will be invaluable resources for the selection of vaccine candidates in future. Further, in Chapter 3, differential gene expression analysis in R. zambeziensis revealed dynamic expression of secretory protein transcripts during feeding, suggestive of stringent transcriptional regulation of these proteins. Knowledge of these intricate expression profiles will further assist vaccine development in future. In Chapter 4, comparative sialotranscriptomic analyses were performed between R. appendiculatus and R. zambeziensis. The ticks have previously shown varying vector competence for T. parva and this chapter presents the search for correlates of this variance. Phylogenetic analyses were performed using these and other publically available tick transcriptomes, which indicated that R. appendiculatus and R. zambeziensis are closely related but distinct species. However, significant expression differences were observed between the two ticks, specifically of genes involved in tick immunity or pathogen transmission, signifying potential bioinformatic signatures of vector competence. Furthermore, nearly four thousand putative long non-coding RNAs (lncRNAs) were predicted in each of the two ticks. A large number of these showed differential expression and suggested a potential transcriptional regulatory function of lncRNA in tick blood feeding. LncRNAs are completely unexplored in ticks. Finally, in Chapter 5, concluding remarks are given on the potential impact the R. appendiculatus and R. zambeziensis sialotranscriptomes may have on future vaccine developments and some future research endeavours are discussed. / Life and Consumer Sciences / Ph. D. (Life Sciences)

<strong>Background:</strong> Our laboratory identified ELTD1, an orphan GPCR belonging to the adhesion GPCR family (aGPCR), as a novel regulator of angiogenesis and a potential anti-cancer therapeutic target. ELTD1 is normally expressed in both endothelial cells and vascular smooth muscle cells and expression is significantly increased in the tumour vasculature. The aim of this project was to analyse ELTD1's function in endothelial cells and its role in breast cancer. <strong>Method:</strong> 62 sequenced vertebrate genomes were interrogated for ELTD1 conservation and domain alterations. A phylogenetic timetree was assembled to establish time estimates for ELTD1's evolution. After ELTD1 silencing, mRNA array profiling was performed on primary human umbilical vein endothelial cells (HUVECs) and validated with qPCR and confocal microscopy. ELTD1's signalling was investigated by applying the aGPCR ‘Stinger/tethered-agonist Hypothesis'. For this, truncated forms of ELTD1 and peptides analogous to the proposed tethered agonist region were designed. FRET-based 2^nd messenger (Cisbio IP-1;cAMP) and luciferase-reporter assays (NFAT; NFÎoB; SRE; SRF-RE; CREB) were performed to establish canonical GPCR activation. To further investigate ELTD1's role in endothelial cells, ELTD1 was stably overexpressed in HUVECS. Functional angiogenesis assays and mRNA array profiling were then performed. To investigate ELTD1 in breast cancer, a panel of cell lines representative of all molecular subtypes were screened using qPCR. Furthermore, an exploratory pilot study was performed on matched primary and regional nodal secondary breast cancers (n=43) which were stained for ELTD1 expression. Staining intensity was then scored and compared with relapse free survival and overall survival. <strong>Results:</strong> ELTD1 arose 435 million years ago (mya) in bony fish and is present in all subsequent vertebrates. ELTD1 has 3 evolutionary variants of which 2 are most common: one variant with 3 EGFs and a variant with 2 EGFs. Additionally, ELTD1 may be ancestral to members of aGPCR family 2. HUVEC mRNA expression profiling after ELTD1 silencing showed upregulation of the mitochondrial citrate transporter SLC25A1, and ACLY which converts cytoplasmic citrate to Acetyl CoA, feeding fatty acid and cholesterol synthesis, and acetylation. A review of lipid droplet (fatty acid and cholesterol) accumulation by confocal microscopy and flow cytometry (FACS) revealed no changes with ELTD1 silencing. Silencing was also shown to affect the Notch pathway (downregulating the Notch ligand JAG1 and target gene HES2; upregulating the Notch ligand DLL4) and inducing KIT, a mediator of haematopoietic (HSC) and endothelial stem cell (ESC) maintenance. Signalling experiments revealed that unlike other aGPCRs, ELTD1 does not couple to any canonical GPCR pathways (Gαi, Gαs, Gαq, Gα12/13). ELTD1 overexpression in HUVECS revealed that ELTD1 induces an endothelial tip cell phenotype by promoting sprouting and capillary formation, inhibiting lumen anastomoses in mature vessels and lowering proliferation rate. There was no effect on wound healing or adhesion to angiogenesis associated matrix components. Gene expression changes following ELTD1 overexpression included upregulation of angiogenesis associated ANTRX1 as well as JAG1 and downregulation of migration associated CCL15 as well as KIT and DLL4. In breast cancer, none of the representative breast cancer cell lines screened expressed ELTD1. ELTD1 breast cancer immunohistochemistry revealed higher levels of vascular ELTD1 staining intensity within the tumour stroma contrasted to normal stroma and expression within tumour epithelial cells. Additionally, ELTD1 expression in tumour vessels was differentially expressed between the primary breast cancer microenvironment and that of the matched regional node. Due to the small size of the pilot study population, survival comparisons between the various subgroups did not yield significant results. <strong>Conclusion:</strong> ELTD1 is a novel regulator of endothelial metabolism through its suppression of ACLY and the related citrate transporter SLC25A1. ELTD1 also represses KIT, which is known to mediate haematopoietic and endothelial progenitors stem cell maintenance, a possible mechanism through which endothelial cells maintain terminal endothelial differentiation. ELTD1 does not signal like other adhesion GPCRS with CTF and FL forms of ELTD1 not signalling canonically. Additionally, ELTD1 regulates various functions of endothelial cell behaviour and function, inducing an endothelial tip cell phenotype and is highly evolutionarily conserved. Lastly, ELTD1 is differentially expressed in tumour vessels between primary breast cancer and regional nodal metastases and is also expressed in a small subset of breast cancer cells in vivo despite no cancer cell lines expressing ELTD1. The pilot study investigating ELTD1 in the primary breast cancer and regional involved nodes will be followed up with a larger study including the investigation of ELTD1 in distant metastases.

Depuis l’émergence du concept de cellules souches cancéreuses (CSC), de telles cellules ont été isolées à partir de diverses tumeurs solides, dont les glioblastomes. Les CSC et les propriétés qui les caractérisent permettent de mieux comprendre l’hétérogénéité tumorale, ainsi que l’agressivité de certaines tumeurs et les récidives après traitement. Avec la mise en évidence des CSC, un nouveau paradigme est apparu dans le domaine de la thérapie anticancéreuse visant à cibler non seulement les cellules de la masse tumorale, mais également les CSC, plus résistantes aux chimio- et radiothérapies, mais aussi capables d’entrer en quiescence et de reformer la tumeur d’origine. L’isolement de CSC à partir des tumeurs, leur physiopathologie et la recherche de molécules capables de les détruire ou de les différencier afin de les rendre plus sensibles aux traitements mobilisent un nombre croissant d’équipes de recherche et certaines industries pharmaceutiques. Cette thèse présente un travail sur des CSC isolées de glioblastomes humains et s’inscrit dans la démarche énoncée ci-dessus. / Since the emergence of the concept of cancer stem cells (CSC), such cells were isolated from various solid tumors including glioblastomas. The CSC and the properties that characterize them allow a better understanding of tumor heterogeneity and aggressiveness of certain tumors and recurrences after treatment. With the highlighting of CSC, a new paradigm has emerged in the field of cancer therapy. New strategies aim at targeting not only the cells of the tumor mass, but also the CSC, more resistant to chemo- and radiotherapy, but also capable of enter into a quiescent state and to reform the original tumor. The isolation of CSC from solid tumors, their pathogenesis and the search for molecules capable of triggering their death or differentiate them to make them more sensitive to treatment, mobilize a growing number of research teams and some pharmaceutical industries.This thesis presents a work on CSC isolated from human glioblastomas and is framed inside the approach set out above.

Infections or diseases are not just stressful for the one who encounters it. The pathogens causing the same also have to deal with the hostile environment present in the host. The maintenance of physiological homeostatic balance is must for survival of all organisms. This becomes a challenging task for the protozoan parasites which often alternate between two different hosts during their life cycle and thereby encounter several environmental insults which they need to acclimatize against, in order to establish a productive infection. Since their discovery as proteins up-regulated upon heat shock, heat shock proteins have emerged as main mediators of cellular stress responses and are now also known to chaperone normal cellular functions. Parasites like Plasmodium falciparum have fully utilized the potential of these molecular chaperones. This is evident from the fact that parasite has dedicated about 2% of its genome for this purpose. During transmission from the insect vector to humans, the malaria parasite Plasmodium falciparum experiences a temperature rise of about 10oC, and the febrile episodes associated with asexual cycle further add to the heat shock which the parasite has to bear with. The exact mechanism by which the parasite responds to temperature stress remains unclear; however, the induction of chaperones such as PfHsp90 and PfHsp70 has been reported earlier. In other eukaryotes, there are three main factors which regulate heat shock response (HSR): heat shock factor (HSF), heat shock element (HSE) and HSF binding protein (HSBP). Bioinformatics analysis revealed presence of HSE and HSBP in P. falciparum genome; however, no obvious homolog of HSF could be identified. Either the HSF homologue in P. falciparum is highly divergent or the parasite has evolved alternate means to tackle temperature stress. Therefore, we decided to biochemically characterize HSBP and understand the heat shock response pathway in the parasite using transcriptomics and proteomics. The expression for PfHSBP was confirmed at both mRNA and protein level and it was found to translocate into the nucleus during heat shock. As previously reported for HSBP in other organisms, PfHSBP also exists predominantly in trimeric and hexameric form and it interacts with PfHsp70-1. Nearly 900 genes, which represent almost 17% of the parasite genome, were found to have HSE in their promoter region. HSE are represented by three repeating units of nGAAn pentamer and its inverted repeat nCTTn; however, the most abundant class of genes in P. falciparum possessed an atypical HSE which had only 2 continuous repeat units. Next, we were interested to find out if these HSE could actually bind to any parasite protein. Therefore, we performed EMSA analysis with the parasite nuclear extracts using HSE sequence as the oligonucleotide. We observed retarded mobility of the oligonucleotide suggesting that it was indeed able to recruit some protein from the nuclear extract. The importance of transcriptional regulation during heat shock was further confirmed when parasite culture subjected to heat shock in the presence of transcription inhibitor did not show induction in the levels of PfHsp70. These evidences suggest that parasite indeed possesses all the components of heat shock response pathway with either a divergent homologue of HSF or an alternate transcription factor which would have taken its role. Next, we performed global profiling of heat shock response using transcriptomic analysis and 2DDIGE based proteomic profiling. Overall, the parasite’s response to heat shock can be classified under 5 functional categories which aim at increasing the folding capacity of the cell, prevent protein aggregation, increase cytoadhesion, increase host cell remodelling and increase erythrocyte membrane rigidity. Out of the 201 genes found to be up-regulated upon heat shock, 36 were found to have HSE in their promoter region. This suggested that HSE-mediated protein up-regulation could be responsible for the induction of only 18% of total number of genes up-regulated upon heat shock. How would the parasite bring about up-regulation of rest of the heat shock responsive genes? It has been previously reported that genes for some of the heat shock proteins in P. falciparum possess G-box regulatory elements in their promoters and recently, it was shown that these elements served as the binding site for one of the transcription factors (PF13_0235) of AP2 family. Therefore, we looked for the status of this AP2 factor and its targets in our transcriptome data. Although, PF13_0235 was itself not up-regulated, we found up-regulation of its target genes which included another AP2 factor gene PF11_0404. The target genes of PF11_0404 were also up-regulated upon heat shock, thereby suggesting the functioning of an AP2 factor mediated response to heat shock. The next major challenge which the malaria parasite has to deal with is the remodelling of the erythrocyte as these cells do not have a cellular machinery which the parasite can take control of. The parasite remodels the erythrocyte with the help of its large repertoire of exported proteins and develops protrusions known as “knobs” on the erythrocyte surface. These protrusions are cytoadherent in nature and constitute the main virulence determinants of malaria. They also represent variable antigens that allow immune escape. Our lab has previously demonstrated an exported PfHsp40, termed as KAHsp40, to be involved in knob biogenesis. Apart from KAHsp40, there are 19 other PfHsp40s which possess the PEXEL motif required for protein export to erythrocytes. Although, Hsp40s work with an Hsp70 partner, none of the parasitic Hsp70s were known to be exported and was always a missing link in the field of malaria chaperone biology. A genomic re-annotation event could fill this gap by re-annotating the sequence for a pseudogene, PfHsp70-x and described it to contain a functional ORF. According to the re-annotated ORF sequence, PfHsp70-x possessed an ER signal peptide and thus could be targeted to the secretory pathway. Following validation of the re-annotation using a PCR-based approach, we confirmed the expression of this protein at the protein level by immunoblot analysis. Using various subcellular fractionation approaches and immunolocalization studies we established that PfHsp70-x indeed gets exported to the erythrocyte compartment; however, it did not contain the PEXEL motif required for protein export. It gets secreted into the vacuole around the parasite via the canonical ER-Golgi secretory pathway. Its trafficking from vacuole into the erythrocyte was mediated by a hexameric sequence which was present just after the signal peptide cleavage site and before the beginning of ATP-binding domain. In the erythrocyte compartment, it was found to interact with KAHsp40 and MAHRP1, proteins previously implicated in knob biogenesis. Most importantly, PfHsp70-x interacted with the major knob component PfEMP1; however, itself did not become part of knobs. Instead, it localized to the Maurer’s clefts in the erythrocyte compartment. Inside the parasite, PfHsp70-x was present in a complex with Plasmepsin V and PfHsp101. These proteins have been shown to be essential for host cell remodelling process. Plasmepsin V recognizes the PEXEL motif and brings about its cleavage and PfHsp101 specifically targets these PEXEL-cleaved exported proteins to the translocon in vacuolar membrane thereby facilitating their export into the erythrocyte. Thus, PfHsp70-x could also be involved in directing the export of knob constituents apart from just facilitating their assembly. Since, we found out that heat shock or the febrile episodes encountered during the asexual cycling of the parasite promote host cell remodelling; we wanted to find out if PfHsp70-x has any specific role under conditions of temperature stress. PfHsp70-x gene expression was not influenced upon heat shock, however, its export into the erythrocyte was inhibited and the protein got accumulated within the parasite compartment. Surprisingly, immunolocalization studies revealed that the accumulated pool of PfHsp70-x localized into the nucleus instead of ER thus suggesting an alternate role to be associated with PfHsp70-x under stress. Overall, our study addresses two major aspects of malaria pathogenesis. First, response to heat shock and second, remodelling of the host cell. We, for the first time describe global profiling of the parasite’s heat shock response and identify a novel P. falciparum specific heat shock protein member to be involved in malaria pathogenesis.

Les Wolbachia sont des alpha-proteobactéries présentes chez de nombreux arthropodes et nématodes filaires. Ces bactéries héritées maternellement induisent chez leurs hôtes des phénotypes allant du parasitisme au mutualisme, avec le long de ce continuum des phénotypes tels que la féminisation (F), l'incompatibilité cytoplasmique (IC) ou la mort des mâles. Wolbachia est ainsi un modèle particulièrement intéressant pour étudier les différents types de relations symbiotiques.Chez Brugia malayi, comme pour les autres nématodes filaires, Wolbachia vit en symbiose obligatoire avec son hôte. L'élimination de la bactérie par des traitements antibiotiques entraîne une perte de fertilité voire la mort du nématode. Chez l'isopode terrestre Armadillidium vulgare, Wolbachia induit la féminisation des mâles génétiques en femelles fonctionnelles entraînant des biais de sex-ratio vers les femelles dans la descendance.Pour comprendre les mécanismes impliqués dans ces deux symbioses, nous avons mis au point une nouvelle méthode de capture pour isoler l'ADN de Wolbachia et séquencer 8 souches de Wolbachia d'isopodes (F et IC). Une étude de génomique comparative a permis d'établir un premier pan-génome des bactéries du genre Wolbachia et d'identifier 2, 5 et 3 gènes présents seulement chez les souches mutualistes, féminisantes ou induisant la mort des mâles. L'expression des gènes potentiellement impliqués dans la féminisation ou le mutualisme a été étudiée au cours du développement de l'hôte. L'étude de l'interactome protéique bactérie-hôte a ensuite été initiée en utilisant comme appât des protéines bactériennes à domaines eucaryotes en vue d'identifier les cibles de Wolbachia chez l'hôte. / Bacteria of the genus Wolbachia are gram-negative alpha-proteobacteria present in many arthropods and filarial nematodes. These obligate intracellular bacteria are maternally inherited and induce a large number of phenotypes across the symbiosis continuum from mutualism to parasitism, including feminization (F), cytoplasmic incompatibility (CI) or male killing. Studying Wolbachia symbioses is therefore of particular interest in the investigation of symbiotic relationships.In Brugia malayi and other filarial nematodes, they are obligate leading to a loss of worm fertility, and eventual death upon their depletion with antibiotic. In arthropods, they rather are parasitic. In the isopod crustacean Armadillidium vulgare they cause feminization when present: genetic males develop as functional female leading to female biased sex-ratio progenies.In order to understand the molecular mechanisms of these two symbioses, we set up a new capture procedure to catch Wolbachia DNA and performed whole-genome sequencing on 8 Wolbachia strains, symbionts of isopods (F & CI). Comparative genomics led to the establishment of the Wolbachia pan-genome as well as the identification of phenotype related gene patterns. We identified 2, 5 and 3 genes that are only found in mutualist, feminizing and male killing strains, respectively. Expression of genes potentially involved in feminization and mutualism were also analyzed throughout host post-embryonic development. Host-symbiont interactome approach was then initiated by protein-protein interaction studies using bacterial proteins with eukaryote like motifs as bait in order to identify Wolbachia host targets involved in symbiosis.

La cardiopathie ischémique incluant l’insuffisance cardiaque est la deuxième cause de mortalité annuelle au Canada. Bien que de nombreuses stratégies préventives et des thérapies pharmacologiques retardent la progression de la maladie, il n’existe aucune solution qui module directement aux les remaniements pathologiques et la perte de cardiomyocytes. Au cours des 25 dernières années, de multiples progrès dans les domaines de la médecine régénérative et de la thérapie cellulaire ont annoncé des résultats prometteurs, mais les résultats d’études cliniques contemporaines demeurent plutôt mitigés. COMPARE-AMI, une étude randomisée-contrôlée de phase II, a évalué l’effet d’injections intracoronariennes de cellules souches hématopoïétiques CD133+ chez des patients souffrant d’infarctus aigu. IMPACT-CABG, une ÉRC de phase II a également évalué l’effet d’injections intramyocardiques de cellules CD133+ chez les patients souffrant de cardiomyopathie ischémique chronique nécessitant une revascularisation chirurgicale. Nous avons émis l’hypothèse que les cellules CD133+ utilisées dans des études cliniques de cardiomyopathies ischémiques aiguës et chroniques des patients répondant à la thérapie cellulaire exhibent des signatures transcriptomiques communes responsables de leur effet thérapeutique. En classant les patients en tant que répondants et non-répondants selon leur fonction cardiaque, nous avons évalué, a posteriori, ces patrons d’expression. Les cellules CD133+ autologues de patients jugés répondants expriment des signatures qui sont hautement conservées entre elles (incluant l’angiogénèse, la régulation de la réponse au stress et la survie cellulaire) et uniques d’un modèle à l’autre et qui pourraient, en partie, exprimer les issus cliniques des patients. Afin de maximiser les effets de la thérapie cellulaire aux cellules souches, nous avons par la suite tenté de reproduire ces phénotypes par stimulation pharmacologique avec des inhibiteurs d’HSP90 pour leurs effets qui semblent reproduire ces signatures. Ainsi, nous avons démontré qu’une stimulation de cellules souches mésenchymateuses humaines (CSMh) au Célastrol (inhibiteur HSP90) pouvait répliquer certains de ces phénotypes. Notamment, des CSMh conditionnées activent des voies de signalisation de type ‘RISK’ et augmentent leur sécrétion de protéines en lien avec la réponse au stress ainsi que d’exosomes contenant des molécules impliquées dans la communication intercellulaire sans être liées à un changement de type cellulaire. De plus, les CSMh traitées semblent améliorer la guérison de plaie par activité paracrine et sont plus résistante à la sénescence oxydative. Ces résultats encourageants nous permettent d’envisager des stratégies plus poussées de pré-conditionnement cellulaire ex vivo de cellules CD133+ avant leur implantation. À terme, cela pourrait mener à une optimisation de la thérapie cellulaire afin d’en maximiser les bénéfices cliniques et d’en exploiter leur plein potentiel. / Ischemic cardiomyopathy and heart failure are the second annual cause of mortality in Canada. Despite rigorous prevention strategies and drug regimens preventing progression, no therapeutic modality can currently reverse the pathologic modifications of the disease. In the last quarter century, numerous advances in the field of regenerative medicine and cell therapy have demonstrated promising effects. COMPARE-AMI, a phase II randomized controlled trial (RCT), evaluated the effect of intracoronary injection of CD133+ cells in acute myocardial infarction following percutaneous intervention. IMPACTCABG, also a phase II RCT, evaluated the effect of intramyocardial injection of CD133+ hematopoietic stem cells in chronic ischemic cardiomyopathy at the time of surgical revascularization. That being said, we believe that the CD133+ cells used in therapy have shared transcriptomic signatures that are responsible for their clinical effects. By classifying patients into responders and non-responders according to an improvement in ejection fraction, we evaluated, a posteriori, those expression patterns. Autologous CD133+ cells of patients classified as responders expressed highly conserved transcriptomic signatures that could be responsible for the clinical outcomes of patients. Notably, these signatures were related to cardioprotective mechanisms including angiogenesis, stress response regulation and cell survival. In order to harness the full potential of stem cell therapy, we attempted to reproduce the identified phenotypes by pharmacological intervention with HSP90 inhibitors which are known to mimic some of these effets. Conditioned human mesenchymal stem cells (hMSC) activated ‘RISK’-type signaling pathways and augmented exosome and protein secretion relating to the response to cellular stress; this activation was unrelated to a switch of cell type. Furthermore, treated hMSC seemed to favour improved wound healing by paracrine activity and were more resistant to oxidative senescence. These encouraging results allow us to develop novel, more advance, strategies of ex vivo cell preconditioning before implantation, including of CD133+ cells. Ultimately, we hope that optimisation of cells through this mechanism and others will allow us to unleash the full potential of stem cell therapy.