The 21st Century has seen a rise in incidence of complex diseases such as cancer and in the quest to develop essential new therapeutic options, the study of drug-cell interactions can yield powerful information. Acute myeloid leukaemia (AML) is an aggressive cancer that causes life-threatening deficits of functional blood cells in humans for which current treatment options are highly toxic and often poorly tolerated. A combination of two existing drugs, bezafibrate and medroxyprogesterone acetate in a drug redeployment situation has shown promise in vitro and in vivo and further investigations are crucial to elucidate the mode of action of this treatment. This project investigated the mechanistic action of BaP at a cellular level. Orthogonal spectroscopic and mass spectrometric platforms were employed to probe the biochemical composition of two AML cell lines, HL60 and K562 in the presence and absence of this combined drug treatment. Analysis was performed on single living cells, dehydrated cells, fixed cells and cell extracts to give a large and detailed data set. A consideration of the main spectral differences obtained by Synchrotron-FTIR and ATR-FTIR in conjunction with multivariate statistical analysis revealed a significant change to the cellular lipid composition with drug treatment; furthermore, this response was not caused by cell apoptosis. In particular, the ratio of CH2:CH3 was observed to increase with BaP treatment and this was determined to be a significant change in both cell lines (p <0.05). An overall increase in lipid unsaturation suggests that BaP targets cellular lipid biosynthesis. Raman microspectroscopy added a further dimension to the spectroscopic study by providing spatial information of lipid distribution which suggested that BaP-induced saturation change is uniform across a single cell. UHPLC-MS was employed for global metabolomics analysis of AML cell extracts and revealed a number of biochemical pathways that were indicated as targets of BaP therapy in both cell lines. Univariate and multivariate analysis determined statistically significant metabolites for which putative identifications were made. Pyrimidine metabolism was the most significant pathway identified for changes consistent in both HL60 and K562 cell lines. The complementarity of ToF-SIMS and UHPLC-MS provided large coverage of the lipidome of AML cells through untargeted and targeted approaches. For data derived by both techniques, a general increase in polyunsaturated species for BaP treated cell extracts was observed which correlated well with findings from spectroscopic investigations. Adopting a multi-disciplinary approach to cell analysis can afford a powerful insight into understanding drug mode of action at a cellular level and novel information regarding BaP mechanistic action in AML cell lines was revealed. This analytical approach could be extended to the future study of drug-cell interactions for other oncological systems.

La peau est un organe sensoriel qui permet notamment à l’organisme de s’adapter à son environnement. Ainsi, de nombreuses fibres nerveuses sont présentes au sein de cette peau permettant de détecter différents stimuli tels que la pression, la douleur ou encore une variation de température. A la suite d’une brûlure profonde, ces terminaisons nerveuses sont détruites et la repousse de ces fibres lors du processus de cicatrisation est inadéquate conduisant à des handicaps souvent sérieux chez les patients. Dans un premier travail, la réalisation d’une brûlure chez des animaux présentant ou non une neuropathie périphérique induite par la résinifératoxine a permis d’étudier le rôle de l’innervation lors de la cicatrisation. Chez les animaux traités, une cicatrisation plus lente et un défaut de réinnervation par rapport aux animaux contrôles étaient observés. Pour approfondir le rôle de l’innervation dans la cicatrisation, des études in vitro ont ensuite été réalisées afin d’évaluer les interactions possibles entre les cellules neuronales et les fibroblastes dermiques. Même en l’absence de contacts directs, il a été montré que les cellules neuronales sont capables d’induire la différenciation des fibroblastes en myofibroblastes. Au vue de l’implication de l’innervation sur la différenciation des fibroblastes et sur la cicatrisation, il semble important de pouvoir proposer aux patients brûlés, pour leur traitement, un nouveau concept de substitut cutané favorisant une repousse axonale fonctionnelle au sein du tissu cicatriciel. / The skin is a sensitive organ which notably allows the body to adapt to its environment. Many nerve fibers are present in the skin and are implicated in the detection of various stimuli such as pressure, pain or temperature variation. After a deep burn injury, these nerve fibers are destroyed and their regrowth during the wound healing process is imperfect which induces serious disabilities for patients. In a first study, a burn model was developed on animals presenting or not a peripheral neuropathy induced with resiniferatoxin and had allowed to study the role of sensory innervation on wound healing. On animals treated with resiniferatoxin, wound healing was delayed and nerve regeneration was imperfect compared to control animals. To further investigate the role of innervation in wound healing, in vitro studies were then performed to evaluate possible interactions between neuronal cells and dermal fibroblasts. Even in the absence of direct contacts, it has been shown that neuronal cells are able to induce the differentiation of fibroblasts into myofibroblasts. Considering the involvement of innervation on the differentiation of fibroblasts and on wound healing, it seems important to provide burned patients with a new concept of skin substitute promoting functional axonal regrowth in the scar tissue.

Viruses are obligate intracellular pathogens. Therefore, their successful replication, at every stage from attachment to assembly and egress, is dependent on host cell functions. The host cell in turn engages mechanisms to counteract virus replication. As a result, viruses have evolved mechanisms to evade these counteracting measures as well as ways to reshape the cellular environment into one that’s favourable for successful replication. Systematic studies offer a platform for unravelling virus-cell interactions and in particular can address three important aspects 1) increase our understanding of basic biology of the virus, 2) identify and characterise novel cellular functions 3) provide important leads for novel targets for antiviral therapy. In this study, I investigated two aspects of virus host interaction; the role of microRNAs (miRNAs) in virus infection and the role of interferon inducible genes in virus infection. Human cytomegalovirus (HCMV) is a β herpes virus that infects humans. HCMV maintains a persistent lifelong infection in the host involving a cycle of latency and reactivation. Infection of healthy individuals with HCMV results in relatively minor symptoms. In contrast, infection of individuals with a compromised immune system, as in the case of organ transplant recipients and AIDS patients, can cause significant morbidity and mortality. In common with other herpes viruses, HCMV expresses multiple small regulatory RNAs called miRNAs. HCMV encodes at least 14 miRNAs. Identifying the targets of these miRNAs will help us understand their functional importance during infection. Recently, a biochemical technique called Cross-Linking, Ligation and Sequencing of Hybrids (CLASH), was developed by Tollervey and colleagues, representing the most advanced systematic technique for the identification of miRNA targets. We adapted this approach to identify high confidence miRNA targets during HCMV infection. However, the protocol was sub-optimal and presented us with technical challenges. Although high quality data sets were not generated, the work was crucial for the establishment of the system which is now generating promising data. Virus-cell interactions can also be elucidated by probing for host factors that are important for virus replication. Type I interferon is a highly effective inhibitor of HCMV replication. Treatment of cells with interferon results in up regulation of multiple effectors known as interferon stimulated genes (ISGs). How these genes block HCMV replication is poorly understood. A library of more than 380 ISG expressing lentiviruses was screened to determine the effects of individual ISGs on HCMV replication. The screen was performed in primary human fibroblast cells and a glioblastoma cell line called U373s. Multiple inhibitory ISGs were identified including well characterised ISGs such as cGAS, STAT2, NOD2, DDX60 and HPSE as well as novel candidates TXNIP, ELF1, FAM46C, MT1H and CHMP5. Five ISGs were identified as HCMV replication enhancers including previously published ISGs BST2 and IFITM1 and novel enhancers ODC1, BCL3 and IL28RA. siRNA screens against top hits demonstrated that STAT2, CPT1A and cGAS are dominant inhibitory factors during HCMV infection and knockdown of these genes can partially rescue HCMV replication following interferon treatment. Finally, using a corresponding rhesus ISG library we show that rhesus SAMHD1 effectively inhibits HCMV replication while human SAMHD1 has no effect, suggesting that HCMV expresses a species-specific inhibitor of SAMHD1. This study defines interferon stimulated pathways important for HCMV replication and identifies multiple novel host factors that both restrict and enhance HCMV replication. These studies demonstrate the effectiveness of using systematic approaches for the identification of novel host virus interactions.

Enteric caliciviruses were detected in humans and animals incoming into food chain such as porcine and bovine species. Caliciviruses have a single stranded, positive, polyadenylated RNA genome. They share properties of high environmental stability, high excretion load and high genetic variation linked to point mutations and recombination. These properties have allowed the formulation of hypothesis about zoonotic transmission or animal reservoir for human strains. The aim of the thesis, composed of four studies, was to investigate the zoonotic risk associated to animal enteric caliciviruses. In a first study, circulation of both sapoviruses and noroviruses was evidenced by molecular detection in Belgian pig farms. They were detected in both asymptomatic animals or in piglets showing clinical signs of enteritis. In a second study, bovine noroviruses were molecularly detected in Belgian cattle. Strains phylogenetically related to those of the genotype 2 were predominant. In the same study, seroprevalence against bovine norovirus infection in cattle was investigated by indirect ELISA. Antigens included in the ELISA were virus-like particules obtained in the baculovirus system by expressing the capsid protein of a strain isolated by the laboratory during a previous study. Apparent seroprevalence was high (93.2%), confirming previous results about apparent molecular prevalence in diarrheic calves (7.5%). In a third study about molecular detection of bovine noroviruses, diagnostic strategy was revised in order to improve the detection of genotype 1 strains and to deal with opportunity of recombination events. Bovine norovirus recombinant strains and also, surprisingly, some sequences genetically related to bovine kobuviruses were detected. In a fourth study, attachment factors and internalization pathways for genotype 2 bovine noroviruses were studied with an original quantitative method based on flow cytometry analysis. Along with a galactosyl residue that seems to be essential, a sialic acid residue was also showed to be implied in the binding of genotype 2 bovine noroviruses or in a posterior step. Internalisation pathways related to lipid rafts and to macropinocytosis were found. Together the results have contributed to the analysis of the zoonotic risk associated to enteric animal caliciviruses in the Belgian epidemiological situation. According to these results, the zoonotic risk seems to be low as no sequences genetically related to the human ones were detected. However, some results suggest to maintain a certain degree of vigilance. Indeed, molecular detection showed the co circulation of both bovine and porcine noroviruses in Belgian farms, implicating that hazard exposition exists and could be high in the Belgian epidemiological situation. Morevover, circulation of recombinant strains in the overall population of the bovine norovirus strains implies that this phenomenon was included in the risk assessment. Infection pressures and high prevalences for human and animal strains in a closed epidemiological context as the Belgian one could increase the risk of interspecific recombination. Finally, the sialic acid residue, possibly involved in the binding of genotype 2 bovine noroviruses, and a poorly specific internalisation pathway as macropinocytosis could also favor interspecies barrier crossing. In the current knowledge, zoonotic risk associated to animal enteric caliciviruses can be communicated as low but a degree of vigilance has to be retained, associated for example to observation of genetic evolution in the populations of human and animal strains.

<p>The gonad is a unique primordial organ that retains the ability to adopt one of two morphological fates through much of mammalian embryonic development. Previous work in our lab found that dimorphic vascular remodeling was one of the earliest steps during sex-specific morphogenesis. In particular, vessels in XY gonads display highly ordered behavior that coincides with testis cord formation. It was unknown how the vasculature may influence testis cord morphogenesis and, if so, how this was mechanistically related to sex determination. The work in this thesis addresses a single over-arching hypothesis: Male-specific vascular remodeling is required for testis morphogenesis and orchestrates differentiation of the XY gonad. </p><p>To address this question we have modified and developed techniques that allow us to isolate aspects of vascular behavior, gene expression, and endothelial influence on surrounding cells. In particular, the application of live imaging was instrumental to understanding the behavior of various gonadal cell-types in relation to remodeling vessels. It is difficult to grasp the complexity of an organ without understanding the dynamics of its constituents. A critical aim of my work was to identify specific inhibitors of the vasculature that do not affect the early stages of sex determination. Combining inhibitors, live imaging, cell sorting, qRT-PCR, mouse models, and whole organ culture has led to a far richer understanding of how the vasculature behaves and the cell-types that mediate its influence on organ morphogenesis. The beauty of our system is that we do not have to settle for a snapshot of the fate of cells in vivo, but can document their journeys and their acquaintances along the way. </p><p>Vascular migration is required for testis cord morphogenesis. Specific inhibitors revealed that in the absence of vessels, testis cords do not form. The work below shows that vessels establish a feedback loop with mesenchymal cells that results in both endothelial migration and subsequent mesenchymal proliferation. Interstitial control of testis morphogenesis is a new model within the field. The mechanisms regulating this process include Vegf mediated vascular remodeling, Pdgf induced proliferation, and Wnt repression of coordinated endothelial-mesenchymal dynamics. Our work also suggests that vascular patterning underlies testis patterning and, again, is mediated by signals within the interstitial space not within testis cords themselves. </p><p>A final aspect of my work has been focused on how vessels continue to influence morphology of the testis and the fate of surrounding cells. Jennifer Brennan, a graduate student in our lab, previously showed that loss of Pdgfr&#945; antagonizes cord formation and development of male-specific lineages. The mechanisms and cell-types related to this defect were not clear. I began to reanalyze Pdgfr&#945; mutants after finding remarkable similarity to gonads after vascular inhibition. This work is providing data suggesting that vessels are not simply responsible for testis morphology but also for the fate of specialized cells within the testis. On the whole, this thesis describes specific roles for endothelial cells during gonad development and mechanisms by which they are regulated.</p> / Dissertation

Tissue engineered human oral mucosa has the potential to fill tissue deficits caused by facial trauma or malignant lesion surgery. It can also help elucidate the biology of oral mucosa and serve as an alternative to in vivo testing of oral care products. The aim of this thesis was to construct a tissue engineered full-thickness human oral mucosa closely mimicking the native tissue. To this end, the feasibility of the concept was tested by co-culturing fibroblasts and epithelial cells isolated from normal human oral mucosa biopsies in a collagen-glycosaminoglycan-chitosan scaffold, developed in our laboratory to construct a skin equivalent. An oral mucosal equivalent closely mimicking the native one was obtained and characterized by histology, immunohistochemistry and transmission electron microscopy. Using the same model, the influence of mesenchymal cells on oral epithelial development was investigated by culturing epithelial cells on lamina propria, corneal stroma and dermal equivalents. They were found to significantly influence the thickness and the ultrastructure of the epithelium. Finally, in order to improve the adhesiveness of conventional scaffolds, an elastin-like recombinamer (ELR) containing the cell adhesion tripeptide, RGD, was used in the production of novel bilayer scaffolds employing lyophilization and electrospinning. These scaffolds were characterized by mercury porosimetry, scanning electron microscopy and mechanical testing. In vitro tests revealed positive contribution of ELR on the proliferation of both fibroblasts and epithelial cells. It was thus possible to construct a viable oral mucosa equivalent using the principles of tissue engineering.

L’ingénierie de la muqueuse orale humaine (MOH) a pour but le comblement des pertes de substances suite à un traumatisme facial ou à la chirurgie des lésions malignes. Elle a aussi des applications en recherche pour élucider les mécanismes biologiques de la MO et en pharmacotoxicologie comme alternative à l’expérimentation animale. L'objectif de cette thèse était de reconstruire une MOH proche du tissu normal. À cette fin, la faisabilité du concept a d'abord été testée par co-culture de fibroblastes de la lamina propria et de cellules épithéliales de MOH dans le substrat de collagène-chitosan glycosaminoglycane, développé pour la production de peaux reconstruites. La caractérisation de la MOH reconstruite par histologie, immunohistochimie et microscopie électronique à transmission a montré la présence d’une LP équivalente avec un épithélium pluristratifié et non kératinisé très proche du tissu d’origine. Grâce à ce modèle, nous avons ensuite démontré que l’origine des fibroblastes (MO, cornée, peau) influence significativement l’épaisseur et l’ultrastructure de l'épithélium obtenu par culture de cellules épithéliales orales. Enfin, afin d'améliorer les propriétés adhésives du substrat à base collagène, nous avons ajouté au collagène, une élastine-like recombinante (ELR) contenant le tri-peptide d’adhésion cellulaire, RGD, et produit un nouveau substrat bicouche, poreux par lyophilisation et recouvert d’une couche fibreuse par électrofilage. Ces substrats ont été caractérisés par porosimétrie au mercure, microscopie électronique à balayage et essais mécaniques. Nous avons démontré l’effet stimulant de ELR sur la prolifération des fibroblastes et des cellules épithéliales / Tissue engineered human oral mucosa has the potential to fill tissue deficits caused by facial trauma or malignant lesion surgery. It can also help elucidate the biology of oral mucosa and serve as an alternative to in vivo testing of oral care products. The aim of this thesis was to construct a tissue engineered full-thickness human oral mucosa closely mimicking the native tissue. To this end, the feasibility of the concept was tested by co-culturing fibroblasts and epithelial cells isolated from normal human oral mucosa biopsies in a collagen-glycosaminoglycan-chitosan scaffold, developed in our laboratory to construct a skin equivalent. An oral mucosal equivalent closely mimicking the native one was obtained and characterized by histology, immunohistochemistry and transmission electron microscopy. Using the same model, the influence of mesenchymal cells on oral epithelial development was investigated by culturing epithelial cells on lamina propria, corneal stroma and dermal equivalents. They were found to significantly influence the thickness and the ultrastructure of the epithelium. Finally, in order to improve the adhesiveness of conventional scaffolds, an elastin-like recombinamer (ELR) containing the cell adhesion tripeptide, RGD, was used in the production of novel bilayer scaffolds employing lyophilization and electrospinning. These scaffolds were characterized by mercury porosimetry, scanning electron microscopy and mechanical testing. In vitro tests revealed positive contribution of ELR on the proliferation of both fibroblasts and epithelial cells. It was thus possible to construct a viable oral mucosa equivalent using the principles of tissue engineering

L’interleukine (IL)-17 et le TNFa sont deux cytokines pro-inflammatoires jouant un rôle important dans diverses maladies inflammatoires systémiques et auto-immunes affectant différents organes et tissus comme le foie et les muscles. Cependant, les rôles de l’IL-17 et du TNFa restent encore mal compris dans les muscles et le foie, qui est impliqué dans la réponse en phase aiguë. En utilisant des cultures de myoblastes, d’hépatocytes et de cellules stellaires hépatiques humaines, nous avons trouvé que l’IL-17 et le TNFa augmentent en synergie la sécrétion de la cytokine pro-inflammatoire IL-6 et de plusieurs chimiokines. Dans les myoblastes, l’IL-17 et le TNFa induisent un stress oxydatif et une dérégulation de calcium montrant ainsi que les processus pathologiques immuns et non-immuns interagissent. Dans les hépatocytes, en augmentant en synergie les niveaux de la CRP et des transaminases, l’IL-17 et le TNFa participent à l’inflammation systémique et aux dommages cellulaires. Etant donné que des infiltrats de cellules immunitaires sont retrouvés lors d’atteintes inflammatoires, les interactions cellulaires contribuent certainement à la chronicité de l’inflammation. Des cellules mononuclées du sang périphérique activées ou non ont ainsi été placées en co-cultures avec les myoblastes, les hépatocytes et les cellules stellaires. Par comparaison aux monocultures, les productions de l’IL-6 et de la chimiokine IL-8 étaient augmentées dans les co-cultures. L’IL-17 et le TNFa contribuaient partiellement à ces effets. Les effets systémiques de l’IL-17 et du TNFa en font donc des cibles thérapeutiques attrayantes pour le traitement des nombreuses maladies inflammatoires systémiques / Interleukin-17A (IL-17) and tumor necrosis factor-a (TNFa) are two pro-inflammatory cytokines playing an important role in various systemic inflammatory and autoimmune disorders affecting different organs and tissues including the liver and the muscles. However, the roles of IL-17 and TNFa are not fully understood in the muscles and also in liver, which is crucial in the acute phase response. By using cultures of human myoblasts, primary human hepatocytes, human HepaRG cells and LX-2 hepatic stellate cells, we found that IL-17 and TNFa increase in synergy the production of the pro-inflammatory cytokine IL-6 and chemokines (IL-8, CCL20, MCP-1). In myoblasts, the IL-17 and TNFa stimulation induces endoplasmic reticulum stress and calcium dysregulation showing that immune and non-immune pathogenic mechanisms interplay. In hepatocytes, IL-17 and TNFa mediate systemic inflammation and cell damage by increasing in synergy the CRP acute-phase protein and transaminase levels through the induction of IL-6. Since active liver and muscle disorders are characterized by inflammatory infiltrates of immune cells, the cell interactions play certainly an important role in the chronicity of the inflammation. Peripheral blood mononuclear cells activated or not were therefore co-cultured with myoblasts, hepatocytes and/or hepatic stellate cells to assess the inflammatory role of the cell-cell interactions. Co-cultures enhance the production of IL-6 and IL-8 compared to monocultures. IL-17 and TNFa contribute partially to these inductions. The systemic effects of IL-17 and/or TNFa make them attractive therapeutic targets for the treatment of various systemic inflammatory disorders

Les cellules neurales expriment des molécules dites immunes qui peuvent exercer des rôles différents de ceux exercés dans le système immunitaire. Les molécules du CMH-I classiques présentent des peptides représentatifs du contenu protéique de chaque cellule aux sentinelles du système immunitaire. Cependant, il est documenté que ces molécules ont aussi des fonctions « non immunes ». En effet, les molécules du CMH-I classiques jouent un rôle dans l'établissement et la plasticité des synapses. Sur divers types cellulaires, elles peuvent aussi interagir avec des récepteurs membranaires en cis, moduler leur stabilité à la membrane et en conséquence leur activité. RAE-1 est un membre de la famille des molécules du CMH-I, décrite initialement dans le système nerveux central embryonnaire. Pour le système immunitaire, RAE-1 est un ligand du récepteur activateur NKG2D, exprimé par les cellules NK, NKT, les lymphocytes T &#947;&#948; et CD8+. RAE-1 est peu ou pas exprimé dans la plupart des tissus adultes. Son expression est induite par le stress génotoxique, la transformation tumorale ou l'infection virale ce qui permet au système immunitaire d'éliminer les cellules « malades » grâce à l'activation des cellules cytotoxiques exprimant NKG2D. Je décris l'expression de RAE-1 par les cellules neurales progénitrices et le rôle non immun de cette molécule dans la prolifération cellulaire. L'expression de RAE-1 est fortement corrélée au niveau de prolifération cellulaire et est dépendante du facteur de croissance EGF. / Neural cells express immune molecules which roles differ from those in the immune system. Classical MHC-I molecules present peptides originated from the proteic content of each cell to patrolling immune cells. However, these molecules can also have nonimmune roles. Indeed, classical MHC-I molecules participate in the establishment of synapses and synaptic plasticity. They can also interact in cis with different membrane receptors on different cell types, and modulate the receptors' membrane stability and activity. RAE-1, a member of MHC-I family, was initially described in the embryonic central nervous system. In the immune system, RAE-1 is a ligand of the activating receptor NKG2D, expressed by NK cells and by NKT, &#947;&#948;T and some CD8+ T lymphocytes. RAE-1 is weakly or not expressed in most adult tissues. Its expression is induced by genotoxic stress, tumoral transformation or viral infection and triggers the elimination of transformed cells by the cytotoxic immune cells which express NKG2D. I describe here the expression of RAE-1 by neural progenitor cells and its role in cell proliferation. RAE-1 expression level is highly correlated with the rate of cell proliferation and depends on the presence of epidermal growth factor (EGF). Exposition to EGF induces the colocalization of RAE-1 and phosphorylated EGF-receptor (EGFR) inside lipid rafts and endocytosed vesicles, which supports a role of RAE-1 as a partner of EGFR. RAE-1 expression is also induced in the nervous tissue in different models of CNS pathologies. In these conditions, RAE-1 could be expressed by proliferating microglia under the control of M-CSF.

Microcystine sind die wohl bekanntesten cyanobakteriellen Toxine. Sie werden im Wesentlichen durch die im Süßwasser weltweit verbreitete, koloniebildende Gattung Microcystis synthetisiert. Die biologische Funktion dieser Peptide ist jedoch ungeklärt. In dieser Studie wurde die Fragestellung erstmals über einen globalen Ansatz auf molekularer Ebene analysiert. Die proteomischen Analysen zwischen M. aeruginosa PCC 7806/ Wildtyp und einigen Microcystin-freien Mutanten deuten auf eine physiologische Rolle der Microcystine. Microcystine beeinflussen die Abundanz zahlreicher Proteine. Prominentester Vertreter ist RubisCO – Schlüsselenzym des Calvin Zyklus. RubisCO und andere im 2D selektierte Proteine konnten außerdem als mögliche zelluläre Bindepartner des Microcystins identifiziert werden. Möglicherweise bindet MC an bestimmte Cysteinreste dieser Proteine. Mit dem Knockout der mcy-Gene geht außerdem eine Überexpression eines Morphotyp-spezifischen Proteins einher, das MrpC genannt wurde. Dieses Protein vermittelt möglicherweise Zell-Zell-Interaktionen in Microcystis. / Microcystins are the most common cyanobacterial toxins found in freshwater lakes and reservoirs throughout the world. They are frequently produced by the unicellular, colonial cyanobacterium Microcystis; however, the role of the peptide for the producing organismen is poorly understood. In this study we describe the first global approach to investigate this topic on a molecular level. Proteomic studies with M. aeruginosa PCC 7806 wild-type and several microcystin-deficient mutants indicated a physiological function for microcystin. Microcystin was shown to influence the abundance of several proteins which have an intra- or extracellular function. A prominent candidate is RubisCO, the key enzyme of the calvin cycle. RubisCO and other proteins, initially selected by 2D analysis, are putative cellular binding partners of microcystin. A potentially interaction mechanismen is the kovalent binding of microcystin to cysteine residues of the protein. Moreover, several knockouts of microcystin biosynthesis genes result in an overexpression of a putative morpho-type specific factor, named MrpC. This protein possibly mediates cell-cell interactions in Microcystis.