Les glioblastomes sont des tumeurs au pronostic défavorable. L’échec des thérapies est lié à la présence de cellules souches cancéreuses (CSCs), résistantes aux traitements ; la caractérisation de ces cellules et l’identification de biomarqueurs sont donc primordiales. Le calcium contrôle de nombreux processus cellulaires ; parmi les éléments majeurs de la signalisation calcique, la Calmoduline (CaM) est impliquée dans différentes pathologies, dont des cancers, et est un puissant régulateur de l’état physiologique d’une cellule. CaM interagit avec de nombreuses protéines impliquées dans la régulation de l’homéostasie calcique de la cellule. Nous avons cherché à identifier et caractériser des antagonistes de CaM, inhibant différentiellement ces interactions. L’utilisation de ces antagonistes en tant que perturbateurs de l’homéostasie calcique a permis de mettre en évidence un marqueur caractérisé des CSCs de glioblastomes. D’autre part, l’étude comparée du protéome membranaire de CSCs issues de glioblastomes a permis de mettre en évidence la surexpression de clusters de différenciation et protéines impliquées dans la signalisation calcique. Ces protéines sont de potentiels marqueurs moléculaires des CSCs de glioblastome. / Glioblastomas are malignant tumor of poor prognosis. Therapeutic failure might be supported by cancer stem cells (CSCs); characterization of these cells and biomarkers identification are of most importance. Calcium controls numerous cellular process; beyond major elements of calcium signaling, Calmodulin (CaM) is involved in different pathologies and tumors, and is a powerful regulator of cell physiological state. CaM interacts with a plethora of proteins involved in cell calcium homeostasis regulation. We aimed at identifying and characterizing CaM antagonists, capable of differentially inhibiting these interactions. The use of these antagonists in calcium homeostasis disturbance led to the identification of a characterized marker of glioblastomas CSCs. In another approach, the comparative study of glioblastomas CSCs membrane proteome uncovered the overexpression of differentiation clusters and proteins involved in calcium signaling. These proteins are potential molecular biomarkers for glioblastomes CSCs.

5 Abstract Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) belongs to the serine/ threonine protein kinase family, which is involved in the calcium signaling pathway. The increase of intracellular calcium concentration induces the activation of calmodulin (CaM), which then activates its binding partners including CaMKII, CaMKIII, CaMKK1 and CaMKK2. CaMKK2 activates CaMKI, CaMKIV and AMP-dependent kinase, AMPK, by phosphorylation. CaMKK2 is naturally present in cells in an autoinhibited state, which is caused by the steric hindrance of the active site by the autoinhibitory domain. When calmodulin binds to the calmodulin-binding domain, the autoinhibitory domain is removed and the active site becomes accessible. Upon activation, CaMKK2 undergoes autophosphorylation, which increases its enzyme activity. Negative regulation of CaMKK2 is mediated by cAMP-dependent protein kinase A (PKA)- and GSK3-dependent phosphorylation. Sites phosphorylated by PKA have been identified for both CaMKK1 and CaMKK2. Two of them are also motifs recognized by scaffolding 14-3-3 proteins. Previous studies have shown that the 14-3-3 protein binding maintains phosphorylated CaMKK2 in an inhibited state by blocking the dephosphorylation of S495, which prevents the binding to calmodulin. However, it is unclear if it is the...

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Les molécules naturelles de différentes origines et essentiellement celles issues des algues marines conçoivent une panoplie de principes bioactifs naturels à fort potentiel thérapeutique. Actuellement, plusieurs recherches sont consacrées à l’isolement de nouveaux composés bioactifs à partir des ressources marines qui renferment des effets bénéfiques prometteurs pour la santé. Parmi les ressources marines, les composés bioactifs présents dans de nombreuses espèces d’algues ont des propriétés nutritionnelles et pharmaceutiques particulièrement intéressantes. Cette thèse s’intéresse aux effets bénéfiques de l’algue Ulva rigida. Elle concerne plus particulièrement les activités des extraits protéiques de l’algue verte Ulva rigida. En effet, dans la présente étude, nous avons étudié l'effet préventif d'un extrait de protéine (PE) d'Ulva rigida contre les dommages cellulaires induits par les UVB. Nous avons démontré qu'un traitement des astrocytes corticaux avec 50 μg de PE induit un fort effet glioprotecteur et supprime les effets nocifs des rayonnements UVB, augmentant en particulier la viabilité cellulaire. D’autres essais supplémentaires nous ont permis d’affirmer que cet extrait stimule l'activité de la catalase et de la superoxyde dismutase et inhibe la production de marqueurs de stress, tels que le H2O2 ou la peroxydation lipidique. Par ailleurs, la digestion protéolytique ou le traitement thermique de l'extrait d'algue ont inhibé cet effet préventif, plaidant pour le rôle prédominant de la fraction protéique. Par la suite, des études protéomiques ont permis d'identifier plusieurs protéines de l'extrait, parmi lesquelles la calmoduline s'est avérée représenter un contributeur important aux activités de protection observées à la fois sur des cultures d’astrocytes que sur des neurones en grain. / Natural molecules from different origins present a high therapeutic potential. Among them, molecules extracted from marine algae are particularly interesting. Several studies are currently being devoted to the isolation of new bioactive compounds from marine resources that have promising health benefits. Among the marine resources, the bioactive compounds present in many species of algae have particularly interesting nutritional and pharmaceutical properties. This thesis deals with the beneficial effects of the alga Ulva rigida. It relates more particularly to the activities of the protein extract of the green alga Ulva rigida. In this study, we studied the preventive effect of a protein extract (PE) of Ulva rigida against UVB-induced cell damage. We have demonstrated that treatment of cortical astrocytes with 50 μg of PE induces a strong glioprotective effect and suppresses the harmful effects of UVB radiation, in particular increasing cell viability. Other extra tests allowed us to say that this extract stimulates the activity of catalase and superoxide dismutase and inhibits the production of stress markers such as H2O2 or lipid peroxidation. Moreover, the proteolytic digestion or the heat treatment of the algal extract inhibited this preventive effect, arguing for the predominant role of the protein fraction. Subsequently, proteomic studies have identified several proteins in the extract, of which calmodulin has been shown to be an important contributor to the protective activities observed on both astrocyte and neuron cultures.

Each year countless lives are affected and about half a million people die from malaria, a disease caused by parasites originating from the Plasmodium family. The most virulent species of the parasite is Plasmodium falciparum (P. falciparum).   Calmodulin (CaM) is a small, 148 amino acid long, highly preserved and essential protein in all eukaryotic cells. Previous studies have determined that CaM is important for the reproduction and invasion of P. falciparum in host cells. The primary structure of human CaM (CaMhum) and CaM from P. falciparum (CaMpf) differ in merely 16 positions, making differences in their structures and ligand affinity interesting to study. Especially since possible inhibitors of CaMpf in favor of CaMhum, in extension, could give rise to new malaria treatments.   Some antagonists, functioning as inhibitors of CaM, have already been analysed in previous studies. However, there are also compounds that have not yet been studied in regards to being possible antagonists of CaM. This study regards three known antagonists; trifluoperazine (TFP), calmidazolium (CMZ) and artemisinin (ART) and also three recently created fentanyl derivatives; 3-OH-4-OMe-cyclopropylfentanyl (ligand 1), 4-OH-3OMe-4F-isobutyrylfentanyl (ligand 2) and 3-OH-4-OMe-isobutyrylfentanyl (ligand 3).   Bioinformatic methods, such as modelling and docking, were used to compare the structures of CaMhum and CaMpf as well as observe the interaction of the six ligands to CaM from both species. In addition to the differences in primary structure, distinguished with ClustalW, disparities in tertiary structure were observed. Structure analysis of CaMhum and CaMpf in PyMOL disclosed a more open conformation as well as a larger, more defined, hydrophobic cleft in CaMhum compared to CaMpf. Simulated binding of the six ligands to CaM from both species, using Autodock 4.2, indicated that TFP and ART bind with higher affinity to CaMhum which is expected. Ligand 2 and ligand 3 also bound with higher affinity and facilitated stronger binding to CaMhum, which is reasonable since their docking is based on how TFP binds to CaM. However, ligand 1 as well as CMZ both bound to CaMpf with higher affinity. Despite promising results for ligand 1 and CMZ, no decisive conclusion can be made solely based on bioinformatic studies.    To gain a better understanding on the protein-ligand interactions of the six ligands to CaMhum and CaMpf, further studies using e.g. circular dichroism and fluorescence would be advantageous. Based on the results from this study, future studies on the binding of CMZ and ligand 1 to CaM as well as ligands with similar characteristics would be especially valuable. This is because they, based on the results from this study, possibly are better inhibitors of CaMpf than CaMhum and thereby could function as possible antimalarial drugs.

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No / Patients with acute vitiligo have low epidermal catalase expression/activities and accumulate 10 -3 M H 2O 2. One consequence of this severe oxidative stress is an altered calcium homeostasis in epidermal keratinocytes and melanocytes. Here, we show decreased epidermal calmodulin expression in acute vitiligo. Since 10 -3M H 2O 2 oxidises methionine and tryptophan residues in proteins, we examined calcium binding to calmodulin in the presence and absence of H 2O 2 utilising 45calcium. The results showed that all four calcium atoms exchanged per molecule of calmodulin. Since oxidised calmodulin looses its ability to activate calcium ATPase, enzyme activities were followed in full skin biopsies from lesional skin of patients with acute vitiligo (n = 6) and healthy controls (n = 6). The results yielded a 4-fold decrease of ATPase activities in the patients. Computer simulation of native and oxidised calmodulin confirmed the loss of all four calcium ions from their specific EF-hand domains. Taken together H 2O 2-mediated oxidation affects calcium binding in calmodulin leading to perturbed calcium homeostasis and perturbed L-phenylalanine-uptake in the epidermis of acute vitiligo.

Als Kapazitation bezeichnet man den im weiblichen Reproduktionstrakt stattfindenden Reifungsschritt, der Spermien das volle Fertilisierungspotential verleiht. Die molekularbiologischen Grundlagen dieses für eine erfolgreiche natürliche oder auch artifizielle Befruchtung essenziellen Prozesses sind bis heute nur unvollständig verstanden. Im Rahmen der vorliegenden Dissertation wurden die mit der Kapazitation einhergehenden funktionellen und strukturellen spermalen Veränderungen untersucht. Die kapazitative Stimulation führte zu einer gesteigerten Motilität bis hin zur Hyperaktivierung, zu einer vermehrt induzierten Akrosomenreaktion und zu einer deutlich reduzierten Apoptoseaktivität. Anhand von Inhibitionsexperimenten wurde die Rolle der potentiellen Signaltransduktoren Caspase-1, Calpain und Calmodulin analysiert. Dabei wies die Calmodulinantagonisierung auf eine ausgeprägte Calciumabhängigkeit aller untersuchten kapazitationsassoziierten Prozesse hin. Die Hemmung von Caspase-1 und Calpain führte zu einer Beeinträchtigung der Motilität und der Akrosomenreaktion ohne das Ausmaß der Apoptoseinduktion zu beeinflussen. Die vorstehend genannten Erkenntnisse wurden zur Evaluation verschiedener Ejakulataufbereitungsprotokolle genutzt. Dabei konnte gezeigt werden, dass die Kombination des modernen Verfahrens der immunomagnetische Zellseparation mit der etablierten Methode der Dichtegradientenzentrifugation dem einfachen Standard in Bezug auf die Anreicherung hochmotiler Spermien mit minimaler Apoptoseaktivität aus frischen wie auch aus kryokonservierten Ejakulaten deutlich überlegen war. Bedeutsam im Hinblick auf eine mögliche pratische Anwendung der immunomagnetischen Zellseparation erscheint der Befund, dass die durch das kombinierte Anreicherungsverfahren erhaltene Spermatozoensubpopulation im Hamsteroozytenpenetrationstest ein signifikant höheres Fertilisierungspotential zeigte.

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