Les relais de phosphorylation de type histidine/aspartate constituent des voies de signalisation impliquées dans la perception et la transduction des signaux jusqu’à la mise en place de réponses spécifiques. Ils mettent en jeu un récepteur ou Histidine aspartate Kinase (HK), des protéines navettes en charge de la transmission du phosphate (HPt) et des Régulateurs de Réponse (RR). L’implication d’un tel système dans la transduction du signal de la contrainte osmotique est avérée chez la levure et fortement suspectée chez Arabidopsis. Ce travail de thèse visait d’une part à caractériser l’implication de cette voie de transduction de la contrainte osmotique chez le peuplier, avec l’identification de partenaires HPt et RR en aval du récepteur HK1 et d’autre part à caractériser le mode de fonctionnement d’un RR de type-B. HK1, un osmosenseur membranaire détecterait le signal et le transmettrait à trois HPt préférentielles. De plus, un partenariat d’interaction se dégagerait entre ces trois HPt et certains RR-B. La régulation transcriptionnelle observée lors d’une contrainte osmotique pour deux des représentants des RR-B témoigne d’une possible implication de ces RR dans cette voie. Ces protéines sont des facteurs de transcription dont la fonction a été confirmée in planta pour l’un d’entre eux. La dimérisation du domaine receveur du RR et son interaction avec le domaine de fixation à l’ADN ou domaine GARP apparaissent comme des points de contrôle clés dans la régulation de l’activité effectrice des RR-B. De plus, la capacité d’un RR-B à se fixer sur ses motifs de reconnaissance (boîtes AGAT) a pu être vérifiée in vitro et la présence de ces séquences a d’ailleurs été retrouvée dans des gènes régulés par la contrainte osmotique. Ce travail prospectif ouvre des perspectives concernant l’implication des RR-B dans la voie de transduction du signal de la contrainte osmotique, et propose notamment des mécanismes fins pour l’élaboration d’une réponse hautement spécifique. / Multistep His-to-Asp phosphorelay systems are signaling pathways devoted to signal perception and transduction for establishment of specific responses. These systems are composed of three successive partners: Histidine-aspartate Kinases (HKs), Histidine-containing Phosphotransfer proteins (HPts), and Response Regulators (RRs). One of the best characterized corresponding systems is the osmo-responsive pathway in yeast. Such systems are also suspected in Arabidopsis. This work aimed to characterize the involvement of an osmosensing pathway in Populus by identifying HPt and RR elements downstream of HK1 and to reveal the underlying mechanisms for the activity of a RR-B. HK1, membrane osmosensor, is expected to be responsible for signal detection and propagation by triggering the activation of three preferential HPt. Furthermore, an interacting partnership between those HPts and particular B-type RRs was observed. Two of them appear to be regulated by an osmotic stress, suggesting their possible involvement in this pathway. The B-type RR members, the final output elements of the pathway, act as transcription factors, as shown for at least for one of them in planta. Taken together, the dimerization of the RR receiver domain and its interaction with its DNA binding domain (GARP), are likely key checkpoints in the regulation of RR-B activity. Besides, the ability of one RR-B to bind its cognate specific DNA sequences (AGAT boxes) was confirmed in vitro and those were found in promoters of osmotic response genes. This work opens up prospects for the involvement of RR-B in the osmotic stress signaling pathway and suggests mechanisms tuning induction of specific responses.

No description available.

As one of several imidazole-containing dipeptides, carnosine is found primarily in the skeletal muscle, the brain, the olfactory bulb and the kidneys of mammals, fishes and birds. The enzyme Carnosine Synthase 1 regulates its synthesis and the two enzymes responsible for the dipeptide’s cleavage into its constituent amino acids are known as serum carnosinase (CN1) and tissue carnosinase (CN2). The amino acid L-histidine is supposed to be mainly responsible for the dipeptides physiological properties based on its imidazole moiety. Among the physiological properties ascribed to the dipeptide are its ability to scavenge reactive oxygen species and to protect against advanced glycation end products and lipid peroxidation. Furthermore, the biogenic dipeptide regulates intracellular calcium homeostasis, acts as a pH buffer and as a metal ion chelator. Based on these primary functions, the dipeptide supports mitochondrial activity and diminishes proteotoxicity. Current studies mainly consider these benefits in muscle tissue and refer to cardiovascular and neurodegenerative diseases. In 1986, Nagai and Suda first revealed tumor growth inhibition after using carnosine in a sarcoma mouse model. Later, Holliday and McFarland confirmed these observations in HeLa cells in vitro. Afterwards, Renner et al. demonstrated an anti- proliferative effect of carnosine on human glioblastoma cells. Unfortunately, the dipeptide’s exact molecular mechanisms on tumor cells are still not entirely understood. Another unresolved question is, whether the dipeptide itself is required for the anti- neoplastic effect or whether L-histidine with its imidazole moiety is sufficient and has to be released from carnosine by cleavage of the dipeptide. In order to get a better insight into these questions we investigated the response of glioblastoma cells to L-histidine and carnosine in primary cell cultures and cell lines derived from glioblastoma. Glioblastoma multiforme represents the most common and malignant primary brain tumor. Significant risk factors are still unknown. At diagnosis, the median age is 64 years and the disease is usually found in a progressed stage. Histopathologically, glioblastoma is characterized by necrosis and pronounced mitotic activity in slightly differentiated cells. Accordingly, the tumor shows rapid progression, aggressive invasiveness and, morphological variety. Since 2005, standard of care against glioblastoma follows the STUPP-protocol, which comprises microsurgery, adjuvant chemotherapy with temozolomide and radiotherapy. Nevertheless, it remains one of the most treatment-refractory intracranial tumors; the median over survival after standard treatment is only 14.6 months. Experiments by Letzien et al. demonstrated that L-histidine mimics the anti-neoplastic effect of carnosine in three glioblastoma cell lines investigated. In addition, the amino acid also increased expression of pyruvate dehydrogenase kinase 4 (PDK4) mRNA expression. These observations pointed towards the possibility that carnosine could just be a vehicle, delivering L-histidine to target cells, and that release of the imidazole-containing amino acid is required for the observed effects. In order to investigate whether the effects observed in cell lines are of general significance, cells from ten glioblastoma cell lines and 21 primary glioblastoma cell cultures derived from surgically removed tumors were incubated in a medium containing different concentrations of either carnosine or L-histidine. Cell viability assays measuring the amount of ATP in cell lysates and dehydrogenase activity in living cells were performed. Both substances induced a significant loss of viability. In fact, L- histidine appeared to be even more effective than carnosine, at the same concentration. Next, we investigated whether the enzymes known to be able to cleave carnosine into amino acids are expressed in the cell cultures. Using RT-qPCR, the expression of the mRNA encoding the two enzymes serum carnosinase (CN1, extracellular) and cytosolic or tissue carnosinase (CN2, intracellular) were analyzed in all 31 glioblastoma cell cultures.The experiments revealed high expression of mRNA encoding CN2 in all cultures, whereas expression of CN1 mRNA (gene: CNDP1) was only slightly detectable. Immunoblot performed with ten cell lines revealed that CN2 protein was also present in all cell lines investigated. Therefore, it had to be assumed, that carnosine may be cleaved inside the cells. In a next series of experiments, we investigated whether inhibition of CN2 by the dipeptidase-inhibitor bestatin (ubenimex) does attenuate the effect of carnosine on tumor cell proliferation. Therefore, cell viability was analyzed in the presence of carnosine and in the absence or presence of different concentrations of bestatin. Aside from a general effect of bestatin on cell viability, especially at higher concentrations, no attenuation of carnosine’s antineoplastic effect was observed in the two cell lines investigated. Therefore, we concluded that cleavage of the dipeptide does not seem to be a prerequisite for its effect on tumor cell viability. As we could not rule out that other unknown dipeptidases aside from CN2 may cleave carnosine, we finally measured the intracellular abundances of cells incubated in the absence or presence of carnosine. Therefore, cells from ten cell lines and from five primary cultures were incubated in the absence and presence of either L-histidine or carnosine, and their extracts were subjected to high performance liquid chromatography (HPLC-MS) after derivatization. Although the intracellular abundances of L-histidine of cells incubated in the presence of carnosine clearly demonstrated that the dipeptide is cleaved inside the cells, no correlation between the intracellular amount of L-histidine and the response of cells with regard to viability was observed. Furthermore, the abundance of L-histidine in cells incubated in the presence of 50 mM carnosine was considerably lower, compared to that of cells incubated in the presence of 25 mM L-histidine. As both conditions resulted in a comparable loss of viability, this strongly indicates that cleavage of the dipeptide is not required for its anti-tumor effect and may even be not very efficient. In conclusion, we could confirm that cleavage of carnosine does occur in glioblastoma cells, although this does not raise the intracellular abundance of L-histidine when compared to cells incubated in the presence of the free amino acid. More importantly, cleavage is not required in order to deploy carnosine’s antineoplastic effect. In addition, it appears to be very likely that the imidazole-moiety whether bound or not bound to another amino acid may be sufficient for a therapeutic response. These observations raise a number of interesting questions that should be investigated considering exploiting the antineoplastic effect described for a potential therapeutic use. First of all, the simple question has to be asked, whether it would be sufficient to use L- histidine as an antitumor drug. In that case one has to ask whether sufficient concentrations of L-histidine could be achieved at the side of the tumor when the amino acid is supplemented. Given the fact that it is a proteinogenic amino acid one may suggest, that it is rapidly taken up by other cells. On the other hand, this may also be the case for carnosine. In addition, carnosine is rapidly cleaved by the presence of CN1 in serum. Whether this is in fact a problem is difficult to answer as there are different reports of small clinical trials where carnosine was able to attenuate cognitive impairments after oral supplementation. In addition, the recently identified CN1 inhibitor carnostatine could possibly be supplemented together with carnosine. Another consideration would be to identify other imidazole containing compounds that are no substrate of CN1. However, as it appears that the imidazole-moiety needs to enter the cells the question is, whether other compounds could be transported across the cell membrane. With regard to treatment of brain tumors one should also keep in mind that, aside from the fact, that the blood-brain-barrier is impaired in glioblastoma, it may still be limiting sufficient delivery. At this point, it is also interesting to note that no side effects of carnosine aside from a rarely appearing dysesthesia, are known. However, given the fact that the outcome of current treatment of glioblastoma is still disappointing it appears to be worth to further investigate carnosine’s antineoplastic effect. As the primary targets of the dipeptide are also still widely unknown, the observation that the imidazole moiety is the main effector may help to further elucidate the mechanisms responsible for the antineoplastic effect. At this point it is also interesting to note that the recently discovered benzimidazolinum Gboxin, which also contains an imidazole moiety, exhibits antitumor activity in glioblastoma cells, most likely by irreversibly compromising oxygen consumption. In this case, an elevated proton gradient and a lower pH in cancer cell mitochondria appear to be responsible for the inhibition of oxidative phosphorylation.:1. List of Abbreviations 3 2. Introduction 5 2.1. Glioblastoma 5 Risk factors 5 Localization and histopathology of glioblastoma 5 Molecular pathology 6 Clinic 6 Prognosis and treatment 6 2.2. Carnosine 7 Occurrence 7 Enzymes and transporters 7 Functions 9 Carnosine and cancer 9 Carnosine and its possible application for therapy 10 2.3. Histidine and other histidine-containing compounds 11 L-histidine and naturally occurring dipeptides 11 Physiological functions of L-histidine 11 L-histidine in health and disease 12 L-histidine as a precursor of other metabolites 12 2.4. Objectives of the study 14 3. Publication 15 3.1. General informations 15 3.2. Carnosine’s inhibitory effect on glioblastoma cell growth is independent of its cleavage 16 3.3. Supplemental materials 29 4. Summary 35 5. References 39 6. Appendix 47 6.1. Declaration of independent work 47 6.2. Statement of the own contribution 48 6.3. Acknowledgements 51

Several published functions associated with the CHK1 histidine kinase of Candida albicans resemble those of the MAPK Cek1p and its cognate receptor Sho1p (SSU81). To explore this further, we have compared mutants lacking the proteins mentioned above and have constructed a double sho1/chk1Δ null mutant to determine relationships among these proteins. We observed that the sensitivity to Congo red (CR), calcofluor white (CW), as well as clumping of cells, was slightly increased in the double mutant compared to the single chk1Δ or sho1Δ mutants. However, Cek1p phosphorylation via Sho1p, which occurs during log phase growth in the presence or absence of CR in Wt cells, does not require Chk1p. These data suggest that Chk1p and Sho1p are components of parallel but independent signal pathways. In addition, bulk mannan of strains was analyzed by GLC/MS and GPC MALLS and NMR. Compared to Wt and a CHK1 gene-reconstituted strain (CHK23) that contained high, intermediate and low Mw mannan species, we found that the mannan of strains CHK21 (chk1Δ null), the cek1Δ null, and the double mutant consisted only of low Mw mannan. The sho1Δ null mutant only demonstrated a reduced intermediate type of mannan. Alcian blue binding was lower in cek1Δ, chk1Δ, and the double sho1/chk1Δ null mutant lacking high and intermediate Mw mannan than in the sho1Δ null which had a partial loss of intermediate Mw mannan only. We conclude that the Chk1p HK is part of a functionally similar but parallel pathway to the Sho1p-Cek1p pathway that confers resistance to the cell wall inhibitors CR and CW. However, a functional relationship in mannan biosynthesis of Chk1p and Cek1p exists that only partially requires Sho1p.

Recent studies suggest that factor (F) XII, which is dispensable for hemostasis, is important for thrombus stabilization and growth. Therefore, FXIIa inhibition may attenuate thrombosis without disrupting hemostasis. FXII activation is stimulated by polyanions such as polyphosphates released from activated platelets, and nucleic acids released by cells. Previously, we showed that histidine-rich glycoprotein (HRG) binds FXIIa with high affinity, inhibits FXII autoactivation and FXIIa-mediated activation of FXI, and attenuates ferric chloride-induced arterial thrombosis in mice. Thus, HRG has the capacity to downregulate the contact pathway in vitro and in vivo. This thesis aimed to identify the complementary binding domains of HRG and FXIIa, and to further explore the anticoagulants effects of HRG on FXIIa-mediated contact activation. We hypothesized that FXIIa binds to the zinc-binding histidine-rich region (HRR) of HRG and that HRG binds to the non-catalytic heavy chain of FXIIa to exert its anticoagulant activities on FXIIa-mediated contact activation. We have localized the complementary binding sites of HRG and FXIIa to be within the HRR domain of HRG and NH2-FNII-EGF1 (NFE) domains of FXIIa. Moreover, we show that the HRR binds to short chain polyphosphate with high affinity, suggesting a dynamic complex between HRG, FXIIa, and polyphosphate (polyP) on activated platelets. We provide evidence for two potential mechanisms through which HRG modulates the contact system. These include by 1) inhibiting FXIIa activity and 2) attenuating the procoagulant effect of polyanions, such as polyP on FXIIa-mediated reactions. Indeed, we show that the interaction of HRG with FXIIa and polyphosphate is predominantly mediated by the HRR domain and that HRR analogs have the capacity to recapitulate the anticoagulant effects of HRG in purified and plasma systems. Therefore, by modulating FXIIa-mediated contact pathway reactions, like HRG, HRR analogs may attenuate thrombosis without disrupting hemostasis. / Thesis / Doctor of Philosophy (Medical Science)

No description available.

No description available.

Les histidine kinases (HKs) représentent une vaste famille de protéines impliquées dans la perception des signaux environnementaux chez les bactéries, les champignons et les plantes. Ces protéines joueraient notamment un rôle majeur dans l’adaptation aux stresses, mais aussi dans la virulence de nombreux micro-organismes procaryotes et eucaryotes. Si les HKs sont à présent bien connues chez les bactéries et les plantes, tant sur un plan structural que fonctionnel, les connaissances concernant ces protéines chez les autres clades de l’arbre du vivant demeurent plus que fragmentaires. C’est ainsi que le premier objectif de ce travail a consisté en l’exploration in silico de la structure et de la distribution des HKs chez les organismes eucaryotes dans le cadre de plusieurs études bioinformatiques : i) chez les champignons inférieurs, ii)chez les levures bourgeonnantes et enfin iii) à travers l’ensemble des super-groupes eucaryotes. Les HKs n’étant pas retrouvées chez les mammifères, elles suscitent depuis quelques années une attention particulière de la communauté scientifique en tant que nouvelles cibles pour le développement d’antimicrobiens. C’est précisément dans ce contexte que la partie expérimentale de ce projet a été initiée au sein du GEIHP. Cette équipe porte en effet ses efforts sur le filamenteux multi-résistant Scedosporium apiospermum qui se situe au second rang parmi les moisissures capables de coloniser chroniquement les poumons des patients atteints de mucoviscidose. Ainsi, dans l’optique d’identifier de nouvelles cibles thérapeutiques du champignon, la seconde partie de ce projet s’est focalisée sur la caractérisation fonctionnelle des HKs chez Scedosporium apiospermum. En parallèle, cette étude nous a également amenés à développer de nouveaux outils moléculaires adaptés à S.apiospermum en vue de futures études d’imageries de fluorescence et de bioluminescence. / Histidine kinases (HKs) represent a broad family of proteins involved in the perception of environmental signals in bacteria, fungi and plants.These proteins play a major role in stress adaptation, but also in the virulence of many prokaryotic and eukaryotic microorganisms. Although HKs are now well known in bacteria and plants, both structurally and functionally, knowledge about these proteins in other clades of the living tree remains more than fragmentary. Thus the first objective of this work was the in silico exploration of the structure and distribution of HKs in eukaryotic organisms through several bioinformatics studies : i) in the lower fungi, ii)in budding yeasts, and finally iii) across all eukaryotic supergroups. Since HKs are not found in mammals, they have been attracting attention in recent years from the scientific community as new targets for the development of antimicrobials. It is precisely in this context that the experimental part of this project was initiated in the GHEIHP. This team is focusing on the multi-resistant filamentous Scedosporiumapiospermum, which ranks second among the molds capable of chronycally conolizing the lungs of cysticfibrosis patients. Thus, in order to identify new therapeutic targets of the fungus, the second part of this project focused on the functional characterization of HKs in S. apiospermum. In parallel, this study also led us to develop new molecular tools adapted to S. apiospermum for future studies of fluorescence or bioluminescence imaging.

O grande grupo heterogêneo de neuropatias periféricas hereditárias estão entre os casos mais comuns de perda sensitiva e fraqueza muscular em crianças e adolescentes. Pelo menos 84 genes estão envolvidos com neuropatias sensitivo-motoras hereditárias (NSMH), sendo suas formas de herança mais comuns as autossômico-dominantes desmielinizante e axonal e as neuropatias ligadas ao cromossomo X, e as mais raras as autossômicorecessivas desmielinizante e axonal e as formas ainda não classificadas. O gene HINT1, possuinte de 3 exons e localizado no cromossomo 5, codifica a proteína Histidine triad nucleotide binding protein 1, uma variante transcricional (mRNA) regulatória que hidroliza substratos. Recentemente mutações em HINT1 foram também relacionadas à neuropatias axonais com neuromiotonia (ARCMT2-NM), e portanto à CMT. O objetivo deste trabalho foi realizar o screening mutacional do gene HINT1 em uma amostra da população brasileira com quadro clínico de CMT recessivo (CMT2-AR), e foram encontradas 1 mutação silenciosa já previamente descrita, 1 polimorfismo exônico e 1 polimorfismo intrônico, também já conhecidos. Concluiu-se que mutações no gene HINT1 não são portanto responsáveis pela CMT-AR nesta amostra da população brasileira. / The large heterogeneous group of inherited peripheral neuropathies are among the most common causes of sensory loss and muscle weakness in children and adolescents. At least 84 genes are involved in inherited sensorymotor neuropathies (NSMH), being the demyelinating and axonal autosomaldominant and the X-linked neuropathies their most common forms of inheritance, and the demyelinating and axonal autosomal-recessive and not yet classified forms the most rare ones. The HINT1 gene, with 3 exons and located on chromosome 5, encodes the protein Histidine triad nucleotide binding protein 1, a regulatory transcriptional variant (mRNA) that hydrolyzes substrates. Recently, mutations in HINT1 were also related to axonal neuropathy with neuromyotonia (ARCMT2-NM), and therefore to CMT. The objective of this study was the mutational screening of the HINT1 gene in a sample of the Brazilian population with clinical recessive CMT (CMT2-AR), and 1 silent mutation previously described, 1 intronic polymorphism and 1 exonic polymorphism, both also known, were founded. It was then concluded that mutations in the HINT1 gene are not responsible for CMT2-AR in this particular sample of the Brazilian population.

O grande grupo heterogêneo de neuropatias periféricas hereditárias estão entre os casos mais comuns de perda sensitiva e fraqueza muscular em crianças e adolescentes. Pelo menos 84 genes estão envolvidos com neuropatias sensitivo-motoras hereditárias (NSMH), sendo suas formas de herança mais comuns as autossômico-dominantes desmielinizante e axonal e as neuropatias ligadas ao cromossomo X, e as mais raras as autossômicorecessivas desmielinizante e axonal e as formas ainda não classificadas. O gene HINT1, possuinte de 3 exons e localizado no cromossomo 5, codifica a proteína Histidine triad nucleotide binding protein 1, uma variante transcricional (mRNA) regulatória que hidroliza substratos. Recentemente mutações em HINT1 foram também relacionadas à neuropatias axonais com neuromiotonia (ARCMT2-NM), e portanto à CMT. O objetivo deste trabalho foi realizar o screening mutacional do gene HINT1 em uma amostra da população brasileira com quadro clínico de CMT recessivo (CMT2-AR), e foram encontradas 1 mutação silenciosa já previamente descrita, 1 polimorfismo exônico e 1 polimorfismo intrônico, também já conhecidos. Concluiu-se que mutações no gene HINT1 não são portanto responsáveis pela CMT-AR nesta amostra da população brasileira. / The large heterogeneous group of inherited peripheral neuropathies are among the most common causes of sensory loss and muscle weakness in children and adolescents. At least 84 genes are involved in inherited sensorymotor neuropathies (NSMH), being the demyelinating and axonal autosomaldominant and the X-linked neuropathies their most common forms of inheritance, and the demyelinating and axonal autosomal-recessive and not yet classified forms the most rare ones. The HINT1 gene, with 3 exons and located on chromosome 5, encodes the protein Histidine triad nucleotide binding protein 1, a regulatory transcriptional variant (mRNA) that hydrolyzes substrates. Recently, mutations in HINT1 were also related to axonal neuropathy with neuromyotonia (ARCMT2-NM), and therefore to CMT. The objective of this study was the mutational screening of the HINT1 gene in a sample of the Brazilian population with clinical recessive CMT (CMT2-AR), and 1 silent mutation previously described, 1 intronic polymorphism and 1 exonic polymorphism, both also known, were founded. It was then concluded that mutations in the HINT1 gene are not responsible for CMT2-AR in this particular sample of the Brazilian population.