Le repositionnement des médicaments est une stratégie visant à trouver de nouvelles indications pour des médicaments existants. Un composé de type thiomorpholine hydroxamate (TMI-1, un inhibiteur d'ADAM-17 utilisé dans le traitement des maladies chroniques inflammatoires) a récemment été repositionné dans le traitement du cancer. Afin d’élucider son mécanisme d'action, une série de dérivés de type d'arysulfonyl thiomorpholine hydroxamate a été synthétisée et évaluée pour déterminer une relation entre structure et activité . Nous avons démontré que l'activité cytotoxique observée dépend du fragment phényl hydroxamate et n'est pas liée à l'inhibition de l'ADAM-17. Grâce à une approche par protéomique inverse, nous avons identifié la protéine membranaire peroxysomale PMP34 / SLC25A17 comme une nouvelle cible de TMI-1. PMP34 est un transporteur transmembranaire de différents cofacteurs nécessaires à la fonction des peroxysomes. Nous démontrons ici que TMI-1 se lie directement à PMP34 et augmente l’interaction entre PMP34 et PEX19, une protéine chaperone impliquée dans l’assemblage et la biologie des peroxysomes, alors que les expériences sur les ARNi ont révélé que PMP34 est essentielle à la viabilité des cellules tumorales. Enfin, le traitement des cellules tumorales par TMI-1 induit une modification de la composition en protéines du peroxysome et augmente le niveau de la protéine PMP70 / ABCD3. Un taux élevé de PMP70 dans le peroxysome augmente la $\beta$-oxydation des acides gras ce qui pourrait entraîner la mort des cellules cancéreuses. En conséquence, PMP34 représente une nouvelle cible intéressante en oncologie. / Drug repositioning is a strategy to find new indications for existing drugs. A thiomorpholine hydroxamate compound (TMI-1, an ADAM-17 inhibitor used for the treatment of chronic inflammatory diseases) has been recently repositioned in cancer. To further elucidate its mechanism of action, a series of arylsulfonyl thiomorpholine hydroxamate derivatives was synthetized and evaluated to determine a structure-activity relationship. We have pinpointed that the observed cytotoxic activity depends on the hydroxamate phenyl moiety and is not related to ADAM-17 inhibition. Using a reverse proteomic approach, we now identify the peroxisomal membrane protein PMP34/SLC25A17 as a new target of TMI-1. PMP34 is a transmembrane transporter of different cofactors required for peroxisome function. We demonstrate here that TMI-1 directly binds to PMP34 and increases the interaction between PMP34 and PEX19, a chaperone protein involved in peroxisome assembly and biology while RNAi experiments revealed that PMP34 is essential for tumor cell viability. Finally, TMI-1 treatment of tumor cells induces modification of peroxisome protein composition and increases the level PMP70/ABCD3 protein. High PMP70 level in peroxisome increases fatty acid $\beta$-oxidation that could lead to cancer cell death. As a result, PMP34 represents a new valuable target in oncology.

No description available.

Concerns about climate changes and scarcity of fossil fuels are rising. Hence wood is becoming an attractive source of renewable energy and raw material and these new dimensions have prompted increasing interest in wood formation in trees, in both the scientific community and wider public. In this thesis, the focus is on a key process in wood development: programmed cell death (PCD) in the development of xylem elements. Since secondary cell wall formation is dependent, inter alia, upon the life time of xylem elements, the qualitative features of wood will be affected by PCD in xylem, about which there is little information. This thesis focuses on the anatomical, morphological and transcriptional features of PCD during xylem development in both the stem of hybrid aspen, Populus tremula (L.) x tremuloides (Michx.) and the hypocotyl of the herbaceous model system Arabidopsis thaliana (L. Heynh.). In Populus, the progressive removal of organelles from the cytoplasm before the time of death (vacuolar bursts) and the slowness of the cell death process, illustrated by DNA fragmentation assays (such as TUNEL and Comet assays), have been ascertained in the xylem fibres by microscopic analyses. Furthermore, candidate genes for the regulation of fibre cell death were identified either from a Populus EST library obtained from woody tissues undergoing fibre cell death or from microarray experiments in Populus stem, and further assessed in an in silico comparative transcriptomic analysis of Arabidopsis thaliana. These candidate genes were either putative novel regulators of fibre cell death or members of previously described families of cell death-related genes, such as autophagy-related genes. The induction of the latter and the previous microscopic observations suggest the importance of autophagy in the degradation of the cytoplasmic contents specifically in the xylem fibres. Vacuolar bursts in the vessels were the only previously described triggers of PCD in the xylem, which induce the very rapid degradation of the nuclei and surrounding cytoplasmic contents, therefore unravelling a unique previously unrecorded type of PCD in the xylem fibres, principally involving autophagy. Arabidopsis is an attractive alternative model plant for exploring some aspects of wood formation, such as the characterisation of negative regulators of PCD. Therefore, the anatomy of Arabidopsis hypocotyls was also investigated and the ACAULIS5 (ACL5) gene, encoding an enzyme involved in polyamine biosynthesis, was identified as a key regulator of xylem specification, specifically in the vessel elements, though its negative effect on the cell death process. Taken together, PCD in xylem development seems to be a highly specific process, involving unique cell death morphology and molecular machinery. In addition, the technical challenges posed by the complexity of the woody tissues examined highlighted the need for specific methods for assessing PCD and related phenomena in wood. / Oron för klimatförändringar och brist på fossila bränslen har ökat påtagligt under de senaste åren. De enorma möjligheter som skogsråvaran erbjuder som alternativ källa för förnyelsebar energi och råmaterial har väckt ett stort intresse också för den biologiska processen bakom vedbildning i träd. Denna avhandling fokuserar på en viktig process i vedbildning: programmerad celldöd (PCD) i xylemet. Xylemcellernas livstid påverkar bildningen av sekundära cellväggar, vilket i sin tur påverkar vedens kvalitativa egenskaperna, så som veddensitet. Trots dess betydelse för viktiga egenskaper hos vedråvaran existerar fortfarande väldigt lite information om xylem PCD på cellulär eller molekylär nivå. I den här avhandlingen belyses de anatomiska, morfologiska och genetiska aspekterna av PCD i xylemutveckling i både stam av hybridasp, Populus tremula (L.) x tremuloides (Michx.) och hypokotyl av det örtartade modellsystemet Arabidopsis thaliana (L. Heynh.). Xylemet i både Populus och Arabidopsis består av två olika celltyper; de vattentransporterade kärlen och de stödjande fibrerna. Det är känt att celldöd i kärlen pågår mycket snabbt efter att den centrala vakuolen brister och de hydrolytiska enzymer släpps in i cytoplasman. I den här avhandlingen ligger fokus på fibrerna i Populus xylemet. Med hjälp av mikroskopianalyser av cellmorfologin (elektronmikroskopi) och DNA-fragmentering i cellkärnan (TUNEL- och Comet-analyser) kunde vi konstatera att till skillnad från kärlen så uppvisar fibrerna en långsam och progressiv nedbrytning av organellerna och cellkärnans DNA före vakuolbristning. Dessutom har kandidatgener för reglering av fibercelldöd identifierats antingen från ett Populus EST bibliotek från vedartade vävnader som genomgår fibercelldöd eller från mikroarray experiment i Populus stam. Dessa kandidatgener är antingen potentiella nya regulatorer av fibercelldöd eller medlemmar av tidigare beskrivna familjer av celldödsrelaterade gener. Bland de sistnämnda finns autofagi-relaterade gener, vilket stöder funktionen av autofagi i samband med autolys av cellinnehållet i xylemfibrerna. Dessa studier pekar därför på en typ av PCD som har inte tidigare beskrivits för xylemet. Arabidopsis är ett alternativt växtmodellsystem för studier av vissa aspekter av vedbildningen, såsom karakteriseringen av negativa regulatorer av PCD. Därför har också hypokotylanatomin analyserats, och ACAULIS5 (ACL5) genen, som kodar för ett enzym i biosyntesen av polyaminer, har visats vara en viktig regulator av xylemspecifikation genom dess negativa effekt på kärlens celldöd. Sammantaget visar denna avhandling att PCD i xylemutvecklingen verkar involvera unika morfologiska och molekylära mekanismer. Vi visar dessutom att komplexiteten hos de vedartade vävnaderna leder till ett behov av bättre anpassade verktyg för att djupare kunna bedöma PCD och liknande fenomen i veden. / Även med namnet Moreau-Courtois, Charleen L. samt Moreau, Charleen.

The great variation displayed by nucleic acid molecules in human cells, and the continuous discovery of their impact on life, consequently require continuous refinements of molecular analysis techniques. Padlock probes and rolling circle amplification offer single nucleotide discrimination in situ, a high signal-to-noise ratio and localized detection within cells and tissues. In this thesis, in situ detection of nucleic acids with padlock probes and rolling circle amplification was applied for detection of DNA in the single cell gel electrophoresis assay to detect nuclear and mitochondrial DNA. This assay is used to measure DNA damage and repair.  The behaviour of mitochondrial DNA in the single cell gel electrophoresis assay has earlier been controversial, but it was shown herein that mitochondrial DNA diffuses away early in the assay. In contrast, Alu repeats remain associated with the nuclear matrix throughout the procedure. A new twelve gel approach was also developed with increased throughput of the single cell gel electrophoresis assay. DNA repair of three genes OGG1, XPD and HPRT and of Alu repeats after H2O2 induced damage was further monitored. All three genes and Alu repeats were repaired faster than total DNA. Finally, padlock probes and rolling circle amplification were applied for detection of the single stranded RNA virus Crimean Congo hemorrhagic fever virus. The virus was detected by first reverse transcribing RNA into cDNA.. The virus RNA together with its complementary RNA and the nucleocapsid protein were detected in cultured cells. The work presented here enables studies of gene specific damage and repair as well as viral infections in situ. Detection by ligation offers high specificity and makes it possible to discriminate even between closely related molecules. Therefore, these techniques will be useful for a wide range of applications within research and diagnostics.

No description available.

No description available.

Huntington’s disease (HD) is a neurodegenerative disorder caused by the inheritance of one mutant copy of the huntingtin gene. Mutant huntingtin protein (mHtt) contains an expanded polyglutamine repeat region near the N-terminus. Cleavage of mHtt releases an N-terminal fragment (N-mHtt) which translocates, and accumulates in the nucleus. Nuclear accumulation of N-mHtt has been directly associated with cellular toxicity. Decreased transcription is among the earliest detected changes that occur in the brains of HD patients and is consistently observed in all animal and cellular models of HD. Transcriptional dysregulation may trigger many of the perturbations that occur later in disease progression and an understanding of the effects of mHtt may lead to strategies to slow the progression of the disease. Current models of N-mHtt-mediated transcriptional dysregulation suggest that abnormal interactions between N-mHtt and transcription factors impair the ability of these transcription factors to associate at N-mHtt-affected promoters and properly regulate gene expression. We tested various aspects of these models using two N-mHtt-affected promoters in in vitro transcription assays and in two cell models of HD using techniques including overexpression of known N-mHtt-interacting transcription factors, chromatin immunoprecipitation, promoter deletion and mutation analyses and in vitro promoter binding assays. Based on our results and those in the literature, we proposed a new model of N-mHtt-mediated transcriptional dysregulation centered on the presence of N-mHtt at affected promoters. We concluded that simultaneous interaction of N-mHtt with multiple binding partners within the transcriptional machinery would explain the gene-specificity of N-mHtt-mediated transcriptional dysregulation, as well as the observation that some genes are affected early in disease progression while others are affected later. Our model explains why alleviating N-mHtt-mediated transcriptional dysregulation through overexpression of N-mHtt-interacting proteins has proven to be difficult and suggests that the most realistic strategy for restoring gene expression across the spectrum of N-mHtt affected genes is by reducing the amount of soluble nuclear N-mHtt.

Proteins are fundamental structural, enzymatic and regulatory components of cells. Analysis of proteins, such as by measuring their concentrations, characterizing their modifications, and detecting their interactions, provides insights in how biological systems work physiologically or pathologically at the molecular level. To perform such analysis, molecular tools with good sensitivity, specificity, high multiplexing and throughput capacity are needed. In this thesis, four different assays were developed and applied to detect and profile proteins and protein complexes in human body fluids, and in cells or tissues. These assays are based on targeting proteins or protein complexes by oligonucleotide-conjugated antibodies, and subsequent proximity dependent enzymatic reactions involving the attached DNA reporter sequences. In paper I, a solid-phase proximity ligation assay (SP-PLA) was applied to detect synthetic and endogenous amyloid beta protofibrils. The SP-PLA provided better sensitivity and increased dynamic range than a traditional enzyme-linked immunosorbent assay (ELISA). In paper II, in situ PLA was applied to investigate the correlation between MARK2-dependent phosphorylation of tau and Alzheimer’s disease. Greater numbers of MARK2-tau interactions and of phosphorylated tau proteins were observed in brain tissues from Alzheimer’s patients than in healthy controls. In paper III, a multiplex SP-PLA was applied to identify protein biomarker candidates in amyotrophic lateral sclerosis (ALS) disease and in the analgesic mechanism of spinal cord stimulation (SCS). Among 47 proteins in human cerebrospinal fluid (CSF) samples, four were found at significantly lower concentrations (p-values < 0.001) in the samples from ALS patients compared to those from healthy controls (follistatin, IL-1α, IL-1β, and KLK5). No significant changes of the analyzed proteins were found in the CSF samples of neuropathic pain patients in   the stimulated vs. non-stimulated condition using SCS. In paper IV, a new technology termed the proximity barcoding assay (PBA) was developed to profile individual protein complexes. The performance of PBA was demonstrated on artificially assembled streptavidin-biotin oligonucleotide complexes. PBA was also proven to be capable of profiling transcriptional pre-initiation complexes from nuclear extract of a hepatic cell line.

Skin sensitization resulting for allergic contact dermatitis (ACD) is an occupational and environmental health issue. The allergic hazard for workers and consumers is a serious problem for individuals, employers and marketing certain products. Consequently, it is necessary to accurately identify chemicals skin sensitization potential. According to the new EU chemical regulation REACH (Registration, Evaluation, Authorization and Restriction of Chemicals), information of skin sensitization of chemicals manufactured or imported at or above 1 ton/year should be available. Currently, valid approaches assessing skin sensitization rely on animal testing, such as local lymph node assay (LLNA). However, it now ultimately eliminates using animals for this purpose. Based on the fact that a key step in the skin sensitization process is formatting a covalent adduct between allergic sensitizers and proteins and/or peptides in skin, a lot of additional approaches are proposed and developed for replacing or reducing animal used. In this research, three bioassays, 24 h growth inhibition toward Tetrahymena pyriformis, long term (24 h) and short term (30 min) bacterial toxicity (to Vibrio fischeri), and a kinetic glutathione chemoassay are applied for predicting the organic chemicals’ skin sensitization potential. The major results and conclusions obtained are listed as follows: 1. Toxicity enhancement (Te) of 55 chemicals comprising different sensitization potencies were determined and compared with their narcotic toxicity to predict their skin sensitization. Three linear regressions yielded for all allergic sensitizer without nonsensitizers for each bioassay. The linear regressions are improved after classifying sensitizers into five different reaction mechanistic domains. Correspondingly, five different slopes from various reaction mechanisms indicate a decreased sensitivity of toxicity enhancement to skin sensitization potential with order SNAr > SN2 > acylation ≈ Schiff base > aromatic Michael addition. Based on the fact that a key step in the skin sensitization process is forming a covalent adduct between allergic sensitizers and proteins and/or peptides, Te > 10 as a threshold is applied to discriminate these allergic sensitizers, with 100% accuracy for strong (with extreme) and weaker sensitizers, up to 72% accuracy for moderate sensitizers and less than 69% accuracy for nonsensitizers. Compared with these bioassays, a decreasing order of sensitivities is 24 h growth inhibition (Tetrahymena pyriformis) > 24 h growth inhibition (Vibrio fischeri) > 30 min bioluminescence inhibition (Vibrio fischeri). These three bioassays are useful tools for screening sensitization potency of allergic chemicals, and the toxicity enhancement (Te) can be used to discriminate sensitizers from weak or nonsensitizers. However, in this context we should separate aromatic from aliphatic Mas (Michael acceptors). Moreover, metabolic biotransformation should be considered during predicting nonsensitizers’ skin sensitization. 2. Chemical reactivity of selected 55 compounds measuring through kinetic glutathione chemoassay applies to predict their skin sensitization. This chemoassay confirms the fact that the key step of sensitizers eliciting skin sensitization is formatting a covalent adduct between sensitizers and skin proteins or peptides. The chemical reactivity of tested sensitizers strongly relates with their sensitization potential, with strong (extreme) sensitizers presenting the highest reactivity as followed with moderate sensitizers, weak sensitizers as well as nonsensitizers. Moreover, an integrated platform of this chemoassay data and three bioassays data is performed, and this performance shows good sensitivity for monitoring skin sensitization potency, with more rational accuracy for each sensitizing classifications. 3. Thiol reactivity (kGSH) as well as toxicity enhancement (Te) of additional 21 aliphatic α,β-unsaturated compounds are determined for predicting their skin sensitization potential. The linear regressions of skin sensitization versus thiol reactivity and skin sensitization versus toxicity enhancement are significantly improved after classifying these 21 compounds to four chemical subgroups (acrylates, other esters, ketones and aldehydes). Thiol reactivity of these subgroups presented different sensitivity to skin sensitization, with a decreasing order as acrylates (－2.05) > other esters (－1.26) > ketones (－0.43) > aldehydes (－0.21). Moreover, thiol reactivity is confirmed to be a more sensitive tool for predicting skin sensitization, compared with toxicity enhancement. Although the datasets are probably too small to give a definite decision, hydrophobicity reveals contribution to skin sensitization for aliphatic MAs, which is different with literature report. This study suggests that aliphatic MAs should be treated separately into different chemical subgroups for analysis, and their skin sensitization potency can be predicted using kinetic glutathione chemoassay as well as toxicity enhancement bioassay.

Ilex paraguariensis Saint Hilaire (Aquifoliaceae), popularly known as yerba-mate, is a native species of South America and has its area of natural occurrence restricted to Brazil, Paraguay and Argentina. It is a product that still has a lot to be improved, mainly, in terms of its industrial uses, in international level. Antioxidants are composts that work as blockers of the oxide-reducers processes unchained by the free radicals, impeding the damages generated by them, being widely used in food, medicines and cosmetics, and recently, they are also being used in antioxidant therapies in diseases where free radicals are implicated. This work has as objective to evaluate the antioxidant capacity of the product yerba-mate chimarrão type, using samples of the RS and SC, in the months june, july and august, through biological and chemical assays. The biological study was accomplished in eucariotics cells of the yeast Saccharomyces cerevisiae treated with yerba-mate samples in presence of the apomorphine and paraquat dichloride agents during the aerobic metabolism (cellular multiplication). The obtained results indicate that the antioxidant capacity of the samples varies significantly in function of the type and concentration of the stressor agents, the concentrations of the samples, the mate type (native or cultivated) and of the producer state (RS and SC). Between the states, RS state presented higher antioxidant effect in vivo, where reforested mate were more efficient as cellular protectors, the opposite happened in SC state. The chemical evaluation was based on the determination of the Total Antioxidant Activity (Hydrophilic and Lipophilic), where the capacity of the sample in to sequestrate the ABTS radical, the Reducing Power, where the iron ion produced in the redox reaction forms a colored product when it reacts with the DPPH radical, with maximum absorption to 525 nm, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical sequestrating effect, based on the capacity of the antioxidant in donating hydrogen for the DPPH radical provoking the sweeping of it of the middle of reaction, modifying the color of the solution were verified. The found values in the analyses in vitro show that the antioxidant capacity in the majority of the samples varied significantly in function of the producer state and of the mate type in the three months of analysis (June, July and August). Hydrophilic Antioxidant Activity was higher in August for all the samples, varying between 0,1336-0,5627 mM equivalent of Trolox, for Lipophilic there was a higher variation among the months of analysis, with the values being between 4,9516-27,6685 mM equivalent of Trolox. For the other chemical analyses, all the samples showed higher antioxidant activity in June, among 4,4373-12,4082 mM equivalent of quercetin to Reducer Power and 4,0221-11,1393 mM equivalent of Trolox the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical sequestrating effect. The results were obtained through the construction of standard curves, treated statisticlly through variance analysis (ANOVA) and Tukey´s post test using Microsoft Excel software. All tests were performed in triplicate. It was concluded that the yerba-mate, ingested in chimarrão form, really has a great antioxidant effect when compared to other natural products with already proven action, in chemical and biological systems, and that this property can be explored seeking the diversification in the consumption ways, fact that would facilitate the expansion of the culture in the country. / Ilex paraguariensis Saint Hilaire (Aquifoliaceae), conhecida popularmente como erva-mate, é uma espécie nativa da América do Sul e tem sua área de ocorrência natural restrita ao Brasil, Paraguai e Argentina. É um produto que ainda tem muito a ser melhorado, principalmente, em termos dos seus usos industriais, a nível internacional. Antioxidantes são compostos que funcionam como bloqueadores dos processos óxido-redutivos desencadeados pelos radicais livres, impedindo os danos gerados por eles, sendo largamente empregados em alimentos, medicamentos e cosméticos, e recentemente, estão sendo usados também em terapias antioxidantes em doenças onde radicais livres estão implicados. Este trabalho tem como objetivo avaliar a capacidade antioxidante do produto erva-mate tipo chimarrão utilizando amostras provenientes do estado do RS e SC, nos meses de junho, julho e agosto, através de ensaios biológicos e químicos. O estudo biológico foi realizado em células eucarióticas da levedura Saccharomyces cerevisiae tratada com amostras de erva-mate em presença dos agentes estressores apomorfina e paraquat durante o metabolismo aeróbico (multiplicação celular). Os resultados obtidos indicam que a capacidade antioxidante das amostras varia significativamente em função do tipo e concentração do agente estressor, das concentrações das amostras, do tipo de erval (nativo ou reflorestado) e do estado produtor (RS e SC). Entre os estados, o RS apresentou maior efeito antioxidante in vivo, onde ervais reflorestados foram mais eficientes como protetores celulares, o contrário ocorreu no estado de SC. A avaliação química se baseou na determinação da Atividade Antioxidante Total (Hidrofílica e Lipofílica), onde se verificou a capacidade da amostra em seqüestrar o radical ABTS, do Poder Redutor, onde o íon ferro produzido na reação redox forma um produto colorido quando reage com o radical DPPH, com absorção máxima à 525 nm, e do Efeito Seqüestrante de Radicais DPPH, baseada na capacidade do antioxidante em doar hidrogênio para o radical DPPH provocando a varredura deste do meio de reação, modificando a cor da solução. Os valores encontrados nas análises in vitro mostram que a capacidade antioxidante da maioria das amostras variou significativamente em função do estado produtor e do tipo de erval nos três meses de análise (junho, julho e agosto). A Atividade Antioxidante Hidrofílica foi maior em agosto para todas as amostras, variando entre 0,1336-0,5627 mM Equivalentes de Trolox, para a Lipofílica houve grande variação entre os meses de análise, com os valores ficando entre 4,9516-27,6685 µM Equivalentes de Trolox. Para as demais análises químicas, todas as amostras mostraram maior atividade antioxidante em junho, entre 4,4373-12,4082 mM Equivalentes de Quercetina para Poder Redutor e 4,0221-11,1393 mM Equivalentes de Trolox para Efeito Seqüestrante de Radicais DPPH. Os resultados foram obtidos através da construção de curvas padrão, tratados estatisticamente através de análise de variância (ANOVA) e pós-Teste de Tukey utilizando o programa Microsoft Excel. Todos os testes foram realizados em triplicata. Concluiu-se que a erva-mate, ingerida na forma de chimarrão, possui realmente um ótimo efeito antioxidante quando comparado à outros produtos naturais de ação já comprovada, em sistemas químicos e biológicos, e que esta propriedade pode ser explorada visando a diversificação das formas de consumo, o que facilitaria a expansão da cultura pelo país.