Rozprawy doktorskie na temat „Molecular Modifications”

The work described in this thesis has been mainly focused on the study of a key molecule involved in blood vessel formation, the tyrosine kinase receptor VEGFR2. Considering that VEGFR2 biology should be tighly regulated to allow proper blood vessel formation and maintenance, we investigated two different mechanims influencing VEGFR2 activity: post translational modification and receptor complex formation. Since VEGFR2 biology is governed through protein modication, mainly phosphorylation, we decided to investigate the possible role of acetylation in VEGFR2 activity. Combining biochemical and proteomic studies, we showed that VEGFR2 is modified by acetylation. Starting from this observation, we further investigated the impact of VEGFR2 acetylation on protein stability and phosphorylation in response to ligand. These findings are of particular interest, since, to our knowledge, this is the first report that a tyrosine kinase receptor might be regulated by acetylation. Additionally, we decided to elucidate the interaction of VEGFR2 with its coreceptor Neuropilin1, with particular attention to the Neuropilin1 molecule, by taking advantage of the FRET imaging technique. Collectively, our work characterizes VEGFR2-Neuropilin1 and Neuropilin1-Neuropilin1 complex formation in response to VEGFs and SEMA3A. Altough we do not provide direct evidence for Neuropilin1 direct signalling, our data suggest that Neuropilin1 oligomer formation might be a key step in Neuropilin1 biology.

Includes bibliographical references (leaves 61-84).<br>The circadian system has a regulatory role in almost all aspects of a plant's life. In Arabidopsis thaliana, almost 36% of the genome has been shown to be circadianly regulated and many genes that are circadianly regulated have been shown to be light responsive or involved in light responses. Rhythmic histone acetylation has been demonstrated in the promoter of TIMING OF CAB EXPRESSION1 (TOC1). Here, I used semi-quantitative Reverse Transcriptase Polymerase Chain Reaction (semi-quantitative RT -PCR) to investigate which enzymes are involved in the rhythmic expression of TOC1. I also determined whether loss-of-function histone acetylation and methylation mutants could affect the overall functioning of the circadian oscillator by measuring their circadian leaf movement and delayed fluorescence (DF) rhythms. GCN5/ HAG1 mutant plants (gcn5) exhibited erratic TOC1 expression in both constant dark (DD) and constant light (LL) conditions. Although TOC1 expression appeared to be rhythmic in both DD and LL conditions, the waveform of the rhythm was altered in TATA-binding protein associated factor 1 (taf1) mutants. This suggested that TAF1 and GCN5 might play different roles in the rhythmic histone acetylation affecting TOC1 expression. DF data and leaf movement data indicated that both TAF1 and GCN5 might play a role in the overall functioning of the A. thaliana circadian clock. Arrhythmic TOC1 expression and DF was observed in histone deacetylase 1 (hd1) mutants, suggesting that HD1 is not only involved in the rhythmic histone deacetylation affecting TOC1 expression but in the overall functioning of the circadian clock. Semi-quantitative RTPCR, DF and leaf movement studies demonstrated that CURLY LEAF (CLF), a histone methylase is involved in both the histone methylation affecting TOC1 expression and in the overall functioning of the A. thaliana circadian clock.

Glasses are amorphous materials which do not exhibit the long-range order or periodicity found in crystalline solids. A glass is formed by cooling a liquid at a sufficient rate such that crystallisation can be avoided. The structural disorder of glasses give them unique properties which make them suitable for a wide range of industrial, pharmaceutical or biological applications. Glass-forming materials generally exhibit several characteristic mechanisms of molecular motion. The physical origins and interrelation between these mechanisms are not well understood. In order to address this, detailed investigations of how glass-transition related dynamics are affected by systematic modification of the molecular structure are needed. This thesis concerns the measurement of the glass-forming properties of three series of molecular glass-formers. These series are comprised of samples which vary systematically in their structure: an alkylbenzene series involving the systematic variation of the length of an alkyl-tail attached to a phenyl-ring and two series involving the successive oligomerisation of styrene and alpha-methylstyrene. The glass forming properties of these series were analysed using Broadband Dielectric Spectroscopy (BDS) and Differential Scanning Calorimetry (DSC). Thermogravimetric Analysis was also employed in order to optimise the sample preparation procedure. The work in this thesis identifies and characterises the detailed molecular weight dependent behaviour of several key relaxation mechanisms in the glassy and supercooled state of three different glass-forming systems. Strong similarities between the relaxation behaviour of the two polymeric and the alkyl chain modified benzene series were found. This demonstrates that much of the observed phenomenology is remarkably general and the work forms a basis for developments of models to address the glass-transition and glassy behaviour. Moreover, it is demonstrated that the glass transition in the three different series of samples behave in a highly similar manner with regards to the system molecular weight and strong support is found for a link between the primary structural relaxation that exists in the supercooled state and the secondary relaxation mechanisms that persist within the glassy state.

Transfer RNA from all organisms has modified nucleosides and position 34 (the wobble position) is one of the most extensively modified positions. Some wobble nucleoside modifications restrict codon choice (e.g. 5-methylaminomethyl-2-thiouridine, mnm5s2U) while some extend the decoding capacity (e.g. uridine-5-oxyacetic acid, cmo5U). In this thesis the influence of wobble nucleoside modification on cell physiology and translation efficiency and accuracy is described. A mutant proL tRNA (proL207) was isolated that had an unmodified adenosine in the wobble position. Surprisingly, the proL207 mutant grows normally and is efficiently selected at the non-complementary CCC codon. The explanation of how an A34 containing tRNA can read CCC codon could be that a protonated A can form a base pair with C. cmo5U (uridine-5-oxyacetic acid) is present in the wobble position of five tRNA species in S.enterica. Two genes (cmoA and cmoB) have been identified that are involved in the synthetic pathway of cmo5U. Mutants were constructed in alanine, valine, proline, and threonine codon boxes which left only a cmo5U containing tRNA present in the cell. The influence of cmo5U on growth or on A site selection rates of the ternary complex was found to be tRNA dependent. During the study of the frameshift suppressor sufY of the hisC3737 frameshift mutation, a dominant mutation was found in YbbB protein, a selenouridine synthetase. The frameshifting occurs at CCC-CAA codon contexts and is specific for CAA codons, which are read by tRNAGlncmnm5s2UUG . The sufY204 mutation is a dominant mutation resulting in a change from Gly67 to Glu67 in the YbbB protein, and mediates the synthesis of several novel modified nucleosides/nucleotides (UKs) with unknown structure. The synthesis of these UKs is connected to the synthesis of cmnm5s2U34. The presence of UK on tRNAGlnU*UG reduced aminoacylation and therefore might account for the slow entry at CAA codons which could result in +1 frameshifting by P site tRNA. The selenourdine synthetase activity is not required for the synthesis of UKs. We hypothesize that an intrinsic activity that is low in the wild type protein has been elevated by the single amino acid substitution and results in the synthesis of UKs.

Cancer was reported as the second leading cause of death in 2018 by the World Health Organisation. Cancer is defined as the set of diseases that proceeds in multiple phases generating a transformation lead by an accumulation of genetic (mutations, copy number), epigenetic (CpG methylation, histone modifications), and epitranscriptomic alterations. Lung cancer is the primary cause of cancer-related deaths. It is commonly diagnosed at advanced stages, leading to a 5-year survival of about 21%. Lung cancer is historically subdivided according to resection specimens in small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). SCLC is an epithelial tumour with neuroendocrine features representing 10-15% of all lung tumours, and the most lethal and aggressive subtype of lung cancer. Epitranscriptomics is the field of study of RNA modifications. RNA modifications are inserted in transcripts derived from both coding and non-coding genes. These modifications are added to ribonucleotide residues on the purine/pyrimidine ring or ribose, and a significant number of enzymes regulates their dynamic. The role of these modifications ranges from provide stability, to be involved in export, maturation, splicing, folding and function of the RNA. Transfer RNAs (tRNAs) tRNA play a fundamental role in protein biosynthesis as an adaptor molecule acting as a biological link between mRNA and protein sequences on the ribosome during the translation process. Transfer RNAs show the highest density of modifications, specifically the anticodon‐loop region is a hotspot of highly diverse modifications. TRIT1 gene encodes the enzyme tRNA-isopentenyltransferase-1 that transfers an isopentenyl group to form the N6‐isopentenyladenosine (i6A) at position 37 of the cytoplasmic tRNA[Ser]Sec and tRNASer(HGA), and mitochondrial (mt) tRNACys(GCA), tRNATyr(GUA), tRNATrp, tRNASer(UGA), and tRNAPhe(GAA). tRNA[Ser]Sec carries the selenocysteine amino acid that is incorporated in 25 proteins in humans called selenoproteins. We investigated genetic and epigenetic alterations that would affect transfer RNAs modifications in cancer. After data-mining the copy number and DNA methylation data for tRNA modifier enzymes in about 1000 cell lines, we identified an outstanding TRIT1 copy number amplification restricted only to small cell lung cancer (SCLC) cell lines (11 of 60). Thus, this was the starting point of an exhaustive study. We focused on evaluating the implication of this event in tumorigenesis, as well as its potential as a drug target candidate. The TRIT1 gene amplification in cancer cell lines was associated to a significant increase at transcriptional and protein levels. To elucidate the functional role of TRIT1 amplification in SCLC, we assessed the consequences of decrease the expression of TRIT1 using the shRNA approach in the TRIT1-amplified DMS-273 SCLC cell line. The level of i6A decreased upon decreasing TRIT1 expression. In vitro assays did not provide keys to elucidate the role of TRIT1 in cancer, and we decided to perform in vivo assays in mice. We observed a significant reduction in tumour growth upon TRIT1 knockdown, providing robust evidence of the impact of TRIT1 amplification in cancer. Moreover, we carried out massive RNA sequencing of the loss-of-function model in the DMS-273 cell line. After computational analysis, 4510 differential expressed mRNA were identified. Notably, 3409 (75.9%) of these mRNAs were found downregulated upon TRIT1 depletion reflecting the significant impact of i6A hypomodification. Gene set enrichment analysis from the downregulated genes showed “regulation of cell differentiation” as the most enriched process upon TRIT1 depletion. Considering that therapeutic options in SCLC are limited, we performed an exhaustive revision of the literature and a computational approach to identify drugs affecting TRIT1-amplified SCLC. The gene amplification-associated overexpression of TRIT1 confers sensitivity to arsenic trioxide and dimethyloxalylglycine (DMOG), as shown in vitro cellular assays and in vivo experiments in mice. Finally, TRIT1 gene amplification was identified in about 10-15% of SCLC primary samples.<br>Las modificaciones del ARN son claves en multitud de procesos celulares que al verse alteradas pueden participar en el proceso de tumorogénesis. En la presente tesis se aborda el efecto de estas alteraciones implicadas en la transformación celular en a las moléculas de los ARN de transferencia (ARNt). Se identificó la amplificación génica de TRIT1 exclusivamente en líneas celulares de cáncer de pulmón microcítico (SCLC). La amplificación de TRIT1 también fue detectada en pacientes de SCLC. La enzima TRIT1 que se encarga de introducir la modificación i6A la posición A37 de ciertos ARNt. La amplificación de TRIT1 lleva a un aumento de su expresión a nivel transcripcional y traduccional. Se generó un modelo celular de pérdida de expresión empleando una línea celular de SCLC con amplificación de TRIT1, y se realizaron ensayos in vitro. También se evaluaron las consecuencias funcionales in vivo mediante la inyección de las células en ratón, se observó una reducción del crecimiento tumoral en los tumores derivados de las células con pérdida de expresión de TRIT1. El análisis transcriptómico del modelo celular, mediante secuenciación masiva de ARN (RNA-seq), mostro una desregulación de procesos biológicos relacionados con la diferenciación celular debido a la amplificación de TRIT1. Este hallazgo condujo a la identificación del trióxido de arsénico como un posible candidato como tratamiento para SCLC por su acción en la diferenciación celular. Ensayos in vitro e in vivo indican que la amplificación de TRIT1 confiere sensibilidad al trióxido de arsénico, proponiendo un nuevo candidato terapéutico para el tratamiento de una enfermedad con tan pocas opciones terapéuticas como es SCLC.

Thesis (Ph.D.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.<br>Vascular endothelial growth factor receptor-2 (VEGFR-2) is an endothelial cell receptor tyrosine kinase (RTK) whose activity is an obligate requirement for both normal development and pathological angiogenesis. A critical post-translational modification (PTM) of RTKs such as VEGFR-2 is tyrosine phosphorylation, which regulates these proteins at multiple levels including, tyrosine kinase activation, signaling, trafficking, and degradation. Similarly, growing evidence now suggests that protein methylation is another important type of PTM that plays a role in protein-protein interaction and signal transduction. In an effort to explore the possibility that methylation plays a role in regulation of VEGFR-2 function, we have employed mass spectrometry analysis coupled with pharmacological inhibitors of the methylation pathway. Our analysis revealed the presence of five methylated residues, three lysine and two arginine. Particularly, methylation of lysine 1041 (K1041), which is proximal to the conserved activation loop of the kinase domain, significantly contributed to VEGFR-2 kinase activation. Mutation of K1041 to multiple different amino acids rendered VEGFR-2 inactive and inhibited the activation of key downstream signaling proteins. Moreover, these mutations reduced VEGFR-2 mediated cell proliferation and capillary tube formation. Single mutations of R817, K856, K861 and R1115 yielded no apparent effect on tyrosine phosphorylation of VEGFR-2, however compounding the methyl deficiencies with triple and quadruple mutations markedly weakened tyrosine phosphorylation and the ligand-mediated downregulation of VEGFR-2. Furthermore, treatment of endothelial cells with global methylation inhibitors including adenosine dialdehyde (AdOx) and 3-deazaneplanocin A (DZNep) decreased ligand mediated tyrosine phosphorylation of VEGFR-2. The study presented here provides evidence that arginine and lysine methylation of VEGFR-2 through both combinatorial and non-combinatorial mechanisms regulate VEGFR-2 phosphorylation and function. This study also demonstrates that RNF121, an endoplasmic reticulum (ER) resident ubiquitin E3 ligase, binds to nascent VEGFR-2 protein and controls the abundance of cell surface VEGFR-2. Taken together, our data describes a novel role for arginine and lysine methylation in the regulation of VEGFR-2 functions and identifies a link between RNF121 ubiquitin E3 ligase and cell surface expression of VEGFR-2.

The attention around selective recognition of G-Quadruplex has steadily grown during the past 10 years. G-4 are non-canonical DNA structures which can be generated from the self-assembling of guanine rich oligonucleotides. Such an interest is justified by the fact that it is widely recognized that the above structures could act as regulators of biological processes in vivo. In fact, there are several examples of G-Quadruplexes stabilization, by small molecules or engineered antibodies, which is translated into a strong influence of the biological process in which the oligonucleotide is involved. The inhibition of telomerase enzyme as well as the gene transcriptional alteration induced by the stabilization of such structures, represent the most important applications, together with the more recently studied effect on translation mediated by G-4 RNA structures. This makes G-Quadruplex a real therapeutic target useful for the developing of new antitumor drugs. The aim of this work is to develop a new generation of G-4 ligands capable to undergo alkylation triggered by biocompatible protocols. The key aspect of this idea is to regulate the alkylation process, exploiting mild chemical or physical modifications, in order to temporally separate it from the reversible recognition. These molecules would therefore act as molecular devices capable of pre-concentration onto the target and stabilizing the complex through non covalent interactions. These ligands after activation of the masked electrophile are capable to generate the alkylating specie and therefore to achieve a strong covalent interaction. These features would induce an irreversible damage which would not be repaired by the common cellular process, enhancing the effectively drug potency in biological response terms. The basic idea is to exploit molecular recognition properties of already described ligands and tethering to them a masked electrophile. The releasing of the alkylating specie will be achieved exploiting physical or chemical triggers. Such molecules would represent a brand new class of irreversible G-4 ligands which has never been conceived before. The common features that such a class of ligands must have are: 1) A wide electron poor and flat surface, which confers molecular recognition properties, exploiting superficial π stacking interactions with the biological target. 2) A precursor of an alkylating specie, triggerable by mild physical and chemical activation protocols, possibly exploitable under physiological conditions 3) A moiety which can be easily modified through substrate interactions or chemical activation. Such moiety will represent the trigger of the alkylating reactivity. In more details, we had focused our attention on the chemical functionalization of Naphthalimide (NI) and Naphthalendiimide (NDI) derivatives, which both are good G-4 binders according to literature. Quinone Methide precurors (QM) electrophiles have been tethered to the above molecules. QM based alkylating species are particularly suitable for this project because they can be generated from very stable precursors through several biocompatible activation protocols. Moreover, their reactivity could be tuned changing the electronic nature of the phenolic precursor. In this PhD thesis we describe the synthesis, the reactivity and the study concerning the interaction of the synthesized molecules with oligonucleotides capable of G-4 folding. Particular emphasis will be given to the consequences induced by the alkylation damage with potentially achievable application both therapeutic and diagnostic. Moreover, we will discuss a parallel project developed during a period spent in the Cambridge University in Prof. Balasubramanian’s reaserch group.<br>Nell’ultimo decennio un sempre più crescente interesse è stato rivolto nei confronti del riconoscimento selettivo dei quartetti di guanina (G-quadruplex), strutture supramolecolari in grado di auto-assemblarsi in condizioni fisiologiche da oligonucleotidi ricchi di residui guaninici. La ragione di ciò risiede nel fatto che tali strutture sembrano agire come regolatori di processi a livello cellulare. Infatti, esistono svariati esempi in cui, soprattutto in vitro, molecole o anticorpi in grado di riconoscere e stabilizzare quartetti di guanina influenzino drasticamente il processo biologico in cui l’oligonucleotide stesso è implicato. L’inibizione indiretta della telomerasi e gli studi dell’effetto sulla trascrizione di oncogeni ne rappresentano le applicazioni più importati, assieme ai più recenti effetti sulla traduzione di RNA. Ciò rende G-quadruplex un vero e proprio target terapeutico per lo sviluppo di nuove terapie antitumorali. Questo lavoro nasce con lo scopo di creare una nuova generazione di leganti di G-4 che manifestino proprietà alchilanti attivabili mediante protocolli biocompatibili. Proprietà alchilanti non intrinseche, ma attivabili attraverso modifiche chimiche e fisiche, permetterebbero un controllo temporale del processo di alchilazione. Tali molecole agirebbero pertanto da veri e propri dispositivi molecolari preconcentrandosi sul target e stabilizzando il complesso attraverso interazioni non covalenti per poi, mediante attivazione, generare la specie alchilante così da ancorare fortemente la molecola all’oligonucleotide. Queste caratteristiche renderebbero il danno indotto irreversibile o non riparabile dai comuni processi cellulari, aumentando notevolmente l’efficacia di azione in termini di effetti farmaco-biologici. L’idea pertanto è quella di sfruttare le proprietà di riconoscimento di alcuni tra i leganti noti in letteratura equipaggiandoli, però, con una specie alchilante silente, il cui rilascio può essere controllato temporalmente mediante azione fisica o chimica. Questo nuovo tipo di molecole rappresenterebbe pertanto una classe di leganti irreversibili di G-4, mai progettata in precedenza. Per far ciò la molecola da sintetizzare deve possedere: 1) una superficie aromatica estesa ed elettron-povera che conferisca le proprietà di riconoscimento molecolare attraverso interazioni di π stacking con il target biologico. 2) un precursore di una specie alchilante che presenti una scarsa o assente reattività intrinseca modulabile mediante attivazione, possibilmente compatibile con condizioni fisiologiche. 3) una porzione molecolare facilmente modificabile per interazioni con il substrato o per attivazione chimica, che funga da “grilletto” della reattività del precursore alchilante. Nella fattispecie ci siamo concentrati sulla derivatizzazione di strutture, che dalla letteratura risultano dei buoni leganti di G-4, come Naftalendiimidi (NDI) o Naftalimmidi (NI), variamente sostituite con dei precursori di alchilanti tipo chinone metide (QM). Questi ultimi risultano particolarmente adatti a questo scopo in quanto posso essere generati da precursori molto stabili, attraverso dei protocolli di attivazione biocompatibili. Soprattutto risultano elettrofili la cui reattività è modulabile variando la natura elettronica del precursore stesso. In questo lavoro di tesi descriviamo la sintesi, la reattività e gli studi di interazione con oligonucleotidi ripegabili a strutture tipo G-4, delle molecole progettate e preparate nel corso del dottorato di ricerca. Particolare enfasi verrà posta sull’effetto indotto dal danno da alchilazione osservato e sulle potenziali applicazioni sia terapeutiche che diagnostiche. Inoltre descriveremo brevemente un progetto parallelo svolto durante il periodo trascorso all’Università di Cambridge, presso il gruppo di ricerca del Prof. Balasubramanian.

<p> The molecular gatekeepers of nearly all gene expression in living cells are the proteins that function in the process of transcription. Transcription occurs when a cell must respond to a signal. These signals can be in the form of metabolic responses, signals for growth or differentiation, signals to defend against stress or pathogenic invasion, to name a few. The fundamentals of transcription have been extensively studied in bacterial systems and model organisms, but technical limitations have hindered their studies in mammalian and human systems. Recent developments in mass spectrometric methodologies, next-generation sequencing and techniques to study difficult-to-detect post-translational protein modifications are extensively reviewed here to highlight an important regulatory network through which gene expression is regulated. In addition, I present two vignettes: the first, a study of the regulatory mechanisms of monomethylation of the HIV-1 Tat protein in regulating HIV-1 gene expression and latency; the second, a study investigating the role of acetylation in regulating RNA Polymerase II protein modifications and gene expression in mammalian systems. Together, these studies combine new mass spectrometric techniques, modification-specific antibodies, protein purification methods, and next generation sequencing to better understand the role of these modifications in regulating the transcriptional response in mammalian systems. These findings can be applied to better understand mechanisms that regulate HIV-1 viral latency, along with fundamentally shifting the field of mammalian transcription by pinpointing unique modes of regulation only found in higher eukaryotes relevant to HIV-1 infection and cancer.</p><p>

Oxidative stress, generated by excessive reactive oxygen species (ROS) and/or decrease in antioxidant defences, is associated with male infertility. A consequence of oxidative stress is the generation of excessive redox dependent protein modifications, such as tyrosine nitration (Nitro-Y) and S-glutathionylation (GSS-R). We hypothesize that excessive Nitro-Y and GSS-R levels impair sperm motility and capacitation (process by which sperm gain fertilizing ability). The aims of this study were to determine: 1) the effect of different ROS on motility, capacitation and the production of Nitro-Y and GSS-R in human spermatozoa and 2) the subcellular localization of Nitro-Y and GSS-R modified proteins. Percoll washed spermatozoa from healthy donors were incubated for 30 minutes at 37oC with increasing concentrations of either hydrogen peroxide (H2O2), tert−butyl hydroperoxide (tBHP), or DaNONOate (a nitric oxide (NO•) donor). Modified sperm proteins were analysed using SDS-PAGE immunoblotting and immunocytochemistry. Levels of GSS-R modified proteins increased dose dependently after exposure to H2O2 and tBHP (0.1 to 1 mM), and were localised mainly in the cytosolic and Triton-soluble fractions of the spermatozoa. Levels of Nitro-Y modified proteins increased dose dependently after exposure to DaNONOate (0.1 to 1 mM), and were mainly localized in the Triton-insoluble fraction. ROS-treated spermatozoa showed a dose dependent decrease of motility and had tyrosine phosphorylation and acrosome reaction levels similarly to that of non-capacitated spermatozoa following incubation under capacitating conditions. These results suggest that excessive Nitro-Y and GSS-R modified sperm proteins may be a culprit for the motility and fertilization failure observed in some conditions of male infertility.In the preparation of this thesis, I participated in the experimental design, performed all experiments and analyzed the resulting data.<br>Le stress oxidatif, causé par un excès des espèces réactives de l'oxygène et/ou une diminution de la capacité anti-oxydante, est associé à une infertilité masculine. Un des conséquence du stress oxydatif est la formation de modifications, telles que la nitration des tyrosine (Nitro-Y) ou la S-glutathionylation (GSS-R) de protéines pouvant être impliquées dans la balance réduction-oxydation. Notre hypothèse est que l'excès de Nitro-Y et GSS-R induit des anomalies de la motilité et de la capacitation des spermatozoïdes. Cette étude a donc pour but de 1- Déterminer les effets des espèces réactives de l'oxygène sur la motilité, la capacitation et la production de Nitro-Y et GSS-R dans le spermatozoïde humain; 2- Localiser les protéines ayant des Nitro-Y et/ou GSS-R dans le spermatozoïde humain. Des spermatozoïdes de donneurs sains, sélectionnés après gradient de Percoll ont été incubés 30 minutes à 37oC en présence de différentes concentrations de H2O2, tert−butyl hydroperoxide (tBHP) ou de Da-NONOate (un donneur de NO•). Les protéines modifiées sont analysées par immuno-buvardage (SDS-PAGE) et immunocytochimie. Nous avons montré une augmentation dose-dépendante des protéines associées au GSS-R en présence de H202 et de tBHP (0.1 to 1 mM). Ces protéines sont localisées dans le cytosol et la fraction soluble au triton du spermatozoïde. D'autre part, nous avons montré une augmentation dose-dépendante des protéines associées au Nitro-Y en présence de DaNONOate (0.1 to 1 mM). Ces protéines sont localisées dans la fraction insoluble au triton du spermatozoïde. De plus, en présence d'espèces réactives de l'oxygène, nous avons observé, une diminution de la motilité des spermatozoïdes et un niveau de réaction acrosomique et de phosphorylation des protéines similaires à des spermatozoïdes non capacités, même après induction de la capacitation. Nos résultats suggèrent qu'un excès de protéines ayant des Nitro-Yet GSS-R peuvent être à l' origine d'anomalies de la motilité et du pouvoir fécondant des spermatozoïdes chez certains hommes infertiles. Dans la préparation de cette thèse, j'ai participé à la conception expérimentale, réalisée toutes les expériences et analysé les données résultantes.

In order to counter pathogen infection while preventing autoimmune responses, the human innate immune system must be precisely regulated to distinguish “self” from “non-self”. Pattern recognition receptors detect “non-self” pathogen RNAs and initiate antiviral signaling. Accumulated evidence suggests that host “self” RNAs contain modified nucleotides that evade or suppress immune signaling; however, the precise mechanisms are not understood. Defining these mechanisms is relevant toward understanding the biology of immunity as well as the applied use of RNAs as therapeutic molecules, where reducing ligand immunogenicity is essential. Evidence from our lab and others’ suggests that the cytosolic RNA helicase RIG-I (retinoic acid inducible gene-I) detects not only the 5’ terminus and double-stranded nature of RNA, but also the presence/absence of modified nucleotides. In the present study, we use a model RNA ligand (polyU/UC), derived from the 3’ untranslated region of the hepatitis C virus RNA, to dissect the mechanisms by which RNAs containing nucleotide modifications suppress or evade RIG-I signaling. Five assays were developed to test our hypothesis that eight different nucleotide modifications, both natural and synthetic, share a common mechanism of innate immune evasion. In vitro transcribed 5’-triphosphate polyU/UC RNA containing canonical nucleotides potently activates the RIG-I signaling pathway in transfected cells, culminating in an antiviral state. When transcribed with any of eight modified nucleotides, the polyU/UC RNA suppressed the RIG-I antiviral response. Unexpectedly, the modified nucleotides had different effects on RIG-I:RNA binding affinity, as well as RIG-I conformational change. The data suggest that multiple RIG-I evasion/suppression mechanisms associated with different modified nucleotides may have evolved to effect a common result of interrupting innate immune signaling responses to “self” RNA. Our findings hold implications for understanding the co-evolution of the innate immune response and RNA modification pathways across domains of life, as well as for defining approaches for testing the multitude of naturally occurring and synthetic nucleotides that may have utility in the design of therapeutic RNAs.<br>Medical Sciences

Background and objectives: Endothelial dysfunction and cancer are two of the important aspects of obesity-related medical complications, the prevalence of which is reaching an epidemic level worldwide. The protein kinase LKB1 has been shown to play opposite roles in these two metabolic diseases by promoting cellular senescence and inhibiting cell proliferation through regulating a series of its downstream targets. However, the molecular mechanisms wherebyLKB1 itself is regulated by its upstream molecules remains poorly understood. The major objectives of this study are to identify novel upstream regulators of LKB1 and to investigate how these upstream regulators modulate the subcellular localization and physiological functions of LKB1 by post-translational modifications. Key findings: 1. Our proteomic analysis demonstrated that LKB1 was modified by both acetylation and phosphorylation. The acetylation sites of mouseLKB1 include Lys48, Lys64and Lys312. The phosphorylation sites of mouseLKB1 include: Ser31, Thr32,Tyr36, Ser69, Thr71, Ser334and Thr336. 2. In both human embryonic kidney 293 (HEK293)cells and primary porcine aortic endothelial cells (PAECs), the nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase SirT1 attenuated the acetylation levels of LKB1,which consequently resulted in enhancedLKB1ubiquitination, thereby leading to the proteasome-mediated degradation of LKB1. 3. In primary PAECs, overexpression of SirT1 protected cells from cell cycle arrest and cellular senescence, whereas overexpression of LKB1 exhibited the opposite effects.SirT1 antagonizedLKB1-induced G1 phase arrest and cellular senescence by promoting the deacetylation and protein degradation of LKB1. 4. The in vitro phosphorylation assay and mass spectrometry analysis demonstrated that LKB1 could be phosphorylated by the Akt kinase at Ser334which was critical for the interaction between LKB1 and 14-3-3. The enhanced association between LKB1 and 14-3-3 subsequently attenuated the interaction between LKB1 and Ste20 related adaptor α(STRADα), which further promoted the nuclear accumulation of LKB1. 5. The cell proliferation and cell cycle distribution analysis of the stably-transfected MDA-MB-231 breast cancer cells demonstrated that overexpression of the LKB1 mutant S334D, which mimicked Ser334 phosphorylation and localized exclusively in the nucleus, completely lost its anti-tumor activities. On the other hand, the S334A mutation enhanced the tumor suppressive functions of LKB1. 6. Nude mice inoculated with the LKB1 S334A stably-transfected MDA-MB-231 cells exhibited delayed tumor onset, decreased tumor growth rate and tumor weight. By contrast, inoculation of nude mice with the MDA-MB-231 cells overexpressing LKB1 S334D mutation showed the opposite effects on these parameters. Conclusions: These results collectively suggest that the deacetylase SirT1 and the protein kinase Aktare the two important upstream regulators of LKB1. SirT1 prevents LKB1-induced cellular senescence and protect endothelial ageing by promoting proteasome-mediated degradation of LKB1. Akt inhibits the tumor-suppressive activity of LKB1 by enhancing the phosphorylation-dependent nuclear translocation. Further investigations on the precise mechanisms whereby SirT1 and Akt regulate LKB1 functions may help to design novel therapeutic strategies for treating obesity-related diseases, such as diabetes, cardiovascular disease and cancer.<br>published_or_final_version<br>Medicine<br>Doctoral<br>Doctor of Philosophy

Thesis (Sc. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, February 2013.<br>"February 2013." Cataloged from PDF version of thesis.<br>Includes bibliographical references (p. 151-168).<br>The thermodynamics and mass transfer kinetics of adsorption and desporption at interfaces play vital roles in chemical analysis, separation processes, and many natural phenomena. In this work, computer simulations were used to design interfacial modifications to alter the physical processes of adsorption and desorption, using two different approaches to molecular design. In the first application, the finite-temperature string method was used to elucidate the mechanism of water's evaporation at its liquid/vapor interface, with the goal of designing a soluble additive that could impede evaporation there. These simulations used the SPC/E water model, and identified a minimum free energy path for this process in terms of 10 descriptive order parameters. The measured free energy change was 7.4 kcal/mol at 298 K, in reasonable agreement with the experimental value of 6.3 kcal/mol, and the mean first-passage time was 1375 ns for a single molecule, corresponding to an evaporation coefficient of 0.25. In the observed minimum free energy process, the water molecule diffuses to the surface, and tends to rotate so that its dipole and one 0-H bond are oriented outward as it crosses the Gibbs dividing surface. As the molecule moves further outwards through the interfacial region, a local solvation shell tends to protrudes from the interface. The water molecule loses donor and acceptor hydrogen bonds, and then, with its dipole nearly normal to the interface, stops donating its remaining donor hydrogen bond. After the final, accepted hydrogen bond is broken, the water molecule is free. An analysis of reactive trajectories showed that the relative orientation of nearby water molecules, and the number of accepted hydrogen bonds, were important variables in a kinetic description of the process. In the second application, we developed an in silico screening process to design organic ligands which, when chemically bound to a solid surface, would constitute an effective adsorption for a pharmaceutically relevant mixture of reaction products. This procedure employs automated molecular dynamics simulations to evaluate potential ligands, by measuring the difference in adsorption energy of two solutes which differed by one functional group. Then, a genetic algorithm was used to iteratively improve a population of ligands through selection and reproduction steps. This procedure identified chemical designs of the surface-bound ligands that were outside the set considered using chemical intuition. The ligand designs achieved selectivity by exploiting phenyl-phenyl stacking which was sterically hindered in the case of one solution component. The ligand designs had selectivity energies of 0.8 to 1.6 kcal/mol in single-ligand, solvent-free simulations, if entropic contributions to the relative selectivity are neglected. This molecular evolution technique presents a useful method for the directed exploration of chemical space or for molecular design.<br>by Nicholas J. Musolino.<br>Sc.D.

In this thesis molecular interactions between polypeptide conjugates and protein targets were investigated. Polypeptides were derivatized with small organic molecules, peptides and oligonucleotides. New strategies were developed with the aim to increase affinities for proteins of biological interest. A 42-residue polypeptide (4-C15L8) conjugated to a small organic molecule 3,5-bis[[bis(2-pyridylmethyl)amino]methyl]benzoic acid (PP1), was shown to bind glycogen phosphorylase a (GPa) in the presence of zinc ions. Under the assumption that hydrophobic interactions dominated the binding energy, the hydrophobic residues of 4-C15L8-PP1 were systematically replaced in order to study their contribution to the affinity enhancement. The replacement of the Nle, Ile and Leu residues by Ala amino acids reduced affinities. The introduction of non-natural L-2-aminooctanoic acid (Aoc) residues into the peptide sequence enhanced the binding affinity for GPa. A decreased KD of 27nM was obtained when Nle5, Ile9 and Leu12 were replaced by Aoc residues, in comparison to the KD value of 280nM obtained for the unmodified 4-C15L8-PP1. It is evident that there are non-obvious hydrophobic binding sites on the surfaces of proteins that could be identified by introducing the more hydrophobic and conformationally flexible Aoc residues. The downsizing of the 42-mer peptide to an 11-mer and the incorporation of three Aoc residues gave rise to a KD of 550 nM, comparable to that of  4-C15L8-PP1 suggesting that bioactive peptides can be downsized by the introduction of Aoc. Aiming to improve in vivo stability, the affinity for human serum albumin (HSA) of hydrophobic, positively and negatively charged polypeptide-PP1 conjugates was evaluated. Increased hydrophobicity due to the introduction of Aoc residues did not significantly increase the affinity for HSA. No binding was observed in the case of the most negatively charged polypeptides whereas the slightly negatively and positively charged polypeptides conjugated to PP1 bound HSA with affinities that increased with the positive charge. It was found that polypeptide-PP1 conjugates target the zinc binding site of the HSA. Affinity enhancement was obtained due to the incorporation of PP1 and increased by charge to charge interactions between the positively charged amino acids of the polypeptide and the negatively charged residues of HSA, in close proximity to the HSA zinc binding site. The survival times of the peptide-PP1 conjugates in human serum were extended as a result of binding to HSA. Zn2+ ion chelating agents can be incorporated in potential peptide therapeutics with a short plasma half-life, without increasing their molecular weights.

Platelets affect vascular integrity by secreting a host of molecules that promote hemostasis and its sequela. Given its importance, it is critical to understand how platelet exocytosis is controlled. Post-translational modifications, such as phosphorylation and acylation, have been shown to affect signaling pathways and platelet function. In this dissertation, I focus on how these modifications affect the t-SNARE proteins, SNAP-23 and syntaxin-11, which are both required for platelet secretion. SNAP-23 is regulated by phosphorylation. Using a proteoliposome fusion assay, I demonstrate that purified IκB Kinase (IKK) phosphorylated SNAP-23, which increased the initial rates of SNARE-mediated liposome fusion. SNAP-23 mutants containing phosphomimetics showed enhanced initial fusion rates. These results, combined with previous work in vivo, confirm that SNAP-23 phosphorylation is involved in regulating membrane fusion, and that IKK-mediated signaling contributes to platelet exocytosis. To address the role(s) of acylation, I sought to determine how syntaxin-11 and SNAP-23 are associated with plasma membrane. Using metabolic labeling, I showed that both proteins contain thioester-linked acyl groups which turn over in resting cells. Mass spectrometry mapping showed that syntaxin-11 is modified on C275, 279, 280, 282, 283 and 285, while SNAP-23 is modified on C79, 80, 83, 85, and 87. To probe the effects of acylation, I measured ADP/ATP release from platelets treated with the acyl-transferase inhibitor, cerulenin, or the thioesterase inhibitor, palmostatin B. Cerulenin pretreatment inhibited t-SNARE acylation and platelet function while palmostatin B had no effect. Interestingly, pretreatment with palmostatin B blocked the inhibitory effects of cerulenin suggesting that maintaining the acylation state of platelet proteins is important for their function. Thus my work indicates that the enzymes controlling protein acylation could be valuable targets for modulating platelet exocytosis in vivo.

Over the past few decades, RNA has emerged as much more than just an intermediary in biology’s central dogma. RNA is now known to play a variety of catalytic, regulatory and defensive roles in living systems as demonstrated through the discoveries of ribozymes, riboswitches, microRNAs, small interfering RNAs, Piwi-interacting RNAs, small nuclear RNAs, clusters of regularly interspaced short palindromic repeat RNAs and long non-coding RNAs. In contrast to the functional diversity of RNA, the chemical diversity has remained primarily limited to canonical polyribonucleotides, the 5’ cap on mRNAs in eukaryotes, modified nucleotides and 3’-aminoacylated tRNAs. This disparity coupled with the powerful functional properties of small molecule-nucleic acid conjugates led us to speculate that novel small molecule-RNA conjugates existed in modern cells, either as evolutionary fossils or as RNAs whose functions are enabled by the small molecule moieties. We developed and applied a nuclease-based screen coupled with high-resolution liquid chromatography/mass spectrometry analysis to detect novel small molecule-RNA conjugates, broadly and sensitively. We discovered NAD-linked RNA in two types of bacteria and further characterized the small molecule and RNA in Escherichia coli. The NAD modification is found on the 5’ end of RNAs between 30 and 120 nucleotides long, and is surprisingly abundant at around 3,000 copies per cell. Subsequent experiments to characterize further NAD-linked RNA have been undertaken, including sequencing the RNAs to which NAD is attached and elucidating the biological functions of the small molecule-RNA conjugate. The development and application of a screen to detect novel nucleotide modifications that is independent of structure or biological context has the potential to increase our understanding of the functional and chemical diversity of RNA. The discovery and biological characterization of NAD-linked RNA can provide new examples of RNA biology and offer insight into the RNA world.

The N-terminal tails of histone proteins protrude from the nucleosome core and are extensively post-translationally modified. These modifications are proposed to affect many DNA-based processes such as transcription, DNA replication and repair. Post-translational modifications on histone tails do not act independently but are subject to crosstalk. One example of crosstalk is on histone H3 between lysine 14 (H3K14) and trimethylated lysine 4 (H3K4me3), a modification found at the 5’ end of most active or poised genes. In this work, Western blots and chromatin immunoprecipitation (ChIP) experiments show that different amino acid substitutions at histone H3 position 14 cause varying degrees of H3K4me3 loss, indicating that H3K14 is not essential for H3K4me3 but acts as a modulator of H3K4me3 levels. A neighbouring residue, H3P16 is also important for H3K4me3 and may operate in concert with H3K14 to control H3K4me3. These crosstalk pathways have gene-specific effects and the levels of H3K4me3 are influenced to different extents on genes that fall into functionally distinct classes. A model is proposed to explain how H3K14/H3P16 may exert these varying effects on H3K4me3 at individual genes. In addition to its ability to regulate H3K4me3, H3K14 also influences the levels of two modifications on H3K18, acetylation and monomethylation. A ChIP-sequencing experiment has shown that H3K18me1, a previously uncharacterised modification in S. cerevisiae, is widely distributed throughout the genome and correlates strongly with histone H3 levels. The potential for a functional acetyl/methyl switch at H3K18 is explored. Together, these data indicate that, with gene-specific effects, crosstalk between histone modifications may be even more complex than originally thought.

Covalent nucleotide modifications in RNAs affect numerous biological processes, and novel functions are continually being revealed even for well-known modifications. Among all RNA species, transfer RNAs (tRNAs) are highly enriched with diverse modifications, which are known to play roles in decoding and tRNA stability, charging, and cellular trafficking. However, studies of tRNA modifications have been limited in a small scale and performed by groups with different methodologies. To systematically compare the functions of a large set of noncoding RNA modifications in translational regulation, I carried out ribosome profiling in 57 budding yeast mutants lacking nonessential genes involved in tRNA modifications. Deletion mutants with enzymes known to modify the anticodon loop or non-tRNA substrates such as rRNA exhibited the most dramatic translational perturbations, including altered dwell time of ribosomes on relevant codons, and altered ribosome density in protein-coding regions or untranslated regions of specific genes. Several mutants that result in loss of tRNA modifications in locations away from the anticodon loop also exhibited altered dwell time of ribosomes on relevant codons. Translational upregulation of the nutrient-responsive transcription factor Gcn4 was observed in roughly half of the mutants, consistent with the previous studies of Gcn4 in response to numerous tRNA perturbations. This work also discovered unexpected roles for tRNA modifying enzymes in rRNA 2’-O-methylation, and in transcriptional regulation of TY retroelements. Taken together, this work revealed the importance and novel functions of tRNA modifications, and provides a rich resource for discovery of additional links between tRNA modifications and gene regulation.

CRISPR/Cas9 induced DNA breaks can be precisely repaired by cellular homology-directed repair (HDR) pathways using exogenously provided template DNA (donor). However, the full potential of precision editing is hindered in many model systems by low cutting efficiencies, low HDR efficiencies and, cytotoxicity related to Cas9 and donor DNA. In this thesis, I address these challenges and present methods that we developed to increase HDR efficiencies in multiple model organisms. In Caenorhabditis elegans, we show that by reducing toxicity high editing efficiencies can be achieved with single stranded oligonucleotide (ssODN) donors. We demonstrate that melting dsDNA donors dramatically improves the knock-in efficiencies of longer (1kb) edits. In addition, we describe 5′-terminal modifications to the donor molecules that further increase the frequency of precision editing. With our methodology a single optimally injected animal can yield more than 100 Green Fluorescent Protein (GFP) positive progeny, dramatically enhancing efficiency of genome editing. Next, we demonstrate the generality of 5′ modified donors by extending our studies to human cell cultures and mice zygotes. In mammalian models, 2′OMe-RNA modifications consistently increase HDR efficiencies by several fold over unmodified donors. Furthermore, end-modified donors exhibited a striking reduction in end-joining reactions including reduced concatemer formation and reduced direct ligation into the host genome. Our study demonstrates that HDR can be improved without inhibiting competing end-joining pathways and provides a platform to identify new chemical modifications that could further increase the potency and efficacy of precision genome editing.

In response to fluctuations in the environment, all living organisms have the ability to sense, respond and adapt to the new conditions. In budding yeast (Saccharomyces cerevisiae) there is a massive and rapid reorganization of the transcriptional program in response to a stressful situation, which is governed by different signaling pathways, transcription factors, chromatin remodelers and histone modifiers. Many examples of histone posttranslational modifications (PTM) have been associated with transcriptional activation or repression under normal growth conditions, however little is known about the role of histones in the cellular adaptive response upon stress. In this study, we systematically analyze by high throughput screens cellular growth and transcription initiation of stress-responsive genes in 569 histone point mutants upon heat and osmostress. These screens provide a novel global map of the histone residues required for cellular survival and transcriptional regulation in response to heat and osmostress. Moreover, we show that the histone residues required in response to stress depend on the type of gene and/or the type of stress. Furthermore we characterized some examples of newly identified histone marks from histones H3 and H4 involved in the transcriptional regulation upon different stress conditions.<br>Todos los organismos vivos tienen la capacidad de adaptarse a condiciones adversas en el medio en el que viven para sobrevivir. En la levadura Saccharomyces cerevisiae se produce una rápida reorganización en el patrón transcripcional en respuesta a una situación de estrés. Esta reorganización transcripcional está regulada por diferentes vías de señalización, factores de transcripción, complejos remodeladores de cromatina y enzimas modificadoras de histonas. Numerosos ejemplos de modificaciones postraduccionales en histonas han sido asociados con la activación o la represión génica en condiciones normales de crecimiento. Sin embargo, se conoce muy poco sobre el papel de las histonas en la adaptación celular frente a una situación de estrés. En este trabajo, hemos analizado de forma sistemática el crecimiento celular y el inicio de la transcripción de genes que se inducen en respuesta a estrés en 569 mutantes puntuales de histonas en condiciones de estrés térmico y osmótico. Nuestros resultados proporcionan un mapa global de los residuos de las histonas esenciales para la supervivencia celular y la activación transcripcional en respuesta a estrés térmico y osmótico. Asimismo, estos análisis revelan que existe una especificidad respecto a los residuos de las histonas que son necesarios en respuesta a estrés dependiendo del tipo de gen y/o del tipo de estrés. Además, en este trabajo hemos caracterizado algunos ejemplos de residuos en las histonas H3 y H4 que tienen un papel importante en la regulación transcripcional en respuesta a estrés.

Synopsis: Understanding the molecular mechanisms regulating cardiac cell proliferation during the embryonic, fetal and adult life is of paramount importance in view of developing innovative strategies aimed at inducing myocardial regeneration after cardiac damage. The Notch pathway plays a key role in the regulation of cardiomyocyte proliferation during mammalian embryonic life. Moreover, it is essentially involved in the cardiac regeneration process after injury in Zebrafish. Therefore, we assessed the efficacy of Notch pathway activation to sustain cardiac regeneration in a model of myocardial infarction in mice. During early postnatal life, cardiomyocytes exit the cell cycle. We demonstrated that this event is paralleled by a decrease of Notch signaling and by the establishment of a repressive chromatin environment at Notch target genes, characterized by Polycomb Group protein 2-mediated silencing. The stimulation of the Notch pathway through Adeno-associated virus-mediated gene transfer of activated Notch1 or of the soluble form of the ligand Jagged1 prolonged the capacity of cardiomyocytes to replicate, which correlated with an increased rate of Notch target gene expression and the maintenance of an open chromatin conformation at Notch target gene promoters. However, the same vectors were ineffective in stimulating cardiac repair in a model of myocardial infarction in adult mice, despite efficient transgene expression. We identified the molecular cause of the lack of action of Notch signaling stimulation in adults in the increased DNA methylation at Notch target gene promoters, which correlated with permanent switch off of the Notch pathway. Our results confirm that the Notch pathway is an important regulator of neonata adults, due to the permanent epigenetic modifications at the DNA level at Notch responsive genes l.

L’électronique organique reste un domaine de recherche prolifique grâce à la diversité de structures moléculaires accessible par la synthèse organique. Les matériaux moléculaires offrent des possibilités de mises en forme inédites comme les techniques de dépôt en solution, utilisables dans la conception de dispositifs organiques sur supports plastiques. Nos travaux de recherche mêlent électrochimie, conception et caractérisations électriques de dispositifs et mesures capteur. Ils abordent deux thématiques : l’élaboration de nouveaux transducteurs à base de polyanilines substituées et de phtalocyanines et l’étude de l’influence de la modification électrochimique de surface sur le comportement de dispositifs connus.Nous avons tout d’abord élaboré des hétérojonctions latérales polymère – phtalocyanine en utilisant les propriétés des polyanilines. De par leur géométrie, ces nouveaux dispositifs diffèrent de l’hétérojonction MSDI (Molecular Semiconductor – Doped Insulator), transducteur conductimétrique bicouche développé au laboratoire pour la détection de gaz comme l’ozone ou l’ammoniac. La caractérisation complète de la poly(2,3,5,6-tétrafluoroaniline) indique que ce polymère est peu conducteur, en comparaison de la polyaniline dont la conductivité peut être augmentée par dopage acido-basique. La présence des fluors empêche l’émergence du régime conducteur que l’on retrouve en milieu acide pour la polyaniline et la poly(2,5-diméthoxyaniline). Ces trois polymères, électrodéposés sur électrodes d’ITO interdigitées ont permis, après sublimation de la bisphtalocyanine de lutécium, de construire les hétérojonctions latérales. Le comportement électrique des différents dispositifs, étudié notamment par spectroscopie d’impédance, diffère en fonction de la nature des substituants de la polyaniline électrodéposée. Enfin, des mesures capteur ont montré la possibilité de détecter l’ammoniac en milieu humide, avec une limite de détection sub ppm.De précédents travaux sur l’élaboration de MSDI ont souligné le rôle primordial des interfaces, notamment dans le cas des n-MSDI qui présentent une hétérojonction p-n. Outre le changement de la nature chimique de la sous-couche employée, une autre manière de jouer sur les interfaces est de modifier électrochimiquement la surface des électrodes interdigitées, par réduction de sels de diazonium. Nous avons ainsi greffé différents benzènes substitués, dont certains ont conduit à la formation de multicouches comme l’ont révélée des mesures de microbalance à quartz électrochimique. Les différentes modifications de surface ont surtout joué le rôle de barrière isolante, amplifiant le comportement non linéaire des caractéristiques courant-tension des MSDI. Le greffage du 2,5-diméthoxybenzène a permis d’améliorer significativement la sensibilité à l’ammoniac de la MSDI à base d’hexadécafluorophtalocyanine de cuivre et de bisphtalocyanine de lutétium, avec une limite de détection de l’ordre de 200 ppb<br>Organic electronics remains a fruitful research field thanks to the diversity of molecular structures reachable by organic synthesis. Molecular materials offer convenient shaping processes, such as solution processing techniques, which can be used for the fabrication of organic devices on plastic substrates.Our works can be summarized as the elaboration of conductometric devices thanks to electrochemistry and the study of their electrical and sensing properties. They deal with two topics: the development of new transducers based on substituted polyanilines and phthalocyanines and the study of the influence of electrochemical modifications on the behavior of known devices.We first developped polymer - phthalocyanine lateral heterojunctions using the properties of polyanilines. Because of their geometry, these new devices differed from the MSDI heterojunction (Molecular Semiconductor - Doped Insulator), a bilayer-based conductometric transducer developed in the laboratory for the detection of gases such as ozone or ammonia. The comprehensive study of poly (2,3,5,6-tetrafluoroaniline) indicated that this material was a poor conducting polymer, compared to polyaniline whose conductivity can be increased by acid-base doping. The presence of fluorine atoms prevented the emergence of the conductive regime found in acidic medium for polyaniline and poly (2,5-dimethoxyaniline). These three polymers, electrodeposited on interdigitated ITO electrodes, allowed us, after sublimation of the lutetium bisphthalocyanine, to build lateral heterojunctions. The electrical behavior of the different devices, studied in particular by impedance spectroscopy, differed according to the nature of the substituents of the electrodeposited polyaniline. Finally, sensing measurements revealed their efficiency to detect ammonia in humid atmosphere, with a sub-ppm limit of detection.Previous works on the development of MSDI emphasized the primary role of interfaces, particularly in the case of n-MSDI that contained a p-n heterojunction. In addition to the modification of the chemical nature of the underlayer, another way to play with the interfaces is to electrochemically modify the surface of the interdigitated electrodes by reducing diazonium salts. Thus, we grafted various substituted benzenes, some of which led to the formation of multilayers as revealed by electrochemical quartz microbalance measurements. The various surface modifications mainly acted as an insulating barrier that amplified the nonlinear behavior of the current-voltage characteristics of MSDI. The grafting of 2,5-dimethoxybenzene significantly improved the ammonia sensitivity of MSDI based on copper hexadecafluorophthalocyanine and lutetium bisphthalocyanine, with a limit of detection of around 200 ppb

Mammalian hair is readily shed and transferred to persons or objects during contact; this property renders hair as one of the most ubiquitous and prevalent evidence type encountered in forensic investigations and at ancient burial sites. The durability and stability of hair ensures their survival for millennia; their status as a privileged repository of viable genetic material consolidates their value as a biological substrate. The aims of this thesis are to showcase the wealth, and breadth, of information that may be gleaned from these unique structures and address the current problem regarding the mis-identification of animal hairs. Despite the similar appearance of human and animal hairs, the expertise required to accurately interpret their respective structures requires significantly different skill sets. Chapter Two in this thesis discusses the consequences of mis-identification of hair structures due to lack of competency or adequate training in regards to hair examiners and discusses some of the myths and misconceptions associated with microscopy of hairs. Hairs are resilient structures capable of surviving for millennia as exemplified by extinct megafauna hairs; however, they are not totally immune to deleterious effects of environmental insults or biodegradation. There is a paucity of morphological data available illustrating the deleterious effects of biological agents on hairs. This void is filled through the comprehensive review of biodegradation of hair in Chapter 3 which showcases, for the first time, a collective visual catalogue of the destructive effects caused by an elite group of biological agents. These effects were evident in hairs from prehistoric, ancient and modern human and animal hairs. During the course of this study hair morphologies were observed which challenged current paradigms in relation to their genesis. The results of the present study unequivocally demonstrate that hair structures, previously characterised as genetic in nature, are due to the effects of biodegradation. Furthermore, this body of work is the first to record that morphological characteristics previously ascribed to taphonomy (post-mortem insults), also occur in hairs from the living. The implications of the interpretation of hair structures in forensic investigations are discussed in greater detail in Chapter 3. Chapter four represents the first demonstration of the advantages of adopting a multidisciplinary approach to hair examination. This chapter presents a detailed microscopical audit of extinct megafauna hair that was the remains of a larger hair sample previously consumed in more destructive analyses (molecular analyses and radio carbon dating). One of the most significant morphological finds of this work was the presence of unusual structural features, not previously recorded, that may have been central to the survival of extinct megafauna. Lastly, Chapter Five introduces the concept of Next Generation Sequencing (NGS also referred to as massive parallel sequencing) to investigate the forensic potential of human hairs on the basis of transfer of their respective bacterial ‘profiles or signatures’. Taken together, this body of work presents fresh approaches to the manner in which mammalian hairs could be processed in the future and demonstrates the benefits of multi-disciplinary approaches to their examination. Bacterial DNA profiles, derived from human hair using NGS technologies, may prove to be a valuable future addition to the forensic molecular toolkit. Furthermore, this present study challenges current paradigms regarding the interpretation of microscopic post-mortem artifacts that occur on ancient and modern mammalian hairs.

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas<br>A pele sofre modificações morfológicas e fisiológicas com o avançar do tempo que podem ser causadas por fatores extrínsecos ao organismo, tais como a exposição à radiação solar, poluição do ar e hábitos tabágicos, e fatores intrínsecos de natureza genómica, bioquímica e celular. Coletivamente, estes fatores contribuem para envelhecimento celular e organismal, incluindo o envelhecimento da pele, podendo também desempenhar um importante papel no processo de envelhecimento prematuro. Ao envelhecimento da pele induzido pela radiação solar foi atribuído um termo próprio, o fotoenvelhecimento. Clinicamente, a pele torna-se mais flácida, mais espessa, hiperpigmentada, e nas áreas mais expostas à radiação solar, como a face e as mãos, observa-se o aparecimento precoce de rugas e outras alterações cutâneas, eventualmente mais graves como o cancro de pele. A prevenção pode ser inicialmente efetuada através da utilização regular de protetor solar e vestuário adequado e, numa idade mais avançada, com cremes que contenham filtros solares e substâncias retardadoras do envelhecimento, tais como antioxidantes, ácido retinóico, entre outros. Existem, porém, outras estratégias mais invasivas de tratamento que serão também analisadas no âmbito do presente trabalho. The skin undergoes morphological and physiological changes with time that may be caused by extrinsic factors, such as exposure to sunlight, air pollution and smoking habits, and intrinsic factors of diverse kind: genomic, biochemistry and/or cellular. Collectively, these factors contribute to cellular and organismal aging including skin aging, and may also play an important role in the premature aging process. Skin aging induced by solar radiation has been assigned a proper term, photoaging. Clinically, the skin becomes more flaccid, thickened, hyperpigmented, and in areas more exposed to solar radiation, such as the face and hands, there is the early appearance of wrinkles and other alterations, which may eventually be associated with more serious skin disorders such as cancer skin. Prevention can be done initially through the regular use of sunscreen and appropriate clothing and, at a later age, with creams that contain sunscreens and aging retardant substances such as antioxidants, retinoic acid, among others. However, there are other more invasive treatment strategies that will also be examined in the context of this work.

Transfer RNA (tRNA) is the adaptor molecule responsible for bringing the correct amino acid to the ribosome during protein synthesis. tRNA contains a number of modified nucleosides, which are derivatives of the four normal nucleosides. A great variety of modifications are found in the anticodon loop, especially at the first (wobble) position of the anticodon. According to Crick’s wobble hypothesis, a uridine at the wobble position of tRNA recognize codons ending with A and G. Uridine-5-oxyacetic acid (cmo5U34), found at the wobble position of six species of tRNA in Salmonella enterica, have been predicted to expand the codon recognition of uridine to include U-ending, but not C-ending codons. To study the function of cmo5U34 we have identified two genes, cmoA and cmoB, which are required for the synthesis of cmo5U34 in tRNA. We have shown that the proline, alanine and valine tRNAs containing cmo5U34 are capable of reading codons ending with any of the four nucleotides, while the threonine tRNA is not, and the importance of having cmo5U is different for the different tRNAs. In addition, we found that cmo5U is important for efficient reading of G-ending codons, which is surprising considering the wobble hypothesis, which states that uridine should read G-ending codons. The dominant +1 frameshift suppressor sufY suppresses the hisC3737 +1 frameshift mutation. We have demonstrated that sufY induces frameshifting at CCC-CAA (Pro-Gln), when tRNAPro[cmo5UGG] occupies the P-site. sufY mutants accumulate novel modified nucleosides at the wobble position of tRNAs that should normally have (c)mnm5s2U34. The presence of an extra sidechain (C10H17) on the wobble nucleoside of tRNAGln[(c)mnm5s2U] leads to slow decoding of CAA codons, inducing a translational pause that allows the P-site peptidyl-tRNAPro[cmo5UGG] to slip into the +1 frame. We have characterized 108 independent frameshift suppressor mutants in the gene encoding tRNAPro[cmo5UGG]. The altered tRNAs are still able to read all four proline codons in the A-site, but induce frameshifts after translocation into the P-site. Some of the mutations are in regions of the tRNA that are involved in interactions with components of the P-site. We hypothesize that the ribosomal P-site keeps a “grip” of the peptidyl-tRNA to prevent loss of the reading frame.

Transforming growth factor-β (TGF-β) signaling is initiated when the ligand binds to type II and type I serine/threonine kinase receptors at the cell surface. Activated TGF-β type I receptors phosphorylate R-Smads which relocate, together with co-Smads, to the cell nucleus and regulate transcription. Enhancement or repression of Smad-specific gene targets leads to intracellular protein compositions which organize functional complexes and thus govern cellular processes such as proliferation, migration and differentiation. TGF-β/Smad signaling relays are regulated by various post-translational modifications. From receptors to gene promoters, intricate interplays between phosphorylation, acetylation, ubiquitination and numerous other modifications, control Smad signaling initiation and duration. However, many steps in the cascade, including receptor internalization, Smad nuclear shuttling and transcriptional termination, still remain elusive. The open gaps in our understanding of these mechanisms most likely involve additional post-translational regulations. Thus, the aim of the present investigation was to identify novel modulators of TGF-β/Smad signaling. In the first part of this thesis, we show the importance of ADP-ribosylation in Smad-mediated transcription. We identified poly(ADP-ribose) polymerase 1 (PARP-1) as a Smad interacting protein. Our work revealed that PARP-1 forms direct interactions with Smad3/4, and PARylates residues in their MH1 domains. This modification restricts Smads from binding to DNA and attenuates Smad-activated transcription. PARylation is reversed by the glycohydrolase PARG. We provide evidence that PARG can de-ADP-ribosylate Smads, which enhances Smad-promoted gene regulation. In the second part, we examine a Smad-dependent gene target of TGF-β signaling, salt inducible kinase 1 (SIK). After induction, SIK cooperates with Smad7 and Smurf2 to downregulate the TGF-β type I receptor. The mechanism relies on both the kinase and UBA domain of SIK as well as the E3-ligase activity of Smurf2. In summary, we have unveiled two enzyme-dependent TGF-β/Smad modulatory mechanisms; SIK promoted receptor turnover and PARP-1/PARG-regulated Smad signaling.

Nous montrons dans cette étude que dans les cellules déplétées pour la nucléoline, une plus faible accumulation de pré-ARNr est associée à une augmentation de marques d’hétérochromatine (H3K9me2) et une diminution de marques d’euchromatine (H4K12ac et H3K4me3) sur la chromatine des gènes ribosomiques. Des expériences de ChIP-seq montrent que la nucléoline est enrichie dans la région codante et promotrice de l’ADNr et est préférentiellement associée avec les gènes non méthylés des ARNr. La déplétion de la nucléoline entraîne une accumulation de l’ARN Pol I au début de l’ADNr et une diminution de UBF sur la région codante et promotrice. La nucléoline interfère avec la liaison de TTF-1 sur le promoteur-proximal T0, inhibant ainsi le recrutement de TIP5 du complexe NoRC, et établissant un état d’hétérochromatine répressive. Ces résultats révèlent l’importance de la nucléoline dans le maintien d’un état euchromatinien des ADNr et dans l’élongation de la transcription. Nous montrons aussi dans cette thèse que l’acétylation est une nouvelle modification post-traductionnelle de la nucléoline. Des études d’immunofluorescence utilisant l’anticorps anti nucléoline acétylée montrent que la nucléoline acétylée est exclue des nucléoles. De plus, par ChIP-seq nous n’avons jamais pu détecter d’association significative de la nucléoline acétylée sur la chromatine des ADNr. Aussi, nous n’avons détecté aucune activation de la transcription de Pol II sur des matrices de chromatine avec la nucléoline acétylée. Nous trouvons une distribution de la nucléoline acétylée majoritairement dans le nucléoplasme où elle co-localise parfaitement avec le facteur d’épissage SC35, et partiellement avec les structures marquées avec un anticorps dirigé contre Y12, mais ne co-localise pas avec des structures contenant la coïline, ce qui suggère que cette fraction de la nucléoline pourrait être impliquée dans la synthèse ou le métabolisme des pré-ARNm<br>Here we have shown that, in nucleolin depleted cells, lower accumulation of pre-rRNA is associated with the increase in heterochromatin marks (H3K9me2) and decrease of the euchromatin histone marks (H4K12Ac and H3K4me3) in rDNA chromatin. ChIP-seq experiments show that nucleolin is enriched in the coding and promoter region of the rDNA and is preferentially associated with the unmethylated rRNA genes. Nucleolin knockdown results in the accumulation of RNAPI at the beginning of the rDNA and a decrease of UBF in the coding and promoter regions. Nucleolin is able to interfere with the binding of TTF-1 on the promoter-proximal terminator T0 thus inhibiting the recruitment of the NoRC subunit TIP5 and HDAC1 and establishing a repressive heterochromatin state. These results reveal the importance of nucleolin in the maintenance of the euchromatin state of rDNA and transcription elongation.In this thesis we have also shown that acetylation is a novel post-translational modification of nucleolin. Immuno-fluorescence studies using anti-acetylated nucleolin antibody illustrated that acetylated nucleolin is excluded from nucleoli and interestingly, neither could we detect any significant binding of ac-nucleolin on rDNA chromatin by doing ChIP-Seq, nor did we detect any activation of Pol II transcription with ac-nucleolin from DNA and chromatin templates. Moreover, we found acetylated nucleolin had a predominant nucleoplasmic distribution where it associates with the splicing factor SC35 and partially with the structures labeled with Y12 antibody, but not with coilin containing structures

We used extreme ultraviolet (XUV) transient absorption spectroscopy to study the autoionizing Rydberg states of oxygen in an electronically- and vibrationally-resolved fashion. XUVpulse initiates molecular polarization and near-infrared pulse perturbs its evolution. Transient absorption spectra show positive optical-density (OD) change in the case of ns sigma(g) and nd pi(g) autoionizing states of oxygen and negative OD change for nd sigma(g) states. Multiconfiguration time-dependent Hartree-Fock (MCTDHF) calculations are used to simulate the transient absorption and the resulting spectra and temporal evolution agree with experimental observations. We model the effect of nearinfrared perturbation on molecular polarization and find that the laser-induced phase-shift model agrees with the experimental andMCTDHF results, while the laser-induced attenuation model does not. We relate the electronicstate- symmetry-dependent sign of the OD change to the Fano parameters of the static absorption line shapes.

Spinal Cord Injury (SCI) is a devastating condition for human and animal health. In SCI particularly, neurons, oligodendrocytes precursor cells, and mature oligodendrocytes are highly vulnerable to the toxic microenvironment after the lesion and susceptible to the elevated levels of noxious stimuli. Thus the regenerative response of the organism in case of SCI is significantly reduced, and only little spontaneous amelioration is observed in lesioned patients during the early phases. This work mainly focuses on studying and characterizing the modification induced by the SCI in a preclinical animal model. We investigated the ECM composition in the spinal cord segments surrounding the primary lesion site at a gene expression level. We found Timp1 and CD44 as a crucial hub in the secondary cascade of SCI in both spinal cord segments surrounding the lesion site. Interestingly, a temporal and anatomical difference in gene expression, indicating a complex regulation of ECM genes after SCI that could be used as a tool for regenerative medicine. We also investigated the modification in synaptic plasticity-related gene expression in spinal and supraspinal areas involved in motor control. We confirmed the anatomical and temporal difference in gene expression in spinal cord tissue. This analysis suggests that a molecular mapping of the lesion-induced modification could be a useful tool for regenerative medicine. In the last part, we evaluated the efficacy of an implantable biopolymer loaded with an anti-inflammatory drug and a pro-myelinating agent on the acute phase of SCI in our preclinical model. We found a consistent reduction of the inflammatory state in the spinal lesion site and the cord's surrounding segments. Moreover, we found increased preservation of the spinal cord tissue with a related upregulation of neuronal and oligodendroglial markers after lesion. Our treatment showed effective ameliorating functional outcome and reducing the lesion extension in the chronic phase.

Alcohol liver disease (ALD) is a major health concern throughout the world. Currently, in the United States, 17 million people suffer from alcoholism, of which 1.4 million people are receiving treatment [1, 2]. The link between ethanol metabolism, reactive oxygen species (ROS) and liver injury in ALD has been well characterized over the last couple decades [3-10]. Ethanol metabolism relies on the availability of the cofactor NAD+ for the oxidation of ethanol into acetate, consequently causing alterations in redox potential. Redox dysfunction within the mitochondria can affect multiple pathways important in maintaining cellular homeostasis. Chapter 1 provides an introduction to the role of ethanol metabolism in oxidative stress and alcohol liver injury (ALI). During ethanol metabolism, both the cytochrome bc1 and NADH dehydrogenase complexes within the mitochondria have been demonstrated to be major contributors to ROS formation and “leak” free radicals [11-13]. As a result, the free radicals superoxide (O2-) and hydrogen peroxide (H2O2) is diffused into the cytoplasm where they can react with other molecules, proteins and DNA and cause tissue injury [4, 14]. Chapter 1 aims to introduce the link between ethanol metabolism and histone post-translational modifications (PTM) such as tyrosine nitration and lysine acetylation using proteomics techniques. Chapter 2 uses a global proteomic study to identify links between gender and ALI. A 10-day chronic-binge mouse model was employed in order to identify gender-specific proteins that may influence the development of ALD. It has previously been established that females are more susceptible to developing ALD, however, the cause is still unknown. This study identifies gender differences in the family of cytochrome P450 proteins using a mouse model for chronic-binge alcohol exposure. The cytochrome P450 family of proteins are important in the metabolism of toxic compounds, such as acetaldehyde, a byproduct of ethanol metabolism. Interestingly, I also identified that female mice expressed naturally higher levels of histone acetylation prior to alcohol exposure when compared to males. Following alcohol exposure, the female mice did not show much change in acetylation, whereas male acetylation levels were raised to similar levels of the female mice. These acetylation changes raised the question, how does alcohol influence epigenetic marks on histone proteins? Recently, new evidence has emerged that supports the role of epigenetics in the pathophysiology of ALD [4, 14-27]. Ethanol metabolism will promote shifts in redox potential and mitochondrial dysfunction, the result is the formation of reactive oxygen and/or nitrogen species (ROS/RNS) [4, 5, 7, 10, 14, 28]. As ethanol is metabolized, the accumulation of ROS/RNS species such as NO- and O2- can induce the post-translational modification nitrotyrosine. Shifts in redox potential will cause the electron transport chain to “leak” the free radical O2-. Another free radical known as nitric oxide (NO-) has been shown to be elevated during times of ethanol consumption [29, 30]. Traditionally, NO has a protective role within the cell at low concentrations, however, in surplus can lead to tissue damage. Ethanol-induced increases in NO- and O2- can instigate to peroxynitrite (ONOO-) formation; a potent oxidant and nitrating agent of tyrosine residues [29, 31-34]. Chapter 3 examines the indirect effect of alcohol metabolism and ROS/RNS formation on histone tyrosine nitration. This project used mass-spectrometry to identify novel targets of histone tyrosine nitration using a mouse-model of chronic-binge alcohol exposure. Interestingly, histone H3 was found to be nitrated on the hinge-region of the N-terminal tail at tyrosine 41. Molecular dynamics of the nitrated and unmodified proteoforms revealed that the DNA prefers a change in conformation upon H3Y41 nitration. Further studies using an antibody synthesized against the nitrated H3y41 region of the protein revealed potential targets within the genome important in fatty acid synthesis and metabolism. Chapter 4 looks at the direct influence of alcohol metabolism and its contribution to histone acetylation via acetate production and acetyl-CoA. Alcohol metabolism has traditionally been thought influence acetylation through the sirtuin family of deacetylase proteins. Sirtuin deacetylases are NAD+-dependent and have been shown to be a regulate protein acetylation within the mitochondria, cytoplasm, and nucleus during times of ethanol exposure [35-37]. Shifts in redox potential attributed to ethanol metabolism can inhibit sirtuin deacetylase activity by out-competing the enzymes for available NAD+, ultimately leading to mitochondrial and nuclear hyperacetylation [17, 28, 38-42]. Currently, there is evidence that ethanol increases acetylation of histone 3 lysine 9, which then targets activation of the alcohol dehydrogenase gene (ADH) [17, 18, 43]. Moreover, Shukla et.al. (2008) support the idea that ethanol can alter epigenetic transcriptional activation based on which modification is selected for a site during times of stress when it can be occupied by more than one modification [22]. Chapter 4 demonstrates the use of mass-spectrometry to metabolically trace 13C2-labeled ethanol in vivo. These new data show clear evidence of 13C2 heavy-labeled ethanol being incorporated into known sites of acetylation on the N-terminal tails of histone H3 and H4. Incorporation of heavy-label was calculated using extracted ion chromatograms (XIC) for the double and singly acetylated and unmodified peptides belonging to H3K9-R17 and H3K18-R23. Total change in acetylation was also assessed for each peptide using the ratio of ratios of total acetylation to unmodified peptide over the fold change in ethanol- to control-fed groups. An interesting observation was observed in that the incorporation of heavy-label suggests site-selectivity of lysine residues over time. Histone 4 contains multiple sites of acetylation on the peptide H4K5-R17, making it hard to quantify manually. MaxQuant evidence files in conjunction with R were used to calculate the 13C2 incorporation on the multiple H4 acetyl-sites over 24-hours. Ethanol-heavy label incorporation at multiple acetyl-sites occurred as a mixture suggesting a role in transcriptional regulation. These new data establish a link between alcohol metabolism and known epigenetic marks on histone proteins. These studies have now established that alcohol metabolism is indirectly linked to histone tyrosine nitration through increased ROS/RNS and directly through acetate production. Understanding how these epigenetic marks fluctuate as ALD progresses will provide potential targets for the development of new drug therapies. The epigenetic marks identified in these studies have previously been established to be important activators in transcription. These data provide novel techniques using proteomics-based metabolic tracing in vivo. Future studies will assess how these marks change after chronic ethanol exposure and whether the changes in epigenetics are heritable. Understanding hereditary of alcoholism will provide insight to those predisposed to the disease.

Les histones linker juguen un paper important en l’organització i manteniment de la cromatina en estructures d'ordre superior i en la regulació transcripcional. La histona H1 en vertebrats té una estructura característica en tres dominis: un domini N-terminal curt i flexible; un domini globular central; i un domini C-terminal llarg. Els dominis N- i C-terminals (CTD) són molt bàsics i es troben desestructurats en solució aquosa. La distribució de càrrega és bastant uniforme al llarg de tot el CTD. La interacció amb el DNA indueix un plegament total i estable del CTD en condicions fisiològiques, fet que permet classificar aquest domini en el grup de les proteïnes intrínsecament desordenades, on el plegament i la unió al lligand estan acoblades. La fosforilació post-traduccional del CTD de la H1 té efectes en l’estructura secundària de la proteïna i en la condensació del DNA. L’estructura secundària de la H10 sencera es va analitzar per espectroscòpia d’infrarroig. La H10, igual que el CTD aïllat, també es plegà degut a la interacció amb el DNA i l’estructura secundària també va ser modulada per fosforilació. El canvi estructural induït per la fosforilació va consistir en un increment de la quantitat d’estructura β, que va esdevenir més significant amb la unió a DNA, on també es va observar una dependència d’aquest increment amb la relació proteïna/DNA. En aquest cas, la proporció d’estructura β va arribar al 54%, suggerint que el CTD en la proteïna sencera presentava una conformació tot-β. Simultàniament, es va observar una reducció significant de l’estructura en hèlix-α, fet que va suggerir una disminució d’aquest element estructural en el domini globular, probablement per la propagació de l’estructura β des del CTD cap a la resta de la proteïna. En presència de SDS, la H10 es va plegar amb percentatges d’estructura secundària similars als observats en la unió al DNA. A una relació molar SDS/proteïna 14:1, la H10 trifosforilada presentava un 55% d’estructura β, indicant que el CTD en la histona H10 també es trobava en una conformació tot-β i formava fibres amiloides. Els nuclis d’eritròcit de pollastre contenen cromatina inerta i altament compacta consistent, principalment, en DNA i proteïnes histona. En aquest estudi, es va emprar aquesta cromatina per analitzar les modificacions post-traduccionals de les histones i l’efecte de la fosforilació per CDK2 en l’agregació de la cromatina. Els nuclis es van digerir amb nucleasa micrococcal i la cromatina es va fraccionar per centrifugació en tampó de baixa força iònica en fraccions soluble i insoluble. Les modificacions post-traduccionals (PTMs) de les histones linker purificades d’ambdues fraccions es van analitzar per Tandem MS. Els sis subtipus d’histona H1 i la histona H5 van ser identificats. En aquest estudi, es van trobar vuit PTMs novells: dues a la histona H5 i sis en els subtipus d’ H1. Algunes de les modificacions van ser identificades específicament en una de les fraccions, suggerint una distribució diferencial d’algunes PTMs en la cromatina. La comparació de les PTMs novells identificades amb altres prèviament descrites en altres espècies va demostrar que la majoria d’elles estan conservades en l’evolució. Atès que les histones linker desenvolupen la seva funció en la cromatina, es va fosforilar ex vivo amb CDK2 els motius S/T-P-X-Z de les histones linker presents en la cromatina d’eritròcit de pollastre; amb l’objectiu d’estudiar l’efecte de la fosforilació en l’agregació de la cromatina. L’anàlisi proteòmic per HPCE i MALDTOF-MS va mostrar que el nombre de grups fosfat va augmentar amb el temps de fosforilació, assolint un 54% d’espècies fosforilades (mono- i di-fosforilades) en el cas de la H5 després de la fosforilació overnight. Experiments de Tandem MS van revelar que, en cap de les histones purificades de la cromatina nativa, cap dels motius S/T-P-X-Z estava fosforilat, indicant que la fosforilació detectada en les mostres tractades amb CDK2 havien estat modificats ex vivo. En H5, nomes S148 va identificar-se en totes les mostres i es trobava fosforilat després d’1 hora. En l’anàlisi per TandemMS de les H1, tots els motius consens de CDK2, excepte S171 (posició en H1.01), es van identificar en els subtipus H1.03, H1.1L i H1.1R. H1.03T16 es va trobar fosforilat després de 15 minuts; H1.1LS192 I H1.1RS186 després d’1 hora; H1.03S155, H1.1LS155 i H1.1RS153 després de 3 hores. Un cop la fosforilació ex vivo de les histones en la cromatina es va comprovar, es va procedir a estudiar l’efecte de la fosforilació en l’agregació de la cromatina induïda per MgCl2 (1.6mM) per Dynamic Light Scattering (DLS). El resultat més significant associat a la fosforilació va ser la disminució del diàmetre hidrodinàmic de les molècules agregades. Aquestes diferències van esdevenir més important amb l’augment del temps de fosforilació i amb la mida dels fragments de cromatina. Els resultats obtinguts van demostrar que la fosforilació de les histones linker impedia l’agregació de la cromatina.<br>Linker histones play an important role in establishing and maintaining chromatin higher-order structure and in transcriptional regulation. Histone H1 in vertebrates has a characteristic three-domain structure consisting of a short flexible N-terminal domain, a central globular domain and a long C-terminal domain. The amino- and carboxyl-terminal (CTD) domains are highly basic and mainly unstructured in aqueous solution. The charge distribution is quite uniform along the CTD. Because of that, chromatin condensation is mediated through charge-neutralization of the negatively charged linker DNA, facilitating chromatin condensation into the 30nm fibre and also intermolecular aggregation. Interaction with DNA induces the complete folding of the CTD under physiological conditions in a very stable manner, which allows to classify this domain as an intrinsically disordered protein, with coupled binding and folding. Post-translational phosphorylation of the CTD of H1 has effects on secondary structure and DNA condensation. Secondary structure of the entire H10 was analysed by infrared spectroscopy. H10, as the isolated CTD, also folded upon DNA interaction and the secondary structure was modulated by phosphorylation. The structural change following phosphorylation was characterized by an increase in the amount of β‐structure that was more significant when bound to DNA and was dependant on the protein/DNA ratio. The proportion of β‐structure reached 54 % suggesting that the CTD was in an all‐β conformation in the entire protein. Concomitant with the increase in β‐structure, there was a remarkable decrease of α‐helix that suggested the loss of some of the α‐helix in the globular domain; probably associated to the propagation of the β‐structure from the CTD towards the rest of the protein. In the presence of SDS, H10 folded with percentages of secondary structure motifs similar to those found when bound to DNA. At a molar ratio 14:1 (SDS/protein) the triphosphorylated protein had 55% of β‐structure indicating that the CTD within histone H10 was also in an all‐β conformation and formed amyloid fibres. Mature chicken erythrocyte nuclei contain highly condensed and inert chromatin, mainly consisting of DNA and histone proteins. Chicken erythrocyte chromatin was used to analyse linker histones post-translational modifications and the effect of phosphorylation by CDK2 on chromatin aggregation. The nuclei were digested with micrococcal nuclease and fractionated by centrifugation in low-salt buffer into soluble and insoluble fractions. Post-translational modifications (PTMs) of the purified linker histones of both fractions were analyzed by Tandem MS. All six histone H1 subtypes (H1.01, H1.02, H1.03, H1.10, H1.1L and H1.1R) and histone H5 were identified. In our study, we identified eight novel post-translational modifications: two were identified in histone H5 and six in histone H1 subtypes. Some of the identified modifications were specific of one chromatin fraction suggesting the differential distribution of some PTMs within chromatin. Comparison of the PTMs found with other previously reported for other species showed that most of them are conserved through evolution. Since histone H1 develops its function within chromatin; chicken erythrocyte chromatin was phosphorylated ex vivo with CDK2 in the S/T-P-X-Z motifs present in linker histones in order to study the effects of ex vivo phosphorylation of linker histones on chromatin aggregation. Proteomic analyses by HPCE and MALDITOF-MS showed that the the number of phosphate groups increased with the time of phosphorylation, reaching, in the case of H5, 54% of phosphorylated species (mono and diphosphorylated) after overnight phosphorylation. Tandem MS after proteolytic digestion revealed that in all linker histones the S/T-PX-Z motifs were unphosphorylated in native chromatin indicating that the phosphorylated peptides found at other times of reaction were modified ex vivo. In H5, only S148 was identified in all samples and was phosphorylated after 1 hour. In the Tandem MS analysis of histone H1 subtypes, all the CDK2 consensus sequences, except S171(H1.1R numbering) were identified for H1.03, H1.1L and H1.1R. H1.03T16 was found phosphorylated after 15 minutes; H1.1LS192 and H1.1RS186 after 1 hour; H1.03S155, H1.1LS155 and H1.1RS153 after3 hours. Once ex vivo phosphorylation of linker histones within chromatin was confirmed, the effect of linker histones ex vivo phosphorylation on chromatin aggregation induced by MgCl2 (1.6 mM) was analysed by Dynamic Light Scattering (DLS). The most remarkable result associated to ex vivo phosphorylation of linker histones within chromatin was a decrease in the hydrodynamic diameter of the aggregated molecules. The differences became greater with the increase of phosphorylation time and with the size of the chromatin fragments. These results indicated that linker histones phosphorylation impaired chromatin aggregation.

Chromatin packages eukaryotic genomes via a hierarchical series of folding steps, encrypting multiple layers of epigenetic information, which are capable of regulating nuclear transactions in response to complex signals in environment. Besides the 1-dimensinal chromatin landscape such as nucleosome positioning and histone modifications, little is known about the secondary chromatin structures and their functional consequences related to transcriptional regulation and DNA replication. The family of chromosomal conformation capture (3C) assays has revolutionized our understanding of large-scale chromosome folding with the ability to measure relative interaction probability between genomic loci in vivo. However, the suboptimal resolution of the typical 3C techniques leaves the levels of nucleosome interactions or 30 nm structures inaccessible, and also restricts their applicability to study gene level of chromatin folding in small genome organisms such as yeasts, worm, and plants. To uncover the “blind spot” of chromatin organization, I developed an innovative method called Micro-C and an improved protocol, Micro-C XL, which enable to map chromatin structures at all range of scale from single nucleosome to the entire genome. Several fine-scale aspects of chromatin folding in budding and fission yeasts have been identified by Micro-C, including histone tail-mediated tri-/tetra-nucleosome stackings, gene crumples/globules, and chromosomally-interacting domains (CIDs). CIDs are spatially demarcated by the boundaries, which are colocalized with the promoters of actively transcribed genes and histone marks for active transcription or turnover. The levels of chromatin compaction are regulated via transcription-dependent or transcription-independent manner – either the perturbations of transcription or the mutations of chromatin regulators strongly affect the global chromatin folding. Taken together, Micro-C further reveals chromatin folding behaviors below the sub-kilobase scale and opens an avenue to study chromatin organization in many biological systems.

Chromatin packages eukaryotic genomes via a hierarchical series of folding steps, encrypting multiple layers of epigenetic information, which are capable of regulating nuclear transactions in response to complex signals in environment. Besides the 1-dimensinal chromatin landscape such as nucleosome positioning and histone modifications, little is known about the secondary chromatin structures and their functional consequences related to transcriptional regulation and DNA replication. The family of chromosomal conformation capture (3C) assays has revolutionized our understanding of large-scale chromosome folding with the ability to measure relative interaction probability between genomic loci in vivo. However, the suboptimal resolution of the typical 3C techniques leaves the levels of nucleosome interactions or 30 nm structures inaccessible, and also restricts their applicability to study gene level of chromatin folding in small genome organisms such as yeasts, worm, and plants. To uncover the “blind spot” of chromatin organization, I developed an innovative method called Micro-C and an improved protocol, Micro-C XL, which enable to map chromatin structures at all range of scale from single nucleosome to the entire genome. Several fine-scale aspects of chromatin folding in budding and fission yeasts have been identified by Micro-C, including histone tail-mediated tri-/tetra-nucleosome stackings, gene crumples/globules, and chromosomally-interacting domains (CIDs). CIDs are spatially demarcated by the boundaries, which are colocalized with the promoters of actively transcribed genes and histone marks for active transcription or turnover. The levels of chromatin compaction are regulated via transcription-dependent or transcription-independent manner – either the perturbations of transcription or the mutations of chromatin regulators strongly affect the global chromatin folding. Taken together, Micro-C further reveals chromatin folding behaviors below the sub-kilobase scale and opens an avenue to study chromatin organization in many biological systems.

The overall goal of my work was to develop and apply new chemical methods for the study of epigenetic DNA and protein modifications. In Chapter 3 the development of Suzuki-Miyaura cross coupling (SMcc) for the post-synthetic modification of DNA is described. DNA modification by SMcc is efficient (4-6h) and proceeds under mild conditions (37°C, pH 8.5). The incorporation of various groups useful for biological investigations is demonstrated using this methodology. Using a photocrosslinker, introduced into the DNA by SMcc capture experiments are performed to identify potential binding partners of modified DNA. In Chapter 4 a dehydroalanine (Dha) based chemical protein modification method is described that enables the introduction of posttranslational modification (PTM) mimics into histones. The PTM mimics introduced by this method are tested using western- and dot-blot and binding and enzymatic assays, confirming they function as mimics of the natural modifications. Chapter 5 describes the use of a generated PTM mimics to elucidate the function of O-linked β-Nacetylglucosamine (GlcNAc) of histones in transcriptional regulation. It is shown that GlcNAcylation of Thr-101 on histone H2A can destabilize nucleosome by modulating the H2A/B dimer – H3/H4 tetramer interface. N- and C-terminal histone tails play an important role in transcriptional regulation. In Chapter 6, nuclear magnetic resonance is used to investigate the structure of the histone H3 N-terminal tail in a nucleosome. The H3 tail, while intrinsically disordered, gains some α-helical character and adopts a compact conformation in a nucleosome context. This H3 tail structure is shown to be modulated by Ser-10 phosphorylation. The effect of a new covalent DNA modification, 5- hydroxymethylcytosine (5hmC), on transcription factor binding is investigated in Chapter 7. 5hmC influences HIF1α/β, USF and MAX binding to their native recognition sequence, implying involvement of this modification in epigenetic regulation.

In the present work we focus into the potential relevance of PUFAs in some models and human samples from patients suffering amyotrophic lateral sclerosis (ALS). Due to its pathological implication, oxidative stress was our first goal. We started from simple oxidative methodology screening to search for an antioxidant substance (among 21 different candidates) available in a Mediterranean diet. The results demonstrated high heterogeneity in carbonyl (measured by DNP) accumulation, regarding the oxidative source, substrate suffering it and the antioxidant structure. Further, thanks to GC/MS and LCQTOF, we detailed the protection over specific accrual of protein and lipid peroxidation markers as well as lipid profile modifications (as % of total fatty acids -FA) in oxLDL thanks to those dietary compounds. Moreover, we demonstrated its in vitro relevance, in terms of survival, when two cell lines (HMEC-1, HepG2) were treated with this oxidized (and protected) compounds, and finally address in vivo importance of those findings, demonstrating decreased carbonyl and oxidative accumulation in hamsters under an atherogenic diet supplemented with antioxidants. Once described the protective effect of antioxidants and specific signatures found regarding lipid oxidation markers, we extend the study focusing in different ALS samples. From previous work, we demonstrated an altered docosohexaenoic acid (DHA) composition in different location for patients suffering sporadic ALS. Hence, we though necessary to define whether the enzymatic machinery aimed to synthesize DHA from its precursors, are affected in sALS. Interestingly, we found a tissue specific variation (spinal cord vs cortex), compatible with our previous FA results. Further, thanks to inmunohistochemistry, differential involvement was unveiled for motor neurons (MN) and surrounding glia. Therefore, trying to depict cellular contribution, we switch to a neuronal model (N2A under oxidative stressors and/or aggregation-prone-TDP-43) and a tissular one (OT). There, we showed decreased desaturase (Δ6) and drebrin expression as well as increased DHA synthesis and an unreported inverse correlation of drebrin loss and aberrant p-TDP-43 expression under oxidative conditions in the cell culture. In the OT model, lipidomic analysis showed specific accretion of 8-iso-PGF2α and NPD1 as well as increased DHA (and dramatically decreased precursors) and reduced AA concentrations (GC measured). Analysis of slice O2 consumption showed decreased O2 levels under excitotoxic treatment and alleviation by antioxidant (tocopherol) addition. Treatment of OT slices with Ω-3 precursors (better than final products) and DHA plus tocopherol ameliorated MNs number. Finally, we wanted to disclose PUFA’s implication and phenothype of an animal model (SODG93A) under a dietary intervention with opposed FA unsaturation levels. Not surprisingly, FA profile was difficult to be altered in nervous system, although subtle specific variations were found. More importantly, differences in survival and clinical manifestations, UPR (Ubiquitin inclusions), mt-DNA (8- oxo-dG) and protein oxidative modifications revealed sex as a relevant factor in lipid handling for this model. Hence, whereas male under a low PUFA diet showed increased survival, females lack this beneficial outcome. Last but not least, we wanted to dig deeper regarding this sexual dimorphism. For this purpose, we focused in mitochondria and analyzed spinal cord oxygen consumption, oxidative damage to proteins and lipid profile along disease progression and also in a neuronal model (N2A overexpressing SODG93A, treated with 17β-estradiol).We could demonstrate a clear sexual implication, with females having late onset clinical symptoms concomitant to an upgraded mitochondrial function and lower protein and mitochondrial damaged proteins compared with males. Finally, to further confirmed steroid potential as a protective element in disease progression, in vitro estradiol pretreatmet of N2A showed increased oxygen consumption, with no relation with the mitochondrial complex expression.<br>En el trabajo que aquí se presenta se ha profundizado en la relevancia que los ácidos grasos poliinsaturados (PUFA) puedan tener en el tratamiento de la esclerosis lateral amiotrófica (ALS). Dada su implicación en el desarrollo de la patología, el estudio del estrés oxidativo asociado fue uno de nuestros primeros objetivos. Para ello hemos empezado por un cribado metodológico simplista, tratando de encontrar una sustancia antioxidante (entre 21), biodisponible en una dieta Mediterránea equilibrada y que fuese capaz de reducir un daño oxidativo generado desde diversos frentes (medido como acumulación de carbonilos) y sobre diferentes substratos. Los resultados demostraron una alta heterogeneidad, dificultando así la elección de un único antioxidante. Aún así, gracias a la GCMS y la LCQTOF pudimos detallar la acumulación específica diferenciada de marcadores de daño oxidativo proteico y daño lipoxidativo producido cuando partículas de lipoproteínas de baja densidad (LDL) son oxidadas bajo la acción de diversos compuestos. Además se pudo objetivar el cambio en la composición lipídica de estas LDL (medida como % del total presente) y su relevancia biológica in vitro, medida en términos de supervivencia, cuando se exponen a dos líneas celulares (HMEC-1, HepG2). Por último se demostró la importancia in vivo, puesto que se pudo observar una menor acumulación de productos carbonílicos en hámsters alimentados con una dieta aterogénica, pero suplementada con antioxidantes. Una vez se demostró el papel jugado por estos antioxidantes en la acumulación diferenciada de productos de oxidación, extendimos el estudio a muestras y modelos de ALS. En trabajos previos habíamos evidenciado una composición tisular diferenciada en diversas localizaciones del sistema nervioso en pacientes diagnosticados de ALS. Por ello consideramos interesante el estudio de la expresión de la maquinaria enzimática necesaria para la síntesis lipídica. De un modo destacado, pudimos ver de nuevo una variación tisular, compatible con niveles reducidos de docosohexaenoico (DHA), y gracias a la inmunohistoquímica también se observaron diferencias entre las motoneuronas (MNs) y la glia circundante. Así pues, para poder revelar las aportaciones diferenciales de los distintos tipos celulares, utilizamos una línea celular (N2A) a la que sometíamos a diferentes estresores (daño oxidativo y sobreexpresión de una versión de TPD-43 que causa agregados) y a un cultivo tisular de médula espinal (OT), dónde se produce una muerta progresiva y selectiva de las MNs. Tras estos experimentos, observamos un descenso tanto en la expresión de la Δ6 desaturasa como de drebrin (marcador presináptico) tras la sobreexpresión de TDP-43, así como una mayor síntesis de DHA y una correlación inversa entre la pérdida de drebrina y la expresión de pTDP- 43 bajo condiciones de estrés oxidativo. Por otro lado, en el modelo OT, el análisis lipidómico reveló la acumulación especifica de 8-iso-PGF2α y NDPD1 (posiblemente en respuesta a un incremento del daño oxidativo) así como el aumento en la concentración de DHA (con un descenso muy marcado de sus precursores) y el descenso de araquidónico. Quisimos analizar también el consumo de oxígeno, tanto en tejido intacto como permeabilizado, pudiendo observar como la excitoxicidad reducía considerablemente su capacidad y como ésta era, en parte, rescatada con el uso de tocoferol. Además, el tratamiento del OT con precursores Ω-3 mejoró también el número de MNs. Por último, quisimos caracterizar la implicación que los PUFA dietarios podrían tener en un modelo animal bien conocido (SODG93A). No fue sorprendente encontrar que el perfil lipídico en el sistema nervioso fue muy difícil de alterar. Aún así, se observaron diferencias en supervivencia, en el devenir clínico, la respuesta UPR (con acumulaciones de Ub), el daño al DNA mitocondrial (8-oxo-dG) y modificaciones oxidativas en las proteínas y cómo éstas tenían un grado de afectación diferencial cuando considerábamos el sexo de los animales. Esto es, mientras que los machos sometidos a una dieta baja en PUFAs de cadena larga demostraron una mayor supervivencia, en las hembras no se apreció mejoría. Por último, pero no menos importante, quisimos profundizar más respecto a este dimorfismo. Centrándonos en la mitocondria, pudimos hacer un seguimiento del consumo de oxígeno a lo largo de la enfermedad, el daño oxidativo a proteínas y el perfil lipídico. Por lo tanto pudimos demostrar una clara implicación sexual, siendo las hembras las que más tarde comienzan su manifestación clínica, con mejores funciones mitocondriales asociadas a un menor daño oxidativo. Finalmente, la relevancia del papel protector de los estrógenos se pudo comprobar in vitro, mediante el pretratamiento con 17β-estradiol en la línea N2A que sobreexpresa SOD1G93A, relacionado con la ALS familiar, proponiéndose el estradiol como un nuevo elemento que juega un papel relevante en el desarrollo de la enfermedad.<br>En aquest treball s’ha intentat profunditzar en la possible rellevància dels àcids grassos poliinsaturats (PUFA) en el tractament de l’esclerosi lateral amiotròfica (ELA). Atesa la seva implicació en el desenvolupament de la patologia, l’estudi de l’estrès oxidatiu associat fou un dels primers objectius. Per això, es va començar amb un cribratge metodològic simplista, intentant trobar una substància antioxidant biodisponible en una dieta mediterrània equilibrada i que fos capaç de reduir el dany oxidatiu generat des de diferents fronts i sobre diferents substrats. Els resultats van demostrar una alta heterogeneïtat, dificultant així l’elecció d’un únic antioxidant. Malgrat això, mercès a tècniques de GC-MS i LC-QTOF, es va poder detallar l’acumulació específica diferenciada de marcadors de dany oxidatiu proteic i dany lipoxidatiu produït quan partícules de lipoproteïna de baixa densitat (LDL) s’oxiden per l’acció de diversos compostos. A més, es va poder objectivar el canvi en la composició lipídica d’aquestes LDL i la seva rellevància in vitro, mesurada en termes de supervivència, quan es cocultiven amb les línies cel·lulars. Per últim, es va demostrar la importància in vivo, atès que es va observar una menor acumulació de productes carbonílics en hàmsters alimentats amb una dieta aterogènica suplementada amb antioxidants. Un cop es va demostrar el paper d’aquests antioxidants en l’acumulació diferenciada de productes d’oxidació, es va estendre l’estudi a mostres i models d’ELA. En treballs previs s’havia evidenciat una composició tissular diferenciada en diverses localitzacions del sistema nerviós central en pacients d’ELA (respecte l’àcid docosahexaenoic (DHA), depleció en medul·la espinal i acumulació en còrtex). Per aquesta raó es va considerar interessant l’estudi de l’expressió de la maquinària enzimàtica necessària per la síntesi lipídica. D’una manera destacada, es va poder veure de nou una variació tissular, compatible amb nivells reduïts de DHA i, per tècniques d’immunohistoquímica, es van observar diferències entre les motoneurones i la glia circumdant. Per tant, per poder revelar les aportacions diferencials dels diferents tipus cel·lulars, es va utilitzar la línia cel·lular N2A, sotmesa a diferents estressos (dany oxidatiu i sobreexpressió d’una forma de TDP-43 que causa agregats) i un cultiu tissular de medul·la espinal, en el que es produeix una mort progressiva i selectiva de les motoneurones. Es va observar un descens en l’expressió de FADS2 i de drebrina, un marcador sinàptic, així com una major síntesi de DHA i una correlació inversa entre la pèrdua de drebrina i l’expressió de TDP-43 sota condicions d’estrès oxidatiu. Per altra banda, en el model organotípic, l’anàlisi lipidòmica va revelar l’acumulació específica de 8-isoPGF2α i NDPD1, així com l’augment de la concentració de DHA i el descens motl marcat del seus precursors a mes del àcid araquidònic. Es va mesurar també el metabolisme oxidatiu, observant-se que l’excitotoxicitat reduïa considerablement la seva capacitat i, en part, es rescatava amb l’ús de tocoferol. A més, el tractament dels cultius organotípics amb precursors d’àcids grassos n-3 va millorar el nombre de motoneurones. Per últim, es va caracteritzar la implicació dels PUFA dietaris en un model animal d’ELA. Malgrat que el perfil lipídic del sistema nerviós central era difícil d’alterar, es van observar diferències en supervivència, fenotip clínic, resposta al malplegament de proteïnes (UPR) amb acumulació d’ubiquitina, dany en el DNA mitocondrial i modificacions oxidatives en les proteïnes, amb un grau d’afectació diferencial quan es considerava el sexe dels animals. En aquest sentit, mentre que els mascles sotmesos a una dieta baixa en PUFA de cadena llarga van mostra una major supervivència, en les femelles nomes va apreciar cap efecte millora. Per profunditzar en el dimorfisme sexual en ELA, i especialment la disfunció mitocondrial, es va analitzar mitjançant respirometria d’alta resolució la medul·la espinal durant tot el desenvolupament de la malaltia. Es va revelar una clara diferència de gènere, amb una manifestació clínica més tardana en femelles que correlaciona amb una millor conservació de la funció mitocondrial i un menor dany oxidatiu. El possible paper protector dels estrògens es va demostrar in vitro mitjançant el pretractament amb estradiol de cèl·lules N2A que sobreexpressen una forma de SOD1 humana mutada associada a l’ELA familiar (G93A-SOD1).

La biogenese des ribosomes se deroule dans un compartiment nucleaire dedie, le nucleole, ou l'arn preribosomique subit de nombreux decoupages et modifications de nucleotides impliquant l'intervention d'un grand nombre de petits arns (snoarns). Les sites des modifications de nucleotides les plus frequentes dans l'arnr (methylations en 2-o du ribose et pseudouridylations) sont specifies par l'appariement au pre-arnr de deux familles distinctes de snoarns, qui forment une structure en duplex reconnue par une enzyme specifique de chaque type de modification. Pour assister le biologiste dans l'analyse des relations structure/fonction des arns, nous avons d'abord developpe essa. C'est un logiciel de visualisation, d'edition, et d'analyse de structure secondaire adapte a des arns de structure aussi complexe que les arnr. Essa est un outil graphique et interactif, utilisable quelle que soit la famille d'arn etudie, qui pourra servir ulterieurement de base a de nouveaux developpements dans le domaine de l'analyse du repliement tridimensionnel des arnr et de leurs precurseurs. Dans la perspective de rechercher dans le genome complet de la levure s. Cerevisiae de nouveaux snoarns a boites h/aca, les guides de pseudouridylation du pre-arnr, nous avons ensuite adapte un logiciel de recherche de motifs arns structures, palingol, en y incorporant des fonctions de detection de motifs en sequence. Nous avons pu detecter de nombreuses sequences positives parmi lesquelles pourront etre experimentalement recherches de nouveaux snoarns h/aca, et aussi obtenir de nouvelles informations sur les cibles probables de plusieurs snoarns deja connus. Enfin, nous avons recherche dans le genome complet de s. Cerevisiae les enzymes susceptibles de catalyser la methylation ribose et la pseudouridylation de l'arn, par analyse de sequences proteiques. Outre la detection de trois pseudouridine synthases putatives, ce travail a conduit a l'identification et la caracterisation experimentale de la premiere 2-o-ribose methylase eucaryotique, qui est specifique d'une position unique dans les arnt.

S. cerevisiae Ada2 and Bre1 has a role in histone post-translational modifications. Deletion of these genes causes deficiency in acetylation (Ada2) or ubiquitination (Bre1) of histones. Further, mutants lacking these genes or homologous genes showed different phenotypes in human and S. cerevisiae while treated with DNA damaging agents 4-NQO and MMS. Bre1 deficient cells showed 4-NQO sensitivity in S. cerevisiae and resistance in human cells. Since it has been shown that S. pombe is more close to mammals in chromatin regulation we wanted to examine S. pombe response against MMS and 4-NQO. By homologous recombination, genes were deleted and mutants were treated with different concentration of both the genotoxins. In accordance with a previous study, Ada2Δ showed sensitivity to MMS while Brl1Δ &amp; Brl2Δ grew as wild type. Surprisingly, unlike S. cerevisiae, S. pombe showed resistance to 4-NQO and has a phenotype similar to the one found in human cells.