In an attempt to develop a Staphylococcus aureus vaccine, we have applied reverse vaccinology approach, mainly based on in silico screening and proteomics. By using this approach SdrE, a protein belonging to serine-aspartate repeat protein family was identified as potential vaccine antigen against S. aureus. We have investigated the biochemical properties as well as the vaccine potential of SdrE and its highly conserved CnaBE3 domain. We found the protein SdrE to be resistant to trypsin. Further analysis of the resistant fragment revealed that it comprises a CnaBE3 domain, which also showed partial trypsin resistant behavior. Furthermore, intact mass spectrometry of rCnaBE3 suggested the possible presence of isopeptide bond or some other post-translational modification in the protein.However, this observation needs further investigation. Differential Scanning Fluorimetry study reveals that calcium play role in protein folding and provides stability to SdrE. At the end we have demonstrated that SdrE is immunogenic against clinical strain of S. aureus in murine abscess model. In the second part, I characterized a protein, annotated as epidermin leader peptide processing serine protease (EpiP), as a novel S. aureus vaccine candidate. The crystal structure of the rEpiP was solved at 2.05 Å resolution by x-ray crystallography . The structure showed that rEpiP was cleaved somewhere between residues 95 and 100 and cleavage occurs through an autocatalytic intra-molecular mechanism. In addition, the protein expressed by S. aureus cells also appeared to undergo a similar processing event. To determine if the protein acts as a serine protease, we mutated the catalytic serine 393 residue to alanine, generating rEpiP-S393A and solved its crystal structure at a resolution of 1.95 Å. rEpiP-S393A was impaired in its protease activity, as expected. Protective efficacy of rEpiP and the non-cleaving mutant protein was comparable, implying that the two forms are interchangeable for vaccination purposes.

The severity of Helicobacter pylori infections largely depends on the genetic diversity of the infecting strain, and particularly on the presence of the cag pathogenicity island (cag-PAI). This virulence locus encodes a type-IV secretion system able to translocate in the host cell at least the cag-encoded toxin CagA and peptidoglycan fragments, that together are responsible for the pathogenic phenotype in the host. Little is known about the bacterial regulators that underlie the coordinated expression of cag gene products, needed to assemble a functional secretion system apparatus. To fill this gap, a comprehensive analysis of the transcriptional regulation of the cag-PAI operons was undertaken. To pursue this goal, a robust tool for the analysis of gene expression in H. pylori was first implemented. A bioluminescent reporter system based on the P. luminescens luxCDABE operon was constructed and validated by comparisons with transcriptional analyses, then it was systematically used for the comprehensive study and mapping of the cag promoters. The identification of bona fide cag promoters had permitted to pinpoint the set of cag transcriptional units of the PAI. The responses of these cag transcriptional units to metabolic stress signals were analyzed in detail, and integrated with transcription studies in deletion mutants of important H. pylori virulence regulators and protein-DNA interaction analyses to map the binding sites of the regulators. Finally, a small regulatory RNA cncR1 encoded by the cag-PAI was identified, and the 5’- and 3’-ends of the molecule were mapped by primer extension analyses, northern blot and studies with lux reporter constructs. To identify regulatory effects exerted by cncR1 on the H. pylori gene expression, the cncR1 knock out strain was derived and compared to the parental wild type strain by a macroarray approach. Results suggest a negative effect exerted by cncR1 on the regulome of the alternative sigma54 factor.

By using the ability of the positive-strand RNA ((+)RNA) virus BMV to replicate in yeast it was previously shown that subunits of the LSm1-7 ring, as well as Pat1 and Dhh1 play an essential role in the transit of the BMV genome from translation to replication. In non-infected cells, these proteins mediate the transition of cellular mRNAs from a translational to a non-translational state by activating decapping in the 5'-3' - deadenylation-dependent mRNA decay pathway. Given the conservation of this pathway from yeast to humans and the common need of all (+)RNA viruses to regulate the transition of their genomes from active translation to a translationally inactive state to allow replication, an exciting possibility, and our working hypothesis, was that LSm1-7, Dhh1 and Pat1 are used not only by BMV to replicate in yeast but also by human (+) RNA viruses, such as HCV, to replicate in mammalian cells. Furthermore, given the key role of these proteins in a common step to all (+)RNA viruses, it is essential to characterize the not yet defined molecular mechanisms associated with such function. In this regard, we also hypothesized that the LSm1-7 complex, as member of the Sm family of proteins, would directly interact with viral genomes of (+)RNA viruses in order to play their role in the virus life cycle in a similar way that other family counterparts directly interact with their RNA targets in order to achieve their different cellular functions. In this work we were able to confirm both hypothesis showing that human homologues of the upper mentioned proteins LSm1-7, Rck/p54 and PatL1, are required for HCV RNA translation and replication. Additionally, we also showed that reconstituted LSm1-7 complexes specifically recognize important signals, either in BMV or HCV genomes, that regulate their translation and/or replication. These observations constitute the first evidence that the LSm1-7 complex is able to directly interact with viral genomes representing also novel LSm1-7 interaction sites. Given the common replication strategies of (+)RNA viruses and the conserved cellular functions of LSm1-7, Pat1 and Dhh1 from yeast to humans, our findings pinpoint a weak spot that may be exploited to generate broad-spectrum antiviral drugs. / Utilizando la capacidad del BMV, un virus de ARN de cadena positiva (ARN(+)), para replicar en levaduras se ha demostrado previamente que las subunidades del anillo LSm1-7, así como Pat1 y Dhh1, desempeñan un papel esencial en la transición del genoma del virus de BMV desde traducción a replicación. En células no infectadas, estas proteínas median la transición de ARNm celulares de la traducción a un estado de no-traducción mediante la activación del proceso de decapping en la via 5'-3' de degradación de los ARNs celulares dependiente de deadenilación. Teniendo en cuenta la conservación de esta vía desde levaduras a humanos y la necesidad común de todos los virus ARN(+) para regular la transición de sus genomas desde un estado activo de traducción a otro no activo para permitir la replicación, una posibilidad interesante, y nuestra hipótesis de trabajo, es que LSm1-7, Dhh1 y Pat1 son utilizadas no solo por BMV para replicar en levaduras, sino también por otros virus ARN(+) que infectan a humanos, como el virus de la hepatitis C, para replicar en células de mamíferos. Por otra parte, dado el papel clave de estas proteínas en un paso común en todos los virus de ARN(+), es esencial caracterizar los mecanismos moleculares aun no conocidos y asociados a dicha función. En este sentido, también estudiamos la hipótesis de que el complejo LSm1-7, como miembro de la familia de proteínas Sm, pueda interactuar directamente con los genomas virales de virus de ARN(+) con el fin de desempeñar su papel en el ciclo de vida del virus de una manera similar a la que otros miembros de su familia interactúan con sus ARN con el fin de lograr sus diferentes funciones celulares. En este trabajo hemos podido confirmar ambas hipótesis demostrando que los homólogos humanos de las proteínas anteriormente mencionadas, LSm1-7, Rck/p54 y PatL1, son necesarios para la traducción y replicación del ARN del virus de la Hepatitis C. Por otra parte, los anillos reconstituidos de LSm1-7 reconocen específicamente señales importantes, tanto en el genoma de BMV como en el de la Hepatitis C que regulan su traducción y/o replicación. Estas observaciones constituyen la primera evidencia de que el complejo LSm1-7 es capaz de interactuar directamente con genomas virales y representan también novedosos patrones de interacción de este complejo con ARN. Teniendo en cuenta las estrategias de replicación en común de los virus de ARN de cadena positiva y las funciones celulares conservadas de LSm1-7, Pat1 y Dhh1 de levaduras a humanos, nuestros resultados señalan la posibilidad de explotar estas proteínas para la generación de medicamentos antivirales de amplio espectro.

Els processos de compostatge estan sovint relacionats amb la generació de males olors degut a les emissions de compostos nitrogenats, principalment amoníac, de compostos sulfurosos, i de compostos orgànics volàtils (COVs). Actualment, un procés de compostatge modern cal que es disseny i operi de manera que minimitzi les emissions i tracti eficaçment els gasos i olors generats. En aquest sentit, la biofiltració és una alternativa simple, econòmica i eficient a l'hora de reduir les emissions oloroses originades en processos de tractament de residus.L'objectiu d'aquesta tesi és l'estudi de les emissions d'amoníac i de COVs, així com la seva posterior biofiltració en processos de compostatge de residus orgànics de composició i propietats diferents (fracció orgànica de residus municipals (FORM), fangs d'estació depuradora d'aigües residuals digerits anaeròbiament i sense digerir, residus carnis i residus de pèl). A la vegada, s'ha dissenyat i construït un aparell per caracteritzar el comportament d'un suport orgànic de rebliment per biofiltrar amoníac en termes de degradació biològica, capacitat d'adsorció i capacitat d'absorció d'amoníac.Els resultats han mostrat com la quantitat d'amoníac volatilitzada en tots el processos de compostatge estudiats està directament influenciada per la relació C/N de la mescla a compostar. A la vegada, s'ha observat que les emissions d'amoníac augmenten exponencialment durant la fase termòfila del procés de compostatge, mentre que durant la fase final les emissions i la temperatura semblen estar relacionades de manera lineal. En el cas de l'estudi de les emissions de COVs, les màximes concentracions s'han detectat durant les primeres 48 hores de procés i s'ha apreciat un augment de les emissions a l'afegir una major quantitat d'estructurant a la mescla inicial a compostar.En el l'estudi de la biofiltració de les emissions d'amoníac s'ha constatat que la biofiltració emprant compost com a medi de rebliment elimina gran part de les emissions d'amoníac derivades del compostaje de FORM i fangs digerits amb eficàcies superiors al 95%. No obstant, s'ha observat una eliminació parcial de l'amoníac durant el compostaje de residus carnis degut a les elevades concentracions d'amoníac emeses i a possibles fenòmens d'inhibició de l'activitat biològica del biofiltre. Pel que fa a la biofiltració de COVs originats en processos de compostatge, s'ha observat que aquesta és altament dependent de la composició de la mescla de COVs volatilitzada i per tant assoleix diferents eficàcies d'eliminació en funció del residu a compostar, obtenint-se les majors eficàcies durant el compostatge de fangs frescos. S'ha constatat que el propi compost com a material de rebliment emet COVs en una concentració aproximadament de 50 mg C·m-3. Finalment, en l'estudi dels mecanismes de biofiltració d'amoníac, s'han considerat adequats els models de Freundlich i de Langmuir per descriure els equilibris dels processos d'adsorció d'amoníac dels materials de rebliment estudiats. S'ha observat que l'absorció d'amoníac es pot representar mitjançant el model lineal regit per la llei de Henry, amb valors de la constant de partició significativament superiors a la constant de Henry quan l'amoníac es dissol en aigua pura, i s'ha comprovat que l'absorció té un pes important com a mecanisme d'eliminació d'amoníac en el rang d'humitat òptim dels biofiltres. Finalment, les velocitats de biodegradació d'amoníac obtingudes han estat considerablement baixes si es comparen amb les dels mecanismes fisicoquímics d'eliminació d'amoníac. Aquest manifesta la importància de controlar les càrregues d'amoníac subministrades als biofiltres que operen en plantes de compostatge, per exemple mitjançant un pretractament emprant un rentador àcid, si es vol garantir un funcionament adequat del sistema a llarg termini. / Composting processes are frequently related to bad odours generation due to the emissions of nitrogen compounds, mainly ammonia, sulphur compounds and volatile organic compounds (VOCs). Currently, modern composting processes have to be designed and operated minimising emissions and treating gases and odours efficiently. In this sense, biofiltration can be adapted to reduce emissions from composting processes. It is also considered a suitable technology in terms of waste recycling, emission reduction and low construction and operating costs. The aim of this thesis is the study of ammonia and VOCs emissions generated during the composting of different organic wastes (organic fraction of municipal solid wastes (OFMSW)), raw sludge, anaerobically digested sludge, animal by-products and hydrolysed hair). The abatement of the ammonia and VOCs generated during the waste composting using the biofiltration technology is also studied. Moreover, a new specific apparatus to characterise organic support media in terms of ammonia biological degradation, adsorption and absorption capacity, has been designed and constructed.Results have shown that the total amount of ammonia emitted during the composting process was directly related to the C/N ratio. Ammonia emissions revealed a strong dependence on temperature, with a distinct pattern in the thermophilic first stage of composting than that of the mesophilic final stage. VOCs emissions were higher during the beginning of the composting process.Biofiltration technology using compost as a biofilter media can effectively remove most of the ammonia content from the composting process of OFMSW and digested sludge, achieving removal efficiencies over 95%. However, in the case of animal by-products, only a partial removal of ammonia was obtained due to the high ammonia emissions. VOCs biofiltration from exhausted gases of composting processes can be performed achieving different efficiencies depending on the waste composted. More sustained removal efficiencies were obtained in the composting of raw sludge. Compost biofilters are emitters of VOCs themselves. Approximately basal emission of 50 mg C·m-3 was measured. Regarding the ammonia biofiltration mechanisms study, adsorption data were successfully fitted to Langmuir and Freundlich isotherms. The results also show that absorption could be represented by a Henry's law linear equation, with values of the Henry coefficient higher than that of pure water. Finally, ammonia biodegradation rates are considerably lower than absorption and adsorption rates. This fact shows the importance of controlling ammonia loading rates supplied to biofilters in composting plants in order to achieve a high efficiency in long term operation.

La demanda mundial de cultivos agrícolas para la alimentación ha experimentado un aumento notorio a través de las últimas décadas como consecuencia de la creciente población humana. Por otra parte, las predicciones globales de cambio climático predicen aumentos de temperatura y períodos de sequía más largos, especialmente en latitudes templadas, donde se encuentra la mayor parte de la producción mundial de cultivos. El presente estudio se llevó a cabo con tres de los cultivos más importantes a nivel mundial: arroz (Oryza sativa L.), trigo (Triticum aestivum L.) y maíz (Zea mays L.). Además de tres de los cultivos más demandados a nivel mundial, estas tres especies también fueron escogidas debido a sus diferentes mecanismos fotosintéticos, y a las diferentes condiciones ambientales a las que están adaptadas. De esta manera, el arroz y el trigo son especies C3 de ambientes cálidos y fríos, respectivamente, y el maíz es una especie C4 de ambientes cálidos. Con el objetivo de evaluar la respuesta de estos tres cultivos a las tensiones derivadas del cambio climático, es decir, alta temperatura y la sequía, las plantas se cultivaron a 38ºC y 25ºC, y dentro de cada temperatura, un lote de plantas era cultivado bajo condiciones de déficit hídrico (WD) y otro, como bajo capacidad de campo (WW). En todos los casos, se midieron los parámetros de crecimiento, fisiológicos y bioquímicos con el fin de evaluar el efecto de los dos estreses impuestos. Con relación a los parámetros fisiológicos, como la asimilación de CO2 y los procesos difusivos, estos se midieron a 25ºC y 38ºC para determinar la capacidad de estos cultivos para adaptarse y aclimatarse a la alta temperatura y el déficit hídrico. Ambas tensiones tuvieron un efecto negativo sobre las plantas, pero el impacto fue diferente dependiendo de los parámetros medidos. La alta temperatura tuvo un mayor efecto perjudicial en la producción de biomasa y la respiración mitocondrial, especialmente en trigo y maíz. En cambio, en las tres especies, la capacidad fotosintética y los parámetros de difusión como gs mostraron una mayor restricción bajo déficit de agua que a alta temperatura. Con respecto a las mediciones bioquímicas, los parámetros de Rubisco in vitro mostraron la misma tendencia que AN, mientras que la actividad Rubisco, la concentración y el estado carbamilación fueron más afectados por las altas temperaturas que por el déficit hídrico. Finalmente, algunos de los parámetros medidos en esta tesis sólo mostraron un efecto negativo cuando ambas tensiones actuaron conjuntamente. Esto indica la importancia de estudiar la interacción entre estos dos estreses.

The Notch signalling is a cellular pathway that results conserved from Drosophila to Homo sapiens controlling a wide range of cellular processes in development and in differentiated organs. It induces cell proliferation or differentiation, increased survival or apoptosis, and it is involved in stemness maintainance. These functions are conserved, but exerted with a high tissue and cellular context specificity. Signalling activation determs nuclear translocation of the receptor’s cytoplasmic domain and activation of target genes transcription. As many developmental pathway, Notch deregulation is involved in cancer, leading to oncogenic or tumour suppressive role depending on the functions exerted in normal tissue. Notch1 and Notch3 resulted aberrantly expressed in human hepatocellular carcinoma (HCC) that is the more frequent tumour of the liver and the sixth most common tumour worldwide. This thesis has the aim to investigate the role of the signalling in HCC, with particular attention to dissect common and uncommon regulatory pathways between Notch1 and Notch3 and to define the role of the signalling in HCC. Nocth1 and Notch3 were analysed on their regulation on Hes1 target and involvement in cell cycle control. They showed to regulate CDKN1C/p57kip2 expression through Hes1 target. CDKN1C/p57kip2 induces not only cell cycle arrest, but also senescence in HCC cell lines. Moreover, the involvement of Notch1 in cancer progression and epithelial to mesenchymal transition was investigated. Notch1 showed to induce invasion of HCC, regulating EMT and E- Cadherin expression. Moreover, Notch3 showed specific regulation on p53 at post translational levels. In vitro and ex vivo analysis on HCC samples suggests a complex role of both receptors in regulate HCC, with an oncogenic role but also showing tumour suppressive effects, suggesting a complex and deep involvement of this signalling in HCC.

The Sox2 transcription factor is modified by sumoylation at the K247 position although the addition of SUMO1 and Pias1 promotes the sumoylation of Sox2 at the additional K123 site. The role of sumoylation on Sox2 biological functions was analyzed by comparing the activity of WT and sumoylation mutants on the transcription of the FGF4 gene in HeLa cells and on the downregulation of the Wnt pathwayvin 293T cells. When SUMO1 and PIAS1 promote the sumoylation of WT Sox2, the transcriptional activity of the FGF4 promoter is inhibited showing that Sox2 sumoylation is necessary for the repression function. However, there is no effect of Sox2 sumoylation on β-Catenin activity. Since we were interested in osteoblast differentiation we set up an inducible system for Sox2 in primary osteoblasts. Following Sox2 doxycycline induction, 158 genes were differentially expressed: 120 up-regulated and 38 down-regulated. We annotated as direct Sox2 targets a number of genes involved in osteoblast biology and we further analyzed 3 of them involved in the BMP pathway. The results show that Sox2 regulates the BMP pathway without affecting SMAD phosphorylation, and that Sox2 sumoylation is not necessary for this function. We also found that genes involved in the Hippo pathway were direct Sox2 targets. As the Hippo pathway is activated by Sox2 and Sox2 interacts with the NF2 promoter, we checked the effect of Sox2 on the expression of NF2. We showed that Sox2 down-regulates the transcriptional activity of the NF2 promoter, allowing the transcription of the YAP/TEAD genes in osteoblasts, thus acting as an upstream regulator of the Hippo pathway. We conclude that Sox2 induction in osteoblasts triggers FGF dependent inhibition of the BMP, Wnt and Hippo pathways.

The first aims of this study were to demonstrate if mitochondrial biogenesis and senescence can be induced simultaneously in cell lines upon exposure to a genotoxic stress, and if the presence of mtDNA mutations which impair the functionality of respiratory complexes can influence the ability of a cell to activate senescence. The data obtained on the oncocytic model XTC.UC1 demonstrated that the presence of mitochondrial dysfunction is involved in the maintenance of a senescent phenotype induced by γ-rays treatment. The involvement of mTORC1 in the regulation of senescence has been shown in this cell line. On the other hand, in cells which do not present mitochondrial dysfunction it has been verified that genotoxic stress determines the activation of both mitochondrial biogenesis and senescence. Further studies are necessary in order to verify if mitochondrial biogenesis sustains the activation of senescence. The second aim of this thesis was to determine the involvement of mTORC1 in the regulation of PGC-1α expression, in order to verify what is the cause of the development of oncocytoma in patients affected by two hereditary cancer syndromes; Cowden and Birt-hogg-Dubé . The study of oncocytic tumors developed by patients affected by these syndromes suggested that the double heterozigosity of the two causative genes, PTEN and FLCN respectively, induce the activation of mTORC1 and therefore the activation of PGC-1α expression. On XTC.UC1 cell line, the most suitable in vitro model, experiments of complementation of PTEN and FLCN were conducted. To date, these results demonstrated that mTORC1 is not involved in the regulation of PGC-1α expression, and PTEN and FLCN seem to have opposite effect on PGC-1α expression.

Top1-DNA cleavage complexes (Top1ccs) trigger an accumulation of antisense RNAPII transcripts specifically at active divergent CpG-island promoters in a replication independent and Top1 dependent manner, leading to transcription-dependent genome instability and altered transcription regulation. Using different cancer cell lines of colon and osteo origins, we show that they display different sensitivity to CPT and G4 binder that is independent from Top1 level. To look at the interactions between Top1 and G4, we show that co-treatment with G4 binders potentiate the cell cytotoxicity of CPT regardless of the treatment sequences. Potentiation is indicated by a reduced inhibition concentration (IC50) with a more profound cytotoxicity in CPT-resistant cell lines, HCT15 and U2OS, hence, indicating an interaction between Top1inhibitor and G4 binders. Moreover, computational analysis confirmed the present of G4 motifs in genes with CPT-induced antisense transcription. G4 motifs are present mostly 5000 bp upstream from transcription start site and notably lower in genes. Comparisons between genes with no antisense transcription and genes with antisense transcription show that G4 motifs in this region are notably lower in the genes with antisense transcripts. Since CPT increases negative supercoils at promoters of intermediate activity, the formation of G4 is also increased in CPT-treated cells. Suprisingly, formation of G4 is regulated in parallel to the transient stabilization of R-loops, indicating a role in response to CPT-induced stress. G4 formation is highly elevated in Pyridostatin treated cells, which previous study shows increased formation of γH2Ax foci. This effect is also seen in the CPT-resistant cell lines, HCT15, indicating that the formation is a general event in response to CPT. We also show that R-loop formation is greatly increased in Pyridostatin treated cells. In order to study the role of R-loops and G4 structures in Top1cc-dependant repair pathway, we inhibited tyrosyl-phosphodiestrase 1 (TDP-1) using a TDP-1 inhibitor.

With this work I elucidated new and unexpected mechanisms of two strong and highly specific transcription inhibitors: Triptolide and Campthotecin. Triptolide (TPL) is a diterpene epoxide derived from the Chinese plant Trypterigium Wilfoordii Hook F. TPL inhibits the ATPase activity of XPB, a subunit of the general transcription factor TFIIH. In this thesis I found that degradation of Rbp1 (the largest subunit of RNA Polymerase II) caused by TPL treatments, is preceded by an hyperphosphorylation event at serine 5 of the carboxy-terminal domain (CTD) of Rbp1. This event is concomitant with a block of RNA Polymerase II at promoters of active genes. The enzyme responsible for Ser5 hyperphosphorylation event is CDK7. Notably, CDK7 downregulation rescued both Ser5 hyperphosphorylation and Rbp1 degradation triggered by TPL. Camptothecin (CPT), derived from the plant Camptotheca acuminata, specifically inhibits topoisomerase 1 (Top1). We first found that CPT induced antisense transcription at divergent CpG islands promoter. Interestingly, by immunofluorescence experiments, CPT was found to induce a burst of R loop structures (DNA/RNA hybrids) at nucleoli and mitochondria. We then decided to investigate the role of Top1 in R loop homeostasis through a short interfering RNA approach (RNAi). Using DNA/RNA immunoprecipitation techniques coupled to NGS I found that Top1 depletion induces an increase of R loops at a genome-wide level. We found that such increase occurs on the entire gene body. At a subset of loci R loops resulted particularly stressed after Top1 depletion: some of these genes showed the formation of new R loops structures, whereas other loci showed a reduction of R loops. Interestingly we found that new peaks usually appear at tandem or divergent genes in the entire gene body, while losses of R loop peaks seems to be a feature specific of 3’ end regions of convergent genes.