Different methods for quality assessment of separated andcommingled plastics from household and electronic waste havebeen developed. Especial attention has been given tospectroscopic methods since they are non-destructive andrequire little or no sample preparation at all. A wide variety of low molecular weight compounds have beenidentified in recycled polyethylene (HDPE) and polypropylenefrom hard packaging waste by gas chromatography- massspectroscopy (GC-MS) after microwave assisted extraction (MAE).Low molecular weight substances such as alcohols, esters,ketones and fragrance and flavour compounds were detected inthe recycled resins. The major category of compounds identifiedin the virgin resins is conformed by aliphatic hydrocarbonssuch as alkanes and alkenes. It was found that theconcentration of aromatic hydrocarbons without functionalgroups, e.g. ethylbenzene and xylenes in recycled HDPE wasapprox. 5 times higher and equal to 120 and 35 ppb,respectively. The potential of near infrared (NIR) and Fourier transformRaman (FT-Raman) spectroscopy in combination with multivariateanalysis as a rapid, non-destructive and accurate analyticalmethod has been studied and the feasibility of these methodsfor at/in line characterisation of several properties ofrecyclates has also been explored. NIR in diffuse reflectance mode has been successfully usedfor quantification of antioxidants in polyethylene, thestandard error of prediction is almost comparable to the errorof wet methods, i.e., extraction plus liquid chromatography.The error of prediction of this method is 35 ppm for Irganox1010 and 68 ppm for Irgafos 168. The inaccuracy in thequantification of Irgafos 168 is due to the fact that thisantioxidant degrades during polymer processing. NIR and Mid-infrared (Mid-IR) worked well for fastdetermination of molecular weight and crystallinity of therecycled HDPE and acceptable errors of prediction, comparableto that of the reference methods, i.e. size exclusionchromatography (SEC) and differential scanning calorimetry(DSC) have been obtained. The present thesis also shows that NIR and Raman are goodcandidates for in/on line compositional analysis of mixedpolymer fractions from recycled plastic waste. Diffusereflectance NIR allows a rapid and reliable measurement ofpellets and requires no previous sample preparation. Thecomposition of binary blends can be determined with highaccuracy. The PP content in the PP/HDPE blends was predictedwith a RMSEP equal to 0.46 %w in the 0-15 %wt region and theRMSEP for PP in the PP/ABS blends was 0.3 %wt. The thermal and thermoxidative stability of recycled PP,HDPE and a 20/80 PP/HDPE blend subjected to multiple extrusionhave been studied by DSC, thermal analysis (TGA) andchemiluminiscence (CL). A decrease in Toxand OIT was observed after each extrusion step.The drop in OIT was sharper after the first two extrusions. TheOIT values produced by DSC and CL were in good agreement.However, CL provided more information about the oxidationprocess taking place in the blends. The moisture content in recycled polyamide 6,6 was readilydetermined by NIR in transmission mode and it could bepredicted with a RMSEP = 0.05 %wt. The accuracy of the methodappeared to be as good as that of the more time consumingthermal methods such as TGA, DSC and loss on dry (LOD), whichwere used as reference methods. The influence of differentamounts of water on the viscoelastic properties of nylon hasbeen investigated. <b>Keywords:</b>Recycling, HDPE, PP, blends, nylon 6,6, ABS,water content, MAE, GC-MS, NIR, FT-Raman, chemiluminiscence,low molecular weight compounds, antioxidant content,crystallinity, molecular weight, thermal stability,characterisation methods, analysis of polymers, blends.

Aquesta tesi és un estudi al voltant de la formació MIR i l'adquisició de les competències professionals en aquest procés. Aquest model formatiu té interès pedagògic perquè encaixa perfectament amb els actuals models de formació per competències al combinar formació i treball.Es plantegen dos grans objectius: es pretén analitzar i contrastar les percepcions de tutors i residents de Catalunya al voltant de l'adquisició de competències professionals en el procés MIR dins l'especialitat de Medicina de Família i Comunitària; posteriorment, la informació obtinguda donarà pistes per suggerir propostes de millora en relació a l'adquisició de competències.Prenent com a punt de partida la hipòtesi que hi ha diferencies entre tutors i residents en les seves percepcions al voltant de l'adquisició de competències es dissenya la recerca. Es tracta d'un estudi descriptiu que combina informació quantitativa i qualitativa. Primerament es treballa amb qüestionaris per obtenir una primera opinió de tots els tutors i residents R3 de Catalunya i posteriorment amb grups focals per poder aprofundir i interpretar les dades obtingudes quantitativament. Aquest procés es fa de forma paral·lela amb tutors i residents. El qüestionari té de dues parts. La primera, consta d'una escala de 42 competències, agrupades en 10 àrees competencials (habilitats clíniques, habilitats instrumentals i habilitats de maneig, comunicació, atenció a la família, atenció a la comunitat, activitat preventiva, docència, aspectes organitzatius i recerca) sobre les que tutors i residents han d'avaluar el grau de capacitació d'aquests últims. La segona part, consta d'altres preguntes sobre la formació MIR en connexió amb aquesta adquisició de competències.Entre les dades obtingudes de forma estadística, entenent que les dades qualitatives no sempre les subscriuen, destaca que les percepcions dels tutors sobre el grau de capacitació per àrees són o molt similars o per sobre de les dels residents i en general força positives llevat de la comunitat que queda clarament "suspesa" i la docència i recerca on els residents els queda molt poc per arribar a "aprovar-se". Com a més valorades destaquen les àrees de clínica, maneig, comunicació i preventiva, mantenint-se la resta en una posició més intermèdia.En quant al model formatiu (estratègies metodològiques, avaluació formativa...) la percepció dels tutors també és més positiva que la dels residents.Per concloure , de tots els resultats obtinguts globalment i de la seva discussió, es destrien del treball algunes aportacions: - L'eix de la formació segueix sent l'assistència individual.- Les àrees competencials vinculades a una medicina més integral (família i comunitat) estan menys consolidades.- Encara s'observen dificultats per practicar una medicina centrada en la persona.- La recerca segueix sent un punt feble.- Hi ha sensibilitat vers la comunicació i el treball en equip.- Les competències es consoliden realment quan es posen en pràctica. El context té un paper fonamental. - Tutorització, autoaprenentatge i modelatge són l'eix metodològic bàsic.- Hi ha poca cultura avaluativa.- La pressió assistencial i la manca de temps entorpeixen el procés formatiu. A partir d'aquí, algunes de les propostes de millora de la formació MIR que es suggereixen són:- Millorar la qualitat docent: formació, espai per docència, reconeixement, criteris d'acreditació amb eines diverses.- Millorar la Tutorització Activa Contínua: centrar-la en el resident i augmentar-ne la freqüència a R1 i R2.- Rotar per diversos tutors de primària.- Potenciar l'aprenentatge cooperatiu.- Desenvolupar mecanismes d'avaluació contínua amb instruments senzills i manejables.- Estimular aquelles àrees que donen un caràcter diferenciador a la primària (família, comunitat, preventiva...).- Buscar el consens en zones frontereres d'actuació. / This thesis is a study of postgraduate medical training and the acquisition of professional competences in this process. This formative model is of pedagogic interest because it fits perfectly well with present training models since it combines education and work. Two great objectives are proposed. Firstly, the objective is to analyse and contrast tutors' and residents' perceptions on the acquisition of professional competences in the postgraduate training process within Family and Community Medicine; secondly, the information obtained will provide clues to suggest proposals for the improvement of acquisition of competences.Research is designed taking as starting point the hypothesis that there are differences between tutors and residents in their perceptions on the acquisition of competences. It is a descriptive study that combines quantitative and qualitative information.Firstly, work is carried out with the help of questionnaires to obtain a first opinion from all tutors and trainees in 3rd year in Catalonia, followed later on by focus groups in order to be able to deepen our understanding and to interpret the data obtained quantitatively. This process is carried out in a parallel way with tutors and residents.The questionnaire consists of two parts. First, it has a scale of 42 competencies, grouped in 10 competency areas (clinical expertise, instrumental abilities and handling abilities, communication, consideration for the family, regard for the community, preventive activity, teaching, management aspects and research) in which tutors and residents have to evaluate the degree of capacitation of the latter ones. The second part consists of other questions regarding postgraduate training in relation to this acquisition of competencies.Among the data obtained statistically, with the prior understanding that qualitative data cannot always be subscribed, we must emphasize that tutors' perceptions on the degree of capacitation in each area are either very similar to or above those of the residents, and generally quite positive except with regards to regard for the community, which is clearly "failed", and to teaching and research skills where residents are close to achieving a "pass". The most valued competency areas are clinical expertise, handling abilities, communications and preventive activity, with the other competency areas maintaining a more intermediate position. With regards to the formative model (methodological strategies, formative evaluation...), tutors' perception is also more positive than the residents' one.To conclude, of all the results obtained globally and from their discussion, some contributions are extracted:- The foundation of the formation continues to be individual attendance. - Competency areas linked to a more global medicine (family and community) are less consolidated.- Difficulties are still observed for practicing a medicine centred on the person.- Research remains a weak point.- There is sensitivity for communication and teamwork.- Competencies really consolidate themselves where they are put into practice. Context plays a crucial role.- Tutorials, self-learning and modelling constitute the basic methodological axis.- There is little evaluative culture.- The pressure on the social security system and the lack of time interrupt the formative process.The following are some of the proposals suggested to improve the postgraduate medical training: - Improve the quality of teaching: formation, teaching space, recognition, accreditation criteria with various tools.- Improve Continuous Active Tutorials: focus them on the resident and increase their frequency for R1 and R2.- Rotate between various primary health care tutors.- Enhance cooperative learning.- Develop continuous evaluation mechanisms with simple tools that are easy to handle.- Enhance those areas which give primary health care a differentiating character (family, community, preventive...).- Seek consensus in border action areas.

Cancer may arise as a result of deregulation of oncogenes and/or tumor suppressors. Although much progress has been made for the identification of such cancer related genes, our understanding of the complex tumorigenesis pathways is still not complete. Therefore, to improve our understanding of how certain basic mechanisms work in normal and in cancer cells, we aimed to characterize two different breast cancer related genes. First part of the study focused on subcellular localization USP32 (Ubiquitin Specific Protease 32) to help understand the function of this uncharacterized gene. USP32 is a member of deubiquitinating enzymes (DUBs) and the gene maps to a gene rich region on 17q23. Genes on 17q23 are known to undergo amplification and overexpression in a subset of breast cancer cells and tumors. DUBs are known to be implicated in a variety of cellular functions including protein degradation, receptor endocytosis and vesicle trafficking. Therefore to elucidate the function of USP32, we localized the full length USP32 protein fused to GFP, in HeLa cells, using Fluorescence Protease Protection (FPP) assay and confocal microscopy. Results suggested a Golgi localization for USP32 as confirmed by co-localization study via BODIPY-TR, a Golgi specific marker. Additional investigations to find the role of USP32 in Golgi will further clarify the function of this candidate oncogene. Second part of the study focused on a potential tumor suppressor. For this purpose, we functionally characterized miR-125b, a microRNA gene as a potential tumor suppressor in breast cancer. microRNAs are regulators of gene expression and their deregulation is detected in cancer cells. miR-125b is reported as a down regulated microRNA in breast cancers. In this study, we investigated the expression, function and possible targets of miR-125b in breast cancer cell lines (BCCLs). Our results revealed a dramatic down regulation of miR-125b in a panel of BCCLs. Restoring the expression of miR-125b in low miR-125b expressing cells decreased the cell proliferation and migration as well as cytoplasmic protrusions, detected by staining of actin filaments. While connection of miR-125b and cell motility based on ERBB2 targeting has been reported earlier, here we present data on ERBB2 independent effects of miR-125b on cell migration in non-ERBB2 overexpressing breast cancer cells. Our results showed involvement of a miR-125b target, ARID3B, in cell motility and migration. Our findings showed miR-125b to be an important regulator of cell proliferation and migration in ERBB2 negative breast cancer cells, possibly through regulating multiple targets.

<p>Different methods for quality assessment of separated andcommingled plastics from household and electronic waste havebeen developed. Especial attention has been given tospectroscopic methods since they are non-destructive andrequire little or no sample preparation at all.</p><p>A wide variety of low molecular weight compounds have beenidentified in recycled polyethylene (HDPE) and polypropylenefrom hard packaging waste by gas chromatography- massspectroscopy (GC-MS) after microwave assisted extraction (MAE).Low molecular weight substances such as alcohols, esters,ketones and fragrance and flavour compounds were detected inthe recycled resins. The major category of compounds identifiedin the virgin resins is conformed by aliphatic hydrocarbonssuch as alkanes and alkenes. It was found that theconcentration of aromatic hydrocarbons without functionalgroups, e.g. ethylbenzene and xylenes in recycled HDPE wasapprox. 5 times higher and equal to 120 and 35 ppb,respectively.</p><p>The potential of near infrared (NIR) and Fourier transformRaman (FT-Raman) spectroscopy in combination with multivariateanalysis as a rapid, non-destructive and accurate analyticalmethod has been studied and the feasibility of these methodsfor at/in line characterisation of several properties ofrecyclates has also been explored.</p><p>NIR in diffuse reflectance mode has been successfully usedfor quantification of antioxidants in polyethylene, thestandard error of prediction is almost comparable to the errorof wet methods, i.e., extraction plus liquid chromatography.The error of prediction of this method is 35 ppm for Irganox1010 and 68 ppm for Irgafos 168. The inaccuracy in thequantification of Irgafos 168 is due to the fact that thisantioxidant degrades during polymer processing.</p><p>NIR and Mid-infrared (Mid-IR) worked well for fastdetermination of molecular weight and crystallinity of therecycled HDPE and acceptable errors of prediction, comparableto that of the reference methods, i.e. size exclusionchromatography (SEC) and differential scanning calorimetry(DSC) have been obtained.</p><p>The present thesis also shows that NIR and Raman are goodcandidates for in/on line compositional analysis of mixedpolymer fractions from recycled plastic waste. Diffusereflectance NIR allows a rapid and reliable measurement ofpellets and requires no previous sample preparation. Thecomposition of binary blends can be determined with highaccuracy. The PP content in the PP/HDPE blends was predictedwith a RMSEP equal to 0.46 %w in the 0-15 %wt region and theRMSEP for PP in the PP/ABS blends was 0.3 %wt.</p><p>The thermal and thermoxidative stability of recycled PP,HDPE and a 20/80 PP/HDPE blend subjected to multiple extrusionhave been studied by DSC, thermal analysis (TGA) andchemiluminiscence (CL). A decrease in T_oxand OIT was observed after each extrusion step.The drop in OIT was sharper after the first two extrusions. TheOIT values produced by DSC and CL were in good agreement.However, CL provided more information about the oxidationprocess taking place in the blends.</p><p>The moisture content in recycled polyamide 6,6 was readilydetermined by NIR in transmission mode and it could bepredicted with a RMSEP = 0.05 %wt. The accuracy of the methodappeared to be as good as that of the more time consumingthermal methods such as TGA, DSC and loss on dry (LOD), whichwere used as reference methods. The influence of differentamounts of water on the viscoelastic properties of nylon hasbeen investigated.</p><p><b>Keywords:</b>Recycling, HDPE, PP, blends, nylon 6,6, ABS,water content, MAE, GC-MS, NIR, FT-Raman, chemiluminiscence,low molecular weight compounds, antioxidant content,crystallinity, molecular weight, thermal stability,characterisation methods, analysis of polymers, blends.</p>

Genomic aberrations are primary contributors to the pathophysiology of cancer [11]. Dysregulated expression of genes located within these aberrations are important predictors of chemoresistance, disease prognosis, and patient outcome [12]. This dissertation is focused on understanding the regulation and/or functions of specific genes located at dysregulated genomic regions such as 3q26 and 14q32 in the biology of ovarian and renal cancer, respectively. Serous epithelial ovarian cancer (EOC) manifest amplification at the 3q26.2 locus [2], an observation consistent with the cancer genome atlas (TCGA) [13]. The most amplified gene in this region is EVI1 which has been extensively studied in hematological malignancies [2]. However, its contribution to the pathophysiology of solid cancers remains unknown. We hypothesized that dysregulated EVI1 and SnoN/SkiL expression (located at the 3q26.2 amplicon) leads to the altered cellular functional response, thereby contributing to the pathophysiology of ovarian cancer. Our group has previously shown that EVI1 splice forms may exhibit altered subcellular localization and functional properties relative to the wild type form [14]. In Chapter 3 of this dissertation, we identified that EVI1 splice forms could modulate epithelial-mesenchymal transition. Our findings indicate that siRNA construct targeting the splice junction between exon 2 of MDS1 to exon 2 of EVI1, (reduces the expression of MDS1/EVI1 and EVI1Del190-515 splice forms) increases epithelial cell markers while decreasing mesenchymal markers and reducing migratory potential of ovarian and breast cancer cells. SnoN/SkiL, another gene overexpressed at the 3q26 is reported by our group to be induced upon As2O3 treatment in ovarian cancer cells via unknown mechanisms [15]. This induction of SnoN opposes the apoptotic cell death pathway induced by the drug treatment [15]. We have previously identified that the PI3K/AKT pathway (also dysregulated in ovarian cancer [16]) contributes to the up-regulation of SnoN upon treatment with As2O3 [17]. However, SnoN is regulated via multiple mechanisms including post-translational modifications [18]. Additionally, c-Ski (a homolog of SnoN) is regulated post-transcriptionally by numerous miRNAs in cancer cells [19-22]. In Chapter 4, we attempted to identify potential miRNAs that could regulate SnoN expression post-transcriptionally. We discovered that miR-494 reduces both SnoN mRNA and protein levels. Our experimental outcomes also demonstrate that miR-494 further sensitizes ovarian cancer cells to drug treatment. Interestingly, miR-494 is located at the 14q32 region which has been shown to be down-regulated in renal cancers [23]. Several reports indicate miR-494 to be involved in tumor suppressive responses including apoptosis and cell cycle arrest in various cancers [24-26]. However, its role in renal cancer biology remains unknown. We hypothesized that miR-494 elicits a tumor suppressive response in renal cancer cells. Through our studies in Chapter 5, we demonstrate that miR-494 reduces cell viability and increases apoptotic response in renal cancer cells. We also show that miR-494 increases LC3B mRNA and protein levels. A 3’UTR luciferase assay indicated that LC3B may be a potential target of miR-494. Intracellular lipid droplets (LDs) increased in miR-494 expressing in a LC3B-dependent manner. This was accompanied with reduced intracellular cholesterol content, increased mitochondrial structural disorganization, and altered Drp1 localization. The outcome of our findings have improved our understanding of the regulation and functional response of these genes/miRNAs (EVI1, SnoN, and miR-494) in ovarian and renal cancers. The studies reported in Chapter 5 identified a novel function of miR-494 in increasing LDs and reducing renal cell survival. However, additional studies are warranted to fully understand the underlying mechanism of increased LDs formation in miR-494 expressing cells and the implication of miR-494 and other miRNAs at the 14q32 region in renal cancer biology. In future, these studies will aid in the development of better treatment strategies which will contribute towards the management of cancer.

High-throughput technology is changing the face of research biology, generating an ever growing amount of large-scale data sets. With experiments utilizing next-generation gene sequencing, mass spectrometry, and various other global surveys of proteins, the task of translating the plethora of data into biology has become a daunting task. In response, functional networks have been developed as a means for integrating the data into models of proteomic organization. In these networks, proteins are linked if they are evidenced to operate together in the same function, facilitating predictions about the functions, phenotypes, and disease associations of uncharacterized genes. In this body of work, we explore different applications of this so-called "guilt-by-association" concept to predict loss-of-function phenotypes and diseases associated with genes in yeast, worm, and human. We also scrutinize certain limitations associated with the functional networks, predictive methods, and measures of performance used in our studies. Importantly, the predictive method and performance measure, if not chosen appropriately for the biological objective at hand, can largely distort the results and interpretation of a study. These findings are incorporated in the development of RIDDLE, a method for characterizing whole sets of genes. This machine learning-based method provides a measure of network distance, and thus functional association, between two sets of genes. RIDDLE may be applied to a wide range of potential applications, as we demonstrate with several biological examples, including linking microRNA-450a to ocular development and disease. In the last decade, functional networks have proven to be a useful strategy for interpreting large-scale proteomic and genomic data sets. With the continued growth of genome coverage in networks and the innovation of predictive methods, we will surely advance towards our ultimate goal of understanding the genetic changes that underlie disease. / text

<p>Epstein-Barr virus (EBV) is a ubiquitous human gamma-herpesvirus which chronically infects >95% of the global population, and can give rise to a number of malignancies in B cells and epithelial cells. In EBV latently infected epithelial cells, such as nasopharyngeal carcinoma (NPC) and gastric carcinoma (GaCa) cells, viral protein expression is low. In contrast, a cluster of viral microRNAs (miRNAs) called miR-BARTs is highly expressed. MiRNAs are small non-coding RNAs which regulate gene expression by binding to complementary sequences in mRNAs. It is likely that miR-BARTs play a crucial role in EBV-infected epithelial cells, however a comprehensive understanding of miR-BARTs is currently lacking. Here, I present two studies utilizing the phenotypic and the target approaches, respectively, to demonstrate that miR-BARTs can inhibit apoptosis and activate the Wnt signaling pathway. To discover miR-BARTs that can inhibit apoptosis, I individually expressed miR-BARTs in the EBV- GaCa cell line AGS, and identified five miR-BARTs that conferred this phenotype. To identify pro-apoptotic genes targeted by the five anti-apoptotic miRNAs, I validated one previously published target and identified nine novel targets by performing photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) in the EBV+ NPC cell line C666. Next, I thoroughly demonstrated that the 10 candidate target genes were substantially suppressed by expression of the relevant miR-BARTs, as measured by 3’UTR-containing firefly luciferase (FLuc) expression, mRNA and protein levels, and knockdown of seven of the 10 candidate genes could suppress apoptosis, mimicking the effects of relevant miR-BARTs. On the other hand, in order to identify miR-BARTs that can activate the Wnt signaling pathway, I analyzed the PAR-CLIP data set of C666 cells and discovered nine anti-Wnt signaling targets of miR-BARTs, including seven novel genes and two pro-apoptotic genes identified above. Using FLuc 3’UTR indicator assays, I proved that the 3’UTRs of all seven newly identified anti-Wnt signaling genes were indeed targeted by the relevant miR-BARTs identified by PAR-CLIP. Utilizing a Wnt signaling FLuc reporter TOPflash which measures the Wnt signaling activation, I confirmed that expression of many miR-BARTs that target Wnt signaling inhibitors can indeed upregulate the Wnt signaling pathway. Together, my results identified and validated a substantial number of novel targets of miR-BARTs involved in apoptosis and the Wnt signaling pathway, indicating that EBV may employ miR-BARTs to heavily target these two pathways to facilitate chronic infection.</p> / Dissertation

Generally Hepatitis C Virus tropism is limited to hepatocytes. This limited tropism is a result of the receptors HCV requires for cellular entry and other host cellular factors including, uniquely, a liver specific miRNA, miR-122. The relationship between HCV and miR-122 is interesting, as commonly, miRNA are associated with suppression of function, but in the case of HCV, miR-122 actively promotes HCV proliferation. In-depth studies have demonstrated that miR-122 along with the RNA induced silencing complex (RISC) protein Argonaute 2 (Ago2) binds directly to two seed sequences separated by 8-9 nucleotides on HCV 5’UTR. Binding to the 5’UTR results in an increase in viral replication and translation. The method by which miR-122 promotes HCV translation and replication is not fully understood but evidence suggests that part of the function of miR-122 is to stabilize the HCV genome and protect it from exonuclease degradation by Xrn1, but other mechanisms remain to be identified. The reliance of HCV on miR-122 is best exemplified by the fact that removal of miR-122 by a miR-122 antagonist drastically impedes HCV viral titers in Chimpanzees and humans with no indication of escape mutants. The observation that HCV augmentation of the HCV life cycle by miR-122 requires Ago2 suggests that other components downstream in the miRNA suppression pathway may also be part of the mechanism of action. Our studies focused specifically on the processing body (p-body) associated DEAD-box helicase DDX6. DDX6 is essential for p-body assembly, required for robust miRNA suppression activity and elevated in HCV associated hepatocellular carcinomas. As such we hypothesized that DDX6 and p-bodies were directly or in-directly associated with the mechanism of action of miR-122. Knocking down DDX6 with siRNA indicated that DDX6 augments both HCV replication and translation. To examine whether DDX6 augmentation of HCV replication was related to the effects of miR-122 on the HCV life cycle, HCV replication and translation were assessed in the presence or absence of miR-122 when DDX6 was knocked down. Our data indicated that HCV replication and translation were augmented equally by miR-122 whether DDX6 was present or not. Our data also demonstrated that HCV replication and translation that was occurring independent of miR-122 was also still affected by DDX6 knockdown. Taken together our observations strongly suggest that the role DDX6 has on HCV is independent of HCV and miR-122’s relationship. In order to better understand miR-122’s relationship with HCV, we hypothesized that targeting the miR-122 binding region with siRNA would inhibit HCV replication initially, but that over the course of several rounds of treatment with the same siRNA, HCV would mutate to escape the siRNA, producing escape mutants that replicate without a dependency on miR-122. These escape mutants could be evaluated on how they replicate without using miR-122, shedding light on miR-122 and HCV’s relationship. Conversely if no escape mutants arose the siRNA could be further studied as a potential therapeutic for HCV. siRNA designed to target the miR-122 binding region inhibited HCV replication, confirming that the designed siRNAs could access the miR-122 binding region and function as an siRNA. Interestingly, when the siRNAs were used against a replication competent HCV RNA having a single nucleotide mutation in the first miR-122 binding site, instead of abolishing siRNA knockdown, two of the siRNA showed enhanced inhibition activity. The target sequences of these siRNAs spanned both miR-122 binding sites and we speculate that their inhibitory activity was due to competition for miR-122 binding to site 2. This observation indicates that siRNA targeting the miR-122 binding region have dual activity, by siRNA induced cleavage, and as a competitive inhibitor of miR-122 binding. Selection for viral escape mutants of the miR-122-binding site targeting siRNAs revealed viral RNAs having mutations within the miR-122 binding sites, in the surrounding region, and to other areas within the HCV IRES. The mutant viruses will be used to assess the influence of miR-122 binding site mutations on HCV replicative fitness, and to determine if the virus can evolve to replicate independent from augmentation by miR-122.

El riesgo sísmico es un concepto que considera la posibilidad de que se produzcan pérdidas de vidas humanas y pérdidas económicas debidas a la acción de un terremoto. Tiene dos componentes principales: la peligrosidad sísmica y la vulnerabilidad sísmica. Para evaluar la peligrosidad sísmica en un punto del territorio se deben considerar conjuntamente la peligrosidad regional y la peligrosidad local. El cálculo de la peligrosidad regional se basa en la estimación del movimiento del suelo producido por el mayor terremoto representativo de la sismicidad de una región o por la contribución de toda la sismicidad regional. La peligrosidad local considera los efectos de suelo, los efectos topográficos, o los efectos inducidos por los sismos como son los deslizamientos o la licuefacción. En la mayor parte de los terremotos destructivos recientes (Michocán en 1985, Loma Prieta en 1989, Kobe en 1995, Turquía en 1999) se ha observado la importancia de los efectos sísmicos locales, como por ejemplo la amplificación del movimiento del suelo debida a efectos de sitio, en la distribución de los daños.El principal objetivo de esta tesis doctoral es exponer y aplicar distintas técnicas y metodologías, tanto experimentales como de simulación numérica, para la evaluación de la peligrosidad sísmica a escala local, adaptadas a distintos grados o niveles de exigencia y para ámbitos con distintas densidades de población. A lo largo de este trabajo se han comprobado las ventajas e inconvenientes de la aplicación de estos métodos en ámbitos rurales y urbanos.En este trabajo se ha realizado un estudio de microzonación sísmica en dos zonas urbanas: la cubeta de Andorra la Vella y la ciudad de Málaga, y en una zona rural: el valle de la Cerdanya. Partiendo de los cálculos de la amplificación del movimiento del suelo en la cubeta de Andorra la Vella, el valle de la Cerdanya y la ciudad de Málaga se propone una metodología avanzada para el cálculo de la amplificación del movimiento del suelo debida a efectos locales, en términos de la intensidad macrosísmica y en términos de la aceleración espectral. Finalmente, se propone un conjunto de metodologías que respondan a distintos requerimientos o exigencias para la obtención de la microzonación o evaluación de la peligrosidad sísmica a escala local.Por otro lado, se ha aplicado el método de Newmark de cálculo de estabilidad de laderas para obtener la peligrosidad de desprendimientos activados por vibraciones del terreno producidas por voladuras. Los resultados obtenidos en este estudio se han aplicado como criterios de prevención durante la excavación de un túnel en el macizo rocoso del Roc del Dui, en el municipio de Queralbs situado en el Pirineo Oriental catalán. / This project is divided in two sections. The first one deals with seismic microzonation for urban and rural areas and the second one evaluates the slope stability under vibratory loads generated by blasting in a tunnel excavation.Seismic risk assessment considers the possibility of human and economic losses due to a seismic motion. This concept has two principal components: the seismic hazard and the seismic vulnerability. Regional and local seismic hazard must be considered to evaluate overall seismic hazard in a specific location. The regional seismic hazard can be calculated assuming a deterministic approach, the soil motion is produced by the largest representative earthquake of the region, or assuming a probabilistic approach that considers the contribution of the regional seismicity in the seismic motion. Local seismic hazard considers the soil effects, the topographic effects, or the induced effects due the seismic motion, as landslides or liquefaction. In most of the recent destructive earthquakes (e.g. Michocán 1985; Loma Prieta 1989; Kobe 1995; Turkey 1999) the high influence of seismic local effects, for example the soil motion amplification, has been observed in the damage distribution.One of the main goals of this doctoral dissertation (PhD) is to explain and to apply different experimental and numerical simulation methodologies for local seismic hazard evaluation, adapting different levels of priority for areas with different population densities. This work verifies advantages and disadvantages of the application of these methods in rural and urban areas.Seismic microzonation studies in urban zones has been performed in Andorra la Vella basin and in the city of Malaga. Microzonation for a rural area has been conducted in the Cerdanya valley. Based on the soil motion amplification calculations in the three pilot areas a more advanced methodology for amplification characterization due to local effects has been proposed, in terms of the macroseismic intensity and spectral acceleration. Finally, a set of seismic microzonation methodologies are proposed depending on different degrees of resolution, evaluation resources, and social requirements.The second main objective of this dissertation is to apply and to validate the Newmark's method for calculations of hillsides stability to obtain the susceptibility of rock falls activated by blasting vibrations. The results obtained have been applied as a criteria of prevention during the excavation at Roc del Dui tunnel rock mass, in the municipality of Queralbs located in the Oriental Catalan Pyrenees.

El objetivo de la tesis ha sido la puesta a punto de un método analítico rápido basado en la espectroscopia FT-MIR para determinar el estado de maduración fenólica de las uvas tintas con el que poder fijar la fecha de vendimia. Para ello se desarrollaron modelos de calibración multivariante PLS que permitieron la cuantificación simultánea de diferentes compuestos fenólicos en extractos de uva de forma rápida y fiable, utilizando la espectrofotometría UV-Vis como técnica de referencia. Para obtener extractos representativos que sirvieran como patrones de calibración, se llevó a cabo un muestreo que contemplaba la variabilidad natural de la uva y se puso a punto un método de extracción rápido y eficaz, cuya capacidad para predecir las características del vino fue comprobada con ensayos de microvinificación. / The aim of this thesis has been the development of a fast analytical method based on the FT-MIR spectroscopy to determine the phenolic ripening state of red grapes and to fix the harvest date. For this purpose, several PLS calibration methods were developed which allow the simultaneous quantification of different phenolic compounds in grape extracts in a fast a reliable way, using the UV-Vis spectrophotometry as reference technique. To get representative extracts which can be used as calibration standards, a sampling which covers the maximum natural variability of grape took place and a fast and efficient extraction method was optimized, whose ability to predict the wine characteristics was checked by microvinification assays.