Rozprawy doktorskie na temat „RVS AS TOOL”

As per the studies conducted in past, it has been stipulated that it is not the earthquakes which kill humans, instead it is the poor and substandard practices involved in the construction of building which leads to its failure in the seismic event. Although, various IS codes for the design of building are available, but due to the rapid urbanization they have been overlooked. Being a seismic zone IV (as Per IS 1893:2016) and a rapidly urbanizing city without considering the standard construction practices, Delhi becomes a soft target for any severe seismic event. Hence, in order to safeguard deficit structures against seismic excitation; a rapid performance evaluation strategy is the need of hour. A lot of effort for the assessment of existing structures has been laid upon, but the basic idea could not be inherited. Eventually the strategy could not be implemented, owing to their technical complexities. Although, Various screening guidelines have been issued by different agencies, but FEMA P-154(2015) and FEMA P-155(2015) supplements the screening in the most comprehensive manner by scoring the screened building for its various attributes, like RVS score has been used to calculate risk of earthquake causing the collapse of building. Adopting the various fundamentals of practices in various guidelines such as FEMA, ATC etc., this dissertation aims to simplify the application of RVS in purview of various Indian Standard Codes, in order to achieve a better probabilistic approach in estimation of probable life building. In this dissertation, a detailed study has been carried out to calculate the probability of collapse of building using RVS score and subsequently that probability of collapse is compared with the probability of MCE shaking. Eventually, the result from comparison is used to decide, if the detailed vulnerability assessment of the building is required or not. Following the methodology prescribed by FEMA 154(2015), four Different types of existing structures have been taken as case study for the calculation of the collapse probability i.e., High-Rise RCC Building, Mid Rise RCC Building, RCC plus Masonry Combined Building and Load Bearing Masonry Building. In addition, RVS score has been used to calculate the Probability of the collapse mechanism to happen in a building under a significant earthquake within next 50 years. Following the calculation of Risk Score, its associated probability of at least one collapse causing earthquake within next 50 years has been calculated. Two different types of buildings i.e., RCC G+3 building and Load Bearing Masonry building, have been taken as a case study for the calculation of Risk Score using RVS score and its associated vulnerability using the technique as per FEMA for any seismic activity in the next 50 years. Following the calculation of Probability of Collapse under the MCE shaking, RCC plus Masonry Combined Building is found to have the least likelihood of being collapsed under MCE ground shaking, i.e., 0.63%, whereas the Mid-Rise RCC Building shows the highest likelihood of being collapsed under MCE ground shaking i.e., 100%. However, the High- Rise RCC Building has 50% likelihood of being collapsed under MCE ground shaking and Load Bearing Masonry Building have been found to have fairly high likelihood of being collapsed under MCE ground shaking i.e., 63%. Following the calculation of at least one collapse causing earthquake within next 50 years, it has been found that the Load Bearing Masonry building has 3.11% chance of being confronted by an earthquake that can cause collapse. And, the probability of an earthquake causing the RCC G+3 building to collapse over the next 50 years has been calculated to be 1.57 %. The risk score is an indicator of the degree of fatality of the building and it has been calculated that Load Bearing masonry building is 100 times more fatal than the newly constructed Load Bearing masonry building. Therefore, detailed structural evaluation for retrofitting the Load Bearing masonry building to be safe under any seismic activity, is required. Hence, as per the study, the RVS guidelines of FEMA P-154(2015) can be used satisfactorily including permissible limits as per Indian standard for preliminary investigation. Based on which a fair decision regarding the necessity of detailed technical evaluation can be done and the methodology will help in prioritizing the building for detailed structural evaluation and retrofitting recommendations.

For every robotics project, choosing a suitable framework and middleware for software and hardware is a challenging task which may influence the entire project. Robotics applications typically are resource constrained when it comes to computations and memory usage. They are built on different hardware platforms and applied in different domains. Therefore it is hard to introduce a common framework for all types of projects. However, in recent years several new attempts have been made and received attention from both researchers and industry. These frameworks are still under development and need to be extended. This paper discusses the different features that are needed for robotics frameworks and compares some of the available middleware and standards.

Cette thèse décrit l'utilisation d'aptamères peptidiques (PA) comme outil d'étude de la signalisation de Ras dans des modèles de lignées cellulaires. Elle décrit ensuite les analyses initiales de l'effet biologique de petites molécules sélectionnées pour leur capacité à inhiber l'interaction entre PAs et H-RasV12. Finalement elle présente la caractérisation de précurseurs de neurones de DRG et que nous espérons pouvoir utiliser pour de futures études. Afin d'analyser les interactions entre Ras et différentes protéines et les perturbations de la voie de signalisation, PAs dirigés contre la protéine Ras sauvage et Ras oncogène avaient été sélectionnés. PA 105R présente un phénotype d'interaction en double hybride de levure (Y2H) avec H-, K-, et N-Ras. PA HR3C présente un phénotype d'interaction avec la protéine Ras sauvage et H-RasV12. Dans les cellules PC12, HR3C bloque aussi bien la signalisation de Ras sauvage que celle de H-Ras oncogène. Par contre dans les cellules AsPC1, la protéine oncogène K-RasD12 endogène est inhibée par 105R, alors que HR3C est bien moins efficace. Des mutations dirigées contre les acides aminés localisés à la surface de HrasV12, suivi d'un test d'interaction en Y2H, ont permis de cartographier les sites de liaison des PAs biologiquement actifs sur H-RasV12. L'activité biologique d'un certain nombre de petites molécules sélectionnées pour leur capacité à dissocier l'un des PAs spécifique de Ras. Ces études ont abouti à l'identification d’une molécules qui pourrait être biologiquement pertinente. Des études supplémentaires seront requises afin d'obtenir, à partir de ces molécules, de possibles candidats aux études pré-cliniques. Finalement, nous avons mis en place la technologie des PAs pour une utilisation dans des cellules primaires dérivées rat DRG, et développé une lignée précurseur de cellules neuronales de DRG que nous avons maintenue en culture 2 ans. Nous espérons ainsi utiliser ces précurseurs pour des études futures dans le PNS
This thesis describes the use of peptide aptamers (PA) for the study of Ras signaling in model cell lines. It further describes the initial analysis of biological effects of small molecules identified for their capacity to displace a biologicallyactive PA from H-RasV12. Finally, it offers characterization of DRGn precursors that we hope to be able to use for future studies. To analyze Ras interactions with other proteins and perturbation of signaling pathways, PAs directed towards wt Ras and oncogenic Ras had been selected. PA 105R elicited an interaction phenotype in yeast two–hybrid (Y2H) with H-, K-, and N-Ras. PA HR3C gave an interaction phenotype with H-Ras wt and HrasV12. In PC12 cells, it blocked both wt and oncogenic H-Ras signaling, whereas 105R had little effect on NGF-induced signaling, but inhibited that stimulated by oncogenic Ras. In contrast, in AsPC-1, endogenous oncogenic K-RasD12 is inhibited by 105R, while HR3C is much less effective. Targeted mutation of amino acids located on the surface of H-RasV12, followed by Y2H interaction mating assays, offered the possibility to map the binding sites of the biologically-active Pas. We evaluated biological activity of a number of small molecules selected for their capacity to displace one of the Ras-specific PAs from the target. These studies resulted in the identification of one molecule that may be biologically pertinent. Further studies will be required to develop possible pre-clinical candidates based on these molecules. Finally, we were to implement the PA technology in primary cells derived from rat DRG, and we developed a DRG precursor line that we have maintained in culture for over two years. It is hoped that we use thes precursors for future studies in the PNS

The goal of this 15 hp thesis in computer engineering was to develop a tool for monitoring and analyzing the data flow on a Controller Area Network (CAN) called Rig Control System (RCS) that is used by Atlas Copco. Atlas Copco develops and manufactures machines for Mining and Rock Excavation. The Rocktec division is responsible for the Rig Control System platform used on machines in all division within the Mining and Rock Excavation Technique (MR) business area. The tool’s primary purpose is monitor and analyze data from the RCS network and present the analyzed data in an easy way to help with development and maintenance of RCS and machines that use RCS. The advantages and how data is sent over the CAN bus are presented as well as the CANopen protocol which is a higher layer protocol based on CAN. Two ways of data acquisition from RCS are presented, a simulated environment and real hardware. Different types of interprocess communication are presented as well as the pros and cons of each of these types. The creation of the tool required a Graphical User Interface (GUI) so different frameworks for this task are also presented and discussed. A version of the tool is presented and discussed in detail. The result of the project is a tool that with further development can be of great use to developers and service engineers working with RCS.
Målet med detta 15 hp examensarbete inom datateknik var att utveckla ett verktyg för att övervaka och analysera dataflödet på ett Controller Area Network (CAN) kallat Rig Control System (RCS) som används av Atlas Copco. Atlas Copco utvecklar och tillverkar maskiner för gruvdrift och bergbrytning. Rocktec-divisionen är ansvarig för Rig Control System-plattformen som används i maskiner från alla avdelningar inom affärsområdet Mining and Rock Excavation Technique (MR). Verktygets primära uppgift är att övervaka och analysera data från RCS-nätverket och presentera den analyserade datan på ett lättöverskådligt sätt för att vara till hjälp vid utveckling och underhåll av RCS och maskiner som använder RCS. Fördelar, hur data skickas över CAN-bussen och även CANopen-protokollet, som är ett högnivåprotokoll baserat på CAN, presenteras. Två olika sätt att samla in data från RCS presenteras, en simulerad miljö och riktig hårdvara. Olika typer av interprocesskommunikation och deras respektive fördelar och nackdelar presenteras. Skapandet av verktyget krävde ett grafiskt användargränssnitt så olika ramverk för denna uppgift presenteras och diskuteras i detalj. Resultatet av projektet är ett verktyg som med vidare utveckling kan vara till stor användning för utvecklare och servicetekniker som arbetar med RCS.

Respiratory syncytial virus (RSV) causes a common infection that is associated with a range of respiratory illnesses from common cold-like symptoms to serious lower respiratory tract illnesses such as pneumonia and bronchiolitis. RSV is the single most important cause of serious lower respiratory tract illness in children < 1 year of age. Host innate and acquired immune responses activated following RSV infection have been suspected as contributing to RSV disease. Toll-like receptors (TLRs) activate innate and acquired immunity and are candidates for playing key roles in the host immune response to RSV. Leukocytes express TLRs including TLR2, TLR6, TLR3, TLR4, and TLR7 that can potentially interact with RSV and promote immune responses following infection. Using knockout mice, we have demonstrated that TLR2 and TLR6 signaling in leukocytes can activate innate immunity against RSV by promoting TNF-α, IL-6, CCL2 (MCP-1), and CCL5 (RANTES) production. As previously noted, TLR4 also contributed to cytokine activation (71, 90). Furthermore, we demonstrated that signals generated following TLR2 and TLR6 activation were important for controlling viral replication in vivo. Additionally, TLR2 interactions with RSV promoted neutrophil migration and dendritic cell activation within the lung. Collectively, these studies indicate that TLR2 is involved in RSV recognition and subsequent innate immune activation and may play a role in modulating acquired immune responses through DCs. Despite the fact that RSV is the single most important cause of infant upper respiratory tract disease, there are no licensed vaccines available to prevent RSV disease. We have developed a virus-like particle (VLP) vaccine candidate for RSV. The VLP is composed of the NP and M proteins of Newcastle disease virus (NDV) and a chimera protein containing the cytoplasmic and transmembrane domains of the NDV HN protein and the ectodomain of the human RSV G protein (H/G). BALB/c mice immunized with 10 or 40 μg total VLP-H/G protein by intraperitoneal or intramuscular inoculation stimulated antibody responses to G protein as good as or better than comparable amounts of UV-inactivated RSV. Furthermore, VLP-H/G induced robust CTL responses in vaccinated animals. Immunization with two or even a single dose of these particles resulted in the complete protection of BALB/c mice from RSV replication in the lungs. Upon RSV challenge of VLP-H/G immunized mice, no enhanced pathology in the lungs was observed, although lungs of mice immunized in parallel with formalin-inactivated RSV (FI-RSV) showed the significant pathology that has been previously observed with FI-RSV vaccination. Thus, the VLP-H/G candidate vaccine was immunogenic in BALB/c mice and prevented replication of RSV in murine lungs with no evidence of immunopathology. These data support further development of virus-like particle vaccine candidates for RSV.

Web 2.0 tools, namely blogs, wikis, podcasts, and RSS were introduced to change teaching practices of in-service high school teachers to improve the collaboration of today's students in the English language classroom. Two high school teachers of English language and their classes participated. The teachers were interviewed about their current teaching practices and provided with training to develop teaching units that use Web 2.0 to engage students as active collaborators in their learning. They integrated blogs, podcasts, wikis, and RSS into their teaching. Additional interviews were conducted during and after the implementation stage. Implementation strategies, changes in teaching practices, challenges encountered, and the impact on student interaction and collaboration were closely examined. Students were surveyed at the conclusion. Teachers found that Web 2.0 tools made them more efficient in teaching. Blogging was the most powerful tool for journal writing and sharing ideas. Wikis were more difficult to use but were useful to facilitate group planning and collaborative construction of knowledge. Podcasts were useful for publishing audio recordings of interviews, speeches, and poetry recitals. RSS feeds made it easy for teachers and students to track updates on websites, posts on blogs, collaborations on wikis, and audio recordings on podcasts. Both teachers and students enjoyed the interactions and collaboration that took place in the English classroom using Web 2.0 tools.

Today's society is characterized by a high degree of change where the manufacturing systems are affected by both internal and external factors. To adapt to current manufacturing requirements in the form of short lead-time, more variants, low and fluctuating volumes, in a cost-efficient manner, new approaches are needed. As the global market and its uncertainties for products and its lifecycles change, a concept called 'reconfigurable manufacturing system' has been developed. The idea is to design a manufacturing system for rapid structural change in both hardware and software to be responsive to capacity and functionality. A company's development towards the concept is often based on a strategy of incremental investments. In this situation, the challenges are to prioritize the right project and maximize the performance as well as the financial efficiency of a multi-approach problem. The report is based on three different issues. Partly how to standardize relevant performance-based metrics to measure current conditions, how new performance-based metrics can be developed in collaboration with reconfigurability characteristics, and set a direction for how decision models can be used to optimize step-based investments. The study is structured as an explorative study with qualitative methods such as semi-structured interviews and document study to get in-depth knowledge. Related literature addresses concepts in search areas such as reconfigurable manufacturing system, key performance indicators, investment decisions, and manufacturing readiness levels. The findings are extracted from interviews and document studies that generate a focal company setting within the automotive industry, which acts as the foundation for further analysis and decisions throughout the thesis. The analysis results in sixteen performance measurements where new measures been created for product flexibility, productionvolume flexibility, material handling flexibility, reconfiguration quality and diagnosability using reconfigurability characteristics. A conceptual decision support model is introduced with an underlying seven-step investment process, analyzing lifecycle cost, risk triggered events in relation to cost, and performance measurements. The discussion chapter describes how different approaches are used during the project that has been revised by internal and external factors. Improvement possibilities regarding method choice and the aspects of credibility, transferability, dependability, and conformability are discussed. Furthermore, the authors argue about the analysis process and how the result has been affected by circumstances and choices. The study concludes that a three-pronged approach is needed to validate the investment decision in terms of system performance changes, cost, and uncertainty. The report also helps to understand which performance-based metrics are relevant for evaluating manufacturing systems based on operational goals and manufacturing requirements.

By combining FEMA's HAZUS-MH (Hazards U.S. Multi-Hazard) flood-loss estimation software and the HEC-RAS hydraulic modeling package, this study was able to quantify potential beneficial and adverse impacts of flood-control and navigational structures along the Middle Mississippi River (MMR; between Mississippi-Missouri River confluence and Thebes, IL). The goal of this investigation was to assess changes in water-surface elevations and associated flood losses to: 1) quantify the potential exposure of flooding under different flood-control configurations along the Middle Mississippi River (MMR), and 2) assess the relative contributions of various engineered structures and flood-loss strategies to potential flood losses. Assessment of the impact of engineering structures was accomplished by modeling five scenarios for the 100- and 500- year floods: 1) current MMR levee configuration (levee protecting for ≤50-year flood); 2) removal of all flood-control structures on the MMR; 3) increasing the height of levees and floodwalls in metropolitan St. Louis to protect urban areas to the 500-year flood level while simultaneously removing all agricultural levees downstream; 4A) a less engineered MMR channel and floodplain with fewer flood control and navigation structures, simulating conditions from 65 years ago (1942-1947) with 1940's levees; and 4B) a less engineered MMR channel and floodplain with fewer flood control and navigation structures, simulating conditions from 65 years ago (1942-1947) with current levee configuration. Comparison of scenarios 2 and 3 relative to scenario 1 allows for quantitative assessment of the flood-control structures on stages and flood losses. Results from scenario 2 revealed that removing all levees along the MMR reduces the average stages from 2.2 m (100-year) to 2.5 m (500-year, but also increased economic and social impacts relative to scenario 1. Scenario 3 revealed that removing agricultural levees downstream of St. Louis on the MMR decreased stages by 1.4 m (100- and 500-year); however, flood losses for the 100-year flood were increased. Flood losses for the 500-year flood were decreased relative to scenario 1. These results suggest that agricultural levees along the MMR protect against medium size floods (50- or 100-year flood) but cause more damage than they prevent during large floods such as the 500-year flood. Comparison of scenarios 4A and 4B relative to scenario 1 allows for a quantitative assessment of river engineering structures and modern buildings constructed over the last 65 years. In scenarios 4A and 4B, a less engineered river decreased stages by 1.2 m (for the 100-year flood) relative to scenario 1. In scenario 4A, the 1940's levees expose modern buildings in the floodplain to flooding, causing economic building losses to increase; however, in scenario 4B, current levee configuration protects modern buildings in the floodplain from flooding causing, economic building losses to decrease. If the current flood-control structures were not built, it is likely that the land in the floodplain for scenarios 4A and 4B would not be developed and the land used would be more flood-tolerant. Sensitivity analyses were run to assess the impact of using the default HAZUS-MH national-level data; this was done by comparing results produced by using aggregate analysis (coarse data) versus results using UDF analysis (detailed data). The aggregate analysis estimated 51% fewer buildings damaged than the UDF analysis. Conversely, the aggregate analysis increased the economic building losses by 51% relative to the UDF analysis. Although collecting local data for a study is not always feasible, the large differences documented here need to be considered when discussing HAZUS-MH results. Overall, this project shows implications for historic and future flood-control and navigational structure projects on the MMR and other rivers. It also emphasizes the importance of studying the impact future engineering structures will have on water-surface elevations and flood losses before implementing them.

Three-dimensional (3D) laser scanners are becoming increasingly more affordable and user-friendly, making 3D-modeling tools more widely available to researchers in various countries and disciplines. In archaeology, 3D-modeling has the particular advantages of facilitating the documentation and analysis of objects that are fragile, rare, and often difficult to access. We have previously shown that 3D-modeling is a highly useful tool for shape analysis of archaeological bone material, due to the high measurement accuracy inherent in the latest generation of 3D laser scanners (Sholts et al. 2010; 2011). In this work, we explore the utility of 3D-modeling as a tool for Viking Age artefact analysis. To test the usefulness of 3D-modeling when analyzing artefacts with a very complex morphology, we chose highly ornate Viking Age baroque shaped brooches as study objects. These baroque shaped brooches constitute a group of dress ornaments mainly encountered in eastern Viking Age Scandinavia. Due to their large cast and/or attached bosses they obtain an almost baroque appearance, hence their name (cf. Jansson 1984: p. 81). They appear in two major versions, i.e. circular or equal armed, and in two kinds of material, i.e. silver- and copper-based alloys. Because of the position of bronze brooches in burial contexts, it appears they were used to fasten the cape or shawl in the female dress (cf. Jansson 1984: p. 75ff., Aagård 1984: p. 96ff.; Neiß 2006, figs. 3, 4; Capelle 1962: p. 106). For the present work a recently excavated brooch from Denmark was analyzed, together with three Russian brooches with nearly iconic status in the field of Viking Age studies. In the three case studies, we investigated possible uses of 3D-modeling for artefact analysis, artefact reconstruction, and tool mark and motif analysis. Exploring the usefulness of 3D-modeling for these purposes allowed us to draw conclusions regarding how 3D-analysis can be best incorporated into future artefact analysis. In addition, the case studies allowed us to gain new insights about the baroque shaped brooches and their uses.

Forskningsfinansiärer: Helge Ax:son Johnsons stiftelse, Svenska institutet (Visby-programmet), Kungliga vitterhets historie och antikvitets akademin (Montelius minnesfond); Svenska fornminnesforeningen

3D-laserskanning som verktyg vid vikingatidsstudier

MyD88 est une protéine adaptatrice du système immunitaire inné, impliquée dans la défense de l’organisme contre les agents microbiens. Elle est recrutée aux « Toll-like receptors » (TLRs) suite à la reconnaissance par ces derniers de motifs microbiens conservés, les PAMPs (Pathogens-associated molecular patterns). La voie de signalisation ainsi déclenchée va aboutir à la production de cytokines pro-inflammatoires, de chimiokines et d’espèces actives de l’oxygène. De cette façon, les TLRs, via MyD88, constituent la première ligne de défense contre les pathogènes.De nombreuses études ont permis de démontrer que MyD88 est nécessaire pour la réponse inflammatoire, qui promeut la carcinogenèse. Dans le cadre d’une étude sur les TLRs et le cancer, l’équipe a démontré, grâce à une étude in vivo, que MyD88 participe au processus de tumorigenèse médiée par l’oncogène ras et est nécessaire à l’activation de la voie canonique des MAPKs, ainsi qu’à la transformation cellulaire in vitro. Nous avons ensuite déterminé le mécanisme par lequel MyD88 intervient dans la voie de signalisation Ras/MAPKs, en permettant le maintien de l’activation de cette voie. En effet, MyD88 interagit avec une MAPK clé de cette voie : la kinase ERK, et protège cette dernière de sa déphosphorylation par sa phosphatase spécifique MKP-3, MyD88 et MKP-3 se liant à ERK par le même domaine. Nous avons démontré la pertinence de ce mécanisme, grâce à la mise en évidence d’une surexpression de la protéine MyD88 et de son interaction avec la forme phosphorylée d’ERK dans des coupes de tissus tumoraux humains (estomac, poumon, colon).L’ensemble des résultats obtenus au cours de ma thèse ont permis de montrer qu’en plus de son rôle bien défini en tant qu’adaptateur des récepteurs de l’immunité innée dans les processus inflammatoires, MyD88 joue un rôle direct, qui semble être crucial dans la signalisation Ras, le contrôle du cycle cellulaire et la transformation cellulaire
MyD88 is an adaptator protein of the innate immune system, implicated in the défense against microbes. MyD88 is recruited by the Toll-Like Receptors (TLRs) upon there interaction with conserved microbial patterns (PAMPs). Therefore, TLR signaling pathway induces the production of pro-inflammatory cytokines, chemokines and reactive oxygen species. TLRs, via MyD88, form the first line of defense against pathogens. Accumulating evidence points to inflammation as a promoter of carcinogenesis. MyD88 is an adaptor molecule in TLR and IL-1R signaling that was recently implicated in tumorigenesis through proinflammatory mechanisms. Here we have shown that MyD88 is also required in a cell-autonomous fashion for Ras-mediated carcinogenesis in mice in vivo and for MAPK activation and transformation in vitro. Mechanistically, MyD88 bound to the key MAPK, ERK, and prevented its inactivation by its phosphatase, MKP3, thereby amplifying the activation of the canonical Ras pathway. The relevance of this mechanism to human neoplasia was suggested by the finding that MyD88 was overexpressed and interacted with activated ERK in primary human cancer tissues. Collectively, these results show that in addition to its role in inflammation, MyD88 plays what we believe to be a crucial direct role in Ras signaling, cell-cycle control, and cell transformation

Oxidative stress contributes to the development and progression of a broad range of syndromes and diseases, especially cancer and neurodegenerative processes. Malignant pleural mesothelioma (MPM) is a highly aggressive tumor originating from the mesothelial cells of the pleura, associated with asbestos exposure. Inhalation of long and thin asbestos fibers induces a chronic inflammatory response at sites of fibers deposition, with accumulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) that over time may lead to malignant cell transformation. MPM is characterized by a long latency period, a poor prognosis, and limited effective therapies. Thus, there is a real need for improvement in prognostic and diagnostic tools for MPM. In recent years the focus in the field of biomarker discovery has moved from intracellular components to secreted factors. Cancer cell secretome provides an useful source in the search for proteins involved in tumor development and can be considered a potential tool to investigate tumor properties. Proteomic approaches are increasingly used for biomarker discovery as proteins produced by cancer cells or their microenvironment may eventually enter the circulation and their expression can be assessed for diagnostic purpose. In order to obtain an inclusive overview of MPM cell protein expression, we examined through a proteomic approach the proteome and secretome profiles of two MPM cell lines (NCI-H28 and NCI-2052) and compared to those of a non malignant mesothelial cell line (Met-5A). In this study, we identified mesothelioma cells-secreted proteins prosaposin (PSAP) and quiescin sulfhydryl oxidase 1 (QSOX1) as novel biomarker candidates for mesothelioma and validated their increase in serum of MPM patients by ELISA. Our results crearly show PSAP and QSOX1 serum levels are significantly increased in MPM patients in respect to asbestos-exposed healthy subjects. The informations gained from this investigation have increased our knowledge on MPM and identified two novel potential biomarkers. Neurodegenerative diseases represent a heterogeneous group of disorders that share common features like abnormal protein aggregation, perturbed Ca2+ homeostasis, excitotoxicity, impairment of mitochondrial functions, apoptosis, inflammation, and oxidative stress. Despite recent advances in the research of biomarkers, early diagnosis and pharmacotherapy, there are no treatments that can halt the progression of these age-associated neurodegenerative diseases. Numerous epidemiological studies indicate that long-term intake of a Mediterranean diet, characterized by a high consumption of extra virgin olive oil, correlates with better cognition in aged populations. Olive oil phenolic compounds have been demonstrated to have different biological activities such as antioxidant, antithrombotic, and anti-inflammatory properties. Oleocanthal, a phenolic component of extra-virgin olive oil, is getting more and more scientific attention due to its interesting biological activities. In this study, we aimed to characterize the neuroprotective effects of oleocanthal against H2O2-induced oxidative stress in neuron-like SH-SY5Y cells. Protein expression profiling, combined with pathways analyses, was used to investigate the molecular events related to the protective effects. Oleocanthal demonstrated to counteract oxidative stress increasing cell viability, reducing ROS production and increasing GSH intracellular level. Proteomic analysis revealed that oleocanthal significantly modulates 19 proteins in the presence of H2O2. In particular, oleocanthal up-regulated proteins related to the proteasome, the chaperone heat shock protein 90, the glycolytic enzyme pyruvate kinase and the antioxidant enzyme peroxiredoxin 1. These data offer new insights in the molecular mechanisms behind oleocanthal protection against oxidative stress.

Abstract Respiratory syncytial virus (RSV) is one of the most common pathogens for early childhood respiratory tract infections. Most children are infected by RSV, approximately 1% of infants require hospital care. RSV infections are often accompanied by otitis media (OM). Surfactant proteins A and D (SP-A and SP-D) are involved in lung function and innate immunity. The proteins are capable of recognizing surface patterns in pathogens and function as a defense before the acquired immunity is developed. The Toll-like receptor (TLR) family is associated to several pathogens. The role of the TLR-family is to recognize pathogens and activate the immune defense. The aim of the thesis was to investigate the genetic association of RSV and OM infections in infants. A candidate gene approach was used study SP-A, SP-D and TLR4 in severe RSV bronchiolitis. Association between SP-A and OM was also studied. A case-control study setup was used for all studies. 1700 samples were collected for the studies. The results revealed genetic association between SP-A gene variation and severe RSV infections. SP-A allele 1A3 was overrepresented in RSV infants, the allele 1A was present more often in the control population. SP-D allele Met11 and genotype Met/Met were predisposing to severe RSV infections. The TLR4 gene did not show direct association with severe RSV. However, we showed for the first time difference in association in two separate epidemics. In the OM study, an association was shown between SP-A gene variations and otitis media in children. The present results have brought new information about innate defense and the genetic variations and associations involved. The results will help understand the mechanisms of innate defense and predisposition to infections. The results also present possibilities to investigate and develop new treatment strategies.

Cancer, which is characterised by aggressiveness and increased capacity for metastatic spread still requires basic researchers and clinicians to direct enormous efforts toward the development of novel therapeutic targets. Potential novel targets can be identified and exploited in combination with currently existing therapeutic approaches to improve their efficacy and overcome treatment resistance of tumour cells, protecting the patient from recurrence. To achieve this, different strategies and techniques can be proposed to identify the most promising candidate molecules for further exploitation as therapeutic targets. Human epidermal growth factor receptors (HERs) and NF-E2-related factor 2 (NRF2) are regulators of cellular proliferation and determinants of cancer initiation and progression. NRF2 and HERs confer cancers with resistance to several therapeutic agents. Nevertheless, there is limited understanding of the regulation of HER expression and activation, and the link between NRF2 and HER signalling pathways. This research has demonstrated that pharmacological activation of NRF2 by tert-butyl hydroquinone (tBHQ) upregulates the expression of HER family receptors, HER1 and HER4, elevates phospho protein kinase B (pAKT) levels, and enhances the proliferation of ovarian cancer cells. Pharmacological inhibition using retinoic acid (RA) and bexarotene and genetic inhibition using small interfering RNA (siRNA), did the opposite. Further, tBHQ caused transcriptional induction of HER1 and HER4 with different levels of expression, while siRNA-mediated knockdown of NRF2 prevented this and further caused transcriptional repression. A panel of potent NRF2 inhibitors were screened with the hope of finding the most potent for further investigation. Bexarotene was found to be the most potent and was used either alone, or in combination with lapatinib or erlotinib. The use of these drugs in combination with bexarotene resulted in the repression of HER1, HER2, HER3 and HER4 expression, inhibition of NRF2, elevation of ROS, depletion of glutathione and enhanced cytotoxicity in PEO1, OVCAR3, SKOV3 and MCF7-AREc32 cell lines. This explained the crosstalk mechanism between HER receptor family and NRF2 and the role of NRF2 in drug resistance and as a relevant anti-cancer target which opens up novel avenues of targeting HER receptor kinase family and NRF2 pathways for improving cancer therapy.

Abscission is a natural self-regulatory mechanism whereby fruit trees shed part of the fruitlets, and is an important agricultural event from the farmer’s point of view because it directly affects the final size and quality of the commodity. In spite of this self-regulatory mechanism, fruit trees set too many fruitlets negatively affecting not only the final quality, but also the returning bloom. To avoid these effects, farmers perform blossom or fruitlet thinning to adjust crop load and ensure a satisfactory fruit quality at harvest for commercial purposes. Chemical thinning is nowadays a common orchard practice in some apple cultivars but its effects on fruit trees depend on environmental factors and genotypes. A widely employed chemical thinner, the carbaryl (Sevin), has been withdrawn from the market and replaced with benzyladenine (BA), a cytokinin that has a milder effect both on the tree and human health. At molecular level, abscission is a coordinated event under the control of auxin and ethylene. Both hormones play their role at the abscission zone (AZ) level. Auxin flux through the AZ target cell layers inhibits the onset of shedding whereas ethylene causes the up-regulation of degrading cell wall enzymes causing the cell separation and the fruitlet shedding. Nevertheless, no information is available about the signalling that causes the decreasing auxin flow and the gain in ethylene sensitivity in AZ. In Arabidopsis thaliana, the isolation of mutants with defects in floral organ abscission allowed the identification and characterization of genes involved in AZ differentiation and/or the signalling pathways. A model has been proposed by characterizing genes encoding receptors and ligands involved in the transfer of the shedding signal. In apple, a model to explain the fruitlet physiological drop is still missing as well as the isolation and characterization of genes involved in abscission. Although most of the researchers focused their attention mainly on the effect of chemicals on fruit quality and returning bloom, few of them considered also the dynamics of the transcriptomic changes occurring as consequence of the abscission induction. A working hypothesis taking into account the carbohydrate status within the fruitlet population and between fruitlets and shoots has been proposed. The recent development of massive gene approaches, based on microarray technologies, allowed to deeply investigate many fundamental biological process from a transcriptomic point of view. A recent work carried out in tomato investigated the genes involved in auxin homeostasis, as a consequence of IAA depletion during flower abscission induction. The model proposed represents a powerful platform for further analyzing the putative regulatory abscission-related genes involved in the gaining of ethylene sensitivity of the AZ. In the present dissertation, two different approaches have been adopted to investigate the early phases of apple fruitlet abscission: 1) A transcriptomic approach to isolate genes involved in the early steps of abscission, and 2) the characterization of volatile organic compounds (VOCs) emitted during abscission induction. 1) Apple fruitlet abscission was induced using BA as thinner. Fruitlets differing in size and position within the cluster were collected over a 4-day time-course, after BA treatment, and their gene expression profiles were analyzed, separately in cortex and seeds, by means of a newly released 30K oligonucleotide microarray. The analysis of the transcriptomic profiles of abscising and non-abscising fruitlets was tested for statistical association with abscission potential to identify molecular signatures strictly related to fruit destiny. Reactive oxygen species (ROS) and carbohydrates (glucose, fructose, sucrose, sorbitol, and starch) were also measured. A hypothetical model for apple fruitlet abscission was obtained by putting together available transcriptomic and metabolomic data. According to this model, BA treatment would establish a nutritional stress within the tree that is primarily perceived by the fruitlet cortex whose growth is blocked by resembling the ovary growth inhibition found in other species. In weaker fruits, this stress is soon visible also at the seed level, likely transduced via ROS/sugar and hormones signalling crosstalks, and followed by a block of embryogenesis and the consequent activation of the abscission zone. 2) Fruitlets of two cultivars (Golden Delicious and Red Chief) with different abscission potentials treated with two different thinning agents (BA and metamitron) were analyzed by means of proton transfer reaction mass spectrometer (PTR-MS) over a 10-day time-course looking for volatile organic compounds (VOCs) differentially emitted. Results pointed out that isoprene is more abundantly emitted by the abscising fruitlets in both cultivars. A relationship between isoprene emission and ABA content in the fruit cortex was also pointed out, along with a specific activation of the corresponding biosynthetic genes. A delayed transcriptional activation of a biosynthetic gene involved in a key step of methyl-erythritol phosphate (MEP) pathway, supplying the precursors for both isoprene and ABA biosynthesis, was also shown. According to the main findings, a role for isoprene as a ROS-detoxifying mechanism mediated and transcriptionally controlled by ABA may be hypothesized. Future perspectives will be focused on all the research lines herein adopted. The biological function of the genes identified by means of the transcriptomic approach will be studied at the cellular level, with in situ hybridization, and their expression pattern further validated. Attention will be focused in particular on transcription factors and other regulatory elements involved in hormonal cross-talk. The abscission-related VOCs (i.e. isoprene) will be validated to assess the possible applications in fruit load prediction systems, in order to fine-tune the use of thinning chemicals according to a more-environment-friendly agriculture.
L'abscissione è un meccanismo auto-regolativo per cui gli alberi da frutto rilasciano naturalmente parte dei frutticini ed è un evento agronomico importante dal punto di vista del produttore perché incide sulla qualità del raccolto a maturazione. Nonostante questo meccanismo auto-regolativo, gli alberi da frutto trattengono troppi frutticini, influendo negativamente non solo sulla dimensione e la qualità finale dei frutti, ma anche sulla fioritura nell’anno successivo. Per evitare questi effetti negativi, i coltivatori utilizzano comunemente diradanti chimici che agiscono su fiori o frutticini, allo scopo di regolare il carico iniziale ed ottenere così una qualità della frutta corrispondente alle esigenze di mercato. In melo il diradamento chimico è una pratica comune la cui efficacia sugli alberi da frutto è dipendente, purtroppo, da fattori ambientali e dai diversi genotipi. Un diradante chimico ampiamente utilizzato, il carbaryl (Sevin), è stato ritirato dal mercato e parzialmente sostituito con la benziladenina (BA), una citochinina che ha un effetto più contenuto sull'albero e sulla salute umana. A livello molecolare, l'abscissione è un processo coordinato principalmente dall'auxina e dall'etilene. Entrambi questi ormoni svolgono il loro ruolo a livello della zona di abscissione (AZ). Il flusso continuo dell'auxina attraverso la AZ inibisce il processo di abscissione, mentre l'etilene induce una regolazione positiva degli enzimi degradanti la parete cellulare provocando la separazione delle cellule della AZ e la caduta dei frutticini. Le informazioni riguardanti il segnale che causa la diminuzione del flusso di auxina e l'aumento nella sensibilità all'etilene nella AZ sono tuttavia ancora parziali e piuttosto carenti. In Arabidopsis thaliana l'isolamento di mutanti con difetti nel processo di abscissione dei fiori ha permesso l'individuazione di geni coinvolti o nel processo di differenziazione della AZ o nella via di trasduzione del segnale. Per quanto riguarda la cascola fisiologica dei frutticini in melo, le informazioni riguardanti il segnale che genera l’evento abscissione sono tuttora carenti e le collezioni di geni legati a tale fenomeno sono ancora molto parziali. La maggior parte delle ricerche, infatti, si è concentrata principalmente sullo studio dell’effetto di prodotti chimici sulla qualità della frutta e sulla fioritura, mentre solamente pochi studi hanno considerato la dinamica dei cambiamenti trascrizionali conseguente all'induzione dell’abscissione. Sulla base dei dati disponibili, è stata proposta l’ipotesi che considera lo stato nutrizionale all’interno della popolazione di frutticini e fra i fruitticini ed i germogli vegetativi come segnale necessario per l’attivazione dell’abscissione. Recentemente, lo sviluppo di approcci trascrittomici di carattere massale, basato sulle tecnologie microarray, ha consentito di studiare in maniera più approfondita questo processo biologico. Un recente studio effettuato in pomodoro ha permesso di studiare geni coinvolti nel mantenimento dell’omeostasi dell'auxina a livello di AZ in seguito ad una diminuzione del flusso della stessa durante l'induzione dell’abscissione nel fiore. Il modello proposto rappresenta un punto di partenza molto rilevante per identificare altri geni coinvolti nella regolazione dell’abscissione e nella sensibilizzazione dell’AZ all'etilene. In questa tesi sono stati impiegati due differenti approcci per studiare l’abscissione in melo: 1) Un approccio massale trascrittomico per isolare i geni strettamente coinvolti nelle prime fasi induttive dell’abscissione e 2) lo studio di composti organici volatili (VOCs) emessi durante l’induzione dell’abscissione. 1) L'abscissione di frutticini di melo è stata indotta usando la BA come agente diradante. Frutticini differenti per dimensione e posizione all'interno del corimbo sono stati raccolti entro i quattro giorni dal trattamento. L'espressione genica è stata analizzata per mezzo di un vetrino 30K recentemente sviluppato. L'analisi dei profili trascrizionali dei frutticini cascolanti e non cascolanti è stata esaminata allo scopo di identificare marcatori molecolari associati al destino del frutto. Il livello di specie reattive dell’ossigeno (ROS) e di alcuni carboidrati (glucosio, fruttosio, saccarosio, sorbitolo e amido) è stato misurato nella cortex degli stessi campioni. Un modello ipotetico per l’abscissione di frutticini di melo è stato ottenuto unendo i dati trascrittomici e metabolomici disponibili. Secondo questo modello, il trattamento con la BA amplificherebbe lo stress nutrizionale già in atto all'interno dell'albero, il quale viene percepito soprattutto dalla cortex di frutticini il cui sviluppo viene quindi bloccato. Nei frutti più deboli, questo stress viene quindi percepito a livello del seme. La traduzione di questo stress avviene probabilmente attraverso il crosstalk tra ROS, zuccheri e ormoni ed è seguito da un blocco dell'embriogenesi e dall'attivazione della AZ. 2) Frutticini di due diverse cultivar (Golden Delicious e Red Chief) con differente potenziale di abscissione trattati con due differenti diradanti chimici (BA e metamitron) sono stati analizzati per mezzo del PTR-MS (proton transfer reaction mass-spectrometer), entro i dieci giorni dal trattamento, allo scopo di identificare composti organici volatili (VOCs) associati all’abscissione. I risultati hanno evidenziato che i frutticini con potenziale di abscissione maggiore in entrambe le cultivar emettono più isoprene rispetto ai frutti persistenti. E’ stata inoltre evidenziata una correlazione significativa tra emissioni di isoprene e contenuto di ABA della cortex, parallelamente all’attivazione specifica dei rispettivi geni biosintetici. Successivamente avviene l’attivazione ritardata dei geni coinvolti nei passaggi chiave della via del metileritritolo fosfato (MEP), che fornisce i precursori per la biosintesi sia del volatile che dell’ormone. Secondo questi risultati, si può ipotizzare per l’isoprene un ruolo di detossificatore di ROS, la cui attivazione è mediata e controllata a livello trascrizionale dall’ABA. Le prospettive future di questa ricerca saranno focalizzate su tutte le linee di ricerca finora perseguite. La funzione biologica dei geni identificati tramite l’approccio trascrittomico sarà ulteriormente studiata a livello cellulare, tramite ibridazioni in situ, e i loro profili di espressione genica saranno ulteriormente validati. Particolare attenzione sarà prestata ai fattori di trascrizione e agli altri elementi regolativi coinvolti nel cross-talk ormonale. Per quanto attiene i composti volatili (isoprene) saranno validati i risultati finora ottenuti allo scopo di verificare possibili applicazioni pratiche in sistemi previsionali che consentano di predire il livello di carica fruttifera e, quindi, di dosare i trattamenti diradanti nell’ottica di un’agricoltura sostenibile.

The guanine nucleotide exchange factors of the Ras protein super family (RasGEFs) are essential cellular components in the process of Ras activation in response to diverse extra cellular stimuli. The rasGEF1b is a highly conserved guanine exchange factor (GEF). In macrophages expression of rasGEF1b mRNA is induced by different TLRs agonists, such as LPS (TLR4), GPImucin (TLR2) and Poli I: C (TLR3). First, by using bioinformatics tolls we analyzed the probable RasGEF1b subcellular localization. The pFLAGCMV2 encoding the recombinant protein FLAGRasGEF1b was used to transfect HEK 293T cells and protein expression evaluated by Western Blot using an anti-FLAG mAb, it showed a protein with apparent molecular weight of 56kDa. By using differential centrifugation, HEK 293T cells were fractioned and the different fractions were recognized using monoclonal antibodies specific to protein of them, using this methodology, we showed that the protein FLAGRasGEF1b was present mostly in the nuclear and heavy membrane fractions. The FLAGRasGEF1b was inserted into a plasmid in fusion with mRFP or YFP fluorescent proteins, and by using confocal microscopy we showed that the proteins were present at early endosome and endolysosome. By using RNA interference (RNAi) we inhibit the expression of FLAGRasGEF1b-YFP, which was capable to activate Ras in HEK 293T cells in vitro.
Os fatores de troca de nucleotídeos guanina da proteína Ras (RasGEFs) são componentes celulares essenciais no processo de ativação das RasGTPases em resposta a diversos estímulos extracelulares. O rasGEF1b é um fator de troca de nucleotídeos guanina (GEF) altamente conservado. A expressão do RNAm do rasGEF1b em macrófagos é induzida por diferentes agonistas de receptores do tipo Toll (TLRs), tais como LPS (TLR4), GPI-mucin (TLR2) e Poli I:C (TLR3). A predição da provável localização subcelular da proteína RasGEF1b foi realizada inicialmente por meio de análises de bioinformática. A expressão da proteína recombinante em células HEK 293T transfectadas com o plasmídio pFLAGCMV2-RasGEF1b, e análise por Western Blotting utilizando anticorpo monoclonal anti-FLAG, demonstrou uma proteína de massa molecular aparente de 56kDa. Pela técnica de centrifugação diferencial, células HEK 293T foram fracionadas e as diferentes frações foram marcadas com anticorpos monoclonais específicos, dirigidos contra proteínas das mesmas; utilizando esta metodologia, demonstramos que a proteína FLAGRasGEF1b está presente em maior abundância nas frações de núcleo e membranas pesadas. O DNA do FLAGRasGEF1b foi inserido em um plasmídio em fusão com as proteínas fluorescentes mRFP ou YFP, e por microscopia confocal demonstramos a localização da proteína em endossomos primários e endolisossomos. Pela técnica de RNA de interferência (RNAi), inibimos a expressão da proteína recombinante FLAGRasGEF1b-YFP. Além disso, demonstramos que RasGEF1B é capaz de atuar como fator de troca de guaninas de Ras quando expressa em células HEK 293T in vitro.

El instrumental relacionado con las actividades domésticas de los cazadores- recolectores es un aspecto al que tradicionalmente se le ha prestado escasa atención. Este exiguo conocimiento me motivó a analizar cantos y fragmentos, un componente habitual pero poco descrito en la mayoría de contextos arqueológicos. Esta conjunto de materiales líticos no tallados se ha denominado comúnmente como instrumental macrolítico o ground stone tools. La presente tesis doctoral aborda el estudio del comportamiento de los últimos grupos cazadores recolectores en un contexto estratigráfico al aire libre. La unidad arqueológica SG del yacimiento de Font del Ros (Berga, Barcelona), con una extensión de más de 1200 m2, presenta un patrón de ocupaciones recurrentes que se registra a lo largo de una horquilla temporal de más de 1800 años (8300-6500 cal BC). Se trata de uno de los escasos registros arqueológicos con estas características que se documentan en la península Ibérica y por tanto es un buen referente para contextualizar la problemática de los cambios organizativos producidos a raíz de la irrupción de los cambios climáticos vinculados a inicios del Holoceno. Esta tesis se recoge los resultados de más 20 años de trabajo de investigación ampliando la información disponible para el conjunto de instrumental macrolitico, los resultados alcanzados evidencian el potencial informativo que encierran estos objetos y alertan de la necesidad de incorporarlos a los estudios de manera sistemática. Esta disertación presenta un compendio de aportaciones científicas publicadas en revistas y congresos internacionales que han sido sometidas a una revisión editorial por pares. La relevancia de estas líneas de investigación ha permitido la aceptación de estos resultados en revistas indizadas en los principales repositorios de publicaciones científicas, como Scopus y Thompson International Science Index. Los dos ejes en los que se centra esta tesis -implicaciones funcionales del instrumental macrolítico y las derivaciones técnicas relacionadas con la talla bipolar- permiten caracterizar el tecnocomplejo recuperado en Font del Ros. Las actividades productivas vinculadas con artefactos no relacionados directamente con la talla, subrayan el papel central que jugaron estos instrumentos en los esquemas organizativos de estos grupos humanos. Los resultados obtenidos subrayan que a pesar de su diversidad tecnomorfológica este grupo presenta atributos propios que podemos sintetizar en los siguientes puntos. ‐ Oportunista (expediency) no existe una selección de soportes (cantos o fragmentos utilizados) ni de materias primas. ‐ Ausencia de preparación de las superficies activas. ‐ Ausencia de mantenimiento (curation) o estandarización en los cantos utilizados. ‐ Artefactos dinámicos: a lo largo de su vida pueden incluirse en varias categorías dentro de la cadena operativa. ‐ Polifuncionalidad: documentada a través de las superposiciones de huellas de uso que corresponden con actividades diversas. ‐ Intercambiabilidad desde un punto de vista cinemática: alternancia entre rol activo y pasivo a lo largo de la vida útil del instrumento. ‐ Reutilización (re-use) de los soportes con nuevas funciones interconectándose diferentes usos. ‐ Reciclaje hacia otras actividades. Formando parte de estructuras de combustión o mediante su transformación en núcleos. ‐ Eficiencia a la hora de realizar las actividades relacionadas con la subsistencia diaria. Los resultados presentados invitan a reflexionar sobre el significado de conjuntos macrolíticos de cronologías similares en el noreste de la península ibérica y establecen un marco referencial para este tipo de utillaje en la transito de las ultimas sociedades cazadores recolectoras a las sociedades agro-pastorales.
The tools related to the domestic activities of hunter-gatherers are a set of materials that traditionally has received little attention. This meager knowledge encourages me to analyze cobbles and fragments, a common component in most archaeological contexts but little described in bibliography. This set of uncarved lithic materials has been commonly referred to as macrolithic instruments or ground stone tools. This PhD addresses the study of the behavior of the last hunter gatherer groups in an open air site stratigraphic context. The SG archaeological unit of the Font del Ros site (Berga, Barcelona), with an surface of more than 1200 m2, presents a pattern of recurring occupations that is recorded along a temporary fork of more than 1800 years (8300-6500 Cal BC). These particular attributes are scarce in the Iberian Peninsula and present this site as a good reference to to contextualize the problem of organizational changes produced as a result of the emergence of climatic changes linked to the beginnings of the Holocene. This thesis collects the results of more than 20 years of research expanding the information available for the set of macrolithic instruments, the results achieved show the informative potential that these objects contain and warn of the need to incorporate them in a systematic way into the studies. This dissertation presents a compendium of scientific contributions published in international journals and conferences that have undergone an editorial peer review. The relevance of these lines of research has allowed the acceptance of these results in indexed journals that are included in the main scientific repositories, such as Scopus and Thompson International Science Index. The two lines of research addressed in the PhD - the functional implications of the macrolithic instruments and the technical derivations related to bipolar size- allow us to characterize the technocomplex recovered in Font del Ros. The productive tasks didn’t relate directly to the knapping activities; underline the central role played by these instruments in the organizational schemes of these human groups. The results obtained underline that despite its technomorphological diversity this group presents its own attributes that we can synthesize in the following points: - Opportunist (expediency) there is no selection of supports (cobbles or used fragments) or raw materials. - Absence of preparation of active surfaces. - Absence of maintenance (curation) or standardization in the selected cobbles. - Dynamic artifacts: throughout their life they can be included in several categories within the chaînes oppératives. - Polifunctionality: documented through overlays use-wear that corresponds to various activities. - Interchangeability from a kinematic point of view: alternation between active and passive role throughout the life of the instrument. - Re-use of the supports: new functions interconnecting different uses. - Recycling towards other activities. These tools become part of combustion structures or are transformed into cores. - Efficiency when carrying out activities related to daily subsistence. The results invite us to reflect on the meaning of macrolithic sets of similar chronologies in the north-east of the Iberian Peninsula and establish a framework for this type of tools in the transit of the last hunter-gatherers to the agro-pastoral societies.

Eligibility for antiretroviral therapy (ART) in HIV-infected patients is defined either by a cluster of differentiation antigen 4 (CD4) count of less than 200cells/mm3 or clinical diagnosis of WHO stage III and IV. Therefore, the decision to start ART becomes difficult when CD4 cell count is not available. With limited laboratory infrastructure, the decision to start ART is usually made based on clinical symptoms leading to late commencement of ART. This calls for alternative criteria to see if nail discoloration (ND) correlates with low CD4 count among untreated HIV infected patients. This will serve as a complementary screening tool for identifying asymptomatic ARV naive HIV patients with a CD4 cell count of less than 200cells/mm3 which signifies 
severe immunosuppression. Study Design and Setting: This was a quantitative cross-sectional descriptive and analytical study involving adult ART naï
ve HIV infected patients in WHO stage I and II. Systematic sampling was used to select the participants from all adult ART naï
ve HIV infected patients attending APIN clinic, located at the Jos University Teaching Hospital (JUTH), Jos, Nigeria. Data Collection: Face-to-face interviews, physical examination and relevant laboratory investigations with selected participants were conducted using a questionnaire guide. Questions on socio-demographic characteristics, clinical data, general physical examinations including finger nail examination and photographing with subsequent laboratory investigations including CD4 count and western blot were employed. Data Analysis: Variables were categorized and data analyzed using descriptive statistics including the frequency, percentage frequency
mean and standard deviation of continuous variables. Association between CD4 count of &le
200cells/mm3 and ND was tested using the chisquare test with an alpha level of 0.05. Prevalence of ND, sensitivity, specificity, positive predictive and negative predictive values and accuracy of the screening test of ND was calculated. Results: 394 patients had their fingernails photographed and assessed. It was shown that distal banded and grey nails were the common types of ND seen with a prevalence of 38%. There was an association between CD4 count &le
200cells/mm3 and ND (p<
0.0001). CD4 count &le
200cells/mm3 was a risk factor for developing ND (RR=2.3[1.8-3.6]). The association has a sensitivity of 78%, specificity of 55%, positive predictive value of 50%, and negative predictive value of 80% and accuracy of test 63%. Conclusion: With a significant association (p<
0.0001) and a sensitivity of 78%, ND can be a useful clinical indicator of immune dysfunction mediated by HIV among patients in WHO stage I or II. ND can either be a clinical sign or a symptom in HIV patients with a CD4 of &le
200cells/mm3 as seen in the study as the specificity and sensitivity of ND compared favourably with other WHO stage III diagnosis. Recommendations: Nail discoloration should complement CD4 count as an additional staging sign to help identify patients likely to benefit from ART especially in resource-limited settings. Finally, all patients with grey or distal banded should be on co-trimoxaxole prophylaxis in line with WHO /national guideline on the use of co-trimoxaxole for all HIV positive patients with a CD4 cell count of &le
350cells/mm3.

L’imagerie scintigraphique des cancers thyroïdiens différenciés (CTD) présente la particularité d’utiliser deux radiopharmaceutiques, l’iode 131 (131I) et le 18-Fluorodésoxyglucose (18FDG). La fixation de ces traceurs dépend habituellement du degré de différenciation et de l’agressivité de la tumeur. L’objectif de ce travail était d’étudier l’apport de différents aspects techniques et d’instrumentation, à savoir l’imagerie hybride par TEMP/TDM et TEP/TDM, la point-spread function (PSF), la taille des voxels et la technologie TEP digitale, et d’explorer si d’autres traceurs TEP pouvaient présenter un intérêt. Le but de la première partie était d’étudier les performances de la TEP/TDM au 18FDG à l’étage cervical pour la détection de la maladie ganglionnaire. Une acquisition TEP/TDM dédiée a amélioré la détection de la maladie tumorale par rapport à l’acquisition classique. L’utilisation de la PSF a permis de détecter des tailles de lésions plus petites et la durée optimale de cette acquisition a été évaluée. Des reconstructions avec des tailles de voxels ultra-fines ont été réalisées sur TEP digitale pour étudier l’impact de la PSF et des voxels ultra-fins sur les données quantitatives. La seconde partie a porté sur l’imagerie 131I-TEMP/TDM et 18FDG-TEP/TDM, afin de quantifier le volume de la maladie persistante. Il a ainsi été montré que la masse tumorale était corrélée au risque post-opératoire et avait un impact sur la réponse au traitement. L’objectif de la troisième partie était d’étudier un autre traceur TEP, la 18-Fluorocholine (FCH), ainsi qu’un marqueur de la néovascularisation, l’antigène membranaire spécifique de la prostate (PSMA). Nos données suggèrent qu’un examen TEP à la FCH négatif au sein d’un nodule thyroïdien à cytologie indéterminée permettrait d’éliminer la malignité, et pourrait éviter des chirurgies inutiles. Par ailleurs, le marquage au PSMA évalué par immunohistochimie dans les néo-vaisseaux est associé à des facteurs de mauvais pronostic. D’autres études sont nécessaires pour confirmer l’intérêt éventuel des examens TEP à la FCH et au 68Ga-PSMA en oncologie thyroïdienne
Radioiodine (131I) and 18-Fluorodeoxyglucose (18FDG) are two radiopharmaceuticals used for scintigraphic imaging in differentiated thyroid cancers (DTC). Tumour uptake of each tracer depends on tumour differentiation and aggressiveness. Our goal was to further assess various technical aspects in DTC imaging workup, such as SPECT/CT and PET/CT, point-spread function (PSF), voxel size, digital PET, and to explore further other PET tracers. The aim of the first part was to assess the performance of 18FDG PET/CT for the detection of neck lymph node involvement. A dedicated PET/CT acquisition improved tumour detection compared to the whole-body acquisition. PSF reconstruction allowed detection of smaller cancer deposits and the optimal acquisition duration time was assessed. Using digital PET acquisitions, ultra-thin voxels reconstructions were performed. The impact of ultra-thin voxels and PSF on quantitative values was evaluated. The second part focused on 131I-SPECT/CT and 18FDG-PET/CT imaging, in an attempt to assess tumour burden of persistent disease. Tumor burden was correlated with the postoperative risk and affected the response to therapy. In the third part, another PET tracer, i.e. 18-Fluorocholine (FCH), and a marker of neovasculature, i.e. prostate-specific membrane antigen (PSMA), were studied. FCH PET/CT offered high negative predictive value to reliably exclude cancer in PET-negative nodules with indeterminate cytology and might prevent unnecessary surgeries. Also, PSMA expression assessed with immunohistochemistry was associated with poor prognosis factors. Further studies are needed to confirm new insights of FCH PET and 68Ga-PSMA PET in DTC

CHIKV is the prototype of Alphaviruses and it causes an acute febrile illness with rash, severely painful arthralgias, and sometimes arthritis. While CHIKV has first been identified in the 1950s in Africa, recent outbreaks of CHIKV in the islands of the Indian Ocean and particular in Italia have re-drawn attention to CHIKV. In the past CHIKV disease was considered self-limiting and non-fatal. However, a number of deaths on Reunion (Anonym, 2006) during the outbreak, which was affected directly or indirectly by CHIKV, have changed this view. To defeat CHIKV outbreaks diagnostic tools and anti CHIKV therapies are urgently needed. In this thesis, we generated tools to investigate CHIKV at the molecular level by serological tests. CHIKV was isolated from a German woman who was infected during her holidays on the Mauritius Island. To characterize this viral isolate the complete viral genome was amplified by PCR and molecular cloned. In order to analyse antibody responses of infected individuals some of the structural and non-structural genes were subcloned in bacterial expression vectors. The NSP2, proteinase, capsid, E1 and E2 were subsequently expressed in E.coli using purified successfully. In this thesis, the structural proteins were used to develop a screening test for anti-CHIKV antibodies in patient derived serum samples. These tests were evaluated with pre-characterized anti-CHIKV sera (30 samples) obtained from the BNI Hamburg and 100 serum samples from German blood donors used as negative controls. Immunoblotting analysis revealed that up to 77% of precharacterised positive sera could recognize the recombinant proteins and there were no detectable reactivity of CHIKV-negative German donor sera. The recombinant proteins were also recognized by 71.4% of positive sera in the newly established ELISA. In order to go further in analyses of the results, an in house IFA was performed. Positive sera (21 samples) were used. The results showed that all of them reacted positive, but this assay was less sensitive than the IFA from BNI. In comparison with the IFA result from BNI Hamburg, the results were not congruent in all test performed. This could be due to various drawbacks of the tests. A cross reaction in Alphaviruses and the different strains are mentioned as well as the denatured forms of the structural proteins. Besides the main structural proteins (E1, E2 and C), other proteins such as non-structural proteins, uncleaved precursor proteins could participate in the different outcomes of serological assays. In order to go further in the CHIKV diagnoses, the CHIKV recombinant proteins were applied to screen the anti-CHIKV antibodies in the Vietnamese population, who are considered to live in the high risk regions. In serological tests, 158 sera of Vietnamese donors were incubated with the recombinant proteins or the fixed CHIKV infected cells. The results showed that 24% of Vietnamese donor sera recognized the recombinant proteins in immunoblot assay, while 36% scored positive in the ELISA assay. In IFA, the sera considered positive were 11.4%. While some discrepancies in serological tests were found, these results showed that the ratio of CHIKV-positive sera seem to be equal to the other regions in the world, which are affected by CHIKV. It is suggested that CHIKV infection in Vietnam has been repeatedly misdiagnosed. This study cohort consisted only of samples originating from Hanoi area of Northern Vietnam, thus, future studies should expand to include samples from other Vietnam areas. To do this the various subtypes of the virus in the different regions should be isolated and the sequences of these viruses should be well characterized.

Dissertação de mestrado em Genética Molecular
Fundamental cellular processes appear to be highly conserved between Saccharomyces cerevisiae and other more complex Eukaryotic species, including humans. “Humanized yeast systems” emerged as a tool to study molecular aspects of human pathologies. The present work aimed at contributing to build and validate a large high throughput platform of yeast strains displaying phenotypes that can enable further testing galectin-related drugs and peptides. This platform was designed to consist of two types of strains, the ones expressing human galectins and the ones expressing these together with the human KRAS cDNA. The rationale behind this relates with the putative dialogue between Galectins and RAS signaling pathway in mammals. Considering that EGFR mediates KRAS signaling and that yeast also harbors a RAS signaling pathway, the “humanized yeasts” expressing KRAS were used to identify the yeast target of anti-EGFR. Furthermore, it was also used for phenotyping the most well-known biological processes known to be controlled by RAS pathway. On the other hand, considering that the deletion of GUP1 in S. cerevisiae increases the resistance to the oncological drug Imatinib, the similarities between the phenotypes associated to the deletion of RAS and GUP genes were also verified. Two Hsp70, Ssa2p and Ssb2p and one glyceraldehyde-3-phosphate dehydrogenase Tdh3p, were identified as EGFR-like proteins. The subsequent alignments analysis between EGFR and these proteins revealed that Ssb2p and its very close homologue Ssa2p present some homology with EGFR sequence, namely at the level of three EGFR conserved amino acids known to be responsible for the interaction with the anti-EGFR antibody Cetuximab used in cancer treatment. This and other lines of evidence support Ssb2p and/or Ssa2p as good candidates for EGFR homology. The phenotypic tests revealed that both the deletions of GUP and RAS genes promote a reduction in chronological life span and cell size, except in the case of Δras2 strain, whose cells were bigger than wild type control. Nutrient depletion (carbon) promoted replication stress in Δras2 cells that failed to enter into G1 arrest, and were blocked in S phase, concurring with the bigger size of Δras2 cells and their short lifespan. Moreover, the cells with GUP genes deleted, in opposition to RAS mutants, showed ability to adhere to solid nitrogen-deficient medium. Neither RAS nor GUP mutants were able to invade or filament under these conditions. With this work we were able to determine the possible homologue of EGFR, many times associated with cancer pathologies, and contributed to gain insights on RAS and GUP genes common phenotypes. In conclusion, the present work opens doors to future discovery of new pathways in yeast, in addition to showing that S. cerevisiae is a suitable model to create a platform to explore therapeutic drugs/antibodies.
Vários processos celulares fundamentais encontram-se conservados entre a levedura Saccharomyces cerevisiae e outras espécies eucariotas mais complexas, incluindo humanos. A “Levedura humanizada” surgiu como uma ferramenta de estudo sobre aspectos moleculares de patologias humanas. Com este trabalho pretendeu-se contribuir para a construção e validação de uma plataforma de estirpes de levedura que exibam determinados fenótipos, permitindo o teste de drogas e péptidos relacionados com as galectinas. Esta foi planeada para incluir duas estirpes a expressar galectinas humanas, assim como o cDNA do KRAS humano. O propósito desta plataforma advém de uma possível interação entre as Galectinas e a via de sinalização dos RAS em mamíferos. Tendo em conta que o EGFR medeia a cascata de sinalização KRAS, e que também a levedura possui uma via de sinalização Ras, usou-se as leveduras humanizadas a expressar o KRAS para identificar o alvo do anti-EGFR. Para além disso, estas foram usadas para a fenotipagem de processos biológicos controlados pela cascata RAS. Por outro lado, tendo em conta que a deleção do GUP1 aumenta a resistência à droga oncológica Imatinib, verificouse também as semelhanças fenotípicas entre as deleções RAS e GUP. Foram identificadas duas proteínas Hsp70, Ssa2p e Ssb2p, e uma gliceraldeído-3-fosfato desidrogenase Tdh3p, como sendo os alvos do anti-EGFR. Subsequentemente, a análise dos alinhamentos entre o EGFR e estas proteínas revelaram que a Ssb2p e a sua homóloga Ssa2p apresentam similaridade com a sequência do EGFR, nomeadamente ao nível de três aminoácidos responsáveis pela interação com o anticorpo anti-EGFR, Cetuximab, usado no tratamento do cancro. Esta informação suporta a hipótese das proteínas Ssb2p e/ou Ssa2p serem boas candidatas a homólogas do EGFR. Os testes fenotípicos revelaram que as deleções dos genes GUP e RAS promovem uma redução da longevidade cronológica e da área celular, com excepção para a estirpe Δras2 cujas células se revelaram maiores do que a wt. A depleção de nutrientes (carbono) induziu stress replicativo nas células Δras2, que por sua vez falharam a entrada na fase G1, ficando bloqueadas na fase S, o que está de acordo com o aumento da área celular e a baixa longevidade cronológica das células Δras2. Além disso, as células com a deleção nos genes GUP, contrariamente aos mutantes RAS, mostraram habilidade para aderir a um meio deficiente em nitrogénio. Nenhum dos mutantes RAS ou GUP foram capazes de invadir ou filamentar nas condições anteriormente descritas. Com este trabalho fomos capazes de determinar o possível homólogo do EGFR, muitas vezes associado a patologias relacionadas com o cancro, assim como contribuir para melhor compreender os fenótipos comuns associados aos genes RAS e GUP. Em conclusão, o presente trabalho abre portas para futuras descobertas de novas vias de sinalização em levedura, além de reforçar a utilização da S. cerevisiae como um bom modelo para criar uma plataforma de exploração de drogas/anticorpos.

Immunsupprimierte Patienten besitzen ein erhöhtes Risiko für opportunistische Infektionen, die hauptsächlich durch das humane Cytomegalievirus (HCMV) und den Schimmelpilz Aspergillus fumigatus verursacht werden. Aufgrund ihrer Lokalisation in den Geweben unterhalb von Lungenepithelien und des Gastrointestinaltraktes werden dendritische Zellen (DCs) als diejenigen Zellen betrachtet, die während der frühen Phase einer Infektion in Kontakt mit HCMV und A. fumigatus kommen und eine Aktivierung von angeborenen und adaptiven Abwehrmechanismen vermitteln. Im Rahmen der vorliegenden Dissertation wurde die Bedeutung von humanen DCs bei der Bekämpfung von HCMV und A. fumigatus näher untersucht. Um mit dem klinisch relevanten HCMV Stamm TB40E arbeiten zu können, musste zuerst ein geeignetes Zellkultursystem zur Anzucht von HCMV etabliert werden. Die aus Fibroblasten aufgereinigten Viren eigneten sich zur erfolgreichen Infektion von DCs, was durch verschiedene Färbemethoden nachgewiesen werden konnte. Aus diesem Grund war es möglich, in Abhängigkeit der Zeit ein Expressionsprofil von Klasse I Interferonen (IFN-alpha, IFN-beta), ausgesuchten Cytokinen (CXCL10, CXCL11, Rantes) und den wichtigen Immunrezeptoren Toll-like Rezeptor 3 (TLR3) und dendritic cell-specific ICAM3-grabbing nonintegrin (DC-SIGN) zu erstellen. Nachdem ein RNA Interferenz (RNAi) System zur erfolgreichen Transfektion von DCs mit small interfering RNA (siRNA) etabliert werden konnte, gelang es die Expression von TLR3 signifikant herunterzuregulieren. Stimulationsexperimente mit dem synthetisch hergestellten Polymer poly I:C identifizierten TLR3 als den Rezeptor, der die Expression von IFN-beta vermittelt. Ferner konnte nachgewiesen werden, dass TLR9 bei ex vivo generierten DCs keine Funktion besitzt. Eine direkte Aktivierung von TLR3 durch HCMV konnte mittels siRNA nicht nachgewiesen werden. Durch den Einsatz von genomweiten Microarray-Analysen konnten eine Vielzahl an Genen gefunden werden, die nach Co-Kultivierung von DCs und lebenden A. fumigatus Keimschläuchen (KS) differentiell exprimiert waren. Dabei wurde ein breites Spektrum an Cytokinen (TNF-alpha, IL-6, IL-10, IL-12), Chemokinen (IL-8, CCL20, CXCL10), Co-stimulatorischen Molekülen (CD40, CD80, CD83, CD86), Prostaglandin Synthese Genen (PTGS2) und Immunrezeptoren (PTX-3, TLR2, TLR4) gefunden, deren zeitabhängiges Expressionsprofil mittels qRT-PCR eindeutig bestätigt wurde. Als Wachen des Immunsystems müssen DCs Krankheitserreger zu einem frühen Zeitpunkt der Infektion erkennen. Die Erkennung von Pilzen wird durch die unterschiedlichen Rezeptoren vermittelt, die TLRs, C-Typ Lektine und Pentraxine umfassen, wobei ihre Bedeutung für humane DCs bisher nur unzureichend geklärt ist. Durch den Einsatz von siRNA konnte die Expression von TLR2, TLR4, myeloid differentiation primary response gene 88 (MyD88), DC-SIGN, Pentraxin-3 (PTX-3) und caspase recruitment domain family member 9 (Card-9) signifikant verringert werden. Für TLR2, TLR4, PTX-3 und DC-SIGN konnte durch den Einsatz der RNAi aufgezeigt werden, dass diese Rezeptoren nicht an der Induktion einer pro-inflammatorischen Immunantwort von DCs nach Infektion mit A. fumigatus beteiligt sind. Sowohl die Stimulierung mit den TLR Liganden Zymosan und LPS, als auch mit A. fumigatus, führte zu einer erhöhten Expression von TNF-alpha und IL-12 (Light Cycler), die sich in einer vermehrten Cytokinfreisetzung (ELISA) bemerkbar machte. Im Gegensatz zur TLR4 siRNA Transfektion und LPS-Stimulation war keine Reduktion der Expression von TNF-alpha und IL-12 nach TLR2 und TLR4 siRNA Transfektion und anschließender Pilzinfektion zu beobachten. Auch der Einsatz von gegen TLRs gerichteten Antikörpern konnte eine mögliche Signaltransduktion bei DCs nicht unterbinden. Anstelle von TLR2 und TLR4 wurde Dectin-1 als DC-Immunrezeptor für A. fumigatus KS identifiziert. Mit Hilfe eines spezifischen Antikörpers gegen Dectin-1 war es möglich, die Freisetzung von TNF-alpha und IL-12 nach Pilzinfektion zu blockieren. In einem unabhängigen Experiment mit siRNA wurde Dectin-1 als Rezeptor für A. fumigatus bestätigt. Wie fortführende Experimente mit Candida albicans KS und Zymosan gezeigt haben, handelt es sich bei Dectin-1 auf humanen DCs um einen generellen Rezeptor für Pilze. Die durchgeführten SNP-Analysen (single nucleotide polymorphism) zur Ermittlung eines Zusammenhanges mit einem erhöhten Virus- und Pilzinfektionsrisiko für Patienten nach Stammzelltransplantation erbrachten die Erkenntnis darüber, dass zwei Marker (rs735240, rs2287886) in DC-SIGN mit einer erhöhten Empfänglichkeit für HCMV, und drei Marker (rs1554013, rs3921, rs4257674) in CXCL10 mit einem vergrößerten Riskio für eine invasive Aspergillose assoziiert waren. Ein Screening von Patienten auf das Vorhandensein dieser definierten SNPs könnte helfen, die individuelle Gefahr für HCMV und A. fumigatus nach nach allogener Stammzelltransplantation abzuschätzen
Patients after allogenic stem cell transplantation (alloSCT) have an increased risk to suffer from viral and fungal infections, which are mainly caused by the human cytomegalovirus (HCMV) and the mold Aspergillus fumigatus. Due to their localization in tissues under lung epithelia and the gastrointestinal tract, dendritic cells (DCs) are considered to be the first cells coming into close contact with HCMV and A. fumigatus for the activation of innate and adaptive immune mechanisms. Within the scope of this dissertation, the role of human monocyte-derived DCs in the abatement of HCMV and A. fumgatus was analyzed. In order to work with HCMV, a cell culture system for effective culturing of the clinical relevant HCMV strain TB40E had to be established first. The viral particles up-cleaned from lung fibroblasts were used for infection of DCs and successful infection was approved by different staining methods. For this reason, it was possible to determine a time-dependent expression profile of class I interferons (IFN-alpha, IFN-beta), selected cytokines (CXCL10, CXCL11, Rantes) and immunoreceptors (TLR3, DC-SIGN). A RNA interference (RNAi) system for human DCs was established to significantly knock-down expression of TLR3 without the induction of an unwanted pro-inflammatory cytokine response. Stimulation experiments with the synthetic polymer poly I:C (which resembles dsRNA of infectious viruses) identified TLR3 as a receptor for triggering expression of IFN-beta. However, whether there is a direct activation of TLR3 through dsRNA intermediates, possibly emerging during replication of HCMV, can not be answered to date definitively, because TLR3 small interfering RNA (siRNA) transfection prior to HCMV infection did not result in minor expression of IFN-beta. Gene expression pattern of DCs after co-cultivation with living A. fumigatus germ tubes was studied by whole genome microarray analysis and real-time PCR, demonstrating an upregulation of a broad spectrum of cytokines (TNF-alpha, IL-6, IL-10, IL-12), chemokines (IL-8, CCL20, CXCL10), co-stimulatory molecules (CD40, CD80, CD83, CD86), prostaglandin synthesis genes (PTGS2), as well as genes involved in fungal recognition (PTX-3, TLR2, TLR4) and cytoskeleton organization / phagocytosis. As the sentries of the immune system, DCs must recognize fungi at an early step of infection. Pathogen detection is mediated by different receptors comprising TLRs, C-type lectins and Pentraxines (PTX), but only little is known about their relevance for DCs. Using specific siRNAs, expression of TLR2, TLR4, myeloid differentiation primary response gene 88 (MyD88), dendritic cell-specific ICAM3-grabbing nonintegrin (DC-SIGN), Pentraxin-3 (PTX-3) and caspase recruitment domain family member 9 (Card-9) was significantly diminished, respectively. In contrast to control experiments with TLR4 siRNA and LPS stimulation, A. fumigatus induced expression of pro-inflammatory cytokines (TNF-alpha, IL-12) was not reduced when TLR2 and TLR4 expression was knocked-down by specific siRNAs prior to infection. However, using siRNAs directed against Dectin-1 allowed demonstration of an interaction between Dectin-1 and A. fumigatus germlings, Candida albicans germ tubes and Zymosan. In an independent approach, cytokine secretion could be blocked by anti-Dectin-1 antibody treatement prior to fungal exposure. In conclusion, Dectin-1 was identified as an important fungal receptor on DCs whereas TLR2 and TLR4 seemed to play a negligible role. Single nucleotide polymorphisms (SNPs) in various cellular receptor genes are associated with the susceptibility to and severity of infectious diseases. In this study, genetic polymorphisms in genes encoding for virus entry receptors have been analyzed for their association to HCMV reactivation and disease in patients after allogeneic stem cell transplantation. A comparison of different genotyping methods highlighted the advantages of the Light Cycler system, the cycle-suequencing and the matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) when using small quantities of patients’ DNA. Two markers (rs735240, rs2287886) in the promoter region of DC-SIGN were found to be significantly associated with an increased susceptibility to HCMV. In addition, three SNPs (rs1554013, rs3921, rs4257674) in CXCL10 elevated the risk for the development of invasive aspergillosis. Screening of patients after alloSCT for the presence of these defined genetic polymorphisms may help to predict the individual risk to suffer from HCMV and A. fumigatus after alloSCT

Les outils de programmation sont des programmes informatiques qui aident les humains à programmer des ordinateurs. Les outils sont de toutes formes et tailles, par exemple les éditeurs, les compilateurs, les débogueurs et les profileurs. Chacun de ces outils facilite une tâche principale dans le flux de travail de programmation qui consomme des ressources cognitives lorsqu’il est effectué manuellement. Dans cette thèse, nous explorons plusieurs outils qui facilitent le processus de construction de systèmes intelligents et qui réduisent l’effort cognitif requis pour concevoir, développer, tester et déployer des systèmes logiciels intelligents. Tout d’abord, nous introduisons un environnement de développement intégré (EDI) pour la programmation d’applications Robot Operating System (ROS), appelé Hatchery (Chapter 2). Deuxièmement, nous décrivons Kotlin∇, un système de langage et de type pour la programmation différenciable, un paradigme émergent dans l’apprentissage automatique (Chapter 3). Troisièmement, nous proposons un nouvel algorithme pour tester automatiquement les programmes différenciables, en nous inspirant des techniques de tests contradictoires et métamorphiques (Chapter 4), et démontrons son efficacité empirique dans le cadre de la régression. Quatrièmement, nous explorons une infrastructure de conteneurs basée sur Docker, qui permet un déploiement reproductible des applications ROS sur la plateforme Duckietown (Chapter 5). Enfin, nous réfléchissons à l’état actuel des outils de programmation pour ces applications et spéculons à quoi pourrait ressembler la programmation de systèmes intelligents à l’avenir (Chapter 6).
Programming tools are computer programs which help humans program computers. Tools come in all shapes and forms, from editors and compilers to debuggers and profilers. Each of these tools facilitates a core task in the programming workflow which consumes cognitive resources when performed manually. In this thesis, we explore several tools that facilitate the process of building intelligent systems, and which reduce the cognitive effort required to design, develop, test and deploy intelligent software systems. First, we introduce an integrated development environment (IDE) for programming Robot Operating System (ROS) applications, called Hatchery (Chapter 2). Second, we describe Kotlin∇, a language and type system for differentiable programming, an emerging paradigm in machine learning (Chapter 3). Third, we propose a new algorithm for automatically testing differentiable programs, drawing inspiration from techniques in adversarial and metamorphic testing (Chapter 4), and demonstrate its empirical efficiency in the regression setting. Fourth, we explore a container infrastructure based on Docker, which enables reproducible deployment of ROS applications on the Duckietown platform (Chapter 5). Finally, we reflect on the current state of programming tools for these applications and speculate what intelligent systems programming might look like in the future (Chapter 6).

Η εργασία αυτή αποτελεί συνέχεια μιας προσπάθειας για την ανάπτυξη της πύλης WEP (Web Engineering Resources Portal). Πρόκειται για ένα Μοντέλο Αναφοράς και μια Πύλη Πόρων που φιλοδοξεί να αποτελέσει ένα σημαντικό εργαλείο για το web engineering, αλλά και αναφορά στην ανάπτυξη εφαρμογών / πληροφοριακών συστημάτων παγκοσμίου ιστού και οδηγό στην ανεύρεση και κατανόηση τεχνολογιών και εργαλείων. Το κομμάτι με το οποίο θα ασχοληθούμε εδώ αφορά το τελευταίο μέρος και τον εκπαιδευτικό χαρακτήρα που επιθυμούμε να προσδώσουμε στην πύλη. Συνοπτικά, η διπλωματική εργασία κινείται σε τρεις άξονες: Πρώτον, στη μελέτη σύγχρονων δυνατοτήτων συλλογής πληροφορίας για την επιλογή των κατάλληλων εργαλείων για εισαγωγή στην πύλη και την ανάπτυξη της δικής μας μεθοδολογίας. Η διαρκής και ραγδαία αύξηση του όγκου των δεδομένων κατέστησε εμφανές το πρόβλημα της ευρέσεως και εξαγωγής της ''χρήσιμης'' ή ''επιθυμητής'' πληροφορίας, καθώς για κάθε κατηγορία ιστοεφαρμογών υπάρχει μια πληθώρα εργαλείων ανάπτυξης (τα οποία μάλιστα διαρκώς ανανεώνονται) καθιστώντας τη διαδικασία επιλογής των καταλληλότερων ιδιαίτερα επίπονη και χρονοβόρα. Δεύτερον, στην εφαρμογή της παραπάνω μεθοδολογίας εργασίας με στόχο την εισαγωγή των αντίστοιχων εργαλείων που προέκυψαν με τη μετατροπή της πληροφορίας σε XML για τη δόμηση της πύλης WEP. Εξηγούμε πως έγινε η εισαγωγή και διαχείριση περιεχομένου με χρήση της τεχνολογίας XML και πως καταλήγουμε στην υλοποίηση των παραπάνω μέσα από την πλατφόρμα του stringbeans. Τρίτον στον εκπαιδευτικό χαρακτήρα της Πύλης. Αναφερόμαστε στις έννοιες του e-learning, και τις διάφορες εναλλακτικές που υπάρχουν στο χώρο της ηλεκτρονικής μάθησης στην εποχή του Web 2.0, όπου οι χρήστες συμμετέχουν ενεργά στη συγγραφή του εκπαιδευτικού υλικού. Αναλύουμε τα χαρακτηριστικά των PLE (Personal Learning Environments) και των LMS (Learning Management Systems) και εκθέτουμε πλεονεκτήματα και μειονεκτήματά τους. Τέλος, καταλήγουμε σε μια γενική αξιολόγηση της προσπάθειάς μας, καταγράφοντας τα αποτελέσματα και τους στόχους που υλοποιήθηκαν, τα συμπεράσματα και τις προτάσεις μας.
This paper is a follow up of the previous work concerning the Web Engineering Resources Portal (shortly WEP). It consists of a Reference Model and a Resources Portal. The aim of WEP is to provide a tool for web engineering as well as a reference for the development of applications / information systems and a guide for finding and understanding web engineering technologies and tools. Here we deal with that last part and the educational aspect of WEP which we are trying to achieve. Briefly, the thesis involves three parts: Firstly, the study of modern possibilities for collecting and importing information regarding the choice of suitable tools and the development of our own methodology. The rapid growth of data volume rendered obvious the problem of finding and extracting of ''useful'' or ''desirable '' information, since there is an abundance of development tools (which are indeed constantly renewed) for every category of web application making the process of choosing the most suitable ones particularly laborious and time-consuming. Secondly, it refers to the enforcement of the above methodology aiming at the import of resultant tools with the transformation of information in XML for the layout of WEP. We explain the way the import and management of content using XML technology is completed and we come to the adaptation and implementation phase through the Stringbeans framework. Thirdly we try to highlight the educational character of WEP portal. The concept of e-learning is described and the various alternatives at the age of Web 2.0, where the users participate actively in the writing of educational material are taken into consideration. Furthermore, we analyze the characteristics of PLE (Personal Learning Environments) and LMS (Learning Management Systems) quoting advantages and disadvantages of both. Finally, we lead to a general evaluation of our effort, mentioning the results and the objectives that were achieved, the conclusions and our proposals for the future.

The thesis entitled "Pushing the Limits of NMR Sensitivity and Chiral Analysis: Design of New NMR Methods and Bio-molecular Tools" consists of six chapters. The research work reported in this thesis is focused on the development of novel chemical and NMR methodological approaches for enantiomeric analysis and mea- surement of residual dipolar couplings (RDCs), and the development of sensitivity enhanced slice selective NMR experiments for obtaining pure shift 1H spectra and the measurement of scalar couplings. The thesis is divided into two parts. The Part I comprises chapters 2-4, where the enantiomeric analysis is discussed, which includes newly developed chiral reagents, two new weak chiral aligning media and design of novel NMR techniques. Part II comprises chapters 5 and 6, which discusses new sensitivity enhanced slice selective NMR techniques. Chapter 1 gives a general introduction to NMR and the problems investigated in the remaining chapters of the thesis. The chapter starts with a brief discussion on the introduction, advancements and general applications of NMR, discussion is also given on the NMR approaches for enantiomeric analysis both in isotropic and anisotropic phases and the measurement of RDCs, including the benefits and limitations associated with each approach. The chapter sets the tone by discussing limitations of the existed NMR enantiomeric approaches and slice-selective techniques, and builds the bridge for the rest of the chapters by addressing these limitations. The chapter also introduces slice selective experiments, their benefits over other conventional methods and limitations. Additional introductory notes are also given on some related concepts. Part I : NMR Chiral analysis and RDCs measurements Chapter 2 discusses chiral sensing properties of RNA nucleosides and their utility as chiral derivatizing agents for the enantio-discrimination of 1o-amines using one dimensional 1H NMR. A three component protocol has been proposed for the complexation of nucleosides with amines, which is rapid, economical and provides maximum diastereomeric conversion. The chiral differentiating ability of nucleosides are examined for different amines based on the 1H NMR chemical shift differences between the diastereomers (∆δ R, S ). Enantiomeric differentiation has been observed at multiple chemically distinct proton sites. It is observed that adenosine and guanosine exhibit large chiral differentiation (∆δ R, S ) due to the presence of a purine ring. The comparison of the diastereomeric excess (de) measured by NMR with those of the gravimetrically prepared ratios are in excellent agreement with each other confirming the robustness of these RNA nucleosides in discriminating primary amines. Chapter 3 establishes the smooth connectivity with the chapter 2 by discussing the limitations of the enantiomeric discrimination using NMR in isotropic solutions. This chapter discusses two new water compatible aligning media that were developed based on self-assembling strategy of small bio-molecules. The self-assembled folic acid, and the binary mixture of 50-GMP and guanosine are introduced as two novel weak aligning media. The properties of these low ordered media have been systematically studied for their easy preparation, physical parameter dependent tunability of their degree of alignment, mesosphere sustainability over a broad range of temperature and the concentration of the ingredients, and the phase reproducibility. The applications of both these new media are demonstrated for chiral and pro-chiral discrimination and also for the measurement of RDCs. Both these liquid crystalline media could be tuned to very low degree of alignment (order parameter of the order of 10−4), which provides simple first order spectra of molecules aligned in them, the analysis provide order dependent NMR spectral parameters. The 50-GMP:guanosine orienting medium can be prepared in less than 1 hour, and has been demonstrated to be an ideal medium for the determination of RDCs that are used as restraints in the structure calculations of small molecules. Chapter 4 describes 1H NMR spectral complexity in isotropic and anisotropic phases and its consequences on enantiomeric analysis. In circumventing such problems, new NMR techniques have been developed and the spin dynamics involved in the designed sequences are discussed. The newly developed 2D 1H NMR experimental method termed as RES-TOCSY, and its applicability for resolving R and S enantiomeric or diastereomeric peaks of all the coupled proton spins in isotropic phase is discussed. The utility of the developed method is demonstrated in diverse situations, such as, for suppressing impurities peaks, resolving the severely overlapped peaks and unraveling the peaks masked due to severe line broadening when metal complexes are used as chiral auxiliaries. The advantages and limitations of the method over other methods available in the literature are discussed and the significant advantage of the present method is illustrated by spectral comparison with J-resolved experiment. The appli- cation of the method for the accurate measurement of enantiomeric excess has also been demonstrated. The chapter also introduces another NMR experimental technique developed for resolving enantiomeric peaks and complete unraveling of R and S spectra in anisotropic phase. The developed 2D NMR method is cited in the literature as CH-RES-TOCSY. In addition to spectroscopic visualization of R and S spectra, the method also yields C-H RDCs. The applicability of the new experiment has been demonstrated on a chosen example. The wide utility of the method has also been demonstrated for the assignment of symmetric cis- and trans- isomers. Part II : Sensitivity Enhancement of Slice selective NMR Experiments Chapter 5 describes applications of slice selective NMR experiments over conven tional NMR methods and their limitations as far as the sensitivity of signal detection is concerned, especially in low concentrated samples. The chapter introduces the implementation of Acceleration by Sharing Adjacent Polarization (ASAP) technique in slice selective experiments. It is convincingly demonstrated that ASAP helps in reducing inter scan relaxation delay and consequently permits acquisition of more number of scans in a given time, resulting in the gain in signal enhancement by a factor of two. The pulse sequences have been suitably designed for obtaining the pure shift 1H spectra and in G-SERF experiment for the measurement of 1H-1H couplings, both with significantly enhanced signal intensities. Chapter 6 describes new sensitivity enhanced slice selective NMR methods for mea- surement of scalar couplings. A new experiment has been developed which is named as Quick G-SERF (QG-SERF). It is a 1D NMR slice selective method developed based on real time spin manipulation technique. The method gives multiple scalar couplings of a selected spin with simplified multiplets, which is analogous to the 2D G-SERF but with considerable saving in instrument time by 1-2 orders of magnitude. The rapidness of the experiment arises due to reduced dimensionality. The spin dynamics involved in the pulse sequence and its working principle have been described. The application of the method is illustrated for the measurement of 1H-1H couplings. The sequence has been further improved to obtain the heteronuclear couplings between two abundant spins in an orchestrated manner and has been demonstrated for measurement of 1H-19F couplings. This sequence cited as HF-QG-SERF has been implemented on the molecules containing number of chemically non-equivalent fluorine atoms.

The thesis entitled "Pushing the Limits of NMR Sensitivity and Chiral Analysis: Design of New NMR Methods and Bio-molecular Tools" consists of six chapters. The research work reported in this thesis is focused on the development of novel chemical and NMR methodological approaches for enantiomeric analysis and mea- surement of residual dipolar couplings (RDCs), and the development of sensitivity enhanced slice selective NMR experiments for obtaining pure shift 1H spectra and the measurement of scalar couplings. The thesis is divided into two parts. The Part I comprises chapters 2-4, where the enantiomeric analysis is discussed, which includes newly developed chiral reagents, two new weak chiral aligning media and design of novel NMR techniques. Part II comprises chapters 5 and 6, which discusses new sensitivity enhanced slice selective NMR techniques. Chapter 1 gives a general introduction to NMR and the problems investigated in the remaining chapters of the thesis. The chapter starts with a brief discussion on the introduction, advancements and general applications of NMR, discussion is also given on the NMR approaches for enantiomeric analysis both in isotropic and anisotropic phases and the measurement of RDCs, including the benefits and limitations associated with each approach. The chapter sets the tone by discussing limitations of the existed NMR enantiomeric approaches and slice-selective techniques, and builds the bridge for the rest of the chapters by addressing these limitations. The chapter also introduces slice selective experiments, their benefits over other conventional methods and limitations. Additional introductory notes are also given on some related concepts. Part I : NMR Chiral analysis and RDCs measurements Chapter 2 discusses chiral sensing properties of RNA nucleosides and their utility as chiral derivatizing agents for the enantio-discrimination of 1o-amines using one dimensional 1H NMR. A three component protocol has been proposed for the complexation of nucleosides with amines, which is rapid, economical and provides maximum diastereomeric conversion. The chiral differentiating ability of nucleosides are examined for different amines based on the 1H NMR chemical shift differences between the diastereomers (∆δ R, S ). Enantiomeric differentiation has been observed at multiple chemically distinct proton sites. It is observed that adenosine and guanosine exhibit large chiral differentiation (∆δ R, S ) due to the presence of a purine ring. The comparison of the diastereomeric excess (de) measured by NMR with those of the gravimetrically prepared ratios are in excellent agreement with each other confirming the robustness of these RNA nucleosides in discriminating primary amines. Chapter 3 establishes the smooth connectivity with the chapter 2 by discussing the limitations of the enantiomeric discrimination using NMR in isotropic solutions. This chapter discusses two new water compatible aligning media that were developed based on self-assembling strategy of small bio-molecules. The self-assembled folic acid, and the binary mixture of 50-GMP and guanosine are introduced as two novel weak aligning media. The properties of these low ordered media have been systematically studied for their easy preparation, physical parameter dependent tunability of their degree of alignment, mesosphere sustainability over a broad range of temperature and the concentration of the ingredients, and the phase reproducibility. The applications of both these new media are demonstrated for chiral and pro-chiral discrimination and also for the measurement of RDCs. Both these liquid crystalline media could be tuned to very low degree of alignment (order parameter of the order of 10−4), which provides simple first order spectra of molecules aligned in them, the analysis provide order dependent NMR spectral parameters. The 50-GMP:guanosine orienting medium can be prepared in less than 1 hour, and has been demonstrated to be an ideal medium for the determination of RDCs that are used as restraints in the structure calculations of small molecules. Chapter 4 describes 1H NMR spectral complexity in isotropic and anisotropic phases and its consequences on enantiomeric analysis. In circumventing such problems, new NMR techniques have been developed and the spin dynamics involved in the designed sequences are discussed. The newly developed 2D 1H NMR experimental method termed as RES-TOCSY, and its applicability for resolving R and S enantiomeric or diastereomeric peaks of all the coupled proton spins in isotropic phase is discussed. The utility of the developed method is demonstrated in diverse situations, such as, for suppressing impurities peaks, resolving the severely overlapped peaks and unraveling the peaks masked due to severe line broadening when metal complexes are used as chiral auxiliaries. The advantages and limitations of the method over other methods available in the literature are discussed and the significant advantage of the present method is illustrated by spectral comparison with J-resolved experiment. The appli- cation of the method for the accurate measurement of enantiomeric excess has also been demonstrated. The chapter also introduces another NMR experimental technique developed for resolving enantiomeric peaks and complete unraveling of R and S spectra in anisotropic phase. The developed 2D NMR method is cited in the literature as CH-RES-TOCSY. In addition to spectroscopic visualization of R and S spectra, the method also yields C-H RDCs. The applicability of the new experiment has been demonstrated on a chosen example. The wide utility of the method has also been demonstrated for the assignment of symmetric cis- and trans- isomers. Part II : Sensitivity Enhancement of Slice selective NMR Experiments Chapter 5 describes applications of slice selective NMR experiments over conven tional NMR methods and their limitations as far as the sensitivity of signal detection is concerned, especially in low concentrated samples. The chapter introduces the implementation of Acceleration by Sharing Adjacent Polarization (ASAP) technique in slice selective experiments. It is convincingly demonstrated that ASAP helps in reducing inter scan relaxation delay and consequently permits acquisition of more number of scans in a given time, resulting in the gain in signal enhancement by a factor of two. The pulse sequences have been suitably designed for obtaining the pure shift 1H spectra and in G-SERF experiment for the measurement of 1H-1H couplings, both with significantly enhanced signal intensities. Chapter 6 describes new sensitivity enhanced slice selective NMR methods for mea- surement of scalar couplings. A new experiment has been developed which is named as Quick G-SERF (QG-SERF). It is a 1D NMR slice selective method developed based on real time spin manipulation technique. The method gives multiple scalar couplings of a selected spin with simplified multiplets, which is analogous to the 2D G-SERF but with considerable saving in instrument time by 1-2 orders of magnitude. The rapidness of the experiment arises due to reduced dimensionality. The spin dynamics involved in the pulse sequence and its working principle have been described. The application of the method is illustrated for the measurement of 1H-1H couplings. The sequence has been further improved to obtain the heteronuclear couplings between two abundant spins in an orchestrated manner and has been demonstrated for measurement of 1H-19F couplings. This sequence cited as HF-QG-SERF has been implemented on the molecules containing number of chemically non-equivalent fluorine atoms.

Le CD40 est une glycoprotéine transmembranaire de type I, appartenant à la famille des TNFRs, exprimée à la surface des cellules immunitaires, hématopoïétiques, vasculaires, épithéliales, et d’autres types de cellules, y compris les cellules tumorales. Le CD40 ne possédant pas de domaine kinase, pour induire un signal il interagit directement ou indirectement avec des protéines adaptatrices telles que les TRAFs et les JAKs. L’interaction du CD40 avec son principal ligand, le CD154, joue un rôle primordial dans la régulation de la réponse immunitaire et le maintien de l’homéostasie. L’activation du CD40 à la surface des cellules B augmente leur capacité de présentation d’antigène, en plus d’induire la prolifération, la commutation isotypique et l’apoptose. Les patients souffrant de mutations au niveau du gène codant pour le CD40 ou de son ligand sont immunosupprimés et sensibles à des infections opportunistes. Des études ont montré que le CD40 comme d’autres membres de la famille des TNFRs est capable de former des homodimères. Plus récemment, on a montré que la formation du CD40 homodimère est le résultat de son engagement sur les cellules B. En plus, cette homodimérisation du CD40 est importante pour la phosphorylation de l’Akt. L’interaction CD40/CD154 peut avoir un rôle direct dans l’immunothérapie par l’induction de l’apoptose de certaines cellules cancéreuses ou un rôle indirect en activant les cellules présentatrices d’antigènes (CPA) afin d'augmenter l’efficacité de l’activation des cellules T cytotoxiques. Nos résultats montrent que l’induction de la mort cellulaire par le CD40 requiert la perméabilisation du lysosome, la libération de la cathepsine B, la présence de ROS et une interaction avec le TRAF6, cette mort cellulaire programmée est plus importante en présence de la forme monomérique du CD40, muté au niveau de la cystéine 238. Par ailleurs, l’homodimérisation du CD40 requerrait sa translocation vers les radeaux lipidiques et nécessiterait la présence des ROS. Cette homodimérisation du CD40 semble être importante pour l’activation des cellules B par le biais de l’induction de l’expression du CD23, CD69 et CD80. De plus, nos résultats montrent pour la première fois une implication du CD40 homodimère dans l’induction du CD23 par le biais du TLR4. Nos résultats soulignent l’importance du CD40 homodimère dans certaines voies de signalisation. Ainsi, ils mettent en évidence le rôle de la Cys-238 dans la coopération entre des récepteurs de la réponse immunitaire innée et adaptative. Toutes ces données permettraient une meilleure compréhension de certaines voies de signalisation impliquées dans plusieurs maladies auto-immunes et faisant objet de plusieurs essais thérapeutiques.
CD40 is a type I transmembrane glycoprotein belonging to the TNFRs family, which is expressed on the surface of immune, hematopoietic cells, vascular, epithelial, and other cell types, including a wide range of tumour cells. CD40 does not have a kinase domain. Thus, to induce a signal, CD40 interacts directly or indirectly with adapter proteins such as TRAFs and Jaks. The interaction of CD40 with its main ligand, CD154, plays an important role in regulating the immune response and homeostasis. The activation of CD40 on the surface of B cells increases its ability to promote antigen presentation, in addition to inducing proliferation, isotype switching, and apoptosis. Patients affected by mutations in the gene encoding the CD40 or its ligand are immunosuppressed and susceptible to opportunistic infections. Studies have shown that CD40, as other members of the family of TNFRs is capable of forming homodimers. More recently, it was shown that the formation of the CD40 homodimer is the result of the engagement of CD40 on B cells by CD154. In addition, the homodimerization of CD40 is important for the phosphorylation of Akt. The CD40/CD154 interaction can have a direct role in immunotherapy by inducing apoptosis of some cancer cells or an indirect role in activating antigen-presenting cells (APCs), thereby increasing the effectiveness of activation of cytotoxic T cells. Our results show that the induction of cell death by CD40 requires permeabilization of the lysosome, the release of cathepsin B, the presence of ROS and interaction with TRAF6, this programmed cell death is greater in the presence of the monomeric form of CD40, due to a mutation at the level of the cysteine 238. Moreover, the homodimerization of CD40 requires its translocation to lipid rafts and the presence of ROS. This homodimerization is necessary for the CD40 B-cell activation via the induction of expression of CD23, CD69 and CD80. In addition, our results show for the first time the involvement of the CD40 homodimer in the induction of CD23 expression via TLR4. Our results emphasize the importance of CD40 homodimer in signaling pathways and highlight the role of Cys-238 in the cooperation between receptors of the innate and adaptive immune response. All together our results will allow a better understanding of CD40 signaling pathways involved in several autoimmune diseases, which give a rise to a better therapeutic trial design.

Renewable Energy Sources (RES) are the most promising resources of energy production for everyday life. Therefore, the precise combination of RES based technologies into hybrid systems could provide the solution to several energy problems facing the planet. The motivation of the present research study is the total understanding of the prevailing phenomena by using RES equipment in several projects. This thesis will focus on standalone hybrid RES based systems. By presenting the RES systems the necessity of buffering systems will become apparent as the most crucial parts of off-grid systems. Therefore, the most well-established buffering technologies will be analytically presented in order to be subsequently embodied into the simulated RES applications. Following the above theoretical approach of RES based equipment and hybrid systems in general, this thesis will focus on a more applied research study comprising the energetic and economical simulation and optimization of a RES based stand alone system that is already installed in Leicestershire, UK. Based on local meteorological data, an optimization strategy has been developed to identify the most economical and efficient scenarios for electricity generation to cover the desirable load on an annual basis. Furthermore, the environmentally-friendly character of the system was highly concerned with emissions reduction; therefore the capability of an off-grid system was also investigated. The feasibility of RES based systems for electricity supply will then be presented for four different Greek Islands. Three specific typical loads have been selected to be covered and the grid connection was considered optional. Up to this point the simulation and optimization procedures were applied by using the HOMER software tool in order to investigate the most suitable well-established platform in the world. After the theoretical research study on the most well-known platform of HOMER an innovative optimization theory based on the energy part of a hybrid system will be presented in order to select the most efficient system according to the desired requirements and the location of a RES based project. This thesis will then focus on the design and operation of an autonomous hybrid system under real-life meteorological conditions which is capable of simulating several loads assumed to cover the electricity demands of small buildings. The specific hybrid system embodies technologies that use photovoltaic and wind energy in combination with an electrochemical storage bank. Experiments on the coverage of annual loads regarding a typical house, a typical country house and a small company were also performed to prove the feasibility of the stand-alone system. The same established RES project was then simulated on a yearly basis using the HOMER software platform to determine real-time results. The above analysis revealed that HOMER software cannot successfully simulate the operation of such a system, therefore the design of a new mathematical model to produce results similar to those of the experimental process was considered essential based on a new optimization strategy.
Οι Ανανεώσιμες Πηγές Ενέργειας (ΑΠΕ) αποτελούν τις πιο πολλά υποσχόμενες πηγές στον τομέα της παραγωγής της ηλεκτρικής ενέργειας μέσα στην ανθρώπινη καθημερινότητα. Έτσι ο ακριβής συνδυασμός των ΑΠΕ σε υβριδικά συστήματα θα μπορούσε να αποτελέσει τη λύση στο μεγάλο ενεργειακό πρόβλημα που αντιμετωπίζει ο πλανήτης τα τελευταία χρόνια και όσο περνάει ο καιρός αυτό φαίνεται να διογκώνεται. Το κίνητρο για την εκπόνηση της παρούσας διδακτορικής διατριβής στηρίζεται στην ανάγκη για απόλυτη κατανόηση των φαινομένων που λαμβάνουν χώρα κατά τη χρήση των ΑΠΕ σε διάφορα συστήματα για την παραγωγή ηλεκτρικής ενέργειας. Επιπλέον, μέσα από αυτή την έρευνα θα φανεί πως οι καιρικές συνθήκες επηρεάζουν τη συμπεριφορά ενός υβριδικού συστήματος και σε ποιό ποσοστό. Ακόμα περιμένουμε να γίνει φανερό το πόσο σημαντική είναι η σωστή επιλογή των τεχνολογιών σύμφωνα με τις ηλεκτρικές ανάγκες που πρέπει να καλυφθούν από ένα εγκατεστημένο σύστημα. Στη συνέχεια της παρούσας εργασίας μελετήθηκε κάτω από ποιες συνθήκες ένα αυτόνομο υβριδικό σύστημα μπορεί να είναι εφικτό καθώς και πόσο ακριβή αποτελέσματα μπορούν αν δώσουν τα θεωρητικά μαθηματικά μοντέλα επάνω στην πρόβλεψη της λειτουργίας ενός συστήματος. Τέλος, παρουσιάστηκε πως μπορεί να ενισχυθεί ο οικολογικός χαρακτήρας ενός συστήματος ενώ την ίδια στιγμή αποκαλύφθηκε η κύρια αδυναμία του κατά τη λειτουργία καθώς και πως αυτή μπορεί να λυθεί με τη χρήση καινοτόμων συσκευών για την αποθήκευση ενέργειας. Μέσω της παρούσας διδακτορικής διατριβής αποδείχθηκε πως ένα υβριδικό σύστημα υποστηριζόμενο από ΑΠΕ μπορεί να μετατραπεί σε εντελώς αυτόνομο με ενισχυμένο τον οικολογικό του χαρακτήρα και με την οικονομική και ενεργειακή βιωσιμότητά του να κυμαίνεται σε υψηλά επίπεδα. Το παραπάνω συμπέρασμα προέκυψε μέσω θεωρητικών αλλά και πειραματικών προσομοιώσεων διάφορων υβριδικών μονάδων. Αυτό αποτελεί ίσως το πιο ενθαρρυντικό στοιχείο για πλήρη αξιοποίηση των ΑΠΕ προκειμένου να καλυφθούν οι παγκόσμιες ενεργειακές ανάγκες με τρόπους εντελώς φιλικούς προς το περιβάλλον στο άμεσο μέλλον.

Tumor-induced immunosuppression can occur by multiple mechanisms, each posing a significant obstacle to immunotherapy. Evidence presented in this dissertation suggests that aberrant cytokine signaling, as a result of altered metabolism of Chronic Lymphocytic Leukemia (CLL) cells, confers a selective advantage for tumor survival and growth. Cells from CLL patients with aggressive disease (as indicated by high-risk cytogenetics) were found to exhibit prolongation in Interferon (IFN)-induced STAT3 phosphorylation, and increased levels of reactive oxygen species (ROS) in these cells reflected these signaling processes. Changes in the relative balance of phospho-STAT3 and phospho-STAT1 levels, in response to combinations of IL-2 + Toll-like receptor (TLR)-7 agonist + phorbol esters, as well as IFN, were associated with the immunosuppressive and immunogenic states of CLL cells. In addition, immunosuppressive leukemic cells were found to express high levels of proteins with O-linked N-acetylglucosamine (O-GlcNAc) modifications, due to increased metabolic activity through the Hexosamine Biosynthetic Pathway (HBP), which caused impaired intracellular signaling responses and affected disease progression. A conclusion of the studies presented here is that the intrinsic immunosuppressive properties of leukemic cells may be overcome by agents such as Resveratrol that target metabolic pathways of these cells.