Rozprawy doktorskie na temat „New variant”

Type I PIP kinases (phosphatidylinositol 4-phosphate 5-kinases, PIP5Ks) catalyse the majority of cellular synthesis of PI(4,5)P2. To date, three mammalian isoforms (r1, r2, r3) have been found. PIP5KIr is subject to complex C-terminal splice variation, enhancing its transcriptional diversity through evolution and producing at least 5 known spliceoforms in the mammals. This study addresses several important questions. (1) Several remarkable differences have been discovered between the neuronal splice variant PIP5KIr_i3 and its close variant, Ir_i2, whose peptide lacks a 26-AA insert near its C-terminus. This study attempts to map these behavioural differences onto motifs within the peptide insert. Furthermore, a site of point mutation is identified near the activation loop, which amplifies the above differences. (2) This study documents properties of the more recently discovered PIP5KIr_i3, about which relatively little is known, for example, the regulation of its subcellular localisation, kinase activity and post-translational modifications. By site-directed mutagenesis and examining more closely several crucial motifs, insight is gained into the putative relationship between the enzyme’s phosphorylation state, cellular localisation, lipid kinase activity and autophosphorylation. (3) The discovery of a new PIP5KIr splice variant, Ir_v6, is described. First discovered in rodents, PIP5KIr_i6 encompasses the 26-AA insert of Ir_i3, but lacks the common C-terminus of Ir_i2 and Ir_i3 which contains peptide motifs that have several roles in vivo. A polyclonal antibody against the C-terminus of Ir_i6 was also developed. Preliminary characterisation of Ir_i6 demonstrates a similar subcellular localisation, but a wider expression profile than its close relative, Ir_i3, suggesting potentially differential functions across tissues and at various developmental stages. (4) The existence of Ir_v3 and Ir_v6 is also confirmed in humans. In light of recent findings of other novel human spliceoforms, this is shown to be a case of intra-exonic splicing producing “alternative 5’ splice site” exons in the human. Overall, this thesis should help to better understand the regulation and physiological roles of PIP5KIr and, specifically, its different splice variants.

Recent technological advances have revolutionised the conduct of animal genetics. Refinements to high‐throughput genotyping and sequencing platforms have brought these technologies within reach of animal geneticists, providing exciting research opportunities. Yet to apply them experimentally at the scale benchmarked by human genetics studies remains financially prohibitive for most animal genetics projects. Limited opportunities for funding along with restricted sampling due to animal ethics constraints or rarity of the trait combine to effect low‐powered datasets. This thesis explores methods of maximising research outcomes in the face of these challenges and the limitations posed by the technologies through adaptable experimental design and creative approaches to data mining. Methodological contributions to the application of three key technologies are made. In addition, novel findings provide new insights to canine genetics.

The topic of my PhD project is the Hereditary Haemorrhagic Telangiectasia (HHT), also known as Rendu-Osler-Weber syndrome, a rare genetic disease with an incidence of about 1:5000, inherited as an autosomal dominant trait. Up to date, mutations in four genes (ENG, ACVRL1, MADH4 and GDF2) have been described in HHT, however, ENG and ACVRL1 mutations account for the 85% of patients. All these genes encode for proteins belonging to the TGF-β/BMPs signalling pathway and are involved in the regulation of angiogenesis. HHT is clinically diagnosed if a patient presents at least three out of four criteria, known as “Curaҫao criteria”: (i) spontaneous and recurrent epistaxis; (ii) telangiectases at characteristic sites, as lips, oral cavity, nose, fingertips and gastrointestinal mucosa; (iii) arteriovenous malformations at characteristic sites, as liver, lungs and central nervous system, and (iv) family history, i.e. a first degree relative with a diagnosis of HHT according to the same criteria. The aim of this thesis is to further investigate the genetic background of HHT in order to better understand the underlying molecular mechanisms and the genotype-phenotype correlations. In particular, my work involved the mutation analyses of ENG and ACVRL1 coding exons and the study of circulating miRNAs in a group of HHT patients. Mutation analyses were performed in new index cases with a HHT clinical diagnosis and their relatives, to identify the disease-causing mutation. Then, the results collected were added to data previously obtained by the Medical Genetics Laboratory (headed by Prof. C. Danesino) and used to perform a descriptive study of the HHT Italian Population, carried out in collaboration with Prof. C. Sabbà, from the University of Bari. Moreover, during my PhD I attended a three-months period in the laboratory of Prof. C. Bernabeu, in Madrid, in order to study the putative pathogenic effect of a particular ENG variant (c.1852+42 C>T) found in a HHT family. The study of circulating miRNAs was performed in collaboration with Prof. M. Denti (University of Trento). We collected plasma samples from 15 people (5 controls; 5 patients with ENG mutation and 5 patients with ACVRL1 mutation) and evaluated the expression level of 752 human miRNAs to identify those misregulated in patients compared to controls. Firstly, we analysed results grouping the patients according to the mutated gene and we identified about 20 misregulated miRNAs. Then, we evaluated if some miRNAs were differentially expressed in patients according to their clinical symptoms. In conclusion, the obtained results increased the knowledge on the molecular pathogenesis, and shed a light on the role exploited by miRNAs in this disorder, thus suggesting new genotype-phenotype correlations, although a validation of the results in a bigger population is mandatory.

Nurture Groups have experienced rises and falls in popularity since their initial introduction by Marjorie Boxall in 1969 in inner city London (Boxall 1976). At present there are more than 1,500 Nurture Groups in the UK (Nurture Group Network Website) with the Nurture Group Network continuing work to expand and develop the use of these groups in more areas across the country. This research project seeks first to examine the effectiveness or success of nurture groups in preparing children for reintroduction to the mainstream classroom. This was examined by conducting a systematic review of the literature which evaluated studies reporting on outcomes for children attending nurture groups, using quantitative measures. The review concludes that the lack of consistency in the methods of analysis employed between studies, the varying perceptions of ‘success’ and the dearth of studies which examine pupil data longitudinally (only one being present Cooper and Whitebread 2007) does not provide a solid and compelling evidence base for effectiveness of nurture groups in preparing children for reintroduction to mainstream classes. Nurture Groups remain popular however and the author sought to question what it is about nurture groups which schools, staff and pupils value. Through discussion of a process of personal epistemological change and development, the focus of the research project shifts from the quantitative measures described in the Systematic Review to a more qualitative approach. In light of the researchers aim to add a unique perspective to the body of literature a decision was made to conduct an empirical research project with the staff and pupils of a nurture group. The nature of the group in relation to traditional nurture principles is explored and explained. The research project is conducted using a combination of focus groups and semi-structured one to one interviews with nurture group staff, children who attend the nurture group and the mainstream teachers of those children. The interviews were transcribed and analysed using Interpretative Phenomenological Analysis to produce superordinate and subthemes which emerged as particularly pertinent to the participants involved. This method of analysis allowed discussion of these themes by looking at both the way in which the participants made sense of their experiences and the researchers understanding and meaning making of the descriptions produced. The study concludes that many of the reported benefits and value laden aspects of the nurture provision tie in with current psychological 2 knowledge of processes such as attachment, relationships, sense of belonging and self-efficacy and self-worth. There is also discussion of the policy issues mentioned by staff which influence practice within school and the nurture group. Tentative conclusions state that this research can contribute to the field by offering an examination of one case study which may contribute to identifying wider patterns and themes in other IPA studies in this area. It is also concluded that the new variant nurture group involved in this study shows evidence of enriching the children’s educational experience; helping to develop skills both in learning and in social and emotional functioning. However, future suggestions for development of the group could include work on developing these skills in a way which can be transferred outside of the safety of the nurture group. Future studies could also look at the views and perceptions of parents and ways in which to implement a nurturing ethos throughout school.

This thesis is an exploration of how the use of temporary agency work in French car plants modifies the experience and mechanisms of labour control in the labour process. Over the last decade, car manufacturers in France have made extensive use of this form of employment, despite regulations which restrict the use of agency labour to exceptional circumstances. Legal challenges aimed at reclassifying temporary agency contracts into permanent employment contracts have revealed that some agency workers have accumulated many years of employment as an agency worker with user-company. The presence of significant proportions of agency workers on assembly lines for long periods of time has implications for the labourcapital relation on the shopfloor. Precarious working conditions for low-skilled workers are assumed to affect the capacity of workers to negotiate relations on the shop-floor. The thesis employs a conceptual framework based upon Burawoy’s (1985) theory of production politics to examine the specific way in which the triadic relationship between the temporary agency worker, temporary employment agency and user-organisation modifies the factory regime within which temporary agency workers labour. Starting from an analysis of the macro- and meso-level development of the post-war French state and of the key economic sectors that constitute the “politics of production”, the thesis focuses on the PSA Peugeot-Citroën plant in Aulnay-sous-Bois as a case study, and combines interview data with other qualitative (textual) data. The research finds that temporary agency workers in the car sector respond to their employment situation in a more complex way than studies of coercion and consent in the labour process suggest. Employment insecurity and the “duality of control” which flows from the triadic relationship upon which the temporary agency contract rests gives rise to a factory regime more conducive to compliance/coercion than consent. However, the “traces of consent” identified by the research illustrate the complex nature of hegemony and despotism in the labour process. Drawing on the findings of the empirical data in the context of France, the thesis develops the concept of hegemonic despotism by examining how hegemonic despotism is expressed across a variety of employment contexts. The thesis identifies a tension between adverse conditions of employment and hegemonic practices, such as the formal adhesion to “soft” models of HRM, alongside the recasting of norms of employment to fit the requirements of contemporary capital accumulation.

The aim of this study is focused on two main areas of NGS analysis data: RNA-seq(with a specific interest in meta-transcriptomics) and DNA somatic mutations detection. We developed a simple and efficient pipeline for the analysis of NGS data derived from gene panels to identify DNA somatic point mutations. In particular we optimized a somatic variant calling procedure that was tested on simulated datasets and on real data. The performance of our system has been compared with currently available tools for variant calling reviewed in literature. For RNA-seq analysis, in this work we tested and optimized STAble, an algorithm developed originally in our laboratory for the de novo reconstruction of transcripts from non reference based RNA-seq data. At the beginning of this study, the first module of STAble was already been written. The first module is the one which reconstructs a list of transcripts starting from RNA-seq data. The aim of this study, particularly, consisted in adding a new module to STAble, developed in collaboration with Cambridge University, based on the flux-balance analysis in order to link the metatranscriptomic analysis to a metabolic approach. This goal has been achieved in order to study the metabolic fluxes of microbiota starting from metatranscriptomic data.

Chlamydia trachomatis is an obligate intracellular bacterium of major human health concern, causing urogential chlamydia infections, lymphogranuloma venereum (LGV) and trachoma. Chlamydia is one of the most common sexually transmitted infections worldwide and can cause infertility. In the first four papers described herein we used a high resolution multilocus sequence typing (MLST) system to investigate the epidemiology of C. trachomatis, and showed that MLST is superior to conventional ompA genotyping with respect to resolution. In the fifth paper we simplified the methodology by developing and validating a multilocus typing (MLT) DNA microarray based on the MLST system. In more detail, MLST analysis of consecutive specimens from 2006 in Örebro County in Sweden, and comparison to specimens from 1999-2000, showed that the new variant C. trachomatis (nvCT) is monoclonal and likely has appeared in recent years. MLST analysis of LGV specimens from men who have sex with men (MSM) showed that the increase of LGV in Europe in the last decade indeed was a clonal outbreak, contrary to the USA where LGV might have been present all along. In the third paper, clinical symptoms could not be correlated with the MLST genotypes, suggesting, together with the combined results of all previous studies, that bacterial factors, if important, need to be understood in the context of host factors. MLST analysis of specimens from a high incidence C. trachomatis area in North Norway revealed interesting epidemiological details concerning unusual genetic variants, the nvCT and MSM, but found no significant difference in genetic diversity compared to two other geographic areas in Norway. Lastly, we developed a MLT array that provides high resolution while being rapid and cost-effective, which makes it an interesting alternative for C. trachomatis genotyping. In conclusion, the MLST system and the MLT array have proven to be useful tools and should now be applied in further investigations to improve our understanding of C. trachomatis epidemiology.

This thesis deals with the issue of investment decision. The goal of thesis is to evaluate for the company owners optimal investment variant of predetermined investment opportunities. Thesis is divided into theoretical-methodological part, which summarizes all the methods and procedures and practical part. The practical part consists mainly of net present value calculations, which chosen input data will be afterwards analyzed by sensitivity test. Application of these apparatuses will reveal the best investment opportunity, respectively their combination

Inherited thrombocytopenias (IT) are a group of rare diseases characterized by a low platelet count (less than 150.000 / mL) and high degree of heterogeneity at both clinical and genetic point of view. To date, mutations in at least 30 different genes have been identified, however about 50% of patients still remain without molecular diagnosis, due to the complexity of the diagnostic framework and because many patients are probably affected by forms of IT not characterized yet. The spread in the last decade of next generation sequencing techniques (Next Generation Sequencing, NGS) allow us to simultaneously analyse many candidate genes and identify thousands of variants in a single analysis. However, understanding the effects of these variants remains a major problem in disorders such as thrombocytopenia, which are mainly autosomal dominant diseases caused by "private" mutations, often missense. In fact, while for "deleterious" mutations, such as large deletions, nonsense or frameshift the relationship between the variant and their pathogenicity is often immediate due to the important structural and functional consequences on the protein, determining the pathogenic role of a missense variant is a complex process that requires targeted functional studies. My PhD work, carried out at the Medical Genetics laboratory of Professor Anna Savoia at Burlo Garofolo hospital in Trieste, fits into this context and consists in the pathogenicity analysis, through functional assays, of the variants identified in three transcription factor genes (MECOM, GFI1B and RUNX1), whose mutations are responsible for MECOM-associated syndrome, platelet - type bleeding disorder - 17 (BDPLT17) and FPD/AML thrombocytopenia, respectively. My PhD work indicates that functional assays are essential, in combination with genetic analysis, to discriminate between pathogenic and non-pathogenic variants. This aspect is very important especially in case of diseases characterized by a wide spectrum of variants that are predicted to be of uncertain significance, as ITs. The functional characterization of variant, in fact, allow us to provide patients correct molecular diagnoses, which is fundamental for an appropriate therapeutic approach and a properly follow-up of patients, especially when they are carriers of a mutation that increase the risk of developing further, even more serious, diseases.

Intellectual Disability (ID) is one of the most common neurodevelopmental disorders, affecting between 1.5-2% of individuals in the general population. This pathology has a serious impact on the affected individuals, their families and also on the health care system. Understanding the genetic mechanisms implicated in the disease is challenging, since ID includes a wide spectrum of possible underlying disorders and the genetic variants determining the disease are highly heterogeneous, requiring the application of different approaches and techniques, from cytogenetic analysis to the application of most recent NGS strategies. In the last decades, big steps forward have been done in the search of the genetics determinants for ID, however today less then half of the patients receive a molecular diagnosis. Moreover, the continue reporting of new ID-genes suggests that many of the genetic variants causing ID still need to be discovered and understood. The aim of the thesis is to identify new genes and genetic variants involved in ID, with the use of diverse approaches and methodologies. The first stage of the study has been the screening of a cohort of nonsyndromic ID patients with a gene-panel specific for non-syndromic ID. This approach has led to the discover of 6 novel putative mutations and, when possible, the impact of the variation has been evaluated in silico, with an analysis of the protein structure. The screening also led to the selection of two patients that underwent to whole-exome sequencing analysis with the aim to identify the causative variant beyond the most common genes for the disease. Notably, we have discovered 2 X-linked mutations having likely a pathogenetic role. One is a missense mutation in CLCN4, a gene member of the chloride channel family that only recently has been demonstrated to cause ID. Another missense mutation has been found in ALG13 gene that has been associated with X-linked non-syndromic ID only once but without a clear explanation of its functioning. The second part of the thesis concerns the investigation on genetic variants having a mild impact on the phenotype. We performed a Runs of Homozygosity study to investigate the role of distant inbreeding in ID. The analysis has revealed that the global amount of homozygosity and the number of homozygous stretches are associated with the degree of the impairment. Finally, we also designed an association study for the Y chromosome to investigate the presence of variants implicated in the development of cognitive function in this genomic region. We analysed the general cognitive function measured in 5 cross-sectional cohorts but did not find evidence for an association on the Y chromosome.

The argument of this paper is that certain salient passages in the New Testament concerning Christology, as it was defined in the Nicene creed in A.D. 325, reflect such orthodoxy better in the Textus Receptus Greek texts and the English translations made from them than do the Alexandrian texts. Arian theology, which was condemned as heretical at Nicea, is examined. Patristic quotations, historical texts, and arguments of the scholars are cited and traced, along with a comparison of Christological verses.

Many real-world optimisation problems are discrete in nature. Although recent rapid developments in computer technologies are steadily increasing the speed of computations, the size of an instance of a hard discrete optimisation problem solvable in prescribed time does not increase linearly with the computer speed. This calls for the development of new solution methodologies for solving larger instances in shorter time. Furthermore, large instances of discrete optimisation problems are normally impossible to solve to optimality within a reasonable computational time/space and can only be tackled with a heuristic approach. In this thesis the development of so called matheuristics, the heuristics which are based on the mathematical formulation of the problem, is studied and employed within the variable neighbourhood search framework. Some new variants of the variable neighbourhood searchmetaheuristic itself are suggested, which naturally emerge from exploiting the information from the mathematical programming formulation of the problem. However, those variants may also be applied to problems described by the combinatorial formulation. A unifying perspective on modern advances in local search-based metaheuristics, a so called hyper-reactive approach, is also proposed. Two NP-hard discrete optimisation problems are considered: 0-1 mixed integer programming and clustering with application to colour image quantisation. Several new heuristics for 0-1 mixed integer programming problem are developed, based on the principle of variable neighbourhood search. One set of proposed heuristics consists of improvement heuristics, which attempt to find high-quality near-optimal solutions starting from a given feasible solution. Another set consists of constructive heuristics, which attempt to find initial feasible solutions for 0-1 mixed integer programs. Finally, some variable neighbourhood search based clustering techniques are applied for solving the colour image quantisation problem. All new methods presented are compared to other algorithms recommended in literature and a comprehensive performance analysis is provided. Computational results show that the methods proposed either outperform the existing state-of-the-art methods for the problems observed, or provide comparable results. The theory and algorithms presented in this thesis indicate that hybridisation of the CPLEX MIP solver and the VNS metaheuristic can be very effective for solving large instances of the 0-1 mixed integer programming problem. More generally, the results presented in this thesis suggest that hybridisation of exact (commercial) integer programming solvers and some metaheuristic methods is of high interest and such combinations deserve further practical and theoretical investigation. Results also show that VNS can be successfully applied to solving a colour image quantisation problem.

In this thesis, new variants of nonnegative matrix factorization (NMF) based ona convolutional data model, -divergence and sparsication are developed andanalyzed. These NMF variants are collectively referred to as -CNMF. Commonsparsication techniques such as L1-norm minimization and elastic net arediscussed and a new regularizer is proposed. It is shown that the new regularizer,unlike the above-mentioned sparsication techniques, has control overthe number of active bases in the NMF dictionary. Moreover, the -CNMF isextended to multichannel signals: it learns a common dictionary by exploitingthe correlation between channels through a multichannel coecient matrix. Asa result, an algorithm for source separation based on multichannel -CNMF isdeveloped. The algorithm is further tested in a multilayer setting, in which thefrequency-shifted coecient matrices serve as input to the next higher layer.Finally, three variants of the algorithm are evaluated in the context of speechenhancement, focusing on the problem of speech extraction from complex auditoryscenes. Figures obtained from the SiSEC 2016 data show that the proposedalgorithms perform comparably or better than the state of the art.
Den här rapporten behandlar utveckling och analys av nya varianter av icke-negativ matrisfaktorisering (eng: nonnegative matrix factorization, NMF), som baseras på en datormodell med faltning, β-divergens och glesa matriser. Dessa varianter av NMF:er kallas allmänt för β-CNMF:er, där C:et står för “convolutional”. Vidare diskuteras vanliga tekniker för regularisering, såsom L1-normminimering och elastiska nät, och en ny formulering för regularisering föreslås. Det visar sig att denna nya formulering, till skillnad från ovan nämnda regulariseringstekniker, möjliggör kontroll av antalet aktiva basfunktioner i NMF:ens bibliotek. Utöver detta så utökas även β-CNMF:en till att behandla multikanalsignaler genom att tränas på en gemensam bibliotek som utnyttjar korskorrelationen mellan kanalerna. Detta möjliggör utveckling av en algoritm för källseparation av multikanalsignaler. Vidare så testas algoritmen i multipla led, där frekvensskiftade koefficientmatriser i ett led utgör indata till nästa led. Slutligen så bedöms tre olika varianter av algoritmen för talförbättring, med fokus på extrahering av tal ur komplexa ljudmiljöer. Mätningar från SiSEC 2016 visar att den föreslagna algoritmen presterar lika bra eller överträffar nu-varande befintliga algoritmer.

In this research, the Modified Weighted Mean iterative methods are developed and investigated for solving Integro-differential equations of Fredholm type. The developed methods are further investigated by implementing computational complexity-reduction techniques on three combination sets of Central Difference-Composite Closed Newton-Cotes dense systems. All new variants of proposed methods are presented with strong convergence theorems and proofs. The effectiveness and efficiency of the methods are investigated and validated with some existing methods, and via vigorous numerical analysis.

L'herpèsvirus humain 8 (HHV-8) est impliqué dans toutes les formes de maladie de Kaposi (MK) mais également dans certaines formes de maladie de Castleman multicentrique (MCM) et dans le lymphome primitif des séreuses (LPS). Ce travail avait pour objectif d’étudier les déterminants cliniques et virologiques impliqués dans l’infection HHV-8 et les maladies qui lui sont liées. Ainsi dans l’étude 1, la charge virale HHV-8 associée à la MK était inférieure à celles retrouvées dans les hémopathies, probablement dû à la physiopathologie de chaque maladie, mais d’autres facteurs pourraient également intervenir. En effet dans l’étude 2, le sous-type HHV-8 influençait la charge virale et la sévérité du tableau clinique dans la MK. De plus, nous avons identifié un nouveau variant systématiquement associé à des formes cliniques graves, renforçant le postulat de déterminants virologiques intervenant dans la physiopathologie des différentes maladies. Malgré l’ère des antirétroviraux, l’immunité jouerait également toujours un rôle important puisque dans l’étude 1, le taux de CD4 était faible et inversement corrélé à la charge virale HHV-8 dans la MK et la MCM alors que dans l’étude 3, les MK épidémiques avec une réponse immunologique et virologique soutenue pour l’infection VIH, étaient associées à un ratio CD4/CD8 inférieur à 1. Enfin dans l’étude 4, l‘analyse in vitro de l’effet du poppers sur le HHV-8 a montré qu’il stimulait la réplication virale. Ce résultat suggère que son utilisation in vivo pourrait être un facteur environnemental favorisant la transmission HHV-8 mais également le développement de MK chez des patients avec une immunité apparente normale ou restaurée
The human herpesvirus 8 is an oncogenic virus involved in the development of all forms of Kaposi’s sarcoma (KS), but also in certain forms of multicentric Castleman disease (MCD) and in primary effusion lymphoma (PEL). The aim of this work was to study the clinical and virological determinants in HHV-8 infection and associated with its three main diseases. Thus, in the study 1, the HHV-8 DNA viral load associated with KS was lower compared with that found in hemopathies, probably due to the pathophysiology of each disease, but other factors could also be involved. Indeed, in the study 2, the HHV-8 subtype influenced the viral load and the severity of the clinical presentation of KS. In addition, we have identified a new variant associated with severe clinical forms, reinforcing the postulate of virological determinants involved in the pathophysiology of the different diseases. Despite the era of antiretrovirals, immunity would still play an important role because in the study 1, the CD4 count was low and inversely correlated with the HHV-8 viral load in KS and MCD whereas in study 3, epidemic KSs with a sustained immunological and virological response for HIV infection were associated with a CD4 / CD8 ratio of less than 1. Finally, in the study 4, in vitro analysis of the effect of poppers on BC-3 cells chronically infected by HHV-8 has been shown to stimulate viral replication. This result suggests that poppers used in vivo could be an environmental factor favoring the transmission of HHV-8 but also the development of MK in patients with apparent normal or restored immunity

My project has been focused on the identification and validation of new mitochondrial gene variants and new mitochondria-related genes. The first report is about a woman presenting with a stroke-like episode and an history of severe hearing loss, frequent migraines, exercise intolerance, myalgias and limb-girdle muscle weakness indicating a slowly progressive myopathy and secondary amenorrhea with low gonadotropin levels. A muscle biopsy showed ragged-red, cytochrome c oxidase-negative fibers, and an isolated defect of cytochrome c oxidase activity in muscle mitochondria and sequence analysis of muscle mtDNA revealed a new heteroplasmic m.6597C>A transversion in the MTCOI gene, encoding subunit I of cytochrome c oxidase. Analysis on transmitochondrial cybrids demonstrated that the mutation is indeed associated with COX deficiency, i.e. pathogenic. The second report is about a new phenotype associated to mutations in the AARS2 gene encoding for the mitochondrial aminoacyl tRNA synthetase, identified in six patients presenting with primary ovarian failure, cerebellar and pyramidal signs and cognitive or behavioural disturbances. Two patients underlined a muscle biopsy which showed a severe complex IV defect at histochemical and biochemical analyses. The third report is about the clinical and biochemical phenotypes associated with mutations in two new mitochondrial aminoacyl tRNA synthetases (ARSs2) genes. In the first patient, an 8 years old child presenting with psychomotor delay, seizures, facial dysmorphisms and hyperlactacidemia and a brain MRI showing hyperintense lesions in the insula and fronto-temporal right cortex, whole exome sequencing (WES) identified a homozygous missense mutation in VARS2, encoding the mitochondrial valyl tRNA-synthetase. In two siblings presenting with a phenotype characterized by hypotonia and psychomotor retardation, high plasma and liquor lactate, both died at few months of age WES revealed two variants in TARS2, encoding the mitochondrial threonyl tRNA-synthetase: a missense and a splice site mutation. VARS2 and TARS2 mutations segregate within patients families. Patients’ clinical- biochemical phenotype and in silico and in vitro analyses of VARS2 and TARS2 mutations clearly indicate these genes as disease-causative. Expression of the corresponding wild-type enzymes led to recovery of the biochemical impairment of mitochondrial respiration in immortalized mutant fibroblasts; yeast modelling of the VARS2 mutation confirmed its pathogenic role.

Autism spectrum disorder (ASD) and Intellectual Disability (ID) are complex neuropsychiatric disorders characterized by extensive clinical and genetic heterogeneity and with overlapping risk factors. The aim of my project was to further investigate the role of Copy Numbers Variants (CNVs), identified through genome-wide studies performed by the Autism Geome Project (AGP) and the CHERISH consortium in large cohorts of ASD and ID cases, respectively. Specifically, I focused on four rare genic CNVs, selected on the basis of their impact on interesting ASD/ID candidate genes: a) a compound heterozygous deletion involving CTNNA3, predicted to cause the lack of functional protein; b) a 15q13.3 duplication containing CHRNA7; c) a 2q31.1 microdeletion encompassing KLHL23, SSB and METTL5; d) Lastly, I investigated the putative imprinting regulation of the CADPS2 gene, disrupted by a maternal deletion in two siblings with ASD and ID. This study provides further evidence for the role of CTNNA3, CHRNA7, KLHL23 and CADPS2 as ASD and/or ID susceptibility genes, and highlights that rare genetic variation contributes to disease risk in different ways: some rare mutations, such as those impacting CTNNA3, act in a recessive mode of inheritance, while other CNVs, such as those occurring in the 15q13.3 region, are implicated in multiple developmental and/or neurological disorders possibly interacting with other susceptibility variants elsewhere in the genome. On the other hand, the discovery of a tissue-specific monoallelic expression for the CADPS2 gene, implicates the involvement of epigenetic regulatory mechanisms as risk factors conferring susceptibility to ASD/ID.

Autism spectrum disorder (ASD) and Intellectual Disability (ID) are complex neuropsychiatric disorders characterized by extensive clinical and genetic heterogeneity and with overlapping risk factors. The aim of my project was to further investigate the role of Copy Numbers Variants (CNVs), identified through genome-wide studies performed by the Autism Geome Project (AGP) and the CHERISH consortium in large cohorts of ASD and ID cases, respectively. Specifically, I focused on four rare genic CNVs, selected on the basis of their impact on interesting ASD/ID candidate genes: a) a compound heterozygous deletion involving CTNNA3, predicted to cause the lack of functional protein; b) a 15q13.3 duplication containing CHRNA7; c) a 2q31.1 microdeletion encompassing KLHL23, SSB and METTL5; d) Lastly, I investigated the putative imprinting regulation of the CADPS2 gene, disrupted by a maternal deletion in two siblings with ASD and ID. This study provides further evidence for the role of CTNNA3, CHRNA7, KLHL23 and CADPS2 as ASD and/or ID susceptibility genes, and highlights that rare genetic variation contributes to disease risk in different ways: some rare mutations, such as those impacting CTNNA3, act in a recessive mode of inheritance, while other CNVs, such as those occurring in the 15q13.3 region, are implicated in multiple developmental and/or neurological disorders possibly interacting with other susceptibility variants elsewhere in the genome. On the other hand, the discovery of a tissue-specific monoallelic expression for the CADPS2 gene, implicates the involvement of epigenetic regulatory mechanisms as risk factors conferring susceptibility to ASD/ID.

This thesis is based on five papers addressing variance reduction in different ways. The papers have in common that they all present new numerical methods.

Paper I investigates quantitative structure-retention relationships from an image processing perspective, using an artificial neural network to preprocess three-dimensional structural descriptions of the studied steroid molecules.

Paper II presents a new method for computing free energies. Free energy is the quantity that determines chemical equilibria and partition coefficients. The proposed method may be used for estimating, e.g., chromatographic retention without performing experiments.

Two papers (III and IV) deal with correcting deviations from bilinearity by so-called peak alignment. Bilinearity is a theoretical assumption about the distribution of instrumental data that is often violated by measured data. Deviations from bilinearity lead to increased variance, both in the data and in inferences from the data, unless invariance to the deviations is built into the model, e.g., by the use of the method proposed in paper III and extended in paper IV.

Paper V addresses a generic problem in classification; namely, how to measure the goodness of different data representations, so that the best classifier may be constructed.

Variance reduction is one of the pillars on which analytical chemistry rests. This thesis considers two aspects on variance reduction: before and after experiments are performed. Before experimenting, theoretical predictions of experimental outcomes may be used to direct which experiments to perform, and how to perform them (papers I and II). After experiments are performed, the variance of inferences from the measured data are affected by the method of data analysis (papers III-V).

Neuf herpèsvirus sont connus pour infecter les équidés. Parmi eux, l’herpèsvirus équin 1 (HVE-1) induit les formes de la maladie les plus graves. En effet, ce virus provoque des troubles respiratoires, des avortements, des morts néonatales et des troubles nerveux qui mènent souvent à l’euthanasie de l’animal. La prophylaxie, reposant sur les bonnes pratiques sanitaires et la vaccination, demeure le meilleur moyen de lutte contre l’ensemble des herpèsvirus équins. Des vaccins qui réduisent efficacement les troubles respiratoires et la dissémination de l’HVE-1 ont été développés. Cependant, ces derniers ne préviennent pas les avortements et n’ont aucun effet démontré contre la forme nerveuse. De plus, la couverture vaccinale demeure insuffisante en France. Les traitements aux antiviraux constituent donc une approche complémentaire dans la lutte contre l’HVE-1. Cependant, trop peu d’études ont évalué l’effet de molécules contre ce virus, limitant les perspectives d’utilisation. Pour répondre à cette problématique, nous avons développé un protocole de criblage à moyen/haut débit à l’aide de la technologie RTCA xCELLigence®, basée sur la mesure d’impédance cellulaire. Suite au criblage de 2891 molécules, 21 candidats ont été identifiés pour leur efficacité contre l’HVE-1. Parmi ceux-ci, l’aphidicoline, la décitabine, le ganciclovir, l’idoxuridine, le pritelivir et le valganciclovir ont présenté les meilleurs effets. L’activité de ces molécules a été confirmée sur différents modèles cellulaires en présence de différentes souches d’HVE-1. Cette étude a conduit à l’identification et à l’étude du mode d’action d’une nouvelle molécule efficace contre l’HVE-1, la décitabine. Cet analogue de la déoxycitidine a également montré un effet synergique in vitro lorsqu’elle est associée au valganciclovir. Lors de la seconde phase de ce travail, nous avons testé l’efficacité d’un traitement au valganciclovir lors d’un challenge expérimental avec infection par nébulisation d’une souche d’HVE-1 (C2254) récemment isolée au cours de l’épizootie de 2018. Cette étude a permis de démontrer qu’une dose de 6,5 mg/kg de valganciclovir, administrée 2 fois par jour, permettait de maintenir un bon niveau de protection avant la mise en place de la réponse immunitaire humorale. En effet, ce traitement permet de réduire les signes cliniques, l’excrétion virale et la virémie cellulaire induits par l’HVE-1. Ces travaux réalisés in vivo démontrent l’efficacité du valganciclovir contre l’HVE-1 et le criblage réalisé in vitro ouvre de nouvelles perspectives de traitement, en particulier avec des associations de molécules
Nine herpesviruses are known to infect the equine population. Among them, the equid herpesvirus 1 (EHV-1) induces the most severe forms of diseases. Indeed, this virus causes respiratory symptoms, abortions, neonatal foal deaths and nervous diseases, often leading to their euthanasia. Prophylaxis, relying on good sanitary practices and vaccination remains the best way to avoid epizooties of herpesviruses. Vaccines reducing efficiently respiratory disorders and EHV-1 dissemination are currently available. However, they do not prevent abortions and have no proven effect against nervous symptoms. In addition, the vaccine coverage is insufficient in France. Antiviral therapy is therefore an interesting complementary approach in the fight against EHV-1. However, there is a lack of studies evaluating the antiviral effect of compounds against EHV-1, limiting the prospects of use. To resolve this issue, we have developed a medium/high throughput screening protocol using the RTCA xCELLigence® technology based on cell impedance measurements. Following the screening of 2891 compounds, 21 candidates were identified for their efficacy against EHV-1. Among them, aphidicolin, decitabine, ganciclovir, idoxuridine, pritelivir and valganciclovir showed the best efficacy. The activity of these compounds was confirmed on different cell lines in the presence of different EHV-1 strains. This study led to the identification and the understanding of the mode of action of decitabine. This deoxycitidine analogue, also showed a synergistic effect when combined with valganciclovir. In the second part of this work, we evaluated the effect of valganciclovir treatment during an experimental infection by nebulisation with a new EHV-1 strain (C2254) recently isolated during the epizootic of 2018. This study demonstrated that a dose of 6.5 mg/kg body weight of valganciclovir, administrated orally twice a day, allowed to maintain a good protection prior the establishment of the humoral immune response. Indeed, this treatment allows to reduce significantly clinical signs, viral excretion and cell-associated viremia induced by EHV-1 on ponies. This work carried out in vivo demonstrated the efficiency of valganciclovir treatment against EHV-1, while the in vitro screening opens up new perspectives of treatment, in particular with compounds association

Specific language impairment (SLI) is a complex neurodevelopmental disorder defined as an unexpected failure to develop normal language abilities for no obvious reason. Copy number variants (CNVs) are an important source of variation in the susceptibility to neuropsychiatric disorders. Therefore, a CNV study within SLI families was performed to investigate the role of structural variants in SLI. Among the identified CNVs, we focused on CNVs on chromosome 15q11-q13, recurrently observed in neuropsychiatric conditions, and a homozygous exonic microdeletion in ZNF277. Since this microdeletion falls within the AUTS1 locus, a region linked to autism spectrum disorders (ASD), we investigated a potential role of ZNF277 in SLI and ASD. Frequency data and expression analysis of the ZNF277 microdeletion suggested that this variant may contribute to the risk of language impairments in a complex manner, that is independent of the autism risk previously described in this region. Moreover, we identified an affected individual with a dihydropyrimidine dehydrogenase (DPD) deficiency, caused by compound heterozygosity of two deleterious variants in the gene DPYD. Since DPYD represents a good candidate gene for both SLI and ASD, we investigated its involvement in the susceptibility to these two disorders, focusing on the splicing variant rs3918290, the most common mutation in the DPD deficiency. We observed a higher frequency of rs3918290 in SLI cases (1.2%), compared to controls (~0.6%), while no difference was observed in a large ASD cohort. DPYD mutation screening in 4 SLI and 7 ASD families carrying the splicing variant identified six known missense changes and a novel variant in the promoter region. These data suggest that the combined effect of the mutations identified in affected individuals may lead to an altered DPD activity and that rare variants in DPYD might contribute to a minority of cases, in conjunction with other genetic or non-genetic factors.

Specific language impairment (SLI) is a complex neurodevelopmental disorder defined as an unexpected failure to develop normal language abilities for no obvious reason. Copy number variants (CNVs) are an important source of variation in the susceptibility to neuropsychiatric disorders. Therefore, a CNV study within SLI families was performed to investigate the role of structural variants in SLI. Among the identified CNVs, we focused on CNVs on chromosome 15q11-q13, recurrently observed in neuropsychiatric conditions, and a homozygous exonic microdeletion in ZNF277. Since this microdeletion falls within the AUTS1 locus, a region linked to autism spectrum disorders (ASD), we investigated a potential role of ZNF277 in SLI and ASD. Frequency data and expression analysis of the ZNF277 microdeletion suggested that this variant may contribute to the risk of language impairments in a complex manner, that is independent of the autism risk previously described in this region. Moreover, we identified an affected individual with a dihydropyrimidine dehydrogenase (DPD) deficiency, caused by compound heterozygosity of two deleterious variants in the gene DPYD. Since DPYD represents a good candidate gene for both SLI and ASD, we investigated its involvement in the susceptibility to these two disorders, focusing on the splicing variant rs3918290, the most common mutation in the DPD deficiency. We observed a higher frequency of rs3918290 in SLI cases (1.2%), compared to controls (~0.6%), while no difference was observed in a large ASD cohort. DPYD mutation screening in 4 SLI and 7 ASD families carrying the splicing variant identified six known missense changes and a novel variant in the promoter region. These data suggest that the combined effect of the mutations identified in affected individuals may lead to an altered DPD activity and that rare variants in DPYD might contribute to a minority of cases, in conjunction with other genetic or non-genetic factors.

The thesis deals with the evalutation, which of the variants of the project investment is the most advantageous to the developer. The thesis is divided to the theoretical and practical part. The theoretical part is dedicaded to the explanation of terms associated with the investment, the investment project, the price determination and valuation of the real estates. There are also explained the terms related to the development projects and the real estate market. At last but not least, the theoretical part clarifies, how is the tax burden applicated to the practical part. The practical part is dedicated to the options such as sale, rent and partical sale of the real structural object, which is located in Prague, Brno and Ostrava. In conclusion, all of the variants are appraised for every city.

Spectral computed tomography (CT) is a novel medical imaging technique that involves simultaneously counting photons at several energy levels of the x-ray spectrum to obtain a single multi-variate dataset. Visualisation of such data poses significant challenges due its extremely large size and the need for interactive performance for scientific and medical end-users. This thesis explores the properties of spectral CT datasets and presents two algorithms for GPU-accelerated real-time rendering from compressed spectral CT data formats. In addition, we describe an optimised implementation of a volume raycasting algorithm on modern GPU hardware, tailored to the visualisation of spectral CT data.

Uno studio filologico condotto sulle tre edizioni di Apocalypse Now di Francis Ford Coppola e delle rispettive varianti, e di come esse influiscano sul messaggio filmico partendo dalla letteratura di riferimento.

The transformed rejection method uses inversion to sample from the dominating density of a rejection algorithm. But in contrast to the usual method it is enough to know the inverse distribution function F^(-1)(x) of the dominating density. This idea can be applied to various continuous (e.g. normal, Cauchy and exponential) and discrete (e.g. binomial and Poisson) distributions with high acceptance probabilities. The resulting algorithms are short, simple and fast. Even more important is the fact that the quality of the method when used in combination with a linear congruential uniform generator is high compared with the quality of the ratio of uniforms method. In addition transformed rejection can be easily employed for correlation induction. (author's abstract)
Series: Preprint Series / Department of Applied Statistics and Data Processing

We explored the missing heritability in a cohort of 140 patients affected by Neurodegenerative disorders (NDDs), including Amyotrophic Lateral Sclerosis (ALS), Frontotemporal dementia (FTD)) Parkinson disease (PD) and Spinocerebellar Ataxia (SCA). We performed Whole Genome Sequencing (WGS) after excluding pathogenic variants in the main disease-relevant causative genes and investigated 3 classes of potentially pathogenic variants: a)Coding/non-coding SNV/Indels in a panel of 696 genes involved in NODs. Using standard annotation, we identified pathogenic/likely pathogenic variants (ACMG) in genes causative of tare forms of each disease (N=15) and in gene causing a NND different from patient clinical presentation (N=15). In addition to the standard annotation we used SpliceAl, a deep leaming tool predicting an effect on splicing mechanism. We found 48 rare variants with a possible splicing Impact (ASTM 0.4). We performed in vitro studies for 9 variants (highest AS) and for 6 of them, we confirmed their role in splicing alteration. b)Genome-wide structural variants Using CNVkit, we identified a 15925 deletion (1.2Mb) in a PD case. Similar deletions has been associated with mild intellectual disability and dysmorphisms but never reported in PD. c)Genome wide Tandem Repeat (TR). Using literature (ExpansionHunter, GangSTR) and novel tools, we Identified 4 novel loci in ALS cohort with a possible TR expansion and replicated the results in larger independent cohorts from Italy (763 ALS, 1018 controls) and International Mine project (3121 ALS, 1217 controls). For 3 of them (FRA1DAC1, RFC1, HK1) the result was not replicated. For the last locus (TFG2) the preliminary data are promising since the TR was observed only in patient and in none controls (1/352 vs 0/292 in ITFG2). In conclusion, using a multilevel WGS data analysis we were able to find missed pathogenetic variants In genes associated with different NDDs, reinforcing the idea of a shared genetic cause among these diseases.

[ES] La estandarización de la Quinta Generación de redes móviles o 5G, ha concluido este año 2020. No obstante, en el año 2014 cuando la ITU empezó el proceso de estandarización IMT-2020, una de las principales interrogantes era cuál sería la forma de onda sobre la cual se construiría la capa física de esta nueva generación de tecnologías. El 3GPP se comprometió a entregar una tecnología candidata al proceso IMT-2020, y es así como dentro de este proceso de deliberación se presentaron varias formas de onda candidatas, las cuales fueron evaluadas en varios aspectos hasta que en el año 2016 el 3GPP tomó una decisión, continuar con CP-OFDM (utilizada en 4G) con numerología flexible. Una vez decidida la forma de onda, el proceso de estandarización continuó afinando la estructura de la trama, y todos los aspectos intrínsecos de la misma. Esta tesis acompañó y participó de todo este proceso. Para empezar, en esta disertación se evaluaron las principales formas de onda candidatas al 5G. Es así que se realizó un análisis teórico de cada forma de onda, destacando sus fortalezas y debilidades, tanto a nivel de implementación como de rendimiento. Posteriormente, se llevó a cabo una implementación real en una plataforma Software Defined Radio de tres de las formas de onda más prometedoras (CP-OFDM, UFMC y OQAM-FBMC), lo que permitió evaluar su rendimiento en términos de la tasa de error por bit, así como la complejidad de su implementación. Esta tesis ha propuesto también el uso de una solución armonizada como forma de onda para el 5G y sostiene que sigue siendo una opción viable para sistemas beyond 5G. Dado que ninguna de las forma de onda candidatas era capaz de cumplir por sí misma con todos los requisitos del 5G, en lugar de elegir una única forma de onda se propuso construir un transceptor que fuese capaz de construir todas las principales formas de onda candidatas (CP-OFDM, P-OFDM, UFMC, QAM-FBMC, OQAM-FBMC). Esto se consiguió identificando los bloques comunes entre las formas de onda, para luego integrarlos junto con el resto de bloques indispensables para cada forma de onda. La motivación para esta solución era tener una capa física que fuese capaz de cumplir con todos los aspectos del 5G, seleccionando siempre la mejor forma de onda según el escenario. Esta propuesta fue evaluada en términos de complejidad, y los resultados se compararon con la complejidad de cada forma de onda. La decisión de continuar con CP-OFDM con numerología flexible como forma de onda para el 5G se puede considerar también como una solución armonizada, ya que al cambiar el prefijo cíclico y el número de subportadoras, cambian también las prestaciones del sistema. En esta tesis se evaluaron todas las numerologías propuestas por el 3GPP sobre cada uno de los modelos de canal descritos para el 5G (y considerados válidos para sistemas beyond 5G), teniendo en cuenta factores como la movilidad de los equipos de usuario y la frecuencia de operación; para esto se utilizó un simulador de capa física del 3GPP, al que se hicieron las debidas adaptaciones con el fin de evaluar el rendimiento de las numerologías en términos de la tasa de error por bloque. Finalmente, se presenta un bosquejo de lo que podría llegar a ser la Sexta Generación de redes móviles o 6G, con el objetivo de entender las nuevas aplicaciones que podrían ser utilizadas en un futuro, así como sus necesidades. Completado el estudio llevado a cabo en esta tesis, se puede afirmar que como se propuso desde un principio la solución, tanto para el 5G como para beyond 5G, la solución es la armonización de las formas de onda. De los resultados obtenidos se puede corroborar que una solución armonizada permite alcanzar un ahorro computacional entre el 25-40% para el transmisor y del 15-25% para el receptor. Además, fue posible identificar qué numerología CP-OFDM es la más adecuada para cada escenario, lo que permitiría optimizar el diseño y despliegue de las redes 5G. Esto abriría la puerta a hacer lo mismo con el 6G, ya que en esta tesis se considera que será necesario abrir nuevamente el debate sobre cuál es la forma de onda adecuada para esta nueva generación de tecnologías, y se plantea que el camino a seguir es optar por una solución armonizada con distintas formas de onda, en lugar de solo una como sucede con el 5G.
[CA] L'estandardització de la Quinta Generació de xarxes mòbils o 5G, ha conclòs enguany 2020. No obstant això, l'any 2014 quan la ITU va començar el procés d'estandardització IMT-2020, uns dels principals interrogants era quina seria la forma d'onda sobre la qual es construiria la capa física d'esta nova generació de tecnologies. El 3GPP es va comprometre a entregar una tecnologia candidata al procés IMT-2020, i és així com dins d'este procés de deliberació es van presentar diverses formes d'onda candidates, les quals van ser avaluades en diversos aspectes fins que l'any 2016 el 3GPP va prendre una decisió, continuar amb CP-OFDM (utilitzada en 4G) amb numerología flexible. Una vegada decidida la forma d'onda, el procés d'estandardització va continuar afinant la frame structure (no se m'ocorre nom en espanyol), i tots els aspectes intrínsecs de la mateixa. Esta tesi va acompanyar i va participar de tot este procés. Per a començar, en esta dissertació es van avaluar les principals formes d'onda candidates al 5G. És així que es va realitzar una anàlisi teòrica de cada forma d'onda, destacant les seues fortaleses i debilitats, tant a nivell d'implementació com de rendiment. Posteriorment, es va dur a terme una implementació real en una plataforma Software Defined Radio de tres de les formes d'onda més prometedores (CP-OFDM, UFMC i OQAM-FBMC), la qual cosa va permetre avaluar el seu rendiment en termes de la taxa d'error per bit, així com la complexitat de la seua implementació. Esta tesi ha proposat també l'ús d'una solució harmonitzada com a forma d'onda per al 5G i sosté que continua sent una opció viable per a sistemes beyond 5G. Atés que cap de les forma d'onda candidates era capaç de complir per si mateixa amb tots els requeriments del 5G, en compte de triar una única forma d'onda es va proposar construir un transceptor que fóra capaç de construir totes les principals formes d'onda candidates (CP-OFDM, P-OFDM, UFMC, QAM-FBMC, OQAM-FBMC). Açò es va aconseguir identificant els blocs comuns entre les formes d'onda, per a després integrar-los junt amb la resta de blocs indispensables per a cada forma d'onda. La motivació per a esta solució era tindre una capa física que fóra capaç de complir amb tots els aspectes del 5G, seleccionant sempre la millor forma d'onda segons l'escenari. Esta proposta va ser avaluada en termes de complexitat, i els resultats es van comparar amb la complexitat de cada forma d'onda. La decisió de continuar amb CP-OFDM amb numerología flexible com a forma d'onda per al 5G es pot considerar també com una solució harmonitzada, ja que al canviar el prefix cíclic i el número de subportadores, canvien també les prestacions del sistema. En esta tesi es van avaluar totes les numerologías propostes pel 3GPP sobre cada un dels models de canal descrits per al 5G (i considerats vàlids per a sistemes beyond 5G), tenint en compte factors com la mobilitat dels equips d'usuari i la freqüència d'operació; per a açò es va utilitzar un simulador de capa física del 3GPP, a què es van fer les degudes adaptacions a fi d'avaluar el rendiment de les numerologías en termes de la taxa d'error per bloc. Finalment, es presenta un esbós del que podria arribar a ser la Sexta Generació de xarxes mòbils o 6G, amb l'objectiu d'entendre les noves aplicacions que podrien ser utilitzades en un futur, així com les seues necessitats. Completat l'estudi dut a terme en esta tesi, es pot afirmar que com es va proposar des d'un principi la solució, tant per al 5G com per a beyond 5G, la solució és l'harmonització de les formes d'onda. dels resultats obtinguts es pot corroborar que una solució harmonitzada permet aconseguir un estalvi computacional entre el 25-40% per al transmissor i del 15-25% per al receptor. A més, va ser possible identificar què numerología CP-OFDM és la més adequada per a cada escenari, la qual cosa permetria optimitzar el disseny i desplegament de les xarxes 5G. Açò obriria la porta a fer el mateix amb el 6G, ja que en esta tesi es considera que serà necessari obrir novament el debat sobre quina és la forma d’onda adequada per a esta nova generació de tecnologies, i es planteja que el camí que s’ha de seguir és optar per una solució harmonitzada amb distintes formes d’onda, en compte de només una com succeïx amb el 5G.
[EN] The standardization of the Fifth Generation of mobile networks or 5G is still ongoing, although the first releases of the standard were completed two years ago and several 5G networks are up and running in several countries around the globe. However, in 2014 when the ITU began the IMT-2020 standardization process, one of the main questions was which would be the waveform to be used on the physical layer of this new generation of technologies. The 3GPP committed to submit a candidate technology to the IMT-2020 process, and that is how within this deliberation process several candidate waveforms were presented. After a thorough evaluation regarding several aspects, in 2016 the 3GPP decided to continue with CP-OFDM (used in 4G) but including, as a novelty, the use of a flexible numerology. Once the waveform was decided, the standardization process continued to fine-tune the frame structure and all the intrinsic aspects of it. This thesis accompanied and participated in this entire process. To begin with, this dissertation evaluates the main 5G candidate waveforms. Therefore, a theoretical analysis of each waveform is carried out, highlighting its strengths and weaknesses, both at the implementation and performance levels. Subsequently, a real implementation on a Software Defined Radio platform of three of the most promising waveforms (CP-OFDM, UFMC, and OQAM-FBMC) is presented, which allows evaluating their performance in terms of bit error rate, as well as the complexity of its implementation. This thesis also proposes the use of a harmonized solution as a waveform for 5G and argues that it remains a viable option for systems beyond 5G. Since none of the candidate waveforms was capable of meeting on its own with all the requirements for 5G, instead of choosing a single waveform, this thesis proposes to build a transceiver capable of building all the main waveforms candidates (CP-OFDM, P-OFDM, UFMC, QAM-FBMC, OQAM-FBMC). This is achieved by identifying the common blocks between the waveforms and then integrating them with the rest of the essential blocks for each waveform. The motivation for this solution is to have a physical layer that is capable of complying with all aspects of beyond 5G technologies, always selecting the best waveform according to the scenario. This proposal is evaluated in terms of complexity, and the results are compared with the complexity of each waveform. The decision to continue with CP-OFDM with flexible numerology as a waveform for 5G can also be considered as a harmonized solution, since changing the cyclic prefix and the number of subcarriers, changes also the performance of the system. In this thesis, all the numerologies proposed by the 3GPP are evaluated on each of the channel models described for 5G (and considered valid for beyond 5G systems), taking into account factors such as the mobility of the user equipment and the operating frequency. For this, a 3GPP physical layer simulator is used, and proper adaptations are made in order to evaluate the performance of the numerologies in terms of the block error rate. Finally, a sketch of what could become the Sixth Generation of mobile networks or 6G is presented, with the aim of understanding the new applications that could be used in the future, as well as their needs. After the completion of the study carried out in this thesis, it can be said that, as stated from the beginning, for both 5G and beyond 5G systems, the solution is the waveform harmonization. From the results obtained, it can be corroborated that a harmonized solution allows achieving computational savings between 25-40% for the transmitter and 15-25% for the receiver. In addition, it is possible to identify which CP-OFDM numerology is the most appropriate for each scenario, which would allow optimizing the design and deployment of 5G networks. This would open the door to doing the same with 6G, i.e., a harmonized solution with different waveforms, instead of just one as in 5G.
Flores De Valgas Torres, FJ. (2020). Study on Air Interface Variants and their Harmonization for Beyond 5G Systems [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/164442
TESIS

This thesis consists of three studies that centre around forecasting realised volatility based on high-frequency financial data. Accurate volatility forecasts are used extensively in many financial applications. The methods used here draw on econometric models and machine learning techniques. The empirical studies are based on fifty stocks and two stock indices. The thesis has established new perspectives on forecasting the realised volatility asset returns to improve financial decision-making.

Thesis (MSc)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: Grapevine leafroll-associated virus 3 (GLRaV-3) is the type member of the genus Ampelovirus in the family Closteroviridae and is considered to be the main contributing agent of grapevine leafroll disease (GLD) worldwide. A metagenomic sequencing study of a grapevine leafroll-diseased vineyard led to the discovery of a new variant of GLRaV-3 in South Africa. This new variant was most related to a New Zealand isolate, NZ-1. In this study, we sequenced two isolates, GH11 and GH30, of the new variant group of GLRaV-3. These isolates have less than 70% nucleotide (nt) identity to other known GLRaV-3 variants, indicating that they should be considered variants of a different strain of GLRaV-3. We propose that the GLRaV-3-like virus identified in this study be grouped together with NZ-1 and some Napa Valley isolates as Group VI of GLRaV-3. This study also provided further evidence that next-generation sequencing is an invaluable approach to identify novel viruses and variants, in that the draft sequence generated with bioinformatic tools in this study was 98% identical to the GH11 sequence generated using Sanger sequencing. The study further confirmed that the industry standard ELISA is still an effective GLRaV-3 diagnostic method and that it is able to detect all known variant groups of GLRaV-3. However, this assay is not able to differentiate between GLRaV-3 variant groups. In the current study therefore, a real-time RT-PCR was designed that is able to detect GLRaV-3 variant groups I, II, III and VI, using a single primer pair targeting the Hsp70h gene of GLRaV-3. If high-resolution melting (HRM) curve analysis is added to the real-time RT-PCR, it is possible to differentiate between variant groups based on three melting point intervals. The RT-PCR HRM assay provides a more sensitive and rapid tool to detect and differentiate between different GLRaV-3 variant groups. Finally, a multiplex RT-PCR was designed to differentiate between the variant groups present in South Africa. This multiplex RT-PCR offers a validation method for the RT-PCR HRM and provides an end-point PCR alternative for variant identification. In order to investigate the spread and impact of different GLRaV-3 variants in vineyards, sensitive diagnostic techniques are a necessity. The abovementioned tools will contribute to the understanding of the pathogenesis of GLD and aid epidemiological studies to investigate how these different GLRaV-3 variant groups are spreading, the association of specific GLRaV-3 variants to disease symptoms and the mealybug vector transmission efficiency for each GLRaV-3 variant.
AFRIKAANSE OPSOMMING: Grapevine leafroll-associated virus 3 (GLRaV-3) is ’n lid van die genus Ampelovirus in die familie Closteroviridae en word beskou as die hoof bydraende faktor van wingerd-rolbladsiekte wêreldwyd. ’n Metagenomiese studie het bewys dat daar ’n nuwe variant van GLRaV-3 bestaan wat nog nie voorheen in Suid Afrika opgespoor kon word met die huidige opsporingsmetodes nie. Hierdie nuwe variant was naaste verwant aan ’n Nieu-Seelandse isolaat, NZ-1. In hierdie studie is die genoomvolgorde van twee isolate, GH11 en GH30, van hierdie nuwe GLRaV-3 variant groep bepaal. Hierdie twee isolate was minder as 70% identies aan ander GLRaV-3 variante, wat daarop dui dat hulle as variante van ’n nuwe virus-ras beskou behoort te word. Ons beveel aan dat hierdie GLRaV-3-verwante virus geklassifiseer word saam met die NZ-1 isolaat en ander isolate uit Kalifornië, as groep VI van GLRaV-3. Hierdie studie het ook verdere bewyse verskaf dat volgende-generasie volgordebepalingstegnologie ’n waardevolle benadering is om nuwe virusse en variante te identifiseer, deurdat die huidige studie gewys het dat die voorlopige volgorde, wat gegenereer is deur bioinformatika-instrumente, 98% identies was aan die GH11 volgorde wat met Sanger volgordebepaling verkry was. Hierdie studie het ook gevind dat die industrie-standaard ELISA, nog steeds ’n effektiewe GLRaV-3 diagnostiese metode is en wel infeksies, veroorsaak deur al die variant-groepe, sal kan identifiseer. Die ELISA toets is egter nie in staat om te onderskei tussen GLRaV-3 variant-groepe nie. In hierdie studie is ’n variant-identifiseerbare in-tyd tru-transkripsie polimerase ketting reaksie (PKR) ontwerp wat GLRaV-3 variant-groepe I, II, III en VI kan identifiseer deur middel van ’n enkele inleier-stel wat die GLRaV-3 Hsp70h-geen teiken. As hoë-resolusie smeltingskurwe-analise bygevoeg word by die in-tyd tru-transkripsie PKR, is dit moontlik om te onderskei tussen variant-groepe op grond van drie smeltingspunt intervalle. Die tru-transkripsie hoë-resolusie smeltingskurwe-toets verskaf meer sensitiewe en geoutomatiseerde metodes om GLRaV-3 variant-groepe te identifiseer en te onderskei. ’n Veelvuldige tru-transkripsie PKR is ook ontwerp om tussen variante wat tans in Suid-Afrika aangetref word, te onderskei en te dien as ’n valideringsmetode vir die in-tyd tru-transkripsie hoë-resolusie smeltingskurwe-toets. Sensitiewe en akkurate toetse, soos bogenoemde, is noodsaaklik vir die bestudering van die verspreiding en impak van die verskillende GLRaV-3 variante in wingerd. Hierdie metodes kan gebruik word om kennis ten opsigte van rolblad patogenese te verbreed en om by te dra tot epidemiologiese studies wat ondersoek hoe hierdie variant-groepe versprei, of daar ’n assosiasie bestaan tussen ’n spesifieke variant en siekte-simptome en of daar ’n verskil is in die witluisvektor oordragseffektiwitiet vir elke GLRaV-3 variant.

Porphyrias are a group of inherited metabolic disorders of heme biosynthesis. Each porphyria derives from an alteration in the heme biosynthetic pathway resulting in a specific accumulation of heme precursors. These rare diseases are characterized by an extended heterogeneity of mutations affecting the coding region of the genes directly or indirectly involved in the pathway. In this study, we assessed three new variants identified in the regulatory regions of the PPOX gene using human hepatocarcinoma (Hep3B) and erythroleukemia (K562) cell lines. We demonstrated a lower expression of the PPOX gene through luciferase assays and RNA analysis for the c.1-883G>C promoter mutant and we suggested a post-transcriptional role for the c.1-413G>T and c.1-176G>A variants in the 5′ UTR of PPOX mRNA variant 2. Transfection experiments of mutant -413T reporter plasmid indicate that this variant inhibits translation of the downstream firefly luciferase mRNA. In fact, the reduced firefly luciferase activity did not correlate with the proportional reduction in firefly luciferase mRNA expression. Normal values of PPOX mRNA level validated with Digital PCR in the patient carrying the c.1-413G>T substitution support this evidence. Data for the c.1-176G>A variant show a possible role in the spicing regulation for it. The qualitative RNA analysis confirms that this variant is involved in the alteration of the normal splicing between exon 1 and exon 2 of PPOX due to a 4 bp deletions in exon 1. The relation between these post-transcriptional alterations and the variegate porphyria remains to be investigated. These results suggest that the regulatory regions have to be considered in the diagnostic process but more studies are required to clarify their role in the disease. To model hepatic porphyrias, we derived hepatocyte-like cells from hiPSC generated from blood of two patients affected by acute intermittent porphyria, a hepatic porphyria. We hypothesized that this can serve as an in vitro model to study different pathways linked with acute intermittent porphyria. The data suggest a general comparable profile of the heme biosynthec pathway between hiPSC-HLC and primary hepatocytes. Although some immature features, probably linked with the in vitro condition, could directly or indirectly affect the metabolism network links with hepatic porphyrias, now hiPSC-HLCs are one of the most useful model available for hepatic diseases. The patient derived hepatocyte-like cells showed a basal overexpression of ALAS1 and its regulator PPARA and PGC1A with induction of oxidative damage response genes. Moreover, the induction of ketogenetic and lipid metabolic genes highlighted a different metabolic profile in the acute intermittent porphyria patient lines. The in vitro simulation of acute attack mediated by ALA administration, one of the principal compound accumulated during the attack, showed a negative feedback regulation on ALAS1 with massive induction of HMOX1 in the control cell line. On the other hand, phenobarbital response in control line suggested the link between cholesterol synthesis and gluconeogenesis with the heme biosynthetic pathway. The drug metabolism in fact, induces the heme biosynthetic pathway through ALAS1, and this gene results constantly overexpressed in the derived patient cell lines. In conclusion, this study focus the attention on the importance of the regulatory regions in the diagnostic process of porphyrias and supply an alternative in vitro model to study the metabolic alterations linked with porphyrias. Further experiments are required to better understand the direct effect of alteration in the regulatory regions of porphyria genes and are necessary to analyse the involvement of different pathways in the onset of the disease.

X-linked intellectual disability (XLID) is a heterogeneous disorder, and mutations causing monogenic XLID have now been reported in over 100 genes. We report a five-generation Sardinian family in which seven affected male family members had intellectual disability and craniofacial dysmorphisms. Large-scale next generation exome resequencing of X chromosome genes detected a rare missense variant (c.995G>A, p.Arg332His) located within a highly conserved domain in the RBM10 gene at Xp11.23. Sanger sequencing confirmed the presence of the variant in affected males and in their mothers. The variant was not present in non affected male family members, has not been reported in variant databases and is disease-causing according to a web-based prediction tool. RBM10 is an RNA binding protein involved in the regulation of transcription, alternative splicing and message stabilization. RBM10 nonsense and frameshift mutations are associated with TARP syndrome characterized by Talipes equinovarus, Atrial septal defect, Robin sequence and Persistence of the left superior vena cava and pre- or postnatal lethality in affected males. RBM10 has not been reported in XLID patients until now. Genic intolerance score suggested that RBM10 may be “intolerant” of functional mutations. Although our finding suggest that RBM10 is a reasonable candidate gene for XLID, functional studies and mutations screening in other patients are needed to prove a definite causal relationship between the variant and the phenotype.