Since the dawn of computing, all practical computing systems, from small laptops and cellphones to large mainframes and supercomputers, have been based on the Von Neumann architecture. In this architectural model, computing units and storage units are physically separated, requiring the computing system to spend most of its time and energy on moving data around. As many recently developed applications are driven by large volumes of digital data, the Von Neumann architecture does not scale well with today’s computational demands. In-memory computing using emerging non-volatile device technologies is a promising orthogonal approach as it mitigates the adverse effects of the physical separation, also known as the Von Neumann bottleneck, by merging storage and memory units. The first part of this thesis is focused on synthesis, verification, and fault-tolerance techniques for flow-based in-memory computing. In synthesis, computations are mapped onto nanoscale crossbars of non-volatile memory devices with the objective of minimizing energy, latency, and/or area for flow-based computations. The objective of verification is to show equivalence or non-equivalence between the computational model and a specification, as logical errors can be introduced during the complex stages of synthesis. Finally, fault-tolerance techniques are explored to ensure correctness of the computations at runtime while handling device errors and to elongate the computing system's lifetime. Lastly, path-based computing is introduced, a novel in-memory computing paradigm. In contrast with flow-based computing, which is a WRITE-based computing paradigm, path-based computing is READ-based. This entails that reprogramming of the non-volatile memory devices is not required during computation. As WRITE operations have higher energy cost than READ operations, path-based computing is more energy-efficient than WRITE-based digital computing paradigms. In summary, this thesis presents a wide variety of synthesis, verification, and fault-tolerance techniques (design automation tools) for in-memory computing, paving way for a new era of energy-efficient data-intensive computing.

Pseudorandom generators belong to the primary focus of cryptology. The key to every cipher has to be generated at random, otherwise the security of the whole cipher is threatened. Another point of importance is the pseudorandom generators' close relationship to the stream ciphers. In this work, we first introduce statistical theory related to randomness testing. Then, we describe 8 classical statistical tests. We introduce a concept of next bit testing and derive variants of previous tests. Moreover, with this new battery of tests we examine the randomness of SHA-3 second round candidates and present the results. Also a sensitivity of tests is investigated and several useful transformations are shown. Powered by TCPDF (www.tcpdf.org)

Next-Generation Sequencing (NGS) technologies have been evolving at an unparalleled pace. The ability to generate millions of base pairs of data in a short time and at lower cost than previously has led to a dramatic expansion of technologies within the field. This dissertation discusses the development and validation of new methods for assessing genomic variation, dynamic changes in gene expression, high-accuracy sequencing, and analysis of recombination events. By reducing the cost of analyzing many samples for genetic divergence by genotyping the same region of the genome in multiple samples, researchers can pursue investigations on a larger scale. Next-RAD (Nextera fragmentation with Restriction-Associated Digestion) allows analysis of a uniform subset of loci between organisms for comparison of populations by genetic differences with reduced burdens of cost and data analysis. This method was applied to the Anopheles darlingi mosquito to identify three distinct species that were thought to be a uniform population. The lowering cost of large-scale sequencing investigations allows for massively parallel analysis of genomic function in a single assay. Regulation of gene expression in response to stress is a complex process which can only be understood by analyzing many pathways in tandem. A novel method is described which quantifies on a genome-wide scale the expression of millions of randomer tags driven by associated transcriptional enhancers. This method provides novel data in the form of high-resolution analysis of gene regulation. Aside from generating novel data types, another force behind development of new technologies is to improve data quality. One limitation of NGS is the inherent error rate. PELE-Seq (Paired End Low Error Sequencing) was developed to address this problem, by employing completely overlapping paired-end reads as well as a dual barcoding strategy to eliminate incorrect sequences resulting from final library amplification. This new tool improves data quality dramatically. Finally, the rapid expansion of tools necessitates the identification of new applications for these technologies. To this end, 10x Genomics Linked-Read sequencing was employed to identify recombination events in multiple species. The haplotype-resolved nature of the data generated from such assays has many promising applications.

Made available in DSpace on 2014-08-20T13:32:45Z (GMT). No. of bitstreams: 1 dissertacao_marcus_redu_eslabao.pdf: 801425 bytes, checksum: d5a120076fe65d76b21da14d5db5817b (MD5) Previous issue date: 2012-02-27 / Leptospirosis is a neglected zoonosis with global distribution. The disease is caused by pathogenic bacteria of the genus Leptospira, which affect humans and various domestic and wild animals, causing serious problems to human health and damage to livestock. The objective of this study was to determine the genome sequence of Leptospira borgpetersenii serogroup Ballum strain 4E, isolated from domestic mice (Mus musculus), one of the main reservoirs of this genus. The complete genome sequence was determined using SOLiDTM system, which generated over 85 million 50 bp reads. These reads were used to obtain scaffolds of the two chromosomes present in this organism through the ab initio sequence assembly with Velvet and Edena softwares and orientation of contigs with G4All software. With completion of the assembly process, the large chromosome was 3,071,053 bp, GC content of 40.58%, 36 tRNA, 4 rRNA and 2,908 open reading frames (ORF). The small chromosome has 305,940 bp, GC content of 40.25%, 277 ORFs, no tRNA or rRNA. A reduction in the large chromosome of 4E strain was observed compared to the large chromosome of L550 strain, where 99 genes of L550 strain are not present in the 4E strain and about 394 kb of non-coding region was also lost. The main hypothesis for this reduction is the effect of the presence of a large number of mobile genetic elements. Genome reduction has been observed in other strains of L. borgpetersenii. The Applied Biosystems SOLiD 4 method allowed determination of the genome sequence of L. borgpetersenii strain 4E, with wide coverage and accuracy. The ab initio assembly methods used allowed for complete utilization of the sequences generated. / A leptospirose é uma zoonose negligenciada com distribuição global. A doença é causada por bactérias patogênicas do gênero Leptospira, as quais acometem humanos e vários animais domésticos e silvestres, acarretando graves problemas à saúde humana e prejuízos na pecuária. O presente trabalho teve como objetivo sequenciar o genoma da Leptospira borgpetersenii sorogroupo Ballum cepa 4E, isolada de camundongo doméstico (Mus musculus), um dos principais reservatórios deste gênero. A sequência completa do genoma foi determinada através do sistema SOLiDTM, onde foram obtidas mais de 85 milhões de leituras com tamanho de 50 pb cada. Essas leituras foram utilizadas para obtenção de scaffolds dos dois cromossomos presente neste organismo, através de montagem ab initio com os softwares Velvet e Edena; e posterior orientação das contigs com o software G4All. Com a conclusão da montagem, o cromossomo maior apresentou o tamanho de 3.071.053 pb, 40,58% de conteúdo GC, 36 tRNA, 4 rRNA e 2.908 fases de leitura abertas (ORF). Para o cromossomo menor o total de bases foi de 305.940 pb, conteúdo GC de 40,25%, 277 ORFs, nenhum tRNA e rRNA foram preditos. Foi observada uma redução do cromossomo maior da cepa 4E em ralação ao cromossomo maior da cepa L550, onde 99 genes da cepa L550 não estão presentes na cepa 4E e cerca de 394 kb de região não codificante também foi perdida. A principal hipótese para a redução é o efeito da presença de um grande número de elementos móveis, processo observado no genoma de outras cepas da espécie L. borgpetersenii. O método Applied Biosystems SOLiD 4 permitiu a determinação da sequência do genoma de L. borgpetersenii cepa 4E, com ampla cobertura e acurácia. Os metodos de montagem ab initio utilizados proporcionaram aproveitar ao máximo as sequencias geradas.

Über das Next Generation EU Programm ist eine gemeinsame Schuldenaufnahme unter dem Dach der EU-KOMMISSION erstmals in großem Umfang ermöglicht worden. Auch wenn stets betont worden ist, dass NGEU eine Maßnahme einmaliger Natur ist, so beschreiben einige EU-Amtsträger:innen, wie etwa der EU-Kommissar für Wirtschaft, PAOLO GENTILONI, sowie EMMANUEL MACRON und MARIO DRAGHI, welche fiskalischen Möglichkeiten die Verstetigung eines solchen Programms bieten könnte. Der Beitrag untersucht daher, welche Bedingungen mit der gemeinsamen Schuldenaufnahme verbunden sind und ob diese Konditionalität für eine künftige Schuldenaufnahme aus fiskalpolitischer Sicht adäquat ist. Daraus sollen Bedingungen abgeleitet werden, die bei einer künftigen gemeinsamen Schuldenaufnahme zu beachten sind. / Through the Next Generation EU Program, joint borrowing under the umbrella of the EU Commission has been made possible on a large scale for the first time. Although it has always been stressed that NGEU is a one-off measure, some EU officials, such as EU Commissioner for Economic Affairs PAOLO GENTILONI, as well as EMMANUEL MACRON and MARIO DRAGHI, describe the fiscal opportunities that the continuation of such a program could offer. The paper therefore examines the conditions associated with joint debt borrowing and whether this conditionality is adequate for future debt borrowing from a fiscal policy perspective. From this, the paper aims to derive conditions that need to be observed for future joint debt borrowing.

In an era where mens violence against women is increasingly recognized as a growing societal issue, this study focuses on the role of next of kins. These next of kins, often overshadowed by the victims of violence, face daily challenges arising from the violence. The study aims to explore the challenges adult next of kins experience in supporting their abused loved ones, the strain on their well-being, and their experiences and needs for support. Using qualitative interviews with ten adult next of kins and an abductive approach, results were analyzed alongside previous research and theories of secondary and vicarious traumatization, codependency, and social exchange theory. The empirical data yielded three main themes: the next of kins care, external support, and the silent burden, further developed into four analytical themes: conditional support, loss of a relationship, involuntary impact on well-being, and in the shadow of the abused.  The findings reveal significant challenges next of kins face in supporting the abused, especially in adapting support to the victims' wishes. The adaptation by next of kins is examined through codependency theory, highlighting strategies used to manage the situation. The study also indicates the psychological and physical impact on next of kins, with symptoms like anxiety, worry, and sleeplessness. Although some symptoms align with secondary traumatization, they more closely fit vicarious traumatization, emphasizing the personal internal change of helping a traumatized loved one. Furthermore, the study underscores the need for increased societal awareness of violence in intimate relationships and its impact on next of kins. Many relatives do not prioritize seeking help for themselves, focusing instead on the victim, and those who seek support often find it lacking due to misunderstanding or lack of knowledge.          This study aims to deepen the understanding of the challenges next of kins face in supporting their loved ones and how they perceive their own support needs. By doing so, it contributes to a relatively new field of knowledge by providing deeper insights into the experiences of next of kin’s and suggesting areas for improvement. / I en tid där mäns våld mot kvinnor alltmer uppmärksammas som ett växande samhällsproblem, fokuserar denna studie på de anhörigas roll. Dessa anhöriga, som ofta är i skuggan av de våldsutsatta, hanterar dagligen den belastande situationen som uppstår av våldet. Studiens syfte är att utforska vilka utmaningar vuxna anhöriga upplever av att stötta sin våldsutsatta närstående, vilka påfrestningar på måendet anhöriga upplever, samt deras erfarenheter och behov av stöd. Genom kvalitativa intervjuer med tio vuxna anhöriga och en abduktiv forskningsansats har resultaten analyserats tillsammans med tidigare forskning och teorierna sekundär och vikarierande traumatisering, medberoende och sociala utbytesteorin. Studiens empiriska data resulterade i tre huvudteman; den anhöriges omsorg, det utomstående stödet och den tysta bördan, vilka ytterligare utvecklades till fyra analytiska teman; det villkorande stödet, förlusten av en relation, den ofrivilliga påverkan på måendet och i skuggan av den våldsutsatta. Resultaten påvisar att anhöriga ställs inför betydande utmaningar i sitt stöd till de våldsutsatta, särskilt när det gäller att anpassa stödet efter de våldsutsattas önskemål. Anhörigas anpassning till situationen undersöks genom medberoendeteorin, som belyser de strategier anhöriga använder för att hantera situationen. Studien visar också hur anhörigrollen innebär en psykisk och fysisk påverkan, med symptom som ångest, oro och sömnlöshet. Även om anhörigas symtom delvis överensstämmer med sekundär traumatisering, matchar de i större utsträckning kriterierna för vikarierande traumatisering, vilket betonar den inre personliga förändringen av att hjälpa en närstående som upplevt trauma. Vidare framhåller studien vikten av en ökad samhällelig kunskap om våld i nära relationer och dess påverkan på anhöriga. Många anhöriga prioriterar inte att söka hjälp för sig själva, då fokus oftast ligger på den våldsutsatta och de som söker stöd upplever generellt ett bristande stöd i form av oförståelse eller okunskap.  Denna studie syftar till att fördjupa förståelsen om de utmaningar anhöriga möter när de stöttar sina närstående, samt hur de själva upplever behovet av stöd. Med detta bidrar studien till ett relativt nytt kunskapsområde genom att ge en djupare inblick i anhörigas erfarenheter och förslag till förbättringsområden.

Ewing sarcoma, an aggressive primary bone and soft tissue tumour is characterised by the expression of the chimeric transcription factor EWS-FLI1 in 90% of patients. This alters expression of many genes including activation of the Insulin Growth Factor (IGF) pathway via IGFBP3 supression. Phase I/II trials with an IGF-1 inhibitor have demonstrated tumour regression in a modest number of Ewing sarcoma patients. The aim of this thesis was to identify mechanisms contributing to the heterogeneity of resistance in Ewing sarcoma following inhibition with OSI-906, a dual kinase inhibitor of IGF-1 (IGF-1R) and Insulin (IR) receptors. The hypothesis was that mechanisms of resistance relate to heterogeneity of responses to signalling pathway activation and inhibition. Through selection, disruption of the pathway would identify subpopulations of cells both sensitive and resistant in their response allowing for interrogation of resistance mechanisms. A genome wide approach was taken to model the resistance profile of cell lines. Through developing a method of unbiased quantification, a panel of validated Ewing sarcoma cell lines (EuroBoNet) were imaged and segmented to assess the responses of biomarkers on signalling pathway activation. Heterogeneity was confirmed between cell lines. The application to diagnostic biopsies led to the identification of prognostic classifiers and cellular subpopulations with clinical prognostic significance. The distribution of Ki67 was found to be predictive of survival and cells with lower levels of CD99 in the cytoplasm were most discriminative. Parallel sequencing strategies (RNA-seq, whole exome sequencing, and aCGH/ SNP array) for genome-wide screening was carried out for point mutations, copy number changes and rearrangements. Systematic detection was used to characterise genomic rearrangements and functional validation performed. Resistant clones, formed via ENU mutagenesis of cell lines, were sequenced in order to demonstrate the resistance profile of OSI-906. In summary heterogeneity of Ewing sarcoma at the genomic and proteomic level can influence the signalling dependency of tumours and response to inhibitors. Genomic and proteomic profiling of tumour cells may be relevant to future developments of novel therapies.

Microorganisms play central roles in planet Earth’s geochemical cycles, in food production, and in health and disease of humans and livestock. In spite of this, most microbial life forms remain unknown and unnamed, their ecological importance and potential technological applications beyond the realm of speculation. This is due both to the magnitude of microbial diversity and to technological limitations. Of the many advances that have enabled microbiology to reach new depth and breadth in the past decade, one of the most important is affordable high-throughput DNA sequencing. This technology plays a central role in each paper in this thesis. Papers I and II are focused on developing methods to survey microbial diversity based on marker gene amplification and sequencing. In Paper I we proposed a computational strategy to design primers with the highest coverage among a given set of sequences and applied it to drastically improve one of the most commonly used primer pairs for ecological surveys of prokaryotes. In Paper II this strategy was applied to an eukaryotic marker gene. Despite their importance in the food chain, eukaryotic microbes are much more seldom surveyed than bacteria. Paper II aimed at making this domain of life more amenable to high-throughput surveys. In Paper III, the primers designed in papers I and II were applied to water samples collected up to twice weekly from 2011 to 2013 at an offshore station in the Baltic proper, the Linnaeus Microbial Observatory. In addition to tracking microbial communities over these three years, we created predictive models for hundreds of microbial populations, based on their co-occurrence with other populations and environmental factors. In paper IV we explored the entire metagenomic diversity in the Linnaeus Microbial Observatory. We used computational tools developed in our group to construct draft genomes of abundant bacteria and archaea and described their phylogeny, seasonal dynamics and potential physiology. We were also able to establish that, rather than being a mixture of genomes from fresh and saline water, the Baltic Sea plankton community is composed of brackish specialists which diverged from other aquatic microorganisms thousands of years before the formation of the Baltic itself. / <p>QC 20150505</p>

Ubiquitin C-terminal hydrolase L1 (UCHL1) is a multifunctional protein primarily expressed in neuronal cells and involved in numerous cellular processes. UCHL1 has been linked with neurodegenerative diseases and a wide range of cancers but its specific role remains unknown. Previous UCHL1 knockdown studies have shown that UCHL1 controls the expression of pro- and anti-apoptotic genes as well as genes involved in cell cycle regulation but it is unknown how UCHL1 regulates these genes. We have shown that UCHL1 is cross-linked to DNA in DU145 but not in LNCaP or PC3 prostate cancer cells. Therefore, we hypothesized that UCHL1 regulates the expression of pro- or anti-apoptotic genes as well as the genes involved in the cell cycle through its interaction with DNA. By utilizing ChIP and ChIP-seq analyses it is possible to determine the UCHL1 target sequences on the genomic DNA. It was shown that UCHL1 is only expressed in DU145 but not in LNCaP, PC3 or C4-2 prostate cancer cell lines. Additionally, UCHL1 is expressed and cross-linked to DNA in HEK293T cells. It is believed that UCHL1 is silenced by upstream promoter methylation when it is not expressed. However, treatment with the epigenetic drugs 5-aza-2′-deoxycytidine and trichostatin A (TSA) did not result in induction of UCHL1 expression in LNCaP, PC3 or C4-2 prostate cancer cell lines. UCHL1 is also associated with p53. However, ChIP assay results have shown that UCHL1 and p53 do not bind to genomic DNA of upstream promoter regions CDKN1A and BAX genes. Additionally, through UCHL1 ChIP-seq analyses in DU145 and HEK293T cells, we discovered that UCHL1 co-localizes to the DNA with the shelterin complex shedding light on a new role of UCHL1 that has never been described before. / October 2014

The first metazoan to be sequenced was a nematode (Caenorhabditis elegans), and understanding the genome of this model organism has led to many insights about all animals. Although eleven nematode genomes have been published so far and approximately twenty more are under way, the vast majority of the genomes of this incredibly diverse phylum remain unexplored. Next-generation sequencing has made it possible to generate large amounts of genome sequence data in a few days at a fraction of the cost of traditional Sanger-sequencing. However, assembling and annotating these data into genomic resources remains a challenge because of the short reads, the quality issues in these kinds of data, and the presence of contaminants and co-bionts in uncultured samples. In this thesis, I describe the process of creating high quality draft genomes and annotation resources for four nematode species representing three of the five major nematode clades: Caenorhabditis sp. 5, Meloidogyne floridensis, Dirofilaria immitis, and Litomosoides sigmodontis. I describe the new approaches I developed for visualising contamination and co-bionts, and I present the details of the robust workflow I devised to deal with the problems of generating low-cost genomic resources from Illumina short-read sequencing. Results: The draft genome assemblies created using the workflow described in this thesis are comparable to the draft nematode genomes created using Sanger sequencing. Armed with these genomes, I was able to answer two evolutionary genomics questions at very different scales. The first question was whether any non-coding elements were deeply conserved at the level of the whole phylum. Such elements had previously been hypothesised to be responsible for the phylum body plan in vertebrates, insects, and nematodes. I used twenty nematode genomes in several whole-genome alignments and concluded that no such elements were conserved across the whole phylum. The second question addressed the origins of the highly destructive plant-parasitic root-knot nematode Meloidogyne incognita. Comparisons with the newly sequenced Meloidogyne floridensis genome revealed the complex hybrid origins of both species, undermining previous assumptions about the rarity of hybrid speciation in animals. Conclusions: This thesis demonstrates the role of next-generation sequencing in democratising genome sequencing projects. Using the sequencing strategies, workflows, and tools described here, one can rapidly create genomic resources at a very low cost, even for unculturable metazoans. These genomes can be used to understand the evolutionary history of a genus or a phylum, as shown.