CoenzymeQ10 (CoQ10) functions as an electron carrier in mitochondria transporting electrons from CI and CII to CIII in the respiratory chain (RC) for normal cellular energy (ATP) production. Mutations in genes of a complicated and not yet well understood CoQ10 biosynthesis cause primary CoQ10 deficiency, a rare autosomal recessive mitochondrial disorder (MD) with diverse heterogeneous clinical phenotypes. Although the major function of CoQ10 is to serve as electron transfer molecule it furthermore possesses multiple metabolic functions which can result in secondary CoQ10 deficiency. Five main clinical phenotypes are associated with CoQ10 deficiency although distinct genotype-phenotype associations are still absent due to the limited molecular genetic diagnoses of most reported CoQ10 deficiency cases. A correlation was found between reduced levels of CoQ10 in muscle tissue and deficient CII + III RC enzyme activities in a South African patient cohort, the current indicators for potential CoQ10 deficiency. The aim of the study was therefore to identify nuclear-encoded mutations in genes associated with CoQ10 deficiencies in a cohort of South African patients diagnosed with respiratory chain deficiencies (RCDs). A high throughput target enrichment strategy was performed in order to identify previously reported and/or possible novel CoQ10-assosciated disease-causing variants using Ion Torrent next generation sequencing (NGS) and an in-house developed bioinformatics pipeline. The data obtained were compared to clinical presentations of the patients to interpret the results of the identified variants considered to be possibly pathogenic. Targeted genes associated with primary CoQ10- and secondary CoQ10 deficiency was successfully sequenced in 24 patients, identifying 16 possible disease-causing variants. Of these variants three compound heterozygous variants were identified in three patients in genes ETFDH, COQ6 and COQ7, which were considered to be pathogenic according to the available data provided. Further validation of these three variants supported its pathogenicity in at least two of these variants (ETFDH and COQ6). In conclusion: This study contributed to better understanding the aetiology of a South African cohort of patients diagnosed with MDs. It also highlighted the valuable role of NGS for such investigations, and furthermore identified areas in the biochemical and molecular diagnostic strategy where improvements could be made in the future. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

CoenzymeQ10 (CoQ10) functions as an electron carrier in mitochondria transporting electrons from CI and CII to CIII in the respiratory chain (RC) for normal cellular energy (ATP) production. Mutations in genes of a complicated and not yet well understood CoQ10 biosynthesis cause primary CoQ10 deficiency, a rare autosomal recessive mitochondrial disorder (MD) with diverse heterogeneous clinical phenotypes. Although the major function of CoQ10 is to serve as electron transfer molecule it furthermore possesses multiple metabolic functions which can result in secondary CoQ10 deficiency. Five main clinical phenotypes are associated with CoQ10 deficiency although distinct genotype-phenotype associations are still absent due to the limited molecular genetic diagnoses of most reported CoQ10 deficiency cases. A correlation was found between reduced levels of CoQ10 in muscle tissue and deficient CII + III RC enzyme activities in a South African patient cohort, the current indicators for potential CoQ10 deficiency. The aim of the study was therefore to identify nuclear-encoded mutations in genes associated with CoQ10 deficiencies in a cohort of South African patients diagnosed with respiratory chain deficiencies (RCDs). A high throughput target enrichment strategy was performed in order to identify previously reported and/or possible novel CoQ10-assosciated disease-causing variants using Ion Torrent next generation sequencing (NGS) and an in-house developed bioinformatics pipeline. The data obtained were compared to clinical presentations of the patients to interpret the results of the identified variants considered to be possibly pathogenic. Targeted genes associated with primary CoQ10- and secondary CoQ10 deficiency was successfully sequenced in 24 patients, identifying 16 possible disease-causing variants. Of these variants three compound heterozygous variants were identified in three patients in genes ETFDH, COQ6 and COQ7, which were considered to be pathogenic according to the available data provided. Further validation of these three variants supported its pathogenicity in at least two of these variants (ETFDH and COQ6). In conclusion: This study contributed to better understanding the aetiology of a South African cohort of patients diagnosed with MDs. It also highlighted the valuable role of NGS for such investigations, and furthermore identified areas in the biochemical and molecular diagnostic strategy where improvements could be made in the future. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

BACKGROUND:Next-generation DNA sequencing (NGS) technologies have made huge impacts in many fields of biological research, but especially in evolutionary biology. One area where NGS has shown potential is for high-throughput sequencing of complete mtDNA genomes (of humans and other animals). Despite the increasing use of NGS technologies and a better appreciation of their importance in answering biological questions, there remain significant obstacles to the successful implementation of NGS-based projects, especially for new users.RESULTS:Here we present an 'A to Z' protocol for obtaining complete human mitochondrial (mtDNA) genomes - from DNA extraction to consensus sequence. Although designed for use on humans, this protocol could also be used to sequence small, organellar genomes from other species, and also nuclear loci. This protocol includes DNA extraction, PCR amplification, fragmentation of PCR products, barcoding of fragments, sequencing using the 454 GS FLX platform, and a complete bioinformatics pipeline (primer removal, reference-based mapping, output of coverage plots and SNP calling).CONCLUSIONS:All steps in this protocol are designed to be straightforward to implement, especially for researchers who are undertaking next-generation sequencing for the first time. The molecular steps are scalable to large numbers (hundreds) of individuals and all steps post-DNA extraction can be carried out in 96-well plate format. Also, the protocol has been assembled so that individual 'modules' can be swapped out to suit available resources.

This thesis is a comparative study of two Next-Generation Firewalls (NGFWs) with the aim to conclude which one is the most suitable for a small enterprise network. The network in question is Company A’s Office A1. Office A is in the process of upgrading their internal network and with the upgrade a new NGFW will be implemented. The two NGFW platforms that have been researched per Company A’s request are Juniper Networks’ SRX-series firewalls and Palo Alto Networks’ (PAN) PA-series, with focus on the SRX1500 and PA-3020 for a fair comparison. To be able to evaluate different platforms and appliances, the concept of NGFW and what it constitutes has been researched and presented. Both of the NGFW platforms have been tested and compared in terms of ease-of-use and cost analysis. The testing focused on the respective web-interfaces and shows no significant differences between the two NGFWs at a first glance in terms of functionality. However, PAN’s web-interface does objectively feel more up-to-date and provides application visibility natively, which Juniper offers as a separate service as part of the centralised management platform, which is excessive for Office A’s network. The research and collection of data has been conducted based on Office A’s needs and requirements. Third-party research has been collected from NSS Labs and Gartner and serves as a basis for the evaluation. The future network of Office A introduces new services and the general usage will mainly consist of office oriented application based traffic. The evaluation of the research of the two NGFWs and the collection of data, in the context of Office A’s network, shows that the PA-3020 would be favoured. The key points are as follows: PAN’s NGFWs are built specifically for application awareness whereas Juniper are new in the NGFW market and has recently started to add the more advanced application awareness features. PAN offers a one-box solution suited for smaller networks such as Office A whereas a Juniper implementation would require additional hardware (VM’s) to obtain similar features. PAN offers more features in terms of user identification which is a key factor in enabling a true context aware security environment seamlessly integrated and invisible to the users. No major difference in cost if a similar set of features are to be implemented, based on non-rebated list prices (additional hardware not included). 1 Note: Due to confidentiality, the name and details of the company has been anonymised throughout the report.

Understanding the genetic basis of adaptive evolution, and how natural selection has shaped patterns of polymorphism and divergence within and between species are enduring goals of evolutionary genetics. In this thesis, I used whole genome re-sequencing data to characterize the genomic signatures of natural selection along different evolutionary timescales in three Populus species: Populus tremula, P. tremuloides and P. trichocarpa. First, our study shows multiple lines of evidence suggesting that natural selection, due to both positive and purifying selection, has widely shaped patterns of nucleotide polymorphism at linked neutral sites in all three species. Differences in effective population sizes and rates of recombination largely explain the disparate magnitudes and signatures of linked selection that we observe among species. Second, we characterize the evolution of genomic divergence patterns between two recently diverged aspen species: P. tremula and P. tremuloides. Our findings indicate that the two species diverged ~2.2-3.1 million years ago, coinciding with the severing of the Bering land bridge and the onset of dramatic climatic oscillations during the Pleistocene. We further explore different mechanisms that may explain the heterogeneity of genomic divergence, and find that variation in linked selection and recombination likely plays a key role in generating the heterogeneous genomic landscape of differentiation between the two aspen species. Third, we link whole-genome polymorphic data with local environmental variables and phenotypic variation in an adaptive trait to investigate the genomic basis of local adaptation in P. tremula along a latitudinal gradient across Sweden. We find that a majority of single nucleotide polymorphisms (SNPs) (&gt;90%) identified as being involved in local adaptation are tightly clustered in a single genomic region on chromosome 10. The signatures of selection at this region are more consistent with soft rather than hard selective sweeps, where multiple adaptive haplotypes derived from standing genetic variation sweep through the populations simultaneously, and where different haplotypes rise to high frequency in different latitudinal regions. In summary, this thesis uses phylogenetic comparative approaches to elucidate how various evolutionary forces have shaped genome-wide patterns of sequence evolution in Populus. / <p>The research in this thesis was supported by the Swedish research council (to Pär K. Ingvarsson) and the JC Kempe Memorial Scholarship Foundation (to Jing Wang). The PhD study of Jing Wang in Sweden was funded by the State Scholarship from China Scholarship council.</p>

Next Generation Networks (NGNs) aim to provide a unified network infrastructure to offer multimedia data and telecommunication services through IP convergence. NGNs utilize multiple broadband, QoS-enabled transport technologies, creating a converged packet-switched network infrastructure, where service-related functions are separated from the transport functions. This requires significant changes in the way how networks are managed to handle the complexity and heterogeneity of NGNs. This thesis proposes a Service Oriented Architecture (SOA) based management framework that integrates heterogeneous management systems in a loose coupling manner. The key benefit of the proposed management architecture is the reduction of the complexity through service and data integration. A network management middleware layer that merges low level management functionality with higher level management operations to resolve the problem of heterogeneity was proposed. A prototype was implemented using Web Services and a testbed was developed using trouble ticket systems as the management application to demonstrate the functionality of the proposed framework. Test results show the correcting functioning of the system. It also concludes that the proposed framework fulfils the principles behind the SOA philosophy.

<p>Advances in sequencing technologies constantly improves the throughput andaccuracy of sequencing instruments. Together with this development comes newdemands and opportunities to fully take advantage of the massive amounts of dataproduced within a sequence run. One way of doing this is by analyzing a large set ofsamples in parallel by pooling them together prior to sequencing and associating thereads to the corresponding samples using DNA sequence tags. Amplicon sequencingis a common application for this technique, enabling ultra deep sequencing andidentification of rare allelic variants. However, a common problem for ampliconsequencing projects is formation of unspecific PCR products and primer dimersoccupying large portions of the data sets.</p><p>This thesis is based on two papers exploring these new kinds of possibilities andissues. In the first paper, a method for including thousands of samples in the samesequencing run without dramatically increasing the cost or sample handlingcomplexity is presented. The second paper presents how the amount of high qualitydata from an amplicon sequencing run can be maximized.</p><p>The findings from the first paper shows that a two-tagging system, where the first tagis introduced by PCR and the second tag is introduced by ligation, can be used foreffectively sequence a cohort of 3500 samples using the 454 GS FLX Titaniumchemistry. The tagging procedure allows for simple and easy scalable samplehandling during sequence library preparation. The first PCR introduced tags, that arepresent in both ends of the fragments, enables detection of chimeric formation andhence, avoiding false typing in the data set.</p><p>In the second paper, a FACS-machine is used to sort and enrich target DNA covered emPCR beads. This is facilitated by tagging quality beads using hybridization of afluorescently labeled target specific DNA probe prior to sorting. The system wasevaluated by sequencing two amplicon libraries, one FACS sorted and one standardenriched, on the 454 showing a three-fold increase of quality data obtained.</p> / QC20100907

This thesis is about new and improved computational methods to analyze complex biological data produced by advanced biotechnologies. Such data is not only very large but it also is characterized by very high numbers of features. Addressing these needs, we developed a set of methods and tools that are suitable to analyze large sets of data, including next generation sequencing data, and built transparent models that may be interpreted by researchers not necessarily expert in computing. We focused on brain related diseases. The first aim of the thesis was to employ the meta-server approach to finding peaks in ChIP-seq data. Taking existing peak finders we created an algorithm that produces consensus results better than any single peak finder. The second aim was to use supervised machine learning to identify features that are significant in predictive diagnosis of Alzheimer disease in patients with mild cognitive impairment. This experience led to a development of a better feature selection method for rough sets, a machine learning method.  The third aim was to deepen the understanding of the role that STAT3 transcription factor plays in gliomas. Interestingly, we found that STAT3 in addition to being an activator is also a repressor in certain glioma rat and human models. This was achieved by analyzing STAT3 binding sites in combination with epigenetic marks. STAT3 regulation was determined using expression data of untreated cells and cells after JAK2/STAT3 inhibition. The four papers constituting the thesis are preceded by an exposition of the biological, biotechnological and computational background that provides foundations for the papers. The overall results of this thesis are witness of the mutually beneficial relationship played by Bioinformatics in modern Life Sciences and Computer Science.

In this thesis work, I have used a comparative genomics approach to study a fungal model organism, Neurospora tetrasperma. My specific focus has been on genomic introgression, intron evolution, chromosomal structural rearrangements and codon usage. All of the studies are based on large-scale dataset generated by next-generation sequencing technology (NGS), combined with other techniques, such as Optical Mapping. In the introgression study, we detected large-scale introgression tracts in three N. tetrasperma lineages, and the introgression showed allele-specific and chromosomal-specific pattern. In the study of introns, we found indications of mRNA mediated intron loss and non-homologous end joining (NHEJ) mediated intron gains in N. tetrasperma. We found that selection is involved in shaping intron gains and losses, and associated with intron position, intron phase and GC content. In the study of chromosomal structural rearrangements, we found a lineage specific chromosomal inversion pattern in N. tetrasperma, which indicates that inversions are unlikely to associate with the origin of the suppressed recombination and the mating system transition in N. tetrasperma. The result suggests inversions are the consequences, rather than the causes, of suppressed recombination on the mating-type chromosome of N. tetrasperma. In the final study, analyses of codon usage indicated that the region of suppressed recombination in N. tetrasperma is subjected to genomic degeneration, and selection efficiency has been much reduced in this region.

Myoclonus-Dystonia (MD) is an inherited, rare, autosomal dominant movement disorder characterized by quick, involuntary muscle jerking or twitching (myoclonus) and involuntary muscle contractions that cause twisting and pulling movements, resulting in abnormal postures (dystonia). The first MD locus was mapped to 7q21-q31 and called DYT11; this locus corresponds to the SGCE gene. Our group previously identified a second MD locus (DYT15) which maps to a 3.18 Mb region on 18p11. Two patients were chosen to undergo next-generation sequencing, which identified 2,292 shared novel variants within the critical region. Analysis of these variants revealed a 3 bp duplication in a transcript referred to as CD108131, which is believed to be a long non-coding RNA. Characterization of this transcript determined that it is 863 bp in size, it is ubiquitously expressed, with high expression in the cerebellum, and it accounts for ~3% of MD cases.