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Herpes simplex virus type 1 (HSV-1) is a double stranded DNA virus that causes epithelial skin infections and persists through the life of the host by infecting neurons, where it can switch to a latent state to evade an immune response. In rare cases during primary infection or after reactivation, instead of undergoing lytic infection at the epithelial surface, it instead travels to the brain and causes herpes simplex virus encephalitis (HSVE) which can have a ≥70% mortality rate if untreated. As the virus takes over its host cell, it gains control of the host cell machinery and manipulates host gene expression in order to evade the immune system and to pool its resources into the replication of the virus. One aspect of the dysregulated gene expression involves microRNAs (miRNAs). MiRNAs are short, non-coding RNAs that bind to the 3' untranslated region (3'UTR) of messenger RNAs (mRNAs), leading to translational repression of the target. Dysregulated miRNAs are often down-regulated during infection as the virus takes over, but many miRNAs have also been found to be up-regulated as well1–5. The aim of this study is to observe the full cellular miRNA changes in the context of an acute viral encephalitic infection using HSV-1, and to further characterize selected up-regulated miRNAs to determine their function in the context of the disease state. Of particular note were miR-141 and miR-200c which showed anti-apoptotic effects on neuronal cell culture and did not impact cell viability during an over-expression of the miRNAs. MiR-141, miR-183 and miR-200a expression was enriched within specific areas of the brain during infection. In addition, the potential for miR-150 to bind to a bioinformatically predicted target site within the shared 3'UTR of the HSV-1 UL18, UL19 and UL20 genes was explored. Examining the changes in expression of this class of regulatory RNAs and investigating their potential functions may yield new insight into the relationship between host and virus during infection.

In human microbiome analysis, sequencing of bacterial 16S rRNA genes has revealed a role for the gut microbiota in maintaining health and contributing to various pathologies. Novel community analysis techniques must be evaluated in terms of bias, sensitivity, and reproducibility and compared to existing techniques to be effectively implemented. Next- generation sequencing technologies offer many advantages over traditional fingerprinting methods, but this extensive evaluation required for the most efficacious use of data has not been performed previously. Illumina libraries were generated from the V3 region of the 16S rRNA gene of samples taken from 12 unique sites within the gastrointestinal tract for each of 4 individuals. Fingerprint data were generated from these samples and prominent bands were sequenced. Sequenced bands were matched with OTUs within their respective libraries. The results demonstrate that denaturing gradient gel electrophoresis (DGGE) represents relatively abundant bacterial taxa (>0.1%) beta-diversity of all samples was compared using Principal Coordinates Analysis (PCoA) of UniFrac distances and Multi-Response Permutation Procedure (MRPP) was applied to measure sample cluster strength and significance; indicator species analysis of fingerprint bands and Illumina OTUs were also compared. The results demonstrate overall similarities between community profiling methods but also indicate that sequence data were not subject to the same limitations observed with the DGGE method (i.e., only abundant taxa bands are resolved, unable to distinguish disparate samples). In addition, the effect of stochastic fluctuations in ???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? differ for DGGE and next-generation sequencing. I compared pooled and individual reactions for samples of high and low template concentration for both Illumina and DGGE using the combined V3-V4 region of the 16S rRNA gene, and demonstrated that template concentration has a greater impact on reproducibility than pooling. This research shows congruity between two disparate molecular methods, identifies sources of bias, and establishes new guidelines for minimizing bias in microbial community analyses.

The study of variation found in DNA is fundamental in human genetic studies. Single nucleotide polymorphisms (SNPs) are simple to document because they can be captured in single DNA sequence reads. Larger structural variation including duplications, insertions, deletions, termed as copy number variation (CNV), inversions and translocations are more challenging to discover. Recent studies using microarray and sequencing technologies have demonstrated the prevalence of structural variation in humans. They can disrupt genic and regulatory sequences, be associated with disease, and fuel evolution. Therefore, it is important to identify and characterize both SNPs and structural variants to fully understand their impact. This thesis presents the analysis of structural variation in the human genome. The primary DNA sample used for my experiments is the DNA of J. Craig Venter, also termed HuRef. It was the first personal human genome sequenced. I combined computational re-analysis of sequence data with microarray-based analysis, and detected 12,178 structural variants covering 40.6 Mb that were not reported in the initial sequencing study. The results indicated that the genomes of two individuals differed 1.3% by CNV, 0.3% by inversion and 0.1% by SNP. Structural variation discovery is dependent on the strategy used. No single approach can readily capture all types of variation, and a combination of strategies is required. I analyzed the formation mechanisms of all HuRef structural variants. The results showed that the relative proportion of mutational processes changed across size range: the majority of small variants (<1kb) were associated with nonhomologous processes and microsatellite events; median size variants (<10kb) were commonly related to minisatellites and retrotransposons; and large variants were associated with nonallelic homologous recombination. Eight new breakpoint-resolved HuRef inversions were genotyped in populations to elucidate these understudied variants. I discovered that the structures of inversion could be complex, could create conjoined genes, and their frequencies could exhibit population differentiation. The data here contributes to our understanding of structural variation in humans. It shows the need to use multiple strategies to identify variants, and it emphasizes the importance to examine the full complement of variation in all biomedical studies.

Ο όρος ‘Δίκτυα Επόμενης Γενιάς’ αναφέρεται σε μελλοντικά δίκτυα πρωτοποριακής λογικής και δομής, προσανατολισμένα στην υποστήριξη σύγχρονων απαιτητικών εφαρμογών και στη βελτίωση της λειτουργικότητας της τερματικής συσκευής, όπως την αντιλαμβάνεται ο χρήστης. Πολύ σημαντικά στοιχεία της νέας τηλεπικοινωνιακής πραγματικότητας που επιφέρουν τα δίκτυα αυτά, και με τα οποία σχετίζεται άμεσα η παρούσα εργασία, είναι η διάθεση προώθησης της γενικευμένης κινητικότητας των ασύρματων χρηστών και η ενθάρρυνση της σύγκλισης επιμέρους τεχνολογιών διαφορετικών δικτύων και της δημιουργίας υβριδικών ετερογενών δικτύων, με στόχο την επίτευξη καλύτερης αξιοποίησης του φάσματος και βελτίωσης των ρυθμών μετάδοσης δεδομένων. Σκοπός της παρούσας διπλωματικής εργασίας είναι η παρουσίαση της δομής και των λειτουργιών των Δικτύων Επόμενης Γενιάς, καθώς και ορισμένων υπαρχόντων τύπων ασύρματων δικτύων, η συνεργασία των οποίων θα μπορούσε να προσφέρει τα επιθυμητά πλεονεκτήματα. Δύο τέτοια είδη δικτύων είναι το δίκτυο κινητής τηλεφωνίας UMTS και τα τοπικά δίκτυα τεχνολογίας WLAN, τα οποία εξετάζονται ενδελεχώς ως προς τα χαρακτηριστικά τους και, κυρίως, ως προς τους μηχανισμούς διευθέτησης της περιαγωγής των χρηστών. Περιλαμβάνεται επίσης μία περιγραφή της λειτουργίας του Mobile IP, πρωτοκόλλου που θεωρείται ιδιαίτερα χρήσιμο για τη διαχείριση της κινητικότητας χρηστών ανάμεσα σε περιοχές εξυπηρέτησης διαφορετικών δικτύων. Η εργασία καταλήγει στη μοντελοποίηση των παραπάνω συστημάτων σε περιβάλλον εξομοίωσης, επιδιώκοντας την αξιολόγηση της αποτελεσματικότητας του ενδεχόμενου συνδυασμού των συστημάτων UMTS και WLAN και της χρησιμότητας του Mobile IP. Το λογισμικό που χρησιμοποιείται γι’ αυτόν το σκοπό είναι το OPNET Modeler ®, ένα πρόγραμμα που αναδεικνύεται τα τελευταία χρόνια σε εργαλείο όλο και περισσότερο πολύτιμο, τόσο σε ερευνητικό όσο και σε επιχειρησιακό επίπεδο. / The term ‘Next Generation Networks’ refers to future networks of revolutionary concept and structure, oriented to the support of demanding applications and the upgrade of the terminal device’s functionality, as perceived by the user. Some very important aspects of the new telecommunications reality that is brought on by these networks, and to which this project is directly related, is the intention of promoting generalized mobility for the wireless users and the encouragement of the convergence of distinct network technologies and of the foundation of new hybrid heterogeneous networks, in order to achieve better spectrum utilization and improvement of data transmission rates. The purpose of the current diploma thesis project is to present the structure and the functions of the Next Generation Networks, as well as of some existing types of wireless networks, the cooperation of which could provide the desirable advantages. Two such network types are the UMTS mobile telephony network and the local networks of WLAN technology, that are examined thoroughly towards their characteristics and, foremost, towards their roaming arrangement mechanisms. Also included is a description of the operation of Mobile IP, a protocol that is considered particularly convenient for the management of users’ mobility between service areas of different networks. The project concludes to the modeling of the above mentioned systems in a simulation environment, aiming to evaluate the efficiency of the prospective combination of the UMTS and WLAN systems and the utility of Mobile IP. The software used for this purpose is OPNET Modeler ®, a program that has emerged during the last years as an increasingly valuable research and business tool.

Η διδακτορική διατριβή με τίτλο «Σχεδιασμός και υλοποίηση συστήματος διαχείρισης και ενοποίησης διαφορετικών ταυτοτήτων χρηστών σε δίκτυα νέας γενιάς» πραγματεύεται την οργάνωση και διαχείριση των ταυτοτήτων χρηστών σε ένα ενοποιημένο δικτυακό περιβάλλον αποτελούμενο από διαφορετικά δίκτυα, τεχνολογίες και υπηρεσίες. Η διατριβή, αρχικά εξετάζει τις προτεινόμενες αρχιτεκτονικές και υπηρεσίες του Μελλοντικού Διαδικτύου και ανάμεσα στα προβλήματα που παρουσιάζονται επικεντρώνεται στην επίλυση της Διαχείρισης Ψηφιακών Ταυτοτήτων (Identity Management – IdM). Μέχρι τώρα οι προτεινόμενες λύσεις είναι λειτουργικές μόνο κάτω από συγκεκριμένες συνθήκες, όπως π.χ. την εφαρμογή νέων παγκόσμιων προσδιοριστικών ή τη δημιουργία μεγάλων ομοσπονδιών εμπιστοσύνης. Στην πράξη όμως τέτοιες πρακτικές δεν μπορούν να υιοθετηθούν από μεγάλης κλίμακας δίκτυα. Σε αντίθεση με αυτές τις προσεγγίσεις, η συγκεκριμένη διατριβή προτείνει τον σχεδιασμό ενός συστήματος που θα έχει ως σκοπό να βοηθάει τα εκάστοτε πλαίσια (δίκτυα, ομοσπονδίες, παρόχους κ.τ.λ.) να αντιμετωπίζουν μόνα τους τα προβλήματα διαχείρισης ταυτοτήτων που παρουσιάζονται στις υπηρεσίες τους. Για να επιτευχθεί αυτό, αρχικά αποδεικνύεται πως είναι αναγκαίος ο διαχωρισμός της διαδικασίας ανακάλυψης δεδομένων που σχετίζονται με τις ψηφιακές ταυτότητες, από τις υπόλοιπες διαδικασίες διαχείρισης (π.χ. ανταλλαγή δεδομένων μεταξύ παρόχων). Με βάση αυτή την προσέγγιση δημιουργήθηκε το σύστημα DIMANDS, το οποίο δίνει την δυνατότητα στους παρόχους να ανακαλύπτουν τα δεδομένα ταυτότητας που απαιτούνται για την ολοκλήρωση μιας δεδομένης υπηρεσίας, ενώ παράλληλα τους επιτρέπει να διαχειρίζονται αυτόνομα τις υπόλοιπες διαδικασίες που σχετίζονται με την υπηρεσία αυτή. Το σύστημα πληροί όλες τις απαιτήσεις ασφάλειας, εμπιστοσύνης, διαφύλαξης ιδιοαπόρρητου και απόδοσης ενός μεγάλης κλίμακας συστήματος διαχείρισης ψηφιακών ταυτοτήτων. Αυτό αποδεικνύεται από προσομοιώσεις που έγιναν στο περιβάλλον εξομοίωσης OPNET. Τέλος σημειώνεται πως ένα δοκιμαστικό σενάριο χρήσης του συστήματος υλοποιήθηκε σε περιβάλλον Ruby on Rails. / This PhD thesis entitled “Design and implementation of a system for the management and unification of users’ diverse identities in next generation networks” examines the organization and management of users’ identity data in a unified network environment composed by diverse networks, technologies and services. The dissertation, initially examines the proposed future Internet architectures and services and among the identified problems, it focuses on the management of Digital Identities (Identity Management – IdM). Until now, the proposed solutions are only functional if specific conditions are met, such as the implementation of new global identifiers or creating large scale federations of trust. In practice though, these conditions cannot be enforced in large-scale networks. Contrary to these approaches, this dissertation proposes the design of a system capable of helping the individual contexts (federations, networks, service providers, etc.) to independently deal with their own identity management problems that appear in their services. To achieve this, initially we prove that it is necessary to separate the process of discovering identity related data from the all the rest of the identity management procedures (e.g. data exchange between providers). Based on this approach we created DIMANDS, a system which allows providers to discover the necessary data required to complete a specific service, while enabling them to manage autonomously the remaining procedures associated with this service. The system meets all the requirements of security, trust, privacy and performance of a large scale identity management system. This is evidenced by simulations made in the OPNET simulation environment. Finally it must be noted that a demo based on the system’s functionality was implemented in Ruby on Rails environment.

Στην παρούσα διατριβή προτείνονται Μηχανισμοί Ασφάλειας για την ανάπτυξη ασφαλών και αξιόπιστων διάχυτων υπηρεσιών υγείας πάνω σε Δίκτυα Επόμενης Γενιάς (Next Generation Networks – NGN). Οι προτεινόμενοι Μηχανισμοί Ασφάλειας έχουν ως στόχο να λειτουργήσουν προσθετικά στο επίπεδο ασφάλειας που προσφέρουν οι υπάρχοντες μηχανισμοί ασφάλειας που υποστηρίζονται από το NGN. Αυτό είναι αναγκαίο καθώς οι διάχυτες υπηρεσίες υγείας εμπεριέχουν ιδιαιτέρως ευαίσθητη πληροφορία. Επιπρόσθετα, στην παρούσα διατριβή προτείνεται ένα γενικό πλαίσιο εφαρμογής, το οποίο υποστηρίζει τους προτεινόμενους Μηχανισμούς Ασφάλειας, προκειμένου να επιτυγχάνεται γρήγορη και αποτελεσματική ανάπτυξη ασφαλών και αξιόπιστων διάχυτων υπηρεσιών υγείας πάνω σε NGN. Πιο συγκεκριμένα, το προτεινόμενο πλαίσιο βασίζεται στην αρχιτεκτονική του προτύπου ETSI/Parlay και επεκτείνει το σύνολο των Διεπαφών των Χαρακτηριστικών Ικανότητας Υπηρεσίας (Service Capability Features Interfaces – SCFs Interfaces) και το σύνολο των μηχανισμών που υποστηρίζει το Πλαίσιο ETSI/Parlay. Το προτεινόμενο πλαίσιο επεκτείνει το σύνολο των Διεπαφών των Χαρακτηριστικών Ικανότητας Υπηρεσίας προκειμένου αυτό να περιλαμβάνει όχι μόνο τις διεπαφές που σχετίζονται με τις υπηρεσίες του υποκείμενου δικτύου (NGN), αλλά και επιπλέον διεπαφές που δίνουν τη δυνατότητα σε διάχυτες υπηρεσίες υγείας να έχουν πρόσβαση σε ικανότητες επαίσθησης (sensing capabilities) δικτύων αισθητήρων που είναι υπεύθυνα για τη συλλογή πληροφορίας περιβάλλοντος καθώς και βιοπληροφορίας. Επίσης, το προτεινόμενο πλαίσιο επεκτείνει το σύνολο των μηχανισμών που υποστηρίζει το Πλαίσιο ETSI/Parlay προκειμένου να είναι δυνατή η παροχή σε διάχυτες υπηρεσίες υγείας όχι μόνο των βασικών μηχανισμών, που υποστηρίζονται από το προτυποποιημένο Πλαίσιο ETSI/Parlay, αλλά και των Μηχανισμών Δικτύων Αισθητήρων καθώς και των Μηχανισμών Ασφάλειας, οι οποίοι προτείνονται στην παρούσα διατριβή. Οι Μηχανισμοί Ασφάλειας, οι οποίοι προτείνονται, στοχεύουν στην παροχή ασφάλειας στα δεδομένα των τελικών χρηστών καθώς και στην ασφαλή πρόσβαση στις διάχυτες υπηρεσίες υγείας και στην ασφαλή χρήση τους. Πιο συγκεκριμένα, οι προτεινόμενοι Μηχανισμοί Ασφάλειας επικεντρώνονται στη διασφάλιση της εμπιστευτικότητας των δεδομένων, της ακεραιότητας των δεδομένων, της πιστοποίησης αυθεντικότητας καθώς και του ελέγχου πρόσβασης των οντοτήτων που συμμετέχουν σε διάχυτες υπηρεσίες υγείας. Για τη διασφάλιση της εμπιστευτικότητας των δεδομένων προτείνεται ένα γενικό σχήμα κρυπτογράφησης. Αυτό το σχήμα επιτρέπει το σχεδιασμό και την υλοποίηση ασφαλών εξατομικευμένων κρυπτογραφικών αλγορίθμων τμήματος για την κρυπτογράφηση δεδομένων διάχυτων υπηρεσιών υγείας, όπως οι ιατρικοί φάκελοι των ασθενών. Επίσης, προτείνεται ένας μηχανισμός για διασφάλιση της ακεραιότητας των δεδομένων για σύστημα ιατρικής τηλε-παρακολούθησης. Αυτό το σύστημα τηλε-παρακολούθησης λειτουργεί σε περιβάλλον έξυπνου σπιτιού και υποστηρίζει τη μεταφορά βιοσημάτων του ασθενή από τον ασθενή στη Μονάδα Παροχής Υπηρεσιών Υγείας. Επιπρόσθετα, προτείνονται δύο μηχανισμοί για διασφάλιση της πιστοποίησης αυθεντικότητας. Ο πρώτος μηχανισμός είναι ένας ευφυής μηχανισμός πιστοποίησης αυθεντικότητας για εφαρμογές e-Hospital πάνω σε WLAN μέσα σε νοσοκομείο. Ο δεύτερος μηχανισμός είναι ένας μηχανισμός συμφωνίας κλειδιού ομάδας και ανάκτησης σε ad hoc δίκτυα, που χρησιμοποιούνται κατά τη διαχείριση ιατρικών συμβάντων έκτακτης ανάγκης σε περιοχές στις οποίες δεν υπάρχει σταθερή τηλεπικοινωνιακή υποδομή. Τέλος, προτείνεται μία υποδομή PKI σε ένα ιατρικό δίκτυο μεγάλης κλίμακας που συνδέει ένα ευρύ φάσμα από Μονάδες Παροχής Υπηρεσιών Υγείας. Η προτεινόμενη υποδομή PKI εστιάζεται στη διασφάλιση της πιστοποίησης αυθεντικότητας και του ελέγχου πρόσβασης των επαγγελματιών του χώρου της υγείας που επιθυμούν να αποκτήσουν πρόσβαση σε υπηρεσίες που σχετίζονται με αυτούς καθώς και σε υπηρεσίες υγείας που σχετίζονται με τον ασθενή. / In this dissertation, Security Mechanisms are proposed for the development of secure and reliable pervasive healthcare services over Next Generation Networks (NGN). The proposed Security Mechanisms aim at increasing the security level provided by the existing security mechanisms supported by NGN. It is essential since pervasive healthcare services include extremely sensitive information. Furthermore, in this dissertation, a generic application framework is proposed supporting the proposed Security Mechanisms in order the rapid and efficient development of secure and reliable pervasive healthcare services over NGN to be achieved. In particular, the proposed framework is based on the ETSI/Parlay architecture and extends the set of the Service Capability Features Interfaces (SCFs Interfaces) as well as the set of mechanisms supported by the ETSI/Parlay Framework. The proposed framework extends the set of the SCFs Interfaces in order to integrate not only the interfaces related to the services of the underlying network (NGN), but also additional interfaces enabling pervasive healthcare services to access sensing capabilities of sensor networks which are responsible for gathering context and bio information. Moreover, the proposed framework extends the set of mechanisms supported by the ETSI/Parlay Framework to provide pervasive healthcare services not only with the basic mechanisms supported by the standardized ETSI/Parlay Framework, but also with the Sensor Networks Mechanisms and the Security Mechanisms proposed in this dissertation. The proposed Security Mechanisms aim at securing the end-user data as well as the access to the pervasive healthcare services and the use of them. In particular, the proposed Security Mechanisms focus on ensuring data confidentiality, data integrity, authentication and access control of entities participating in pervasive healthcare services. To ensure data confidentiality, a generic encryption schema is proposed. This schema enables the design and implementation of secure personalized block ciphers for encryption of data included in pervasive healthcare services such as patients’ medical records. Moreover, a data integrity mechanism for a tele-monitoring system is proposed. This tele-monitoring system operates in a smart home environment and supports transmission of patient’s biosignals from the patient to the Healthcare Center. Additionally, two authentication mechanisms are proposed. The first mechanism is an intelligent authentication mechanism for e-Hospital applications over WLAN in a hospital. The second mechanism is a group key agreement and recovery mechanism in ad hoc networks used for handling emergency medical incidents in areas without fixed telecommunications infrastructure. Finally, a PKI infrastructure in a large-scale healthcare network connecting a wide spectrum of Healthcare Centers is proposed. The proposed PKI infrastructure focuses on ensuring authentication and access control of healthcare professionals willing to access services related to them as well as healthcare services related to patient.

Thesis (MSc (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / Title page: Dept. of Genetics, Faculty of Science / ENGLISH ABSTRACT: Next-generation sequencing technologies are increasingly used in metagenomic studies, largely due to the high sequence data throughput capacity and unbiased approach in determining the genetic composition of an unknown environmental sample. This study investigated the applicability of the Illumina next-generation sequencing platform for metagenomic sequencing of grapevine viruses to provide the first complete viral profile, or virome, of a diseased vineyard. Leaf material was harvested from 44 randomly selected vines in a leafroll-diseased vineyard in South Africa. Sample material was pooled and double-stranded RNA extracted. The dsRNA was sequenced as a paired-end sequencing run using the Illumina sequencing-by-synthesis technique, and more than 19 million sequence reads, equivalent to approximately 837 megabases of metagenomic sequence data, were obtained. Of these data, approximately 400 megabases could be assembled into 449 scaffolds, using the de novo assembler Velvet. These scaffolds were subjected to BLAST searches against the NCBI databases and top hit scores were used for virus identification. Based on the BLAST results, suitable sequences were selected from the NCBI database and used as reference sequence in MAQ mapping assemblies. The bioinformatic analyses allowed for the determination of the virus species present, the most prominent variants, and the relative abundance of each. Four known grapevine viral pathogens were identified. Grapevine leafroll-associated virus 3, representing 59% of the analyzed short read sequence data, was identified as the most prominent virus species. Three variants of this virus were detected: GP18 was the most abundant, followed by a minor Cl766/NY1 variant and a potential novel grapevine leafroll-associated ampelovirus. A single Grapevine rupestris stem pitting ]associated virus variant, similar to SG1, and a Grapevine virus A variant, a member of molecular group III, were identified. This study is also the first to report the presence of Grapevine virus E (GVE) in South African vineyards. Grapevine virus E was further genetically characterized and the genome sequence of GVE isolate SA94 determined. The GVE SA94 genome sequence, 7568 nucleotides in length, is the first complete genome sequence for the virus species. The genome organization of GVE SA94 is typical of vitiviruses, but in contrast to other RNA viruses, the AlkB domain is located within the helicase domain in open reading frame 1 (ORF 1). Grapevine virus E SA94 shares nearly 100% nucleotide identity with the Japanese TvP15 isolate and GVE 3404, a de novo scaffold generated from the metagenomic sequence data. Bioinformatic analysis of metagenomic sequence data further revealed the presence of three fungus-infecting viral families, Chrysoviridae, Totiviridae and the unclassified dsRNA virus, Fusarium graminearum dsRNA mycovirus 4. A virus from the family Chrysoviridae, similar to Penicillium chrysogenum virus, was the second most abundant virus detected. We demonstrated the successful application of a short read sequencing technology, such as the Illumina platform, for viral profiling of an infected vineyard. To our knowledge this is the first application of the Illumina technology for this purpose. / AFRIKAANSE OPSOMMING: Volgende-generasie tegnologie om basis volgordes van nukleiensure te bepaal, word al meer gebruik in metagenomiese studies. Dit is veral weens die hoe data-omset kapasiteit en onbevooroordeelde aanslag in die bepaling van die genetiese samestelling van onbekende omgewingsmonsters. Hierdie studie het die aanwending van die Illumina volgende-generasie volgorde-bepalingsplatform in 'n metagenomiese studie van wingerdvirusse, ondersoek. Dit het ten doel gehad om die eerste volledige virus profiel, of viroom, van 'n geinfekteerde wingerd saam te stel. Blaarmateriaal is verkry vanaf 44 lukraak-gekose wingerdstokke in 'n rolblad-geinfekteerde wingerd in Suid-Afrika. Monster materiaal is saamgevoeg en dubbelstring-RNS geekstraheer. Die dubbelstring-RNS is onderwerp aan gepaarde-ent volgorde-bepaling deur gebruik te maak van die Illumina volgorde-bepaling-deur-sintese tegniek. Meer as 19 miljoen volgorde reekse, ekwivalent aan ongeveer 837 megabasisse volgorde data, is verkry. Van hierdie data kon ongeveer 400 megabasisse saamgevoeg word in 449 konstrukte ("scaffolds"), deur gebruik te maak van die de novo samesteller Velvet. Hierdie konstrukte is onderwerp aan BLAST soektogte teen die NCBI databasisse en die hoogste trefslag-telling is gebruik vir virus identifikasie. Op grond van die "BLAST" resultate is geskikte volgordes geselekteer vanaf die NCBI databasis en gebruik as verwysingvolgordes in MAQ kartering-analises. Met die bioinfomatika analises kon die virus spesies teenwoordig, asook die mees prominente variante en relatiewe voorkoms van elk, bepaal word. Vier bekende virus wingerdpatogene is geidentifiseer. Grapevine leafroll-associated virus 3, verteenwoordig deur 59% van die geanaliseerde kort-reeks volgorde data, is identifiseer as die mees prominente virus spesie. Drie variante van die virus is in die wingerdmonster opgespoor: GP18 kom die mees algemeen voor, gevolg deur 'n CL-766/NY1 variant en 'n potensiele nuwe wingerd rolblad-geassosieerde ampelovirus. 'n Enkele Grapevine rupestris stem pitting-associated virus variant, soortgelyk aan SG1, en 'n Grapevine virus A variant, 'n lid van molekulere groep III, is geidentifiseer. Hierdie studie is ook die eerste om die teenwoordigheid van Grapevine virus E (GVE) in Suid-Afrikaanse wingerde te rapporteer. Grapevine virus E is verder geneties gekarakteriseer en die genoomvolgorde van GVE isolaat SA94 is bepaal. Die GVE SA94 genoomvolgorde, 7568 nukleotiede lank, is die eerste volledige genoomvolgorde vir hierdie virus spesie. Die genoomorganisasie is tipies van vitivirusse, maar in kontras met ander RNA virusse is die AlkB domein binne-in die helikase domein van oopleesraam 1 (ORF 1) geleë. Grapevine virus E SA94 deel byna 100% nukleotied identiteit met die Japannese TvP15 isolaat en GVE 3404, 'n de novo konstruk gegenereer vanaf die metagenomiese volgorde data. Bioinformatika analises van die metagenomiese volgorde data het verder die teenwoordigheid van drie swam-infekterende virus families, die Chrysoviridae, Totiviridae en ongeklassifiseerde dubbelstring-RNS virus, Fusarium graminearum dsRNA mycovirus 4, aangetoon. 'n Virus van die Chrysoviridae familie, soortgelyk aan Penicillium chrysogenum virus, het die tweede meeste voorgekom in die wingerd monster. Hierdie studie demonstreer die suksesvolle toepassing van 'n kort reeks volgorde-bepalingstegnologie soos die Illumina platform, vir die opstel van 'n virusprofiel van 'n geinfekteerde wingerd. Sover ons kennis strek is hierdie die eerste aanwending van die Illumina tegnologie vir hierdie doel.

Orientador: Erick da Cruz Castelli / Resumo: O Complexo Principal de Histocompatibilidade (MHC) é um complexo gênico que está intimamente envolvido com a regulação do sistema imune. Esse complexo comporta o sistema de Antígenos Leucocitários Humano (HLA), cuja principal importância está relacionada com o reconhecimento do que é próprio ou não do organismo. HLA-C é o gene polimórfico menos variável dos genes HLA clássicos e o que tem menor expressão nos tecidos, exceto na interface materno-fetal, em que é o único gene clássico expresso. A molécula codificada por esse gene possui significante função na apresentação antigênica e regulação da atividade de células NK, o que permite uma íntima associação com situações fisiológicas, como gestação, e patológicas, como doenças infecciosas, autoimunes, inflamatórias, neoplasias e rejeições a enxertos transplantados. Sua porção gênica mais estudada é a que codifica a fenda de ligação a peptídeos antigênicos, devido sua destacada importância na apresentação de antígenos a células T citotóxicas. No entanto, outras regiões do gene, que são negligenciadas nos estudos de variabilidade, também merecem destaque por influenciarem na sinalização e modulação da citotoxicidade de células efetoras, na ancoragem e estabilidade da molécula na membrana plasmática e na internalização e reciclagem da molécula HLA-C. Desta maneira, nós exploramos a variabilidade dos segmentos que codificam α3 (éxon 4), transmembrana (éxon 5) and cauda citoplasmática (éxon 6 and éxon 7) da molécula HLA-C em uma popu... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Major Histocompatibility Complex (MHC) is a gene complex closely involved in the regulation of the immune system. This complex includes the Human Leukocyte Antigen (HLA) system, whose main role is related to the recognition of self/non-self structures of humans. HLA-C is the least variable polymorphic gene of classical HLA genes and has the lowest expression in tissues, except at the maternal-fetal interface, where it is the only classical HLA class I expressed gene. The molecule encoded by this gene has a significant role in the antigen presentation and regulation of NK cells activities, which allows an intimate association with physiological conditions, such as pregnancy, and pathological conditions like infectious, autoimmune, and inflammatory diseases, cancer, and transplantation rejection. The most studied HLA-C portion is that encoding the peptide-binding groove, due to its outstanding importance in presentation of antigens to cytotoxic T cells. However, other regions of the gene, which are neglected in the variability studies, are also important in influencing the signaling and modulation of effector cell cytotoxicity, in the anchorage and stability of the molecule on the cell surface, and in the internalization and recycling of the HLA-C molecule. Here, we explore the variability of the segments encoding the α3 (exon 4), transmembrane (exon 5) and cytoplasmic tail (exon 6 and exon 7) domains of the HLA-C molecule in an admixed population sample from Southeastern B... (Complete abstract click electronic access below) / Mestre

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