The predominant nuclear fuel used globally at present is uranium which is a finite resource. Thorium has been identified as an alternative nuclear fuel source that can be utilized in almost all existing uranium–based reactors and can significantly help in conserving limited uranium reserves. Furthermore, the elimination of proliferation risks associated with thorium–based fuel cycles is a key reason for re–evaluating the possible utilization of thorium in high temperature reactors. In addition to the many advantages that thorium–based fuel has over uranium–based fuel, there are vast thorium resources in the earth's crust that up until the present have not been exploited optimally. This study focuses on determining the amount of uranium ore that can be saved using thorium as a nuclear fuel in HTR's. Four identical 200 MWth high temperature reactors are considered which make use of different fuel cycles. These fuel cycles range from the conventional uranium fuel cycle to a thorium–based fuel cycle in which no U–238 is present and have been simulated using the VSOP–A system of computer codes. This study also considers the effect that protactinium, an isotope that occurs in thorium–based fuel cycles, will have on the decay heat production in the case of a depressurized loss of coolant (DLOFC) accident. / Thesis (M.Ing. (Nuclear Engineering))--North-West University, Potchefstroom Campus, 2011.

The predominant nuclear fuel used globally at present is uranium which is a finite resource. Thorium has been identified as an alternative nuclear fuel source that can be utilized in almost all existing uranium–based reactors and can significantly help in conserving limited uranium reserves. Furthermore, the elimination of proliferation risks associated with thorium–based fuel cycles is a key reason for re–evaluating the possible utilization of thorium in high temperature reactors. In addition to the many advantages that thorium–based fuel has over uranium–based fuel, there are vast thorium resources in the earth's crust that up until the present have not been exploited optimally. This study focuses on determining the amount of uranium ore that can be saved using thorium as a nuclear fuel in HTR's. Four identical 200 MWth high temperature reactors are considered which make use of different fuel cycles. These fuel cycles range from the conventional uranium fuel cycle to a thorium–based fuel cycle in which no U–238 is present and have been simulated using the VSOP–A system of computer codes. This study also considers the effect that protactinium, an isotope that occurs in thorium–based fuel cycles, will have on the decay heat production in the case of a depressurized loss of coolant (DLOFC) accident. / Thesis (M.Ing. (Nuclear Engineering))--North-West University, Potchefstroom Campus, 2011.

The aim of this study is to investigate the resemblance between the developmental stages and developmental tasks of the seven chakras (Judith, 1996) and Erik Erikson’s developmental theory (Bee, 1996). The researcher aims to describe the similarities between the chakra system and developmental psychology. The discussion of literature was divided into two components. Firstly, Stages 1-8 of Erikson’s developmental theory were discussed. Secondly, the chakra system was discussed. The researcher discussed the following concepts to explain the term chakra system: the human and the body; chakra definitions and associations, chakra names, and the colours associated with the chakras. For the purposes of this study, the functions of the separate chakras and the development of the chakra system were focal points. The study was done at a private yoga organisation in Durbanville, Cape Town, where the participants of the study regularly took part in classes. These participants were chosen because of their reliability (adults and regular participation) and their receptivity for alternative healing. Their age and gender was also relevant to the study, because the researcher used non-probability sampling. The main prerequisites for individuals to participate in the study were regular attendance and the individuals’ status as adults. / Dissertation (MA(Couns Psych))--University of Pretoria, 2005. / Psychology / unrestricted

In hierdie ondersoek is daar gepoog om die belange van die minderjarige kind in egskeiding te bevorder deur die egskeidingsevalueringsproses waarvolgens die belange van die kind bepaal word, te ondersoek en te beskryf. Die sisteemteoretiese benadering, wat op holisme as uitgangspunt gebaseer is, is gedefinieer en beskryf ten einde die sistemiese perspektief op die gesin-in-egskeiding uit te klaar. Voorts is egskeiding as internasionale fenomeen en die implikasies van egskeidingsdispute vir die minderjarige kind ondersoek en in terme van die juridiese en maatskaplike implikasies asook die beginsel van die “beste belang” van die kind beskryf en verduidelik. Navorser het `n selfontwikkelde praktykmodel vir egskeidingsevaluering ontwikkel en geëvalueer met die doel om die vasstel van die belange van die minderjarige kind in egskeiding te bevorder. Dit is gevolg deur die empiriese resultate, `n algemene opsomming, gevolgtrekkings en aanbevelings. Die volgende navorsingsvraag is geformuleer om die ondersoekende en beskrywende gedeelte van die empiriese navorsing te rig: Wat is die aard en inhoud van die egskeidingsevalueringsproses wat in die praktyk deur die Kantoor van die Gesinsadvokaat toegepas word ten einde die belange van die minderjarige kind te bevorder? Gelei deur die navorsingsvraag is die volgende hipotese geformuleer: Indien die sisteemteoretiese praktykmodel tydens egskeidingsdispute toegepas word, sal die beste belang van die minderjarige kind bevorder word. Vir hierdie ondersoek, waar twee evalueringsprosesse met mekaar vergelyk is, was die kwantitatiewe navorsingsbenadering die aangewese keuse. Intervensienavorsing as tipe navorsing is volgens die Ontwerp- en Ontwikkelingsmodel toegepas. Die kwantitatief-beskrywende opname navorsingsontwerp is benut ten einde die doel van die studie te bereik, naamlik: <ul>Om `n praktykmodel vir egskeidingsevaluering te ontwikkel en empiries te evalueer ten einde die beste belang van die minderjarige kind te bevorder.</li></ul> Voortspruitend uit die doel is die volgende doelwitte geformuleer: <ul> <li>Om vanuit `n sisteemteoretiese perspektief egskeiding as maatskaplike verskynsel, die gesin as maatskaplike sisteem en die belange van die minderjarige kind, teoreties te konseptualiseer.</li> <li>Om die huidige egskeidingsevalueringsproses, soos deur die Kantoor van die Gesinsadvokaat toegepas, te ondersoek en te beskryf.</li> <li>Om `n praktykmodel vir egskeidingsevaluering vanuit `n sisteemteoretiese perspektief, wat die belange van die minderjarige kind bevorder, te ontwikkel.</li> <li>Om die effektiwiteit van die nuut ontwikkelde praktykmodel vir egskeidingsevaluering empiries te evalueer.</li> <li>Om aanbevelings te maak ten opsigte van die praktykbenutting van die nuut ontwikkelde praktykmodel om die belange van die minderjarige kind in egskeidingsevaluering te bevorder.</li></ul> Ten einde die heersende evalueringsproses wat deur die Kantoor van die Gesinsadvokaat in egskeidingsdispute toegepas word met die evalueringsproses soos deur die nuut ontwikkelde praktykmodel toegepas is, te kon vergelyk, het navorser `n vraelys ontwerp vir evaluering van beide prosesse. Die vraelys het uit die volgende drie afdeling bestaan: (1) die professionele profiel van die deskundige (gesinsraadgewer/navorser); (2) profiel van die gesinsisteem in egskeiding wat geëvalueer is; en (3) die aard en inhoud van die evalueringsproses vir egskeidingsdispute. Voltooiing van die vraelys deur die deskundiges wat die twee egskeidingsevalueringsprosesse toegepas het, het meegebring dat die data vergelykbaar was. Daarvolgens kon die twee evalueringsprosesse dus vergelyk word en die effektiwiteit van die nuut ontwikkelde praktykmodel empiries geëvalueer word. Die empiriese resultate ten opsigte van die toepassing van die twee egskeidingsevalueringsprosesse in die praktyk, het `n wesenlike onderskeid in die aard en inhoud van die twee prosesse met betrekking tot die beste belang van die minderjarige kind aangetoon. Dit het geblyk dat in die toepassing van die egskeidingsevalueringsproses deur die Kantoor van die Gesinsadvokaat die assessering van die behoeftes, wense, ontwikkelingsvlak, verhoudings asook sistemiese verbintenisse van die minderjarige kind nie prioriteit geniet nie. Tydsbesteding aan konsultasies met die minderjarige kind is baie beperk en is slegs op `n ad hoc basis uitgevoer. Kollaterale bronne is ook op `n ad hoc basis betrek sodat dit nie moontlik was om die sistemiese verbintenisse van die minderjarige kind te eksploreer nie. Die evalueringsproses soos deur die nuut ontwikkelde praktykmodel toegepas is, bepaal as `n standaard prosedure individuele konsultasies vir assesseringsdoeleindes met die minderjarige kind ten einde die wense, behoeftes, ontwikkelingsvlak, verhoudings en sistemiese verbintenisse te kan bepaal met die oog op die vasstelling van die beste belang van die minderjarige kind vir die post-egskeidingsfase. Voldoende tydsbesteding aan die assessering van die minderjarige kind het hoë prioriteit volgens die praktykmodel geniet. Die evalueringsproses volgens die nuut ontwikkelde praktykmodel het kollaterale bronne as `n standaard prosedure betrek sodat die sistemiese verbintenisse van die minderjarige kind volledig geëksploreer kon word. Dit het dus uit die empiriese resultate geblyk dat die hipotese wat vir die studie gestel was, bevestig is. ENGLISH The aim of this study was to promote the interests of the minor child in a divorce situation, through an investigation into the process of assessment and evaluation whereby the best interests of the minor child in divorce disputes are determined. The systems theory approach, which sees holism as basic principle, was defined and described to explain the systems perspective of the family system in a divorce situation. Divorce as an international phenomenon and the implications of divorce disputes on the minor child were investigated, described and explained according to the legal and social implications as well as the principle of “the best interests” of the child. The researcher presented a self-developed practice model for divorce evaluation with the aim to promote the best interests of the minor child in divorce disputes. The study was concluded with the empirical results, a general summation, conclusions and recommendations. One research question was formulated to guide the investigative and descriptive part of the study, namely: What is the nature and content of the process of evaluation in divorce disputes to determine the best interests of the minor child which is administered by the Office of the Family Advocate? The following hypothesis was formulated according to the research question: If the practice model in divorce evaluation, based on systems theory, is administered in divorce disputes, the best interests of the minor child will be promoted. The quantitative research approach was selected in this investigation where two processes of evaluation were to be compared and the type of research was identified as Intervention Design and Development. The quantitative-descriptive survey design was selected to achieve the following goal of the study, namely: <ul> <li> To develop a practice model for evaluation in divorce and to evaluate the practice model empirical to promote the best interests of the minor child</li></ul> In order to obtain the goal of the study the following objectives were formulated: <ul> <li>To conceptualise theoretically divorce as social phenomenon, the family as social system and the interests of the minor child from a systems perspective.</li> <li>To investigate and describe the current process of evaluation in divorce disputes as it is administered by the Office of the Family Advocate.</li> <li>To develop a practice model for evaluation in divorce disputes from a systems perspective to promote the interests of the minor child.</li> <li>To evaluate empirically the effectiveness of the newly developed practice model for evaluation in divorce disputes.</li> <li>To make recommendations regarding the implementation of the newly developed practice model to promote the interests of the minor child in divorce disputes.</li></ul> A questionnaire was developed by the researcher to enable the comparison of the two processes of evaluation in divorce disputes. The questionnaire had three sections, namely: (1) the professional profile of the expert investigator (family counsellor/researcher); (2) profile of the family system who was evaluated; and (3) the nature and content of the process of evaluation. Data from the questionnaires completed by the expert investigators was compared to enable researcher to evaluate the effectiveness of the newly developed practice model to promote the interests of the minor child. The empirical results from the two evaluation processes confirmed a distinct difference in the nature and content of the two processes. The evaluation process in divorce as done by the Office of the Family Advocate did not put emphasis on the assessment of the needs, wishes, developmental status, relationships or systemic bonds of the minor child in a divorce situation. The time allocated to assessment of the minor child is extremely limited and consultations with the minor child was only done on an ad hoc basis. Consultations with collateral sources were also done on an ad hoc basis. It was thus not possible for family counsellors to explore in-depth the systemic relationships of the minor child in a divorce situation. The evaluation process according to the new practice model recognised the importance of individual consultations with the minor child. Individual assessment of the minor child was done as a regular procedure to establish the needs, wishes, developmental status, relationships as well as the systemic associations of the minor child in a divorce situation. The practice model recognised the need for adequate time allocation for assessment of the minor child. Exploring collateral sources was done as a standard procedure according to the practice model to allow opportunity for assessment of the systemic bonds of the minor child. The empirical results confirmed the hypothesis which was developed for this study. / Thesis (DPhil)--University of Pretoria, 2008. / Social Work and Criminology / unrestricted

The technique of twelve-tone serialism was developed by Schoenberg to give structure to atonal compositions. The writer was interested in the fact that Dallapiccola’s twelve-tone compositions, as with Berg’s, were easier received by the general public than the works of other serial composers of the time. The reason being that his twelve-tone rows contain tonal elements. The boundaries of the field of study were determined in chapter 1. Terminology and problems encountered with the study were also discussed. To be able to analyse the tonal aspects of Dallapiccola’s rows, the basic outlines of tonality were clearly defined in chapter 2. It was found that tonal suggestions could be achieved by means of using: * tones that form part of a diatonic scale, * explicit triads, * implicit triads, * scale constructions and * tones that outline the tonic. The different types of twelve-tone rows were discussed and illustrated by means of music examples in chapter 3. A list of published works as well as a short discussion of each composition followed in chapter 4. Mention was made whether a work was tonal, atonal or a twelve-tone composition and Dallapiccola’s use of serial procedures in each composition was discussed. Chapter 5 showed where and how the above criteria were used in his rows. The study thus provides evidence that Dallapiccola had tonal references in mind when planning the rows and that tonal suggestions are common in his works. The twelve-tone rows often employ three, four or five of the criteria of implied tonality in a composition. Of the five criteria, tones that form part of a diatonic scale was used the most. This was discussed with all the rows, except Il prigioniero (row of hope) and Cinque frammenti di Saffo’s first and second tone rows. Dallapiccola used tones to outline the tonic in thirty one twelve-tone rows. Implicit triads were used in all but six works. Scale constructions appeared in nineteen works and Dallapiccola’s love for using a few chromatic notes or three consecutive notes of the implied major scale became apparent. Explicit triads only appeared in thirteen compositions. In the other rows, Dallapiccola created a tonal sense by means of other criteria or had a more chromatic approach. Dallapiccola thus used tonal references in the tone rows of his serial compositions and showed that twelve-tone tonality could be used to create a lyrical idiom. In this way the people that were trained in the tonal school could also appreciate twelve-tone compositions. In the opera Il prigioniero, it is especially evident that the Italian tradition of lyricism and dramatic intensity do exist after verismo composers (Morris 1999:257). / Dissertation (MMus(Performing Art))--University of Pretoria, 2005. / Music / unrestricted

Bipolêre versteuring is ‘n kroniese, herhalende en ernstige siekte met ‘n voorkoms van 1 % wêreldwyd. Pasiënte kan psigoties word, selfmoordideasie hê en ook soms aggressief raak. Dikwels moet hulle gehospitaliseer word - ook teen hulle wil, want hulle mag soms geen insig toon nie. Pasiënte mag presenteer met ‘n ko-morbiede toestand, byvoorbeeld substansmisbruik of ‘n persoonlikheidsversteuring. Dit gebeur dikwels dat hulle nie hulle toegewysde rolle, byvoorbeeld dié van gade, broodwinner, ouer of volwasse kind, kan vervul nie. Versorgers van pasiënte gediagnoseer met bipolêre versteuring, word eweneens beïnvloed deur die siekte en die gepaardgaande stigma daarvan. Dit kan vir hulle baie moeilik wees wanneer hulle die verantwoordelikhede en rolle van die pasiënt moet oorneem. Beskou vanuit die oogpunt van ‘n ekologiese sisteemperspektief is die aard van hierdie impak op die maatskaplike funksionering van die versorgers relevant vir maatskaplike werk in die geestesgesondheidsveld. Die verkenning van hierdie impak en die ontwikkeling van ‘n intervensie om die impak aan te spreek het dus die rasionaal van hierdie studie gevorm. Die geïntegreerde biopsigososiale model, binne ‘n ekologiese sisteem perspektief, word aangewend by die psigiatriese instelling waar die navorser werk. Hierdie benadering het dus ook gedien as die vertrekpunt vir die betrokke studie. Die gemengde-metode navorsingsbenadering is tydens hierdie studie benut ten einde ‘n uitgebreide analise van die navorsingsprobleem te verskaf. Die voorgenome studie het binne die kategorie van toegepaste navorsing geval, want die doel daarvan was om ‘n program te ontwikkel om hulp te verleen aan versorgers van individue gediagnoseer met bipolêre versteuring. Die navorser het haar navorsing gerig aan die hand van Rothman en Thomas (1994) se intervensie ontwerp en ontwikkelingsmodel (D&D) deur gebruik te maak van kwalitatiewe en kwantitatiewe metodes. Na afloop van die analise van die kwalitatiewe data, is ‘n unieke psigo-maatskaplike opvoedkundige program, die SEE-SAW program, ontwikkel en toe geïmplementeer. Die konsep van balans versus wanbalans binne die sisteem is essensieel; die navorser se program het ten doel gehad om beter balans binne die sisteem van die pasiënt en versorger te bewerkstellig. Die gevolgtrekkings het duidelik getoon dat die versorger van die bipolêre pasiënt blootgestel is aan ‘n wye spectrum van behoeftes en uitdagings, maar dat intervensies soos die SEE-SAW program van groot waarde mag wees. / Bipolar disorder (BD) is a chronic, recurrent, serious illness that occurs in 1 % of people globally. Patients might become psychotic, suicidal and sometimes violent. They often need to be hospitalised, even against their will because they may have no insight. Patients may have a comorbid condition such as substance abuse or a personality disorder. They are frequently unable to fulfil their assigned roles of spouse, breadwinner, parent or adult child. Caregivers of patients diagnosed with BD are also affected by the illness and the associated stigma. It becomes very difficult for them when they have to take over the responsibilities and roles of the patient. The nature of this impact on the social functioning of the caregivers, understood from an ecological systems perspective, is relevant for social work in the mental health care field. Discovering more about this impact and developing an intervention to curb it therefore formed the rationale for this study. The integrated biopsychosocial model, within an ecological systems perspective, is being used at the psychiatric institution where the researcher works. This approach therefore also served as the point of departure from which this research was conducted. A mixed methods research approach was utilised to provide a comprehensive analysis of the research problem. The proposed study fell into the category of applied research, due to its aim of designing a programme to assist caregivers of people diagnosed with BD. The researcher directed the study according to Rothman and Thomas’s (1994) intervention design and development model (D&D), using qualitative and quantitative methods. After the analysis of the qualitative data a unique psychosocial educational programme, the SEE-SAW programme, was developed and then implemented. The concept of equilibrium versus disequilibrium in the system is central; the researcher’s programme therefore strove towards better equilibrium within the system of patient and caregiver. From the conclusions it is apparent that the caregiver of the BD patient is exposed to a wide spectrum of needs and challenges, but that interventions such as the SEE-SAW programme may be very helpful. / Thesis (PhD)--University of Pretoria, 2014. / Social Work and Criminology / PhD (Social Work) / Unrestricted

Text in Afrikaans / Hierdie studie kontrasteer die Newtoniaanse denkwyse en die konstruktivisme as vertrekpunt. Die beginsels van reduksionisme, neutraliteit en liniere oorsaaklikheid waarop die Newtoniaanse denke gefundeer is, word gekontrasteer met die sirkulariteit en non-objektiwiteit van interveiwante elemente binne die lewende sisteem. Die verskille in aannames van personeel van 'n welsynsvereniging word ondersoek aan die hand van observasie van prosesse van veranderende denke wat oor 'n periode ontwikkel het. Die proses van verandering word aan die hand van 'n gevallestudie bespreek. Binne die veranderende denke, is erkenning verleen aan die sistemiese orientasie waar die interaktiewe intervetwantskap van die elemente binne die sisteem ter sprake raak. Die lewende sisteem en die kenmerke van outonomie, outopo"ise, organisasie en struktuur word binne die filosofie van konstruktivisme bespreek. Uit hierdie filosofie evolueer 'n proses waar binne realiteite geskep word, konsensus binne die sisteem verkry word, en die organisasie van die lewende sisteem reflekteer word / This study focusses on the contrasting thinking patterns of Newtonian thinking and constructivism. The principles of reductionism, neutrality and linear causality on which Newtonian thinking is grounded are compared with circular processes and non objectivity of interrelated elements that are to be found in living systems. The differences in assumptions of personnel in a welfare organisation are investigated through observation of changing processes of thinking that evolved over a period of time. The process of change is discussed by way of a case study. Recognition is given to the systemic orientation where the interactive interrelatedness of elements within the system evolved within changing patterns of thinking. The living system and characteristics of autonomy, autopoiesis, organisation and structure are discussed from a philosophy of constructivism. From this philosophy evolved a process in which realities were created, consensus was reached within the system, and the organisation of the living system was reflected. / Social Work / M.A. (Sosiale Wetenskappe(Geestesgesondheid))

Text in Afrikaans / Hierdie studie kontrasteer die Newtoniaanse denkwyse en die konstruktivisme as vertrekpunt. Die beginsels van reduksionisme, neutraliteit en liniere oorsaaklikheid waarop die Newtoniaanse denke gefundeer is, word gekontrasteer met die sirkulariteit en non-objektiwiteit van interveiwante elemente binne die lewende sisteem. Die verskille in aannames van personeel van 'n welsynsvereniging word ondersoek aan die hand van observasie van prosesse van veranderende denke wat oor 'n periode ontwikkel het. Die proses van verandering word aan die hand van 'n gevallestudie bespreek. Binne die veranderende denke, is erkenning verleen aan die sistemiese orientasie waar die interaktiewe intervetwantskap van die elemente binne die sisteem ter sprake raak. Die lewende sisteem en die kenmerke van outonomie, outopo"ise, organisasie en struktuur word binne die filosofie van konstruktivisme bespreek. Uit hierdie filosofie evolueer 'n proses waar binne realiteite geskep word, konsensus binne die sisteem verkry word, en die organisasie van die lewende sisteem reflekteer word / This study focusses on the contrasting thinking patterns of Newtonian thinking and constructivism. The principles of reductionism, neutrality and linear causality on which Newtonian thinking is grounded are compared with circular processes and non objectivity of interrelated elements that are to be found in living systems. The differences in assumptions of personnel in a welfare organisation are investigated through observation of changing processes of thinking that evolved over a period of time. The process of change is discussed by way of a case study. Recognition is given to the systemic orientation where the interactive interrelatedness of elements within the system evolved within changing patterns of thinking. The living system and characteristics of autonomy, autopoiesis, organisation and structure are discussed from a philosophy of constructivism. From this philosophy evolved a process in which realities were created, consensus was reached within the system, and the organisation of the living system was reflected. / Social Work / M.A. (Sosiale Wetenskappe(Geestesgesondheid))

Reverse genetics is an innovative molecular biology tool that enables the manipulation of viral genomes at the cDNA level in order to generate particular mutants or artificial viruses. The reverse genetics system for the influenza virus is arguably one of the best illustrations of the potential power of this technology. This reverse genetics system is the basis for the ability to regularly adapt influenza vaccines strains. Today, reverse genetic systems have been developed for many animal RNA viruses. Selection-free reverse genetics systems have been developed for the members of the Reoviridae family including, African horsesickness virus, bluetongue virus and orthoreovirus. This ground-breaking technology has led to the generation of valuable evidence regarding the replication and pathogenesis of these viruses. Unfortunately, extrapolating either the plasmid-based or transcript-based reverse genetics systems to rotavirus has not yet been successful. The development of a selection-free rotavirus reverse genetics system will enable the systematic investigation of poorly understood aspects of the rotavirus replication cycle and aid the development of more effective vaccines, amongst other research avenues. This study investigated the importance of co-expressed rotavirus proteins in the development of a selection-free rotavirus reverse genetics system. The consensus sequences of the rotavirus strains Wa (RVA/Human-tc/USA/WaCS/1974/G1P[8]) and SA11 (RVA/Simian-tc/ZAF/SA11/1958/G3P[2]) where used to design rotavirus expression plasmids. The consensus nucleotide sequence of a human rotavirus Wa strain was determined by sequence-independent cDNA synthesis and amplification combined with next-generation 454® pyrosequencing. A total of 4 novel nucleotide changes, which also resulted in amino acid changes, were detected in genome segment 7 (NSP3), genome segment 9 (VP7) and genome segment 10 (NSP4). In silico analysis indicated that none of the detected nucleotide changes, and consequent amino acid variations, had any significant effect on viral structure. Evolutionary analysis indicated that the sequenced rotavirus WaCS was closely related to the ParWa and VirWa variants, which were derived from the original 1974 Wa isolate. Despite serial passaging in animals, as well as cell cultures, the Wa genome seems to be stable. Considering that the current reference sequence for the Wa strain is a composite sequence of various Wa variants, the rotavirus WaCS may be a more appropriate reference sequence. The rotavirus Wa and SA11 strains were selected for plasmid-based expression of rotavirus proteins, under control of a T7 promoter sequence, due to the fact that they propagate well in MA104 cells and the availability of their consensus sequences. The T7 RNA polymerase was provided by a recombinant fowlpox virus. After extensive transfection optimisation on a variety of mammalian cell lines, MA104 cells proved to be the best suited for the expression rotavirus proteins from plasmids. The expression of rotavirus Wa and SA11 VP1, VP6, NSP2 and NSP5 could be confirmed with immunostaining in MA104 and HEK 293H cells. Another approach involved the codon-optimised expression of the rotavirus replication complex scaffold in MA104 cells under the control of a CMV promoter sequence. This system was independent from the recombinant fowlpox virus. All three plasmid expression sets were designed to be used in combination with the transcript-based reverse genetics system in order to improve the odds of developing a successful rotavirus reverse genetics system. Rotavirus transcripts were generated using transcriptively active rotavirus SA11 double layered particles (DLPs). MA104 and HEK293H cells proved to be the best suited for the expression of rotavirus transcripts although expression of rotavirus VP6 could be demonstrated in all cell cultures examined (MA104, HEK 293H, BSR and COS-7) using immunostaining. In addition, the expression of transcript derived rotavirus VP1, NSP2 and NSP5 could be confirmed with immunofluorescence in MA104 and HEK 293H cells. This is the first report of rotavirus transcripts being translated in cultured cells. A peculiar cell death pattern was observed within 24 hours in response to transfection of rotavirus transcripts. This observed cell death, however does not seem to be related to normal viral cytopathic effect as no viable rotavirus could be recovered. In an effort to combine the transcript- and plasmid systems, a dual transfection strategy was followed where plasmids encoding rotavirus proteins were transfected first followed, 12 hours later, by the transfection of rotavirus SA11 transcripts. The codon- optimised plasmid system was designed as it was postulated that expression of the DLP-complex (VP1, VP2, VP3 and VP6), the rotavirus replication complex would form and assist with replication and/or packaging. Transfecting codon- optimized plasmids first noticeably delayed the mass cell death observed when transfecting rotavirus transcripts on their own. None of the examined coexpression systems were able to produce a viable rotavirus. Finally, the innate immune responses elicited by rotavirus transcripts and plasmid-derived rotavirus Wa and SA11 proteins were investigated. Quantitative RT-PCR (qRT-PCR) experiments indicated that rotavirus transcripts induced high levels of the expression of the cytokines IFN- α1, IFN-1β, IFN-λ1 and CXCL10. The expression of certain viral proteins from plasmids (VP3, VP7 and NSP5/6) was more likely to stimulate specific interferon responses, while other viral proteins (VP1, VP2, VP4 and NSP1) seem to be able to actively suppress the expression of certain cytokines. In the light of these suppression results, specific rotavirus proteins were expressed from transfected plasmids to investigate their potential in supressing the interferon responses provoked by rotavirus transcripts. qRT-PCR results indicated that cells transfected with the plasmids encoding NSP1, NSP2 or a combination of NSP2 and NSP5 significantly reduced the expression of specific cytokines induced by rotavirus transcripts. These findings point to other possible viral innate suppression mechanisms in addition to the degradation of interferon regulatory factors by NSP1. The suppression of the strong innate immune response elicited by rotavirus transcripts might well prove to be vital in the quest to better understand the replication cycle of this virus and eventually lead to the development of a selection-free reverse genetics system for rotavirus. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2014

Reverse genetics is an innovative molecular biology tool that enables the manipulation of viral genomes at the cDNA level in order to generate particular mutants or artificial viruses. The reverse genetics system for the influenza virus is arguably one of the best illustrations of the potential power of this technology. This reverse genetics system is the basis for the ability to regularly adapt influenza vaccines strains. Today, reverse genetic systems have been developed for many animal RNA viruses. Selection-free reverse genetics systems have been developed for the members of the Reoviridae family including, African horsesickness virus, bluetongue virus and orthoreovirus. This ground-breaking technology has led to the generation of valuable evidence regarding the replication and pathogenesis of these viruses. Unfortunately, extrapolating either the plasmid-based or transcript-based reverse genetics systems to rotavirus has not yet been successful. The development of a selection-free rotavirus reverse genetics system will enable the systematic investigation of poorly understood aspects of the rotavirus replication cycle and aid the development of more effective vaccines, amongst other research avenues. This study investigated the importance of co-expressed rotavirus proteins in the development of a selection-free rotavirus reverse genetics system. The consensus sequences of the rotavirus strains Wa (RVA/Human-tc/USA/WaCS/1974/G1P[8]) and SA11 (RVA/Simian-tc/ZAF/SA11/1958/G3P[2]) where used to design rotavirus expression plasmids. The consensus nucleotide sequence of a human rotavirus Wa strain was determined by sequence-independent cDNA synthesis and amplification combined with next-generation 454® pyrosequencing. A total of 4 novel nucleotide changes, which also resulted in amino acid changes, were detected in genome segment 7 (NSP3), genome segment 9 (VP7) and genome segment 10 (NSP4). In silico analysis indicated that none of the detected nucleotide changes, and consequent amino acid variations, had any significant effect on viral structure. Evolutionary analysis indicated that the sequenced rotavirus WaCS was closely related to the ParWa and VirWa variants, which were derived from the original 1974 Wa isolate. Despite serial passaging in animals, as well as cell cultures, the Wa genome seems to be stable. Considering that the current reference sequence for the Wa strain is a composite sequence of various Wa variants, the rotavirus WaCS may be a more appropriate reference sequence. The rotavirus Wa and SA11 strains were selected for plasmid-based expression of rotavirus proteins, under control of a T7 promoter sequence, due to the fact that they propagate well in MA104 cells and the availability of their consensus sequences. The T7 RNA polymerase was provided by a recombinant fowlpox virus. After extensive transfection optimisation on a variety of mammalian cell lines, MA104 cells proved to be the best suited for the expression rotavirus proteins from plasmids. The expression of rotavirus Wa and SA11 VP1, VP6, NSP2 and NSP5 could be confirmed with immunostaining in MA104 and HEK 293H cells. Another approach involved the codon-optimised expression of the rotavirus replication complex scaffold in MA104 cells under the control of a CMV promoter sequence. This system was independent from the recombinant fowlpox virus. All three plasmid expression sets were designed to be used in combination with the transcript-based reverse genetics system in order to improve the odds of developing a successful rotavirus reverse genetics system. Rotavirus transcripts were generated using transcriptively active rotavirus SA11 double layered particles (DLPs). MA104 and HEK293H cells proved to be the best suited for the expression of rotavirus transcripts although expression of rotavirus VP6 could be demonstrated in all cell cultures examined (MA104, HEK 293H, BSR and COS-7) using immunostaining. In addition, the expression of transcript derived rotavirus VP1, NSP2 and NSP5 could be confirmed with immunofluorescence in MA104 and HEK 293H cells. This is the first report of rotavirus transcripts being translated in cultured cells. A peculiar cell death pattern was observed within 24 hours in response to transfection of rotavirus transcripts. This observed cell death, however does not seem to be related to normal viral cytopathic effect as no viable rotavirus could be recovered. In an effort to combine the transcript- and plasmid systems, a dual transfection strategy was followed where plasmids encoding rotavirus proteins were transfected first followed, 12 hours later, by the transfection of rotavirus SA11 transcripts. The codon- optimised plasmid system was designed as it was postulated that expression of the DLP-complex (VP1, VP2, VP3 and VP6), the rotavirus replication complex would form and assist with replication and/or packaging. Transfecting codon- optimized plasmids first noticeably delayed the mass cell death observed when transfecting rotavirus transcripts on their own. None of the examined coexpression systems were able to produce a viable rotavirus. Finally, the innate immune responses elicited by rotavirus transcripts and plasmid-derived rotavirus Wa and SA11 proteins were investigated. Quantitative RT-PCR (qRT-PCR) experiments indicated that rotavirus transcripts induced high levels of the expression of the cytokines IFN- α1, IFN-1β, IFN-λ1 and CXCL10. The expression of certain viral proteins from plasmids (VP3, VP7 and NSP5/6) was more likely to stimulate specific interferon responses, while other viral proteins (VP1, VP2, VP4 and NSP1) seem to be able to actively suppress the expression of certain cytokines. In the light of these suppression results, specific rotavirus proteins were expressed from transfected plasmids to investigate their potential in supressing the interferon responses provoked by rotavirus transcripts. qRT-PCR results indicated that cells transfected with the plasmids encoding NSP1, NSP2 or a combination of NSP2 and NSP5 significantly reduced the expression of specific cytokines induced by rotavirus transcripts. These findings point to other possible viral innate suppression mechanisms in addition to the degradation of interferon regulatory factors by NSP1. The suppression of the strong innate immune response elicited by rotavirus transcripts might well prove to be vital in the quest to better understand the replication cycle of this virus and eventually lead to the development of a selection-free reverse genetics system for rotavirus. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2014