Akademisk avhandling--Filosofie doktorsexamen--Uppsala, 1984. / Résumé en anglais.

Diss. Ph. D.--Stockholm university, 1993.

Thèse--Bruxelles, 1973. / Bibliogr. p. 178-183. Index.

Texte remanié de: Tesis doctoral--Universidad complutense de Madrid, 1994-95. Titre de soutenance : El grupo Ah Canul de la ciudad maya yucateca de Oxkintok. / Bibliogr. p. 215-220.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Galactooligosaccharides are produced by the transglycosylation activity of β-galactosidases (β-gal, EC 3.2.1.23) when utilising lactose as a substrate. They have emerged as important constituents used in the food and pharmaceutical industries owing to their prebiotic properties. Although transglycosylation was discovered in 1951 (Wallenfels 1951), and a number of β-gals have had their transglycosylation activity characterised, the activities of these enzymes are not optimal for industrial use. Their tendency to favour the hydrolytic reaction over the transglycosylation reaction, coupled with the production of shorter chain oligosaccharides has driven scientists to investigate altering protein structure both to increase chain lengths and the amount of oligosaccharide produced at lower substrate concentrations. In an attempt to alter the amount of oligosaccharide produced by a metagenomically derived β-gal belonging to the glycosyl hydrolase 2 family, random and site-directed mutagenesis were used. A randomly mutagenised library was screened on SOB agar plates containing 5% (w/v) lactose which should select for clones that synthesise oligosaccharides at relatively low concentrations. No such activity was detected. Site-directed mutagenesis was also utilised to alter protein structure. It was confirmed that the β-gal utilised in this study belonged to the glycosyl hydrolase 2 family through mutation of the predicted catalytic acid/base glutamic acid to a non-catalytic residue, thus removing activity. Another mutation was utilised to investigate if it was possible to increase the degree of polymerisation of oligosaccharides produced by the β-gal. This mutation was successful in increasing the degree of polymerisation. Biochemical characterisation of the β-gal revealed that it exhibited optimal activity at pH 8.0, with a temperature optimum of 30°C. The β-gal exhibited a Km and Vmax of 54.23 mM and 2.26 μmol/minute-1/mg protein-1 respectively, similar to kinetic parameters that have been determined for a number of previously characterised enzymes. / AFRIKAANSE OPSOMMING: Galaktooligosakkariede word geproduseer deur die transglikosileering aktiwiteit van β-galaktosidase (β-gal, EG 3.2.1.23) wanneer hulle laktose as 'n substraat gebruik. Hierdie oligosakkariede het na vore gekom as 'n belangrike bestandeel vir gebruik in die voedsel en farmaseutiese bedryf as gevolg van hulle prebiotiese eienskappe. Alhoewel transglycosylation al in 1951 ontdek is (Wallenfels 1951) en 'n aantal β-gals se transglycosylation aktiwiteit gekenmerk is, is hierdie ensieme nie ideaal vir industriële toepassings nie. Die geneigdheid om die hidrolitiese reaksie oor die transglycosylation reaksie bevoordeel, tesame met die produksie van korter oligosakkariede het wetenskaplikes ondersoek genoop om die proteïenstruktuur te verander om ketting-lengte en die kwantiteit van oligosakkaried geproduseer teen laer substraat konsentrasies te verhoog. In 'n poging om die opbrengs van die oligosakkaried wat deur 'n metagenomiese β-gal wat aan die glycosyl hidrolase 2 familie behoort te verander, is lukraak en terrein gerigte-mutagenese gebruik. Die mutagenese biblioteek is op SOB agarplate met 5% (w/v) lactose gekeur, om klone wat die fenotipe wat verband hou met die produksie oligosakkaried teen relatiewe lae konsentrasies te selekteer. Geen aktiwiteit is opgemerk nie. Terrein gerigte-mutagenese is ook gebruik om die proteïenstruktuur te verander. Deur ‘n bioinformatiese voorspelling, is dit bevestig dat die β-gal wat in hiedie studie gebruik word tot die glycosyl hidrolase 2 familie behoort. Dit is gedoen deur mutasie van die voorspelde katalitiese suur/basis glutamiensuur na 'n nie-katalitiese oorskot, dus die verwydering van aktiwiteit. Nog ‘n mutasie is gebruik om te ondersoek of dit moontlik was om die ketting-lengte van die oligosakkaried wat deur die β-gal geproduseer is te verhoog. Die mutasie was suksesvol in die verhoging van die oligosakkaried wat geproduseer was. Biochemiese karakterisering van die β-gal het getoon dat hierdie β-gal optimale aktiwiteit het by pH 8.0, met 'n optimum temperatuur van 30°C. Die β-gal het 'n Km en Vmax van 54.23 mM en 2.26 μmol/minute-1/mg proteïen-1 onderskeidelik, soortgelyk aan kinetiese parameters wat bepaal word vir ensieme wat voorheen gekenmerk is.

Influenza A is one of the most prevalent and significant viral infections worldwide, resulting in annual epidemics and occasional pandemics. Upon infection, antiviral drugs targeting the neuraminidase protein and M2 protein are the only treatment options available. However, the emergence of antiviral drug resistance is concerning, therefore the aim of this work was to identify inhibitor molecules that may bind to highly conserved regions of selected internal influenza A proteins. Sequences of the non-structural protein 1 (NS1), nuclear export protein (NEP) and polymerase basic protein 2 (PB2) from all hosts and subtypes were aligned and the degree of amino acid conservation was calculated based on Valdar's scoring method. Missing parts of the experimental structures were predicted using the I-TASSER server and ligand binding hot spots were identified with computational solvent mapping. Selected binding sites in conserved regions were subjected to virtual screening against two compound libraries using AutoDock Vina and AutoDock 4. Two out of twelve top hit compounds predicted to target the NS1 protein showed capability of reducing influenza A H1N1 replication in plaque reduction assays at concentrations below 100 μM, although the target protein and mechanism of action could not be confirmed. For the NEP, conservation analysis was based on 3000 sequences and binding hot spots were located in common areas amongst three structures. Docking results revealed predicted binding affinities of up to -8.95 kcal/mol, and conserved amino acid residues interacting with top compounds include Arg42, Asp43, Lys39, Ile80, Gln101, Leu105, and Val109. For the PB2 protein, conservation analysis was based on ~12,000 sequences and fifteen potential binding hot spots were identified. Docking results revealed predicted binding affinities of up to -10.3 kcal/mol, with top molecules interacting with the highly conserved residues Gln138, Gly222, Ile539, Asn540, Gly541, Tyr531 and Thr530. The findings from this research could provide starting points for in vitro experiments, as well as the development of antiviral drugs that function to inhibit influenza A replication without leading to resistance.

γ-aminobutyric acid is the major inhibitory neurotransmitter in the adult mammalian central nervous system (CNS) and may also play a neurotrophic role during CNS development. Diversification of GABA_A receptor mediated responses are in part a result ofvariation in subunit composition in the receptor complex. This variation arises both from the number of different subtypes of GABA_A receptor subunits (α1-6, β1-4, γ1-3, δ1, ρ1-3, ε, ρ), as well as from post-transcriptional processes such as RNA splicing. In this thesis, I have investigated the developmental onset of GABA_A receptor gene expression and the distribution and temporal expression of GABA_A receptor subunit mRNAs and 12 splice variants within the developing and adult murine CNS. Preliminary studies using S 1 nuclease protection analysis demonstrated that α1, β3 and γ2 were the predominant subtypes of GABA_A receptor subunits expressed at embryonic day 14 and in the adult murine CNS. In situ hybridisation analysis demonstrated overlapping but distinct spatial and temporal patterns of GABA_A subunit mRNA expression during postnatal development and in the adult murine CNS. Analysis of γ2 mRNA splice variants demonstrated that the γ2S transcript is the predominant γ2 mRNA expressed during latter stages of embryo genesis, while the γ2L transcript is the predominant γ2 isoform present inthe adult CNS. Since there is a 29 to 47 percent amino acid identity among the various GABA_A receptor subunits, I have also demonstrated through site-directed mutagenesis studies, that changes in a conserved amino acid in the cysteine loop of the bovine a 1 GABA_A receptor subunit resulted in a loss of agonist and antagonist binding (DI49N), while a change in a conserved amino acid in the M1 transmembrane domain of the bovine α1 GABA_A receptor subunit resulted in loss of agonist binding and reduction in the B_max and K_d for antagonist binding (P243A). 'These results are in contrast to the effect of identical mutations in the bovine β1 subunit and suggest that if the pentameric GABA_A receptor assembly is composed of (α1)2(β1)1(γ2)2, then changes in highly conserved amino acids in the α1 receptor subunit would have a greater distortion on the structure of the receptor complex.

O objeto de estudo da presente pesquisa são as comunidades mediadas pela Internet (CMIs). Uma CMI consiste de um conjunto de pessoas que compartilham interesses e que, durante algum tempo, utilizam recursos em comum na Internet (por exemplo, um web site – objeto preferencial deste trabalho) para trocarem informações umas com as outras relativamente aos interesses compartilhados. A pesquisa realizada é exploratória e qualitativa, tendo feito uso de estudos de caso, pesquisa-ação e entrevistas em profundidade para estruturar uma base conceitual para as CMIs e reunir elementos relevantes a serem considerados quando da construção de web sites para as mesmas. Realizou-se estudo de caso de 5 (cinco) web sites de CMIs, a fim de serem identificadas as principais tecnologias e métodos em uso atualmente para a estruturação de web sites para CMIs. Na pesquisa-ação, 7 (sete) grupos de pessoas foram identificados e, para cada um, construiu-se 1 (um) web site, de modo que se ofereceu um espaço na Internet para a interação dos seus integrantes. A observação da interação das pessoas através dos web sites permitiu concluir-se que, dos sete grupos iniciais, apenas 1 (um) poderia ser caracterizado como CMI, conforme critérios de Jones (1997): associação sustentável, variedade de comunicadores, espaço virtual para a comunicação em grupo, e interatividade. Para as entrevistas em profundidade, elaborou-se um questionário com base no referencial teórico, nos estudos de caso e na pesquisa-ação, sendo aplicado a 17 (dezessete) pessoas (da única CMI e de dois dos sete grupos). O objetivo das entrevistas foi levantarem-se percepções sobre os web sites utilizados pelos grupos, percepções essas que, sob análise de conteúdo, ajudaram na formação de um conjunto de 12 (doze) recomendações para a construção de web sites para CMIs. As recomendações são de natureza diversa, mas deixam clara a necessidade de haver um entendimento profundo do contexto de uma CMI previamente ao projeto do seu web site.

In spite of the multisite phenomenon’s apparent “success” and effectiveness in fulfilling the Great Commission, there are looming variables related to leadership structures and organizational complexities that have not been adequately addressed. Perhaps the most significant question in need of further research in the multisite field is simply determining where final decision-making authority is found. Does such authority lie at the campus level, or at the larger church level? For example, what decisions can and cannot a campus pastor make apart from the approval of the senior leadership team? The longevity of the multisite movement will ultimately be determined by the presence of healthy leadership structures that learn the tedious balance between control and freedom. The senior leadership team has the arduous task of being closely enough involved in the operations of each campus to ensure that campuses rightly embody the DNA of the overall church. Yet at the same time, this senior leadership team must empower and legitimize campus leadership in such a way that they do not feel unnecessarily restricted and compromised of their unique calling and giftedness. Such perceptions on behalf of campus leadership can lead to mistrust, conflict, and the ultimate deterrence of the mission of the church. This quantitative study addresses the leadership dynamics and organizational complexities in multisite churches, with a particular emphasis on the relationship between campus pastors and the church’s senior leadership team. Chapter 1 introduces the research problem and its practical significance. It also defines the research questions while indicating the study’s limitations. The examination of the precedent literature in chapter 2 underscores the biblical and theological foundations of this topic, such as the clear examples of shared leadership structures seen in the New Testament. Additionally, this chapter details the contemporary research regarding multisite structures in general, and leadership structures in particular. Chapter 3 presents the research design, defines the population, establishes the selection criteria, and outlines the instrumentation. Chapter 4 analyzes the findings, while chapter 5 presents the researcher’s conclusions. The study’s ultimate aim is to stimulate meaningful dialogue among multisite churches, and to strengthen multisite churches’ leadership structures so as to better ensure biblical fidelity, health, unity, and longevity.

O objeto de estudo da presente pesquisa são as comunidades mediadas pela Internet (CMIs). Uma CMI consiste de um conjunto de pessoas que compartilham interesses e que, durante algum tempo, utilizam recursos em comum na Internet (por exemplo, um web site – objeto preferencial deste trabalho) para trocarem informações umas com as outras relativamente aos interesses compartilhados. A pesquisa realizada é exploratória e qualitativa, tendo feito uso de estudos de caso, pesquisa-ação e entrevistas em profundidade para estruturar uma base conceitual para as CMIs e reunir elementos relevantes a serem considerados quando da construção de web sites para as mesmas. Realizou-se estudo de caso de 5 (cinco) web sites de CMIs, a fim de serem identificadas as principais tecnologias e métodos em uso atualmente para a estruturação de web sites para CMIs. Na pesquisa-ação, 7 (sete) grupos de pessoas foram identificados e, para cada um, construiu-se 1 (um) web site, de modo que se ofereceu um espaço na Internet para a interação dos seus integrantes. A observação da interação das pessoas através dos web sites permitiu concluir-se que, dos sete grupos iniciais, apenas 1 (um) poderia ser caracterizado como CMI, conforme critérios de Jones (1997): associação sustentável, variedade de comunicadores, espaço virtual para a comunicação em grupo, e interatividade. Para as entrevistas em profundidade, elaborou-se um questionário com base no referencial teórico, nos estudos de caso e na pesquisa-ação, sendo aplicado a 17 (dezessete) pessoas (da única CMI e de dois dos sete grupos). O objetivo das entrevistas foi levantarem-se percepções sobre os web sites utilizados pelos grupos, percepções essas que, sob análise de conteúdo, ajudaram na formação de um conjunto de 12 (doze) recomendações para a construção de web sites para CMIs. As recomendações são de natureza diversa, mas deixam clara a necessidade de haver um entendimento profundo do contexto de uma CMI previamente ao projeto do seu web site.