Philosophiae Doctor - PhD / Several lines of cellular and physiological evidence have suggested the presence of a novel class of systemically mobile plant molecules that are recognized by antibodies generated against vertebrate atrial natriuretic peptides (ANPs). Functional characterization of these immunoanalogues, referred to as immunoreactive plant natriuretic peptides (irPNPs) or plant natriuretic peptides (PNPs), has shown that they play important roles in a number of cellular processes crucial for plant growth and maintenance of cellular homeostasis. Although the various biological roles of PNPs in plants are known, their exact mode of action remains elusive. To elucidate the mechanisms of action for these immunoanalogues, we have prepared a biologically active recombinant PNP from Arabidopsis thaliana (AtPNP-A) and the biological activity was demonstrated by showing its ability to induce water uptake into Arabidopsis thaliana protoplasts. In addition, the molecule was shown to downregulate photosynthesis while at the same time up-regulating respiration, transpiration as well as net water uptake and retention capacities in the sage Plectranthus ecklonii. Further analysis of the recombinant AtPNP-A indicated that the peptide can induce systemic response signalling though the phloem. A recombinant Arabidopsis wall associated kinase-like protein (AtWAKL10) that has a domain organization resembling that of vertebrate natriuretic peptide (NP) receptors was also partially characterized as a possible receptor for the recombinant AtPNP-A. Vertebrate NP receptors contain an extracellular ligand-binding domain and an intracellular guanylate cyclase (GC)/kinase domain and signal through the activity of their GC domain that is capable of generating intracellular cGMP from GTP. The structural resemblance of AtWAKL10 to vertebrate NP receptors could suggest a functional homology with receptor molecules and it is conceivable that such a receptor may recognize PNPs as ligands. The characterization of the recombinant AtWAKL10 showed that the molecule functions as both a GC and a kinase in vitro. This strengthened the suggestion that AtWAKL10 could be a possible AtPNP-A receptor especially considering the fact that AtPNP-A applications to plant cells also trigger cGMP transients. Furthermore, a bioinformatic analysis of the functions of AtPNP-A and AtWAKL10 has inferred both molecules in plant pathogen responses and defense mechanisms, thus indirectly functionally linking the two proteins. / South Africa

Magister Legum - LLM / This study aims to evaluate the prospective benefits and challenges of developing a regional competition regulatory framework in SADC. Further, the study seeks to identify important lessons from the EU and COMESA that are crucial for the pursuance of a regional competition regulatory framework.

Malaria is a devastating disease which affects almost half of the world’s population. Since the description of the malaria genome sequence, various aspects of the parasite have been studied, including drug resistance mechanisms, epidemiology and surveillance systems. Alarmingly, very little is known about the basic biological processes such as the regulation of the expression of parasite genes. The parasite, Plasmodium falciparum, has developed highly specialized methods of regulating the transcription of genes, starting at the regulation of genes controlling basic cellular processes such as protein synthesis and erythrocyte invasion, followed by the transcriptional regulation of more specialized genes, such as those aiding in immune evasion and pathogenesis. The description of the P. falciparum transcriptome by Bozdech et al. in 2003 revealed a complex, just-in-time and tightly regulated transcription profile of P. falciparum genes. This suggests that the most probable Achilles heel for Plasmodium may be its unique mechanisms of regulating gene expression. Various cis- and trans-regulatory sequences have been identified in P. falciparum, along with possible DNA (and RNA) binding proteins. The first part of this research focussed on transcriptional regulatory mechanisms in which an in silico search identified cis-regulatory sequences in the 5’ untranslated region of the antigenically variant var gene family. Electrophoretic mobility shift assays (EMSA) were used to identify protein binding partners of these sequences, which could ultimately act as transcription factors in regulating the expression of this essential gene family. The second part of the research investigated the involvement of post-transcriptional regulatory mechanisms in the polyamine biosynthetic pathway of P. falciparum. Polyamines have been proven to be crucial for the parasite’s development and therefore, an RNA interference knock-down strategy was used to verify the importance of the polyamine biosynthetic enzymes S-Adenosylmethionine decarboxylase (AdoMetDC), Ornithine decarboxylase (ODC) and Spermidine synthase. It is clear that various mechanisms for gene regulation are used by the parasite and that this is critical for the survival of this organism. The results of this study suggest the potential presence of both double-stranded and single-stranded DNA regulatory proteins within P. falciparum nuclear extract. As controversial as RNA interference remains in P. falciparum, this technique was used as a plausible knock-down strategy of parasite specific genes and certain trends, regarding the visible decreases in gene transcript level after double-stranded RNA treatment, were observed. However, final conclusions as to the feasibility of using RNA interference in P. falciparum remain to be elucidated. This study therefore ultimately lends insight into the transcriptional and post-transcriptional levels of P. falciparum gene regulation. / Dissertation (MSc)--University of Pretoria, 2010. / Biochemistry / unrestricted

Numerous studies have demonstrated that tumour cells have the ability to alter immune function to create an immune suppressed environment. This allows tumour cells to escape immune surveillance and consequently the tumour can progress. Dendritic and T cells have critical roles in immune activation and tolerance and are thus major targets of tumour-mediated immune suppression. Understanding the mechanism(s) by which tumour cells modulate the immune system will facilitate the development of immune system-based therapies for cancer treatments. In this study we sought to determine the nature of, and cellular and molecular mechanisms underlying, changes in immune status during tumour progression using mouse models of prostate cancer. Detailed analysis of the immunological status in a mouse prostate dysplasia model (12T-7slow) revealed that immune suppression accompanied tumour progression. We found that T cells isolated from tumour-bearing hosts were hypo-responsive to antigen stimulation. Furthermore, we demonstrated that CD4+CD25+ regulatory T cells were responsible, at least in part, for this alteration. Anti-CD25 antibody treatment reduced, but did not prevent, tumour growth in either a transplanted prostate tumour model or a spontaneously developing prostate tumour model. In addition, an altered dendritic cell phenotype and an elevated frequency of CD4+CD25+ regulatory T cells were observed within the tumour mass. Similar alterations were observed in the prostate-specific Pten knockout mice which develop advanced prostate adenocarcinoma. Interestingly, evidence of immune activation, such as an increased frequency of activated T cells, was detected in the tumour microenvironment in both mouse prostate tumour models. To identify factors that may play critical roles in the altered immune cell phenotype observed in the tumour microenvironment, a global gene expression profiling analysis was carried out to evaluate the changes in immune-related gene expression patterns. This analysis provided additional evidence for the co-existence of immune suppression and immune activation. Moreover, subsequent analyses suggested that one differentially expressed transcript, interferon regulatory factor 7, and its target genes might be involved in modulating immune cells and/or tumour progression. Taken together, these studies have important implications for designing specific and effective anti-tumour immune therapy strategies that involve manipulation of tumour cells, dendritic cells and regulatory T cells. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

In the past decade, fast advancements have been made in the sequencing, digitalization and collection of the biological data. However the bottleneck remains at the point of analysis and extraction of patterns from the data. We have developed a method that is aimed at widening this bottleneck by automating the knowledge extraction from the biological data. Our approach is aimed at discovering patterns in a set of DNA sequences based on the location of transcription factor binding sites or any other biological markers with the emphasis of discovering relationships. A variety of statistical and computational methods exists to analyze such data. However, they either require an initial hypothesis, which is later tested, or classify the data based on its attributes. Our approach does not require an initial hypothesis and the classification it produces is based on the relationships between attributes. The value of such approach is that is is able to uncover new knowledge about the data by inducing a general theory based on basic known rules. The core of our approach lies in an inductive logic programming engine, which, based on positive and negative examples as well as background knowledge, is able to induce a descriptive, human-readable theory, describing the data. An application provides an end-to-end analysis of DNA sequences. A simple to use Web interface accepts a set of related sequences to be analyzed, set of negative example sequences to contrast the main set (optional), and a set of possible genetic markers as position-specific scoring matrices. A Java-based backend formats the sequences, determines the location of the genetic markers inside them and passes the information to the ILP engine, which induces the theory. The model, assumed in our background knowledge, is a set of basic interactions between biological markers in any DNA sequence. This makes our approach applicable to analyze a wide variety of biological problems, including detection of cis-regulatory modules and analysis of ChIP-Sequencing experiments. We have evaluated our method in the context of such applications on two real world datasets as well as a number of specially designed synthetic datasets. The approach has shown to have merit even in situations when no significant classification could be determined.

A primary aim of Synthetic Biology is the design and implementation of biological systems that perform engineered functions. However, the assembly of double-stranded DNA molecules is a major barrier to this progress, as it remains time consuming and laborious. Here I present three improved methods for DNA assembly. The first is based on, and makes use of, BioBricks. The second method relies on overlap-extension PCR to assemble non-standard parts. The third method improves upon overlap extension PCR by reducing the number of steps and the time it takes to assemble DNA. Finally, I show how the PCR-based assembly methods presented here can be used, in concert, with in vivo homologous recombination in yeast to assemble as many as 19 individual DNA parts in one step. These methods will also be used to assemble an incoherent feedforward loop, gene regulatory network.

BACKGROUND AND OBJECTIVES: Standard meta-analysis compares two treatments whereas network meta-analysis compares multiple treatments. In light of the increasing number of treatments available, we have seen a shift from using standard meta-analysis towards using network meta-analyses to support regulatory and reimbursement decisions. This thesis (composed of nine separate papers) applied network meta-analysis to a series of real world problems which simultaneously addressed many of these methodological issues while also supporting healthcare decision making. METHODS: In the first chapter, background information on network meta-analysis and outline the rationale for each of the subsequent chapters is provided. In Chapter 2, an applied example is presented where we use FDA Public Summary documents to populate a network meta-analysis of antithrombotic therapies for atrial fibrillation. In Chapter 3, the advantages and disadvantages of rapid network meta-analysis using ClinicalTrials.gov are investigated by re-doing Chapter 3 but only using data available in ClinicalTrials.gov and examining concordance. In Chapter 4, the application of network meta-analysis when events are rare are investigated using an illustrative example investigating the risk of serious infection in biologics. In Chapter 5, the application of network meta-analysis to a complex network – triptans for migraine – are presented where there are a large number of studies and treatments. In Chapter 6 a network meta-analysis which assesses both a benefit and harm and integrates findings using a benefit harm methodology is presented. In Chapter 7, methods to incorporate non-randomized studies into network meta-analysis are investigated using data on riskof myocardial infarction derived from the Mini-Sentinel distributed data network. FINDINGS AND CONCLUSION: Pragmatic research around methodological areas in network meta-analysis were conducted to address real world problems for decision makers. It is my hope that this thesis and the approaches used in this thesis for the application of network meta-analysis will be disseminated to enhance research capacity in conducting network meta-analysis in Canada.

Interferon regulatory factor 3 (IRF3) plays an important role in activating the innate immune response in a variety of conditions, including viral infection. As well as regulating the immune response to viruses, IRF3 is involved in regulating cellular functions including apoptosis. Apoptosis and the inflammatory response to viral infection are very different; therefore, it is obvious that IRF3 plays dramatically different roles in the cell depending on the conditions. We previously identified a non-activating phosphorylation of IRF3 in response to adenovirus (Ad) in which Serine-173 is phosphorylated. In addition to Ad infection, IRF3- S173 is phosphorylated in response to genotoxic stresses including ultraviolet (UV) irradiation and etoposide. In this study, I show that this phosphorylation event is involved in a variety of processes including protein stability, cell survival and IRF3 regulation. Thus, phosphorylation of IRF3-S173 is a novel and important event in a complex regulatory pathway of an integral protein.

The thesis analyses the influence of reimbursement regulatory fee for hospitalization which was introduced in regional hospitals in 2009 in Czech Republic. The difference in differences analysis was used to study the possible relationship between reimbursement of the fee for length of hospitalization in acute care hospitals and in after-care facilities. In acute care the influence was 0,5-1 % of the length and in after-care facilities between 8-12 %. This relationship may be biased because of introduction of DRG system which may decrease the length of stay and may be differently distributed between the treatment and control groups.

This thesis is comparing impacts defined by Regulatory impact assessment (RIA) with real impacts discovered with online survey related to the specific Czech environmental legislative act. There is described RIA, experience with RIA in Czech and comparison of development and experience of RIA in EU and USA. As a result, comparison of the impacts within RIA and the ones identified by the survey is that real impacts are different from the predicted ones within RIA. The summary of this thesis is that process RIA is alright but accuracy of predicted impacts is dependent on RIA processor, rigorousness and complexity of RIA.