A laboratory scale fermenter control system, based on a Z80 microprocessor operating at 4 MHz, has been developed. The system has the following features: 1 -" 8 Kbytes of RAM and 4 Kbytes of EPROM (with extra space for another 4 Kbytes); 2 -" Two serial links (RS232c);3-" An analog/digital converter with an 8 channel multiplexer and an opto-triac system for switching control devices (heater, peristaltic pumps); 4 -" Four interface cards to connect the parameter sensors. This system has the advantage that it is based on well established (though not state-of-the-art) microelectronic technology (Z80 chips) widely available in the world at reasonable prices. This, plus the fact that both hardware and software were locally designed, means that it is truly 'portable' in the sense that, with limited resources, copies can easily be constructed in developing countries (the idea is that Mark-II will be built in North East Brazil to support research into yeast fermentation technology). The control system was evaluated in batch and chemostatic growth modes. Both modes were used to investigate the growth kinetics of a genetically engineered Saccharomyces cerevisiae strain (BC55) carrying the plasmid pCYG4 which directs 10 fold more NADP-GDH activity than wild type cells. Batch culture experiments showed that the presence of plasmid increased ammonia uptake by the cells but did not improve biomass or ethanol yield compared to wild type cells. Under carbon-limitation NADP-GDH activity was in phase with GOGAT and penicillinase activity. NADP-GDH and GOGAT activities were inversely proportional to intracellular ammonia concentration and proportional to intracellular L-glutamate concentration. Under nitrogen limitation (using ammonia as limiting substrate) GOGAT activity increased, but NADP-GDH and penicillinase activity remained at the same level as under carbon limitation. Using L-glutamate as nitrogen source NADP-GDH activity was very low and GOGAT and penicillinase activities were undetectables with an increase in NAD-GDH activity. Oscillations found in enzyme activities and intracellular metabolite concentration under carbon and nitrogen limitation experiments are a consequence of a mixed culture (the presence of cells with plasmid GDH (gdh+ cells) and cells without plasmid GDH (gdh- cells)) with changing concentrations of the 2 populations.

M.Sc. / The global tuberculosis (TB) epidemic has resulted in the development of numerous methods for the enumeration and viability assessment of Mycobacterium tuberculosis (M.tb), as these methods play a key role in TB management and research. In this study the methods of quantitative culture (CFU), the microplate alamar blue assay (MABA), flow cytometry, the green fluorescent protein microplate assay (GFPMA) and quantitative PCR were investigated and compared for the enumeration and viability assessment of mycobacteria in culture. The MABA and the GFPMA were applied to the enumeration and viability assessment of mycobacteria post-infection. Quantitative culture was found to be simple and low in cost but was lengthy. The MABA, an economic and quick assay, was more sensitive for high mycobacterial concentrations. The flow cytometric enumeration of fluorescent mycobacteria was rapid and sensitive, but was dependent on access to a flow cytometer and therefore was costly. Flow cytometry facilitated enumeration but was limited concerning viability assessment. The GFPMA was a simple, rapid and cost effective assay. However, decreased sensitivity was observed for low mycobacterial concentrations. Quantitative PCR, although high in cost, was sensitive and rapid. The MABA and the GFPMA were useful for the enumeration of mycobacteria post-infection, with the former being the more sensitive method. This study serves as a reference of the methods available for the enumeration and viability assessment of M.tb. The advantages and disadvantages established for each of the methods investigated in this study enables an informed selection of the most appropriate method for a specific objective and research environment.

Protein-protein interactions (PPIs) dominate all cellular functions across every domain of life. If PPIs become aberrant, they may result in many human diseases, such as cancer or Alzheimer’s. Despite their clinical significance, modulating aberrant PPIs is a daunting task. Most PPI surfaces are long, hydrophilic and structurally complex. Thus, finding molecules that moderate specific aberrant PPIs is an important goal in drug discovery research. For example, PPIs have been modulated by peptidomimetics, synthetic peptides that assume three-dimensional structures similar to proteins, but unlike natural peptides, they are proteolytically stable. However, building libraries of peptidomimetics is challenging as current methods rely on solid phase peptide synthesis, which limits the size and diversity of peptidomimetic libraries. As such, using the translation machinery to synthesize peptidomimetics is an attractive approach. In Chapter 1, we begin by discussing bacterial protein synthesis. Then, we delve into a detailed discussion of the application of the bacterial translation machinery for the in vitro translation of synthetic peptides. In this discussion, we review the different technologies, their advantages and limitations with respect to the incorporation of amino acids with unnatural backbones. After reviewing the methods used to incorporate backbone analogs, and their compatibility with the bacterial translation machinery, we describe a novel approach for the ribosomal incorporation of -amino acids analogs containing an -substituent, -hydroxy--amino acids (Chapter 2). We demonstrate that the ribosome incorporates this new class of substrates through the formation of an intermediate ester bond that rapidly rearranges to form a native peptide bond. Using this approach, we show that -hydroxy--amino acid single incorporation efficiencies are comparable the incorporation efficiencies obtained with natural amino acids. In Chapter 3, we apply this approach to the synthesis of peptides containing multiple -hydroxy--amino acids. This chapter describes the results obtained with the in vitro synthesis of peptides containing two consecutive -hydroxy--amino acids, three consecutive -hydroxy--amino acids, and alternating -hydroxy--amino acids and -amino acids. Based on these results, we propose experiments to improve these incorporation yields for the application of this technology for the in vitro synthesis of diverse peptidomimetic libraries.