Microbially derived bioactive peptides to improve human health

This thesis describes a study of various methods to produce bioactive peptides. Initially, the generation of anti-Cronobacter spp. peptides by fermentation of milk protein is described. Lactobacillus johnsonii DPC6026 was used to generate two previously described antimicrobial peptides. Phenotypic analysis indicated unsatisfactory casein hydrolysis. The genome of the strain was sequenced and annotated. Results showed a number of unique features present, most notably a large symmetrical inversion of approximately 750kb in comparison with the human isolate L. johnsonii NCC 533. The data suggest significant genetic diversity and intra-species genomic rearrangements within the L. johnsonii spp.. Cronobacter spp. have emerged as pathogens of concern to the powdered infant formula industry. Chapters 3 and 4 of this thesis describe novel methods to generate two antimicrobial peptides, Caseicin A and B. In Chapter 3 a bank of Bacillus strains was generated and investigated for caseicin production. Following casein hydrolysis by specific B. cereus and B. thuringiensis strains the peptides of interest were generated. Chapter 4 describes a sterile enzymatic method to generate peptides from casein. Bioinformatic tools were used to predict enzymes capable of liberating caseicin peptides from casein. Hydrolysates were generated using suitable enzymes, examined and some were found to produce peptides with activity against Cronobacter spp.. This study establishes a potential industrial-grade method to generate antimicrobial peptides. Administration of GLP-1 leads to improved glycaemic control in diabetes patients. Generation of a recombinant lactic acid bacteria capable of producing a GLP-1 analogue is described in Chapter 5. In-vivo analysis confirmed insulinotropic activity. The results illustrate a method using bacteriocin producing cellular machinery to generate bioactive peptides. This thesis describes the generation of bioactive peptides by bacterial fermentation, tailored enzymatic hydrolysis and recombinant bacterial methods. The techniques described contribute to bioactive peptide research with regards novel methods of production and industrial scale-up.

The right to humanitarian assistance in natural and human-made disasters: progress and challenges for an emerging international legal framework

Natural and human-made disasters cause on average 120,000 deaths and over US$140 billion in damage to property and infrastructure every year, with national, regional and international actors consistently responding to the humanitarian imperative to alleviate suffering wherever it may be found. Despite various attempts to codify international disaster laws since the 1920s, a right to humanitarian assistance remains contested, reflecting concerns regarding the relative importance of state sovereignty vis-à-vis individual rights under international law. However, the evolving acquis humanitaire of binding and non-binding normative standards for responses to humanitarian crises highlights the increasing focus on rights and responsibilities applicable in disasters; although the International Law Commission has also noted the difficulty of identifying lex lata and lex ferenda regarding the protection of persons in the event of disasters due to the “amorphous state of the law relating to international disaster response.” Therefore, using the conceptual framework of transnational legal process, this thesis analyses the evolving normative frameworks and standards for rights-holders and duty-bearers in disasters. Determining the process whereby rights are created and evolve, and their potential internalisation into domestic law and policy, provides a powerful analytical framework for examining the progress and challenges of developing accountable responses to major disasters.

Investigation of the genotoxic potential of the marine biotoxins okadaic acid and azaspiracids

The present study investigated the genotoxic potential of the marine biotoxins okadaic acid (OA) and azaspiracids (AZAs). Harmful algae blooms (HABs) are an increasing global problem with implications for the ecosystem, economy and human health. Most data available on human intoxication are based on acute toxicity. To date, limited data has been published on possible long term effects, carcinogenicity and genotoxicity. To investigate genotoxicity in the present study, DNA fragmentation was detected using the COMET assay. In contrast to most other available studies, two further endpoints were included. The Trypan Blue Exclusion assay was used to provide information on possible cytotoxicity and assess the right concentration range. Flow cytometer analysis was included to detect the possible involvement of apoptotic processes. In house background data for all endpoints were established using positive controls. Three different cell lines, Jurkat T cells, CaCo-2 cells and HepG-2 cells, representing the main target organs, were exposed to OA and AZA1-3 at different concentrations and exposure times. Data obtained from the COMET assay showed an increase in DNA fragmentation for all phycotoxins, indicating a modest genotoxic effect. However, the data obtained from the Trypan Blue Exclusion assay showed a clear reduction in cell viability and cell number, indicating the involvement of cytotoxic and/or apoptotic processes. This is supported by data obtained by flow cytometer analysis. All phycotoxins investigated showed signs of early/late apoptosis. Therefore, the combined observations made in the present study indicate that OA and AZA1-3 are not genotoxic per se. Apoptotic processes appear to make a major contribution to the observed DNA fragmentation. The information obtained in this study stresses the importance of inclusion of additional endpoints and appropriate positive controls in genotoxicity studies. Furthermore, these data can assist in future considerations on risk assessment, especially regarding repeated exposure and exposure at sub-clinical doses.