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    Microbiome and host immune responses in colorectal cancer development and radiotherapy response : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Auckland, New Zealand
    (Massey University, 2022) Sulit, Arielle Kae
    Colorectal cancer (CRC) is a highly heterogeneous disease that manifests differently from patient to patient, making prognosis, management, and therapy more complex as no universal solution is available. With the advent of high throughput sequencing, descriptions of CRC tumors have moved from histopathological features and descriptions to molecular characterization, allowing for the subtyping of CRC into groups with similar characteristics. This inter-tumoral heterogeneity affects CRC development and patient response to treatments. Understanding the different mechanisms of CRC development and response to therapy is therefore crucial to personalized healthcare. The majority of CRC tumors do not have a familial background, suggesting the environment plays a large role in their development. Environmental factors include the microbiome, which has been shown previously to affect CRC development. However, the role of the microbiome has largely been overlooked in studies of the CRC subtyping and radiotherapy response in rectal cancer treatment. In this thesis, I show that bacteria in CRC may affect immune responses that drive CRC development in different subtypes, and radiotherapy response. I used RNA sequencing to revisit the consensus molecular subtypes (CMS) of CRCs and identified microbes that have possible contributions to their different characteristics. As microbes have been associated with differing responses to therapy, I also looked at their putative roles in radiotherapy response in a rectal cancer cohort. I first developed a computational pipeline that takes raw sequencing reads as input and yields host gene expression data, microbiome abundances and functional information. I then focused on two subtypes of CRC, CMS1 and CMS4. Analysis of host gene expression in these subtypes confirmed that their expression profiles are enriched in gene sets associated with immune responses. Analysis of the microbiome content found that lipopolysaccharides (LPS) from Fusobacterium periodonticum and Bacteroides fragilis in CMS1, and Porphyromonas asaccharolytica in CMS4 potentially affect the production of the immune infiltrates of their respective subtypes. F. periodonticum LPS enhanced cytokine production while LPS from the latter two bacteria suppressed cytokine production in peripheral blood mononuclear cells (PBMCs) in vitro. These data indicate possible roles of LPS from these microbes in CRC development via immune response. These also indicate possible roles of these molecules in CRC therapy. I also found that in complete responders of radiotherapy, there was an enrichment in host gene functions that are associated with complement activation, response to viruses, and B-cell activation, all of which indicated a link to immunotherapy responses triggered by radiotherapy. Furthermore, bacteria that had previous associations with immunotherapy responses were enriched in complete responders indicating a role in enhancement of these cytotoxic immune responses in radiosensitivity. Immune infiltrates have always been a crucial element in cancers, and the type of infiltrates can have conflicting effects on cancer development and therapeutic responses. In this work, I show that the types of bacterial molecules and how they interact between species can affect specific immune responses in CRC development, that dampening of immune responses in CRC is as crucial as inducing immunogenicity, and that specific bacteria also affect immune responses in a manner that may be similar to immunotherapy that will increase radiosensitivity. This work provides an initial look into mechanisms of how microbes interact with host immune responses affecting CRC development into two different subtypes, and radiosensitivity. It also provides initial experimental evidence for effects of LPS in CRC development and a possible mechanism of radiotherapy sensitivity to test in the laboratory.
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    Molecular and immunological analysis of New Zealand isolates of Mycoplasma ovipneumoniae : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Palmerston North, New Zealand
    (Massey University, 2021) Bridgeman, Benjamin
    Mycoplasma ovipneumoniae is a pathogen of caprinae expected to infect the majority of domestic sheep in early life. M. ovipneumoniae is suspected to be the primary cause of chronic non-progressive pneumoniae (CNP) in lambs. Currently there is no effective vaccine against M. ovipneumoniae, and many commonly used antibiotics have limited effect. Diagnosis of M. ovipneumoniae infections can be difficult, due in part to the limited overt symptoms seen in cases of CNP. Despite the significance of M. ovipneumoniae infections there is limited information on the molecular and immunological data of M. ovipneumoniae. Thus, the major focus of this thesis was to identify potential immunogenic proteins for a vaccine or diagnostic purposes and construct genomes from New Zealand isolates of M. ovipneumoniae. Draft genomes were produced for three New Zealand isolates of M. ovipneumoniae, (isolates 16, 90, and 103), and annotated. Using antisera produced against these isolates, immunogenic proteins were identified from hydrophobic membrane protein fractions of M. ovipneumoniae using Western blotting. Those protein bands were subjected to mass spectrometry analysis, and by comparing mass spectrometry data to the genomes, six novel proteins, GAPDH, P146, MATE, hypothetical protein 1, hypothetical protein 2, hypothetical protein 3, and two previously described immunogenic proteins, EF-Tu and HSP70, were identified. Five of the proteins were produced as recombinant proteins for further study. The nucleotide sequence of EF-Tu was edited using an overlap polymerase chain reaction (PCR) to convert the sequence to Escherichia coli codon preference. This sequence was then cloned into the pET22b (+) vector for expression of histidine-tagged fusion protein. Additionally, EF-Tu, GAPDH, P146, hypothetical protein 3 and an epitope of hypothetical protein 1 were produced as recombinant proteins by GenScript. Antisera were produced in sheep against each of the recombinant proteins for further studies. Enzyme-linked immunosorbent assay (ELISA) confirmed that each protein was immunogenic and elicited antibody responses in sheep. Immunofluorescence microscopy confirmed the localisation of native EF-Tu and GAPDH proteins on the surface of M. ovipneumoniae cells. Antisera produced against whole cell antigens from M. ovipneumoniae cross-reacted with P146 and hypothetical protein 3 suggesting a potential role for these proteins to be used to detect animals infected with M. ovipneumoniae. Sheep vaccinated with M. ovipneumoniae produced weak IFN-γ responses but stronger IL-17 responses in whole blood cultures stimulated with whole cell antigens. Of the five proteins tested as recall antigens in the IL-17A assay, recombinant GAPDH produced a strong IL-17A response in sheep vaccinated with M. ovipneumoniae suggesting a potential role for GAPDH in a diagnostic assay for measuring IL-17A responses in M. ovipneumoniae infected sheep. Mycoplasmas are known to adhere to host epithelial cells as part of the pathogenesis process. A bovine endometrial epithelial cell line was established as a model to study adherence of M. ovipneumoniae to epithelial cells and determine if antibodies against the surface proteins could interfere with adherence. M. ovipneumoniae cells adhered to the endometrial epithelial cells. Adherence was inhibited by antibodies directed against whole cells of M. ovipneumoniae but not against the recombinant proteins.
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    Study of an exported protein of Mycobacterium avium subspecies paratuberculosis : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand
    (Massey University, 2004) Copland, Susan Maree
    Johne's disease is a chronic, progressive enteric disease of ruminants caused by infection with Mycobacterium avium subspecies paratuberculosis (M. ptb) from the MAIS complex (M. avium, M. ptb, M. intracellulare and M. scrofulaceum). The lack of specific and sensitive diagnostic tests often leads to M. ptb infected animals being diagnosed with bovine tuberculosis, a member of the MTB complex (M. tb, M. bovis, M. bovis BCG, M africanum, M. microti and M. canetti). Secreted proteins from pathogenic mycobacteria have been found to be important for the development of protective immunity, namely a cell mediated immune response (CMI). The development of reliable differential diagnostic tests will require the use of species-specific secreted protein antigens and the CMI response. Due to the taxonomic distance between the MAIS and MTB complexes our hypothesis was that the M. ptb genome may encode for secreted proteins that are absent from members of the MTB complex. If such proteins can stimulate an immune response they may be suitable for use as antigens in a differential diagnostic test for Johne's disease. To this end, the secreted protein library clone pJEMIl-M ptb281 was examined and its insert found to contain the 5' region of the hypothetical M.ptb281 ORF fused in frame with phoA. The entire ORF was determined using M. avium and M. ptb database sequences then cloned into E. coli and mycobacterial expression systems. These systems incorporate 6x histidine (His6) affinity tags into recombinant proteins allowing them to be semi-purified by Ni-NTA affinity chromatography. Semi-purified recombinant proteins tested positive by western blot analysis to highly specific anti-His6-tag antibodies. Amino acid sequencing to confirm the identity of these recombinant proteins and screening for their ability to stimulate an immune response were prevented by time constraints. Homologs to M. ptb281 were absent from M. tb, M. bovis and M. bovis BCG but present in the MAIS complex, making this protein unsuitable for use as an antigen to differentiate between MAIS complex species in a diagnostic test. M. ptb281 homologs found in the genomes of two members of the Acetomycetes order corresponded to hypothetical proteins predicted by computer software programs trained to identify genes, which may indicate that the hypothetical M. ptb281 ORF may encode a functional protein.
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    A genetic approach to identify Mycobacterium bovis exported protein antigens : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Biology, Massey University
    (Massey University, 1997) Borich, Suzanne Marie
    A novel approach, combining phoA-fusion technology with T cell screening of a recombinant cosmid library, was used to detect Mycobacterium bovis exported T cell antigens. An M. bovis BCG library of phoA-fusions was constructed in Escherichia coli and Mycobacterium smegmatis using the plasmid vector pJEM11. The M. bovis BCG DNA inserts from ten PhoA+ clones were partially sequenced and used to search databases for similarities to known genes. These revealed similarities to a family of genes coding for high temperature-requirement serine proteases and a Mycobacterium leprae putative exported lipoprotein gene (pel). The DNA inserts from PhoA+ clones were used to probe an M. bovis cosmid library expressed in M. smegmatis 10 identify cosmids containing the full-length genes coding for these exported proteins. Culture filtrates (CFs) prepared from selected M. smegmatis recombinants (cosmids) were assayed for their ability to induce proliferation and IFN-γ-production from peripheral blood mononuclear cells (PBMCs) taken from M. bovis BCG-immunised and non-immunised control cattle. Culture filtrates from two recombinant M. smegmatis (cosmids 44 and 56) induced significant IFN-γ-production and proliferation by PBMCs from immunised animals. An exported protein gene, identified using the phoA-fusion technology, was subcloned from cosmid 56 and its sequence determined and analysed. Database searches using the deduced amino acid sequence of this gene revealed similarities to an M. leprae putative exported lipoprotein (Pel) and a family of MalE maltose-binding proteins. The M. bovis pel gene was shown to be expressed by recombinant M. smegmatis. Preliminary evidence from this study indicates that the M. bovis Pel protein is recognised by antigen-specific lymphocytes from M. bovis BCG-immunised animals. The PBMCs taken from M. bovis challenged and M. bovis BCG vaccinated / challenged cattle also recognised CF from recombinant M. smegmatis expressing the pel gene in in vitro immunoassays. The combined strategy of using phoA-gene fusions and T cell screening of CFs from a recombinant M. bovis cosmid library proved a sensitive and rapid method for the detection of potential M. bovis T cell antigens.