Colonisation by many bacteria and viruses is now thought to depend upon their ability to adhere to host cells via proteinacious surface appendages called adhesins. Information relevant to the prevention and cure of many diseases therefore is supplied by knowledge of this adhesive process, especially the chemistry of the binding and the structure of the binding molecules. At this time, the structure of very few adhesin receptors is known. Similarly, the quaternary and primary structure of only a small number of adhesins is currently available. Those associated with Escherichia coli are known in some cases to be arranged as helical coils with repeating proteinacious subunits with molecular weights of 10-30 kDa, however there is conflicting information on the distribution along these coils of the polypeptide involved in adhesion. Thermodynamic binding studies have not yet been used to clarify this problem because of the size of the receptors for the adhesins. This thesis presents a thermodynamic study of the binding between the adhesin from an F41+ E.coli and its receptor, glycophorin, from the human red blood cell membrane using an aqueous polymer two-phase system. The study shows that 2.4±0.8 glycophorin molecules bind to the predominant subunit of this adhesin, suggesting that this subunit has one binding site, since glycophorin dissolves as a dimer. It is proposed that the assay could be used, in addition, to obtain information on the chemical specificity and the thermodynamics of this particular reaction, in order to obtain a broader understanding of the colonisation and infection by this particular pathogen. / Science, Faculty of / Chemistry, Department of / Graduate

Terminal uridylation of RNA 3ꞌ ends has recently been recognised as an important biological regulatory mechanism. The human terminal uridyl transferase ZCCHC11 was shown previously to uridylate and thereby regulate replication-dependent histone mRNAs and several miRNA precursors and mature miRNAs. Studies in cancer cell lines, mouse models and patient samples suggest that ZCCHC11 is a potential oncogene. However, very little is known about regulation of its expression and subcellular localisation. In this study, the regulation of the expression of ZCCHC11 was investigated, especially in relation to the roles played by the untranslated regions of its mRNA. The second aim of this study was to investigate the subcellular localisation of ZCCHC11, both under physiological conditions and under cellular stress. The results presented show that ZCCHC11 expression is regulated negatively through its 5ꞌ and 3ꞌUTRs. Upstream open reading frames in the 5ꞌUTR decrease its translation, while regulation through the 3ꞌUTR involves the combined activity of positive and negative factors. In this study it was shown that HuR positively regulates ZCCHC11 expression, whereas TTP contributes to its negative regulation. An homologous enzyme, ZCCHC6, negatively regulates ZCCHC11 expression. Regulation through the 3ꞌUTR of ZCCHC11 is particularly marked following S-phase arrest. ZCCHC11 expression increases under cellular stress and both UTRs take part in this regulation. Regulation under cellular stress also involves an alteration in sub-cellular localisation. ZCCHC11 is predominantly cytoplasmic in unstressed cells, but re-localises to p-bodies and stress granules under cellular stress. Mutating one of three zinc knuckle motifs within ZCCHC11 significantly decreased differential co-localisation with stress granule marker TIA-1 under oxidative stress. Taken together, the data presented provide insights into the post-transcriptional regulation of this post-transcriptional regulatory protein and its subcellular localisation, especially under cellular stress.

1 v. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1992

Thesis (Ph. D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1992.

Many plant RNA viruses provide replication and encapsidation functions for one or more subviral RNAs that can modulate the symptoms of the associated helper virus. In this dissertation, I report my studies on symptom modulation in Arabidopsis thaliana by subviral RNAs associated with turnip crinkle virus (TCV), a single-stranded, positive-sense, plant RNA virus. Satellite RNA C (satC) is a virulent satRNA that normally intensifies symptoms of wild-type (wt) TCV but can attenuate symptoms if the TCV coat protein (CP) is either replaced with that of cardamine chlorotic fleck carmovirus (TCV-CPCCFV) (Kong et al., 1995) or if TCV contains an alteration in the CP initiation codon (TCV-CPm) (Kong et al., 1997b). I found that TCV-CPm produced reduced level of a CP (10∼20% of wt level) that contained two additional amino acids at its N terminus and did not form virions in infected protoplasts. SatC did not substantially affect the accumulation of TCV-CPm genomic RNA in protoplasts. These results, along with data reported previously (Kong, 1996), led to the conclusion that satC-mediated symptom attenuation of TCV-CPm involves a reduction in virus long-distance movement (Kong et al., 1997b). By characterizing the promoter for the CP mRNA, i.e., the 1.45-kb sgRNA promoter and defining the sequence and structural elements required for promoter activity, I was able to construct TCV variants expressing a level of wt CP similar to TCV-CPm (10∼20% of wt). I found that these mutants also have their symptoms attenuated by satC, indicating that the level of viral CP, not the mutation in the N terminus, is the crucial factor in determining whether satC is going to attenuate or exacerbate symptoms. Another normally virulent subviral RNA, namely defective interfering RNA G (diG) exhibited different symptom modulation of TCV-CPm compared with satC, and the determinants for this differential symptom modulation were previously localized to the T-terminal 100 bases of the subviral RNAs containing six positional differences (Kong et al., 1997a). In this dissertation I report the further characterization of the determinants in these six positions and the possible mechanism underlying the differential symptom modulation by these two subviral RNAs. My results revealed that two positions located in the 3′-terminal stem-loop structures of satC and diG, which also serve as promoters for complementary strand synthesis, are critical for symptom modulation. Furthermore, the hairpin CP binding capacity correlates with the symptom modulation. Several models for symptom modulation by the subviral RNAs associated with TCV are proposed.

Thesis (M.S.)--Michigan State University. Dept. of Small Animal Clinical Sciences, 2008. / Title from PDF t.p. (viewed on Aug. 10, 2009) Includes bibliographical references (p. 101-125). Also issued in print.

Retinoblastoma binding protein 6 (RBBP6) is a nuclear protein, previously implicated in the regulation of cell cycle and apoptosis. It is a multi-domain protein containing a Zinc finger, a RING finger, an Rb binding domain, a p53 binding domain and a novel N-terminal protein domain, the so called, Domain With No Name or DWNN. The RBBP6 gene encodes three isoforms of this particular protein. A common feature of all three isoforms of RBBP6 is the presence of the N-terminal DWNN domain. RBBP6 isoform 3 is comprised of the DWNN domain only. The DWNN itself has a ubiquitin-like fold, sharing 22% similarity with ubiquitin. It is likely that DWNN regulates intracellular levels of the two tumour suppressors, Rb and p53 through the ubiquitin-proteasome pathway and as such, DWNN may therefore play a role in the deregulation of cell cycle control in cancer cells. A mouse homologue, P2P-R of the gene has been implicated in mitotic apoptosis. The expression of DWNN, RBBP6 and their roles in the cell cycle, apoptosis and human cancer were investigated. RT-PCR and real-time PCR were used to determine the gene expression of DWNN and RBBP6 variants in human cancer cells. An anti-human DWNN antibody was characterized using both Western Blotting analysis and MALDI-TOF mass spectroscopy to determine whether the antibody specifically recognizes DWNN and RBBP6 isoforms, or if it recognizes other proteins. Western blotting was also used to determine the nature of the DWNN in human cell lines. A DWNN probe and the characterized anti-human antibody were used to localize DWNN and RBBP6 gene products at the mRNA and protein levels using ISH/FISH and Immunohistochemistry respectively. Cell labelling was also performed using this antibody to localize RBBP6 products in human cell lines. RNA interference and over-expression of DWNN and RBBP6 gene products was carried out to further investigate the role of RBBP6 products in the cell cycle, apoptosis and carcinogenesis. Cloned RT-PCR products of RBBP6 binding domains, the RING finger domain, pRb-binding and p53-binding domains in human cancers cell lines (Hek 293T, MCF7, HeLa and HepG2 cells) showed no mutations, but MCF-7 cells showed the lowest expression of the RBBP6. Real-time PCR and Western blotting analysis confirmed that MCF-7 cells express very little DWNN (RBBP6 isoform 3) and RBBP6 gene products when compared to Hek 293T, HeLa and HepG2 cells. It was also shown that the anti-human DWNN antibody recognizes the DWNN domain (RBBP6 isoform 3) and the larger RBBP6 isoforms. Using 2D gel electrophoresis and MALDI-TOF spectrometry, it was also found that DWNN is associated with other proteins namely, Recoverin and a hypothetical protein XP_002342450. This result suggested that DWNN may be a ubiquitin-like protein, which may be specific to these proteins in human cells. FISH and IHC demonstrated that the DWNN domain and its relatives are down-regulated in human cancers at both mRNA and protein levels, respectively. In contrast, however, cell staining showed that the expression of the DWNN gene products was high during the G2/Mitosis transition. Knocking-down the DWNN domain or over-expressing it did not sensitise the Hek 293T cells to Camptothecin (CPT)-induced apoptosis but rather slowed down cell growth. These results strongly suggest that the DWNN gene is likely to be involved in cell cycle control. Up-regulation in mitotic cells and down-regulation in cancers also implies that RBBP6 gene products may additionally be involved in cell cycle arrest. Moreover, down-regulation in human cancers particularly indicates that the loss of its function which correlates with loss of cell cycle control in this disease may be involved in the pathogenesis of cancer. This was confirmed by up-regulation of the DWNN in arsenic trioxide induced cell cycle arrested cells specifically at G2/M phase where a p53-dependent cell cycle arrest ensued. It is thus proposed that the DWNN may be implicated both as a p53 stabilizer and additionally as a G2/M progression regulator.

TRAP1 is a Heat Shock Protein (HSP) chaperone to retinoblastoma but also associated to the tumor necrosis factor receptor. HSPs are primarily up regulated in cancer. Work in our lab noted a down regulation of TRAP1 in some non-small cell lung cancers compared to normal lung. The first aim of this project was to evaluate the effect of the loss of TRAP1 on cell proliferation using a spheroid model. The presence of TRAP1 in spheroids promoted cell proliferation and a faster onset of hypoxia. This suggests an oncogenic role for TRAP1 since rapid hypoxia development equates to poor prognosis. Micro array analysis showed that TRAP1’s loss was associated with increased transcrpition of the Junctional Mediating and Regulatory protein (JMY). JMY possesses an oncogenic property due to its ability to facilitate cell motility. Additionally it has tumor suppressor activity in promoting p53 activation. The second aim of this project was to produce an anti-JMY antibody and use it to characterize JMY and additionally verify the association between TRAP1 and JMY. JMY was found to be widely expressed in normal tissues and in many types of tumors. In neoplastic tissues, comparing primary versus metastatic tumors, JMY was found to have significantly higher expression in the metastatic compared with the primary tumors. A pilot study showed that nuclear co-expression of JMY and P53 was associated with shorter overall survival suggesting that a possible tumorigenesis mechanism could be via a deregulation/mutation of JMY/p53 or both. Finally, using 3 dimensional constructions, I demonstrated the distinct morphological difference between an angiogenic tumor and a non-angiogenic tumor. Additionally, I showed a characteristic cytoplasmic p53 sequestration in the non-angiogenic phenotype that is absent in the angiogenic phenotype. This could be the mechanism that the non-angiogenic tumor uses to adapt to hypoxia. This would imply that there is a potential for cancers to escape therapy by switching between these 2 phenotypes.

The molecular basis for the gene-for-gene relationship of Vm-resistance in apple to Venturia inaequalis was investigated. Incompatible reactions involved a hypersensitive response (HR), which was accompanied by the accumulation of dark brown pigments and autofluorescent materials in epidermal and mesophyll cells at the site of invasion. Cell-free culture filtrates of the avirulent isolate elicited an HR in the Vm host (h5) leaves, but not in the susceptible host (h1). The elicitor activity was resistant to boiling but was abolished by proteinase K digestion. Elicitation of HR was used to monitor purification of the avirulence factor, AVRVm, from liquid cultures of the avirulent isolate following ultrafiltration, acetone precipitation and ion-exchange chromatography. The purest fraction contained three major proteins all with low isoelectric points (pI 3.0-4.5). The fraction also elicited HR on the differential host h4, but not on other resistant hosts (h2, h3 and h6) tested. Three candidate AVRVm proteins were identified and amino acid sequences were obtained using Edman degradation and mass spectrometry. Nucleotide sequences corresponding to these proteins were found in databases of V. inaequalis expressed sequence tags. There were no polymorphisms evident between avirulent and virulent isolates (representing races 1 and 5 respectively) either at genomic DNA or cDNA level of the full open reading frames. RT-PCR revealed that all genes were expressed in both avirulent and virulent isolates during in vitro and in planta growth. All three genes showed similar levels of expression between avirulent and virulent isolates during their in vitro growth. However, preliminary RT-PCR experiments showed that two of these genes were likely to be expressed at lower levels in the virulent compared with the avirulent isolate during compatible infection. Implications of this difference in expression and the future experiments to identify the genuine AvrVm gene were discussed.

The molecular basis for the gene-for-gene relationship of Vm-resistance in apple to Venturia inaequalis was investigated. Incompatible reactions involved a hypersensitive response (HR), which was accompanied by the accumulation of dark brown pigments and autofluorescent materials in epidermal and mesophyll cells at the site of invasion. Cell-free culture filtrates of the avirulent isolate elicited an HR in the Vm host (h5) leaves, but not in the susceptible host (h1). The elicitor activity was resistant to boiling but was abolished by proteinase K digestion. Elicitation of HR was used to monitor purification of the avirulence factor, AVRVm, from liquid cultures of the avirulent isolate following ultrafiltration, acetone precipitation and ion-exchange chromatography. The purest fraction contained three major proteins all with low isoelectric points (pI 3.0-4.5). The fraction also elicited HR on the differential host h4, but not on other resistant hosts (h2, h3 and h6) tested. Three candidate AVRVm proteins were identified and amino acid sequences were obtained using Edman degradation and mass spectrometry. Nucleotide sequences corresponding to these proteins were found in databases of V. inaequalis expressed sequence tags. There were no polymorphisms evident between avirulent and virulent isolates (representing races 1 and 5 respectively) either at genomic DNA or cDNA level of the full open reading frames. RT-PCR revealed that all genes were expressed in both avirulent and virulent isolates during in vitro and in planta growth. All three genes showed similar levels of expression between avirulent and virulent isolates during their in vitro growth. However, preliminary RT-PCR experiments showed that two of these genes were likely to be expressed at lower levels in the virulent compared with the avirulent isolate during compatible infection. Implications of this difference in expression and the future experiments to identify the genuine AvrVm gene were discussed.