published_or_final_version / Electrical and Electronic Engineering / Master / Master of Philosophy

Persistence in human macrophages is central to the virulence of Mycobacterium tuberculosis, which is the causative agent of tuberculosis. Although the intracellular parasitism is apparent, molecular determinants of mycobacterial virulence are not well understood. The current investigation identified virulent genes of M. tuberculosis by measuring survivability of Mycobacterium smegmatis recombinants inside a human monocytic cell line THP-1 after acquiring various virulent gene candidates of M. tuberculosis. These gene candidates included nine virulent gene candidates suggested by other studies, five genomic polymorphisms identified in hypervirulent strains of M. tuberculosis using microarray-based comparative genomic hybridization, and ten single nucleotide polymorphisms identified in the hypervirulent strains using full genome sequencing. Interestingly, only recombinants harboring a truncated Rv2820c and a known virulent gene mce1A survived significantly better than vector control after six hours of ex vivo infection. As nucleotide sequencing indicated that the truncated Rv2820c loses around 60% of gene at 3’ end, ex vivo survivability of M. smegmatis recombinants harboring the last 60% of Rv2820c as well as the intact Rv2820c was measured, but was similar to that of vector control. The 3’ truncated portion itself did not alter mycobacterial survivability ex vivo, but its presence did compromise the survival advantage gained due to the truncated Rv2820c. To determine whether the truncated and the intact Rv2820c could enhance mycobacterial virulence in vivo, these two alleles were transformed into Mycobacterium marinum and their recombinants were used to infect zebrafish. In vivo infection showed that zebrafish infected with the recombinant harboring truncated Rv2820c died significantly faster than vector control, whereas the recombinant harboring intact Rv2820c behaved similarly to vector control. Results indicated that the truncated Rv2820c, but not the intact Rv2820c, could enhance mycobacterial virulence both ex vivo and in vivo. Additional nucleotide sequencing revealed that the 3’ truncation in Rv2820c is caused by a Beijing/W-defining deletion RD207 and is commonly found in Beijing/W strains of M. tuberculosis. Non-Beijing/W strains possess the intact Rv2820c conversely. Since Beijing/W strains have proven to be more virulent than non-Beijing/W strains both ex vivo and in vivo, the truncated Rv2820c may be one of the Beijing/W-specific virulence determinants. To confirm that Rv2820c of Beijing/W strains really enhances M. tuberculosis survival in human macrophages, the truncated Rv2820c was transformed into non-Beijing/W M. tuberculosis strains and their recombinants were used to infect THP-1 cells. Ex vivo infection confirmed that the truncated Rv2820c could enhance M. tuberculosis survival inside human macrophages, but is unlikely to induce a different profile of cytokine secretion from infected macrophages. In conclusion, the current study demonstrated that the truncated Rv2820c of Beijing/W strains could enhance mycobacterial virulence both ex vivo and in vivo. Enhanced phenotypic virulence, however, was not observed for the intact Rv2820c of non-Beijing/W strains. The truncated Rv2820c may be one of the Beijing/W-specific virulence determinants and collaboratively contribute to the high phenotypic virulence of this family. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

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To study the function of WdStuAp and WdPacCp in Wangiella dermatitidis, a black, polymorphic fungal pathogen of humans with yeast phase predominance, WdSTUA and WdPACC were cloned, sequenced, disrupted and expressed. WdStuAp was most similar to the APSES proteins of Aspergillus species and its APSES DNA-binding domain was located in its N-terminal half. Deletion of WdSTUA in W. dermatitidis induced convoluted instead of normal smooth colony surface growth on the rich, yeast maintenance agar medium, YPDA, at 37°C. Additionally, deletion of WdSTUA repressed aerial hyphal growth, conidiation and invasive hyphal growth on the nitrogen poor, hyphae-inducing agar medium, PDA, at 25°C. Ectopic expression of WdSTU Arepressed the convoluted colony surface growth on YPDA at 37°C, and also strongly repressed hyphal growth on PDA at 25°C and 37°C. Expression of WdSTUA in S. cerevisiae induced pseudohyphal growth on the nitrogen poor medium. WdPacCp was also most similar to the PacCp proteins of Aspergillus species. Three zinc finger DNA-binding motifs were at the N-terminus, and the C-terminus had the signaling protease cleavage site. WdPACC was more expressed at neutral-alkaline pH than at acidic pH. Truncation of the coding sequence for about 40 residues upstream of the conserved processing protease cleavage site of WdPacCp affected growth on YPDA, increased sensitivity to Na⁺ stress, decreased growth level at neutral-alkaline pH, and repressed hyphal growth on PDA at 25°C. Truncation of the coding sequence for the conserved signaling protease box of WdPacCp impaired growth and reduced RNA expression of class II chitin synthase gene WdCHS1 at acidic pH, and activated hyphal growth on PDA. My results suggested that WdStuAp and WdPacCp play important roles in yeast-hyphal transitions in W. dermatitidis, and that WdPacCp is particularly important for W. dermatitidis to adapt to different ambient pH conditions.

This study examined maternal predictors of children's facial expressions of emotion in mother-child interactions. Ninety-four mothers and their 14- to 27-month old toddlers were observed during a 20-minute interaction. Results demonstrated that two different components of maternal sensitivity, supportive behavior and child-oriented motivation, predicted more facial expressions of joy and sadness and less flat affect in children. Maternal autonomy granting, a third component of maternal sensitivity, predicted more facial expressions of anger in children. This study also examined relations between macrosocial variables (i.e., maternal well-being and demographic factors) and children's facial expressions of emotion and how maternal sensitivity mediated such relations. High maternal education was directly related to fewer facial expressions of sadness and anger, high SES was related to more facial expressions of joy, and both greater marital satisfaction and social support were related to more facial expressions of anger. It was also shown that supportive behavior mediated associations between: maternal depressive symptoms and both low joy and high flat affect, marital satisfaction and low flat affect, maternal education and high joy, and family income and high joy. Child-oriented motivation mediated associations between maternal depressive symptoms and both high flat affect and low sadness. Findings suggest that it is important to consider multiple measures of maternal sensitivity and the broader macrosocial context in which the parent-child relationship is embedded when examining children's facial expressions of emotion in mother-child interactions.

MicroRNAs (miRNAs) play a vital role in regulating gene expression. Detecting conserved and novel miRNAs in very large genomic datasets generated using next generation sequencing platforms is a new research area in the field of gene regulation, but finding useful miRNA information from a large wheat genome is a challenging research project. We propose to design a toolchain that will identify conserved miRNAs using various software tools such as Basic Local Alignment Search Tool (BLAST), Bowtie 2, MAFFT and RNAfold. Our toolchain identified 36 wheat conserved miRNA families that matched with 232 experimental sequences. Moreover, we found 87 plant conserved miRNA families that matched between 613 experimental sequences and the miRBase dataset. In addition, we observed significant differential expression for the wheat exposed to the heat stress compared to those exposed to light and UV stresses or no stress (control). / October 2015

Recombinant Protein G (rPG), an engineered form of streptococcal protein G with a theoretical molecular weight of 22.26 kDa was successfully cloned and expressed in E.coli BL 21(DE3) cells. The albumin binding domain was removed during the gene synthesis to avoid unspecific binding. This recombinant form of protein G contains only the IgG binding domains along with the 6X histidine tag at the N terminal. The removal of non-specific domains maximizes the specificity of IgG binding through the Fc region. The recombinant protein G was purified through heat treatment and using immobilized metal affinity chromatography (IMAC). On an SDS-PAGE gel there was only a single band of the purified preparation which migrated at 32 KDa, however when analyzed by mass spectrometry it was ~22.4 kDa, this phenomenon of retarded migration on SDS-PAGE has been known from previous studies. The production of recombinant protein has also been optimized. The effects of expression temperature, inducer type, inducer concentration and media composition have been investigated. The expression was done at 10 liter scale using the best expression conditions, and the protein was purified to homogeneity, dialyzed and lyophilized. The pure protein was immobilized on a POROS AL (Self Pack® POROS® 20 AL, Applied Biosystems, USA). The immobilized protein IgG binding has been monitored using a VersAFlo system. This system allowed real time monitoring of IgG binding characteristics. / <p></p><p> </p>

Allelic expression imbalance, or AEI, is the term given to differences in the expression levels of the two alleles of a gene. AEI has been previously identified in a number of species using various techniques. Here, the genome-wide extent of allelic expression imbalance in the pathogenic yeast species, Candida albicans, was examined through use of RNA sequencing in combination with a novel computational pipeline based around the diploid reference genome. Techniques for validating these results were investigated, and the difficulties surrounding specificity and quantification are discussed. As C. albicans is a highly heterozygous species, it was hypothesised that polymorphisms within alleles lead to differences in allele expression, which are further linked to differences in allele function. The functional consequences of AEI were therefore interrogated through investigation of Gene Ontology, identification of condition specific responses in AEI, and targeted construction and phenotypic screening of heterozygous knockout strains. Together, these results strongly suggest that divergence in allele expression is not linked to differences in allele function. Investigations of the possible control mechanisms behind the differences in allele expression were considered, with a focus upon structural factors such as chromosomal location, GC content, allele length and codon usage. However, issues with establishing causality are present, and difficulties lie in distinguishing between functional differences and consequences of bias in sequencing technologies. This piece of research has advanced the understanding of gene expression mechanisms within a medically important pathogen, paving the way for further investigations into the functional consequences of allelic expression imbalance in Candida albicans.

I present the results of my analysis of the final stage of gene expression, the translation of messenger RNA. Translation is carried out by large molecular motors called ribosomes, which move along the mRNA molecule in a stepwise fashion, producing a polypeptide chain. Translation can be modelled as a driven diffusion process, and I extend the Totally Asymmetric Simple Exclusion Process (TASEP) to consider the possibility that ribosomes that terminate from the lattice can rejoin the lattice to reinitiate translation, known as ribosome recycling. Inclusion of recycling leads to marked changes in the phase transition boundaries when compared with a non-recycling lattice, with extension and expansion of the so-called maximal current regime, where protein production is at optimal levels. Recycling makes this phase accessible even at low values of de novo initiation. Furthermore, recycling leads to a sharp peak in particle current on the low density-high density phase boundary, with a decrease in current within the high density regime. Simulations of real biological sequences from the budding yeast Saccharomyces cerevisiae suggest that increasing ribosomal availability can, in fact, reduce the efficiency of protein production in a sequence-dependent manner, particularly for mRNAs encoding proteins with a stressresponse function. Consistent with this, separating the yeast transcriptome into phase transition classes brings to light class-dependent features including parameter values and protein functionality. The role ribosome recycling plays in determining the lifetime of a messenger RNA is also studied. Ribosome initiation has a stabilising effect; counteracting this is ribosome termination, which enhances removal of proteins that protect the mRNA 'tail', exposing it to degradation enzymes. Mathematical modelling of this process shows that recycling can affect mRNA lifetimes in a phase-dependent manner that is not straightforward. This indicates that there are additional, important factors involved in the regulation of decay beyond the interplay between termination and initiation. The research reported in this thesis shows that systems biology can offer insight into a complex stage of gene regulation, and has a significant role to play in future research.