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Item Shear stress adaptation of Listeria monocytogenes in mono and dual-species biofilms(Elsevier Ltd, 2025-12-01) Pant K; Palmer J; Flint SWhile the impact of stress on L. monocytogenes associated with food processing has been recognized in planktonic conditions, the available research overlooks the response of this pathogen in the multi-species biofilm, commonly found in food processing and manufacture. The objective of this study was to understand the effect of shear stress on L. monocytogenes in single and dual-species (with P. fluorescens) biofilm formed in a continuous turbulent flow system. In the single-species biofilm, L. monocytogenes was able to form a biofilm under the turbulent flow with cell concentration reaching 5.1 log CFU/cm2 after 48 h, where filamentous cells (27.7 μm in length) were observed. In contrast, there were no visible filaments in the dual-species biofilm, and L. monocytogenes cell concentration was significantly higher (p < 0.001) at 8.7 log CFU/cm2. The cells harvested from single-species L. monocytogenes biofilm formed under turbulent flow showed significantly (p < 0.001) lower motility and higher adhesion compared with cells harvested from planktonic and static conditions. Gene expression analysis showed significant (p < 0.001) downregulation of motB (motility), sigB (stress), and cell division (ftsX and ftsW), and upregulation of mpl (adhesion) and rodA (rod shape), indicating L. monocytogenes adaptation to shear stress. This study provides fundamental information on the multi-species biofilm formation by L. monocytogenes under stress.Item Investigating the impact of tobacco particulate matter and selected components on monoamine oxidase activity, protein expression, and gene expression in brain SH-SY5Y cells : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Health Sciences at Massey University, Wellington, New Zealand(Massey University, 2023) Niraula, PrakshitSmoking addiction is one of the most widely discussed topics today due to a large number of smokers and the millions of lives it claims every year. Though a large population makes effort to quit smoking, the quit attempts mostly end in relapse, indicating the complex nature of smoking addiction. Nicotine is believed to be the major component responsible for the addiction, however, nicotine replacement therapy (NRT) has not proved to be a completely satisfying approach to smoking cessation. The low efficacy of NRT, as well as much research related to smoking addiction, suggest the role of non-nicotinic components in smoking addiction. It is hypothesised that monoamine oxidase inhibitors (MAOIs) present in tobacco smoke play a role in smoking addiction by prolonging nicotine’s reinforcing effect. Based on this hypothesis, Tobacco Research Group, Wellington has identified six candidate MAOIs in cigarette smoke. This PhD project aims to investigate the effect of nicotine, tobacco particulate matter (TPM) and the candidate MAOIs on MAO activity, MAO protein levels, MAO genes expression and global gene expression. A human neuroblast SH-SY5Y cell line was exposed to different regimens, which included ethanol (control), nicotine, TPM and the cocktail of candidate MAOIs for a period of 1, 3, 5 and 7 days. A modified kynuramine assay was performed after SH-SY5Y cells were exposed to the different treatments to determine the effect of the exposure treatments on MAO activity and to identify the optimum period of exposure that would result in maximum MAO inhibition. Exposure for a period of 3 days was chosen as an optimum period of time for exposure and for expression and whole genome experiments. Similarly, a MTT assay was performed to determine if the exposure treatment had any cytotoxic effect. Change in MAO protein and MAO gene expression after exposure to the different treatments for the optimum period of time were then determined using Western blot and qPCR, respectively. Finally, the effect of exposure treatments for an optimum period of time on global gene expression was determined using RNA sequencing (RNAseq) technology. It was observed that nicotine did not have any significant MAO inhibitory effect compared to the control in any of the treatments examined. TPM and MAOIs caused significant inhibition of total MAO activity when exposed for 1 and 3 days. However, no significant inhibition was seen in the exposure for 5 and 7 days. Change in MAO A and MAO B gene and protein expression levels after the exposure treatment for 3 days was not observed. Nevertheless, several genes were found to have differential expression after exposure to the treatments for 3 days. Many of these differentially expressed genes were linked with diseases and conditions related to smoking and addiction. The results suggest that the candidate MAO inhibitors identified by the Tobacco Research Group, Wellington could be the primary contributors of the MAO inhibitory property observed in cigarette smoke. This data could also possibly answer the major question regarding the component responsible for MAO inhibition by cigarette smoke in smokers. Further research is required to fully elucidate and understand the mechanisms behind the MAO inhibition from the MAOIs, and a better understanding of these mechanisms may provide a framework for the development of novel smoking cessation therapies.Item Functional characterisation of coq8 in Drosophila : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Science in Genetics, Massey University(Massey University, 2018) Hura, Angelia JosephineWith the increasing number of novel mutations being discovered by whole genome and whole exome sequencing, functional studies are increasingly required to determine whether specific mutations are responsible for the disease phenotypes. Drosophila, with its vast set of genetic and molecular tools as well as robust behavioural assays, is an ideal model for functional characterisation. Coenzyme Q biosynthesis is highly conserved from yeast to humans and involves a number of genes in the enzymatic pathway including COQ8A. The role of COQ8A in CoQ biosynthesis is not clear. However, mutations in COQ8A have been associated with autosomal recessive cerebellar ataxia, which is characterised by gait ataxia, cerebellar degeneration and CoQ10 deficiency. This project aimed to characterise the phenotypes resulting from the reduction of coq8 expression (the Drosophila homologue of COQ8A) to develop a model of coq8 deficiency that could be used to characterise COQ8A mutations functionally. RNAi knockdown of coq8 resulted in severe developmental delay, larval lethality, locomotor impairment, a decrease in ATP production, as well as developmental deficits and neurodegeneration in the Drosophila eye. Reintroduction of wild-type Drosophila coq8 partially rescued the larval lethality, restored locomotor function and also primarily rescued the necrotic phenotype in the eye. This model could, therefore, be used to determine whether a specific mutation impaired function, such that it would not rescue the deficiency. As a proof-of-principle, two mutant variants of coq8, I295P and L520*, which were modelled on the human COQ8A mutations L277P and c.1506+1G>A (which results in a truncated protein) did not rescue the coq8 deficiency, indicating that they disrupted normal coq8 function. However, the reintroduction of human COQ8A did not restore function but instead exacerbated the necrotic and neurodegenerative phenotype in the eye suggesting that it may be impairing the mitochondrial function of wild-type coq8. Drosophila provides the means to characterise disease-causing genetic mutations functionally. Here we have developed a model that can be used to study the role of coq8 in Drosophila and have found that Drosophila coq8 and human COQ8A differ in function.Item MSL-1 plays a central role in assembly of the MSL complex which mediates dosage compensation in Drosophila melanogaster : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North, New Zealand(Massey University, 1998) Pan, Lewis Li-WeiDosage compensation in Drosophila melanogaster is achieved by a twofold increase of transcription of X-linked genes in males. This involves the binding of four proteins, MSL-1, MSL-2, MSL-3 and MLE (collectively known as the MSLs) which are believed to act as a multi-protein complex, to hundreds of sites along the length of the X chromosome. MOF, a putative histonc acetyl transferase, is thought to be also associated with MSLs and plays a role in hypertrascription of X-linked genes. Overexpression of either a C-terminal or N-terminal domain of MSL-1 leads to male-specific lethality which is probably due to association with other MSLs to form a non-functional complex. One aim of this study was to identify whether any known MSLs and/or unknown protein binds with the C-terminal domain of MSL-1. A second aim was to further define the domain of MSL-1 which interacts to MSL-2. Initial attempts to identify the protein which interacts the C-terminal domain of MSL-1 by either genetics analysis or co-immunoprecipitation were inconclusive. Thus, an alternative approach of affinity chromatography of epitope-tagged MSL-1/MSL-complex was followed. Transgenic flies which express either a FLAG-tagged N-terminal region of MSL-1 or FLAG tagged C-terminal domain following heat shock were generated. These lines were crossed with other transgenic lines to co-express the MSL-1 domain with Either MSL-2, MSL-3, MLE or MOF. FLAG affinity chromatography of protein extracts prepared from these flies showed that MSL-2 co-purifies with the N-terminal domain of MSL-1 (aa 85 - 263), whereas MOF and MSL-3 co-purify with the C-terminal domain of MSL-1 (aa 705 - 1039). MLE docs not appear to associate with either region of MSL-1. Further, the C-terminal domain of MSL-1 also bound specifically to a glutathione S-transferase-MOF fusion protein. Co-expression of MSL-2 rescued males from the lethal effect which was caused by overexpression of the N-terminal domain of MSL-1. However, co-expression of either or both MOF and MSL-3 with the C-terminal domain of MSL-1 did not improve male viability. This suggests that additional factors may bind to the FC/MOF/MSL-3 complex. Finally, MLE also bound to GST-MOF fusion protein, suggesting a direct interaction between MLE and MOF. These findings suggest that MSL-1 plays a central in assembly of the MSL multi-protein complex that is required to achieve dosage compensation.Item Development of a reporter gene assay to identify control elements required for dosage compensation in Drosophila Melanogaster : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University(Massey University, 1998) Fitzsimons, HelenDosage compensation (equalisation of X-linked gene products) occurs in Drosophila melanogaster by a two-fold transcriptional increase of X-linked gene expression in the male. This involves the binding of four proteins, MSL-1, MSL-2, MSL-3 and MLE (collectively known as the MSLs), to hundreds of sites along the length of the male X. The MSLs are thought to recruit MOF, a histone acetyl transferase, which facilitates the increase in transcriptional activity of X-linked genes. The DNA sequences required to target the MSL complex to the X chromosome (known as dosage compensation regulatory elements, or DCREs) remain elusive, despite numerous attempts over the last ten years to identify them. DCREs are thought to be present at multiple sites along the length of the X chromosome, as antibodies to the MSLs bind to hundreds of sites along the X, and autosomal genes transduced to the X usually become dosage compensated. The first objective of this study was to develop a reporter gene assay to screen for DCREs that would minimise problems previously encountered. A construct consisting of the constitutive armadillo promoter fused to the lacZ reporter gene (called arm-lacZ) was flanked by insulator elements which block the repressive effects of the autosomal chromatin environment. Fragments of X-linked DNA were inserted upstream of the armadillo promoter with the premise that males carrying one copy of an autosomal insertion of this construct would express twice the level of ß-galactosidase as females. Transgenic flies carrying autosomal insertions of X-linked fragments plus arm-lacZ were generated and one dose males and females were assayed for ß-galactosidase activity using a spectrophotometric assay. In all cases, males and females expressed the same level of lacZ. This suggests that no DCREs that could confer dosage compensation onto arm-lacZ were present in the X-linked fragments. arm-lacZ is capable of being dosage compensated as males and females carrying one copy of an X-linked insertion of arm-lacZ produce a 2:1 male to female ratio. This implies that DCREs of the 'strength' required to dosage compensate arm-lacZ are rarer than previously thought. A second method of dosage compensation that is independent of the MSLs is thought to occur in Drosophila. The X-linked gene runt is dosage compensated in the absence of the MSLs. It is possible that runt is sex specifically regulated by the female specific Sex lethal protein (Sxl). Sxl down-regulates msl-2 in females by binding to (U)8 or A(U)7 sequences in the msl-2 5' and 3' untranslated regions (UTRs) of the mRNA. runt mRNA contains three Sxl binding sites in its 3' UTR, as do 20 other X-linked genes. The second objective of this project was to determine if Sxl could down regulate a gene in females, purely by the addition of three Sxl binding sites to the 3'UTR. Sxl binding sites were inserted into the 3'UTR of arm-lacZ in the form of a 40 bp synthetic linker containing three of the sites, and also as a 170 bp fragment from the runt 3' UTR. ß-galactosidase assays of flies carrying the Sxl binding sites from runt showed that males expressed an average of 1.31 to 1.46 times the level of lacZ than females. This shows that Sxl can down-regulate a gene if there are Sxl binding sites in its 3' UTR, however, to achieve two-fold regulation, additional factors may be required, or topologically, the sites may not have been in the right position in the 3' UTR for optimal activity of Sxl. Flies carrying the synthetic linker expressed the same level of ß-galactosidase in both sexes which suggests that either additional elements within the 3' UTR are required, or that the spacing between the sites is critical for the action of Sxl.Item Construction of a novel fungal gus expression plasmid, and its evaluation in Aspergillus nidulans : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University(Massey University, 1996) McGowan, Tania LouiseA GUS expression plasmid, pFunGus, was constructed containing a multi-cloning site for the insertion of gene regulatory elements, to be used in fungal reporter gene studies. A derivative of pFunGus (pFG-gpd) was constructed by the insertion of the gpdA promoter (glyceradehyde-3-phosphatc dehydrogenase) into the multi-cloning site of pFunGus for the assessment of the plasmid's transformation and expression properties in Aspergillus niduans. The correct construction of pFunGus and pFG-gpd was verified by analytical restriction digests and by its property of GUS expression in A. nidulans. The plasmid was integrated into the A. nidulans genome via cotransformation with the phleomycin resistance plasmid, pAN8-l. Transformation frequencies of between 3 and 250 transformants per µg of pAN8-l DNA were obtained. Initial screening for cotransformation yielded no pFG-gpd transformants. Attempts to improve cotransformation frequencies by optimisation of cotransformation conditions were unsuccessful. However, large scale screenings of transformants lead to cotransformants being isolated at a very low cotransformation frequency. Approximately 0.45% of pAN8-l transformants possessed the GUS phenotype. The eight pFG-gpd transformants obtained were analysed by Southern hybridisation. Six out of the eight transformants had a single copy integration. Of the remaining two transformants, one had three copies integrated at separate locations, one of which was disrupted, and the other had four copies integrated as tandem repeats, one of which was disrupted. All the transforming DNA appeared to be integrated ectopically. The physiology of the transformants was assessed by dry weight increase, colony extension and total protein content. These showed that the transformants biology was not significantly compromised by the transforming DNA. Finally, high levels of GUS expression were observed in all pFG-gpd transformants and the GUS expression per copy of the GUS expression cassette integrated into the genome was constant. These results showed that the transformed gene copy number determined the levels of gene activity rather than the position of integration in the genome. Overall these results demonstrate the potential application of the versatile GUS expression plasmid, pFunGus for reporter gene studies in filamentous fungi.Item Spatial and temporal localisation of exopolysaccharide gene expression in mucoid and non-mucoid Pseudomonas aeruginosa biofilms : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology, Massey University, Manawatu, New Zealand(Massey University, 2014) Holbrook, Stacey Ann KashelThe biofilm, or surface-associated microbial community, is the preferred method of growth for most bacteria. Pseudomonas aeruginosa is an ubiquitous, opportunistic pathogen capable of biofilm formation in a wide range of natural and clinical environments. In particular, biofilms formed by P. aeruginosa in the lungs of people with cystic fibrosis (CF) are responsible for a significant decline in the health and prognosis of these patients. Once established, P. aeruginosa biofilms begin to excrete an exopolysaccharide (EPS) called alginate which protects the bacterial microcolonies from antimicrobial molecules and confers a mucoid phenotype. Once this phenotypic switch has occurred, the biofilm becomes impossible to eradicate and ultimately leads to the death of the patient. Here, fluorescent signalling systems and confocal laser scanning microscopy (CLSM) have been used to spatially and temporally resolve the expression of three EPSs produced by P. aeruginosa; the pellicle-forming EPS (Pel), the EPS encoded by the polysaccharide synthesis locus (Psl) and alginate. In order to observe the effect (if any) of EPS production on spatial localisation of the cells within the biofilm, the biofilm-associated characteristics of three P. aeruginosa double-knockout mutants, each able to produce only one EPS has been observed. In analysing these biofilm structures, it was found that Pel has a role in facilitating an increased surface area of the biofilm, while Psl-producing mutants form a biofilm structure with a significantly increased biomass. By visualising fluorescent signals throughout a biofilm consisting of a mixture of the three mutants, the spatial localisation of EPS-producing bacterial populations has been observed. Here, Pel-producing mutants tended to aggregate at the attachment surface, suggesting a role in adhesion of the biofilm structure. Spatial and temporal localisation of EPS promoter activity was achieved by transforming the prototypic P. aeruginosa PAO1 strain with one of three plasmids encoding unstable gfp expression under the control of each EPS’s promoter sequence. Overall, this study has demonstrated the applications and limitations of fluorescence-based localisation of bacterial gene expression throughout P. aeruginosa biofilm development. Collectively, this information can help to guide future investigations into the expression and regulation of the genes associated with a biofilm phenotype, with the aim of identifying a target for effective therapy against this important pathogen.Item Development of a tetracycline-inducible lentiviral vector with an instant regulatory system : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science (MSc) in Biochemistry at Massey University, Manawatu, New Zealand(Massey University, 2013) Yang, TianLentiviral vectors, originally derived from human immunodeficiency virus, provide highly efficient viral gene delivery vehicles. Lentiviral vectors often use a constitutive promoter to drive the expression of a therapeutic gene. To regulate the expression of a therapeutic gene, a regulatory system such as Tet-On needs to be established in the target cell lines to produce a regulatory protein, reverse Tet-responsive transcriptional activator (rtTA). The expressed rtTA binds to the tetracycline responsive element (TRE) in the promoter in response to doxycycline and activates transcription of gene of interest. A hypothesis in this study is based on the speculation that a basal leaky expression of rtTA in the bi-directional TRE vectors allows instantly inducible expression of a gene of interest and thereby avoids the time-consuming procedures for generating Tet-On cell lines. Based on this hypothesis, a novel lentiviral vector has been developed to examine an instant induction of PP2Cβ as a target gene. Three instantly inducible bicistronic lentiviral vectors [pLenti-Bi-TRE-Tet-on (V), pLenti-Bi-TRE-Tet-on-PP2Cβ WT (WT), pLenti- Bi-TRE-Tet-on-PP2Cβ MUT (MUT)] were constructed and characterised to assess the usefulness of these vectors. Transient transfection of both WT and MUT vectors into HEK293T cells showed a great induction of PP2Cβ expression upon 24 h of 1 μM doxycycline treatment. The result promises the use of these vectors as a mammalian expression plasmid with a feature of inducible target gene expression. However, viral infection studies involving lentiviral packaging and infection procedures did not show a reproducible expression of rtTA or PP2Cβ in HEK293T cells. Therefore, the inducibility of viral transduction needs to be improved for the future studies of PP2Cβ in primary cells.Item Lactococcal plasmid replicon ; vector construction and genetic organization : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University, Palmerston North New Zealand(Massey University, 1990) Xu, FengfengThe 5.5 kb high-copy number cryptic plasmid pDI25 from Lactococcus lactis subsp. lactis 5136 was isolated and used as the basis to construct a series of vectors. The vector pFX1 (5.5 kb) was first made by ligating the 4.5 kb HpaII-MboI fragment of the lactococcal plasmid to the 1 kb chloramphenicol transacetylase gene from the staphylococcal plasmid pC194. Plasmid pFX1 was further modified by deleting a non-essential 1.9 kb ClaI region to construct pFX2 (3.6 kb). Deletion analysis showed an essential region for plasmid replication was located within a 1.2 kb CfoI-ThaI-CfoI fragment. The vector pFX3 was constructed by incorporating the α fragment of the Escherichia coli lacZ structural gene, a multiple cloning region and the T7 and T3 promoters from pUBS into pFX2. Recombinant plasmids constructed in E. coli using X-gal selection could be subsequently electroporated into lactococci. pFX3 could also be used directly for transcription studies or DNA sequencing of cloned inserts. A set of lactococcal translational gene-fusion vectors was constructed by incorporating the E. coli lacZ gene fusion system (pNM480,481,482) into pFX2. These constructions, pFX4, pFX5 and pFX6, permit the fusion, of cloned genes to lacZ in all three reading frames. Gene expression can be readily and quantitatively monitored by measuring β-galactosidase activity. All the pFX vectors were efficiently transformed into lactococci and E. coli by electroporation (104-106 cfu/μg DNA in each host) and maintained stably in both organisms (> 95% cells carrying the Cm marker after 100 generations growth without drug selection). A cell-wall bound proteinase from Lactococcus lactis subsp. cremoris H2 was isolated and characterized as a PI type proteinase since it preferentially degraded β-casein. A 6.5 kb HindIII fragment of plasmid pDI21 (63 kb) was initially cloned and expressed this enzymatic activity in E. coli using vector λNM1149. The restriction map of this pDI21 prt gene fragment had minor differences from those of other published lactococcal prt fragments. Using pFX1, the pDI21 prt gene fragment was recloned and directly electroporated into lactococci where it was efficiently expressed. The effectiveness of pFX3 was demonstrated by initially cloning a pDI1 4.4 kb EcoRI tagatose 1,6-bisphosphate aldolase gene fragment into E. coli from where it was electroporated into lactococci. Using the translational fusion vectors pFX4, pFX5 and pFX6, the 6.5 kb HindIII prt gene fragment of pDI21 was identified as having two promoters with opposite orientations. The pDI21 2.0 kb EcoRI galactose-6-phosphate isomerase gene fragment was shown to carry a promoter and the direction of gene transcription was determined. The complete DNA sequence of the lactococcal portion of pFX2 (2508 bp) was determined and the genetic organization analyzed. A lactococcal plasmid plus origin and two replication protein coding regions (repA and repB) were located. RepA had an αhelix-turn-αhelix motif, a geometry typical of DNA-binding proteins. RepB showed high homology to the plasmid replication initiation proteins from other Gram-positive bacteria and Mycoplasma. The transcribed inverted repeat sequence between repA and repB could form an attenuator to regulate pFX2 replication. Upstream of the plus origin site, and in a region nonessential for replication, a 215 bp sequence identical to the staphylococcal plasmid pE194 and carrying the RSA site was identified. The genetic organization of this lactococcal plasmid replicon shares significant similarity with the pE194 group of plasmids.Item Does DNA topography coordinate intra- & inter-chromosomal Galactose gene expression? : this thesis is presented in partial fulfilment of the requirements for the degree of Masters of Science in Molecular Biology at Massey University, Albany, New Zealand(Massey University, 2008) Yu, ShuqiaoFor a long time, DNA had been considered as a stabilized, rigid, and “linear” structure, which acts as a platform for molecular regulators to function. However, genome structure in living cells is far more complex than the linear representation of the primary DNA sequence implies. This thesis aims to investigate whether the position of a gene within the genome plays a role in the regulation of its activity. The galactose (GAL) gene family of Saccharomyces Cerevisiae is used as a model. This gene family enables yeast cells to utilize galactose as an alternative carbon source; and it consists of structural and regulatory genes. Structural genes GAL1, GAL10 and GAL7 exist in a cluster on yeast chromosome II. The products of the regulatory genes, GAL3, GAL4, and GAL80, regulate the expression of the GAL structural genes, depending on the availability of carbon sources. Specifically, GAL gene expression is repressed by glucose, paused for induction (noninduced) by glycerol/lactate, and fully induced by galactose. The aim of this project was to study the relative position of the GAL structural genes within the nucleus, and whether any chromosomal interactions at the GAL locus help to regulate their activation. These were tested in accordance with the expression status of the GAL genes (i.e. repressed, noninduced or induced). Followed confirmation of the existence of any chromosomal interactions, protein/protein complexes that mediate these interactions were attempted to identify. The methods applied in this project were Chromosome Conformation Capture (3C) and Circular Chromosome Conformation Capture (4C), which applied in combination to map the positions of the GAL genes in the context of the overall genome structure. The results indicated that the GAL locus on chromosome II was divided into two “interaction zones”. DNA loops formed around these interaction zones to form an S-shape structure in a carbon source-independent manner. Two novel inter-chromosomal interactions between chromosomes II and XVI, i.e. SVL3-GAL7 and HOS1-GAL10, were also identified. Although these interactions occurred regardless of the GAL gene activities, it was suggested by real-time PCR that the interaction frequency for SVL3-GAL7 declined as the GAL genes being activated. Unfortunately no protein/protein complexes were identified to play an important role in mediating either intra- or inter-chromosomal interactions. Future work will be needed to identify the protein/protein complexes that play a role in mediating the S-Shape structure at the GAL locus and the two inter-chromosomal interactions. Additional works could also focus on the understanding of the functional implication of the interactions between chromosomes II and XVI.