Translation factors are essential components of the ribosome, yet it has been reported that many ribosomal proteins (RPs) and other translation factors are found at transcription sites of Drosophila melanogaster (D. melanogaster) polytene chromosomes. Whilst these findings might indicate the presence of ribosomal subunits at transcription sites, it has also been reported that these proteins associate with non-coding RNA in Saccharomyces cerevisiae (S. cerevisiae), suggesting that their localization with transcription sites reflects their non-ribosomal function. However, the functional significance of RPs and translation factors at transcription sites is unclear and may reflect excess protein synthesize unincorporated into ribosomes, leading to a large pool of free proteins able to interact non specifically with other proteins and nucleic acids. The following work investigates these issues further in Schizosaccharomyces pombe (S. pombe). I tagged three RPs (RpL7, RpL11 and RpL25), by homologous recombination and used a Chromatin immunoprecipitation approach to investigate whether the association of RPs occurs across specific genes/transcripts or whether chromatin association is genome wide. In agreement with previous studies in D. melanogaster and S. cerevisiae, I found that RPs preferentially associate to transcriptionally active genes. ChIP followed by analysis on micro-arrays (ChIP-on-chip) revealed that RPs associate with several protein encoding genes. Further analysis of the three RPs showed that they tend to bind a common subset of genes. Whilst RNase sensitivity suggests RPs association with nascent RNA, I found no correlation between the ChIP-on-chip signals of RPs with either Pol II occupancy or transcript level but did show that RPs associate with non-coding-RNA genes most notably with tRNA genes. ChIP of RpL7 in a strain carrying an exogenous wild-type tDNATyr gene or a mutant with a non-functional promoter confirmed that RpL7 associates only to the active tRNA gene. These results suggest a functional role of RPs in tRNAs biogenesis and perhaps in a role in Pol III transcription, as it was recently suggested by the finding that RPs copurify with TFIIIE in S. cerevisiae. Nonsense-mediated mRNA decay (NMD) removes any mRNA containing premature termination codon (PTC), and requires Upf1. Phospho-Upf1 inhibits conversion of 40S/Met-tRNAiMet/mRNA to translationally competent 80S/Met-tRNAiMet/mRNA initiation complexes to repress continued translation initiation. Analysis with Upf1 showed Upf1 also associates with many transcription sites and has a role in DNA replication and/or repair. Notably, Upf1 binds to chromatin mostly during S phase; perhaps indicating a role for its helicase activity during DNA replication in S. pombe. In summary, my data indicate that association of RPs, and at least one NMD factor, to chromatin is a general feature of eukaryotes. The main challenge for future studies is to identify the factors driving this association and the functional significance.

Mycobacterium tuberculosis, the causative agent of the infectious disease Tuberculosis, is one of the most successful human pathogen. The disease remains a global health priority due to the spread of HIV and multidrug resistant strains. Therefore, there is a need to extend the understanding of the physiology and pathogenicity of M. tuberculosis in order to develop new therapies, antimicrobial drugs and vaccines. Mycobacterium possesses a unique cell envelope responsible for the reduced susceptibility to antibiotics and pathogenicity due to the high lipid content. It is composed by the plasma membrane, an unusual lipid-rich cell wall and an outermost layer known as the capsule. Moreover, other complex metabolites play a role in M. tuberculosis virulence such as inorganic polyphosphate, a polymer involved in stringent response and long term survival. In this study, a transposon mutant library was generated in order to identify new genetic determinants related to cell envelope; furthermore, specific mutants strains were generated to investigate the role of MmpL factors in mycolic acids transport, to test the role of a group of ABC-transporters in capsule biosynthesis and also to assess the function of exopolyphosphatases in survival under stress and nutrient limitation condition.

The ability of Escherichia coli to survive in extreme acid conditions is an important component of its physiology. In my study I have profiled the Escherichia coli K-12 BW25113 strain using microarray technology and I have analysed a multi-omics dataset representing the transcriptional and metabolic responses of the MG1655 Escherichia coli strain. An initial high-level model in the BW25113 strain representing the interaction between two component systems regulators and effectors functions was built using the ARACNE methodology. My model supported the view that acid resistance involves a mechanism based on the transcriptional switch between the expression of genes encoding aerobic and anaerobic enzymes and controlled by the two-component system regulator OmpR. Experimental validation of the model confirmed this hypothesis. This model allowed me to predict that the MG1655 strain would be more sensitive to acid than the related BW25113 strain. Acid exposure induced an opposite response in this strain by repressing most of the anaerobic enzymes in favour of the aerobic metabolism. A dynamical model, developed by using State Space Models, revealed three potential regulators of acid adaptation in the MG1655 strain: OmpR, YehT and DcuR. I concluded that OmpR has a key role in acid adaptation in both strains and that the ability to reassess the balance in the expression of bioenergetics genes is more important for survival than proton detoxification.

The chicken (\(Gallus\) \(gallus\) \(domesticus\)) is the most abundant and widely distributed livestock animal with a global population of over 21 bill ion. A newly hatched broiler chick increases its body weight by 25% overnight and 50-fold over five weeks. The symbiotic, complex and variable community of the microbiome forms an important part of the gastrointestinal tract (gut). It is involved in gut development, biochemistry, immunology, physiology and non-specific resistance to infection. This study investigated the chicken gut microbiota using high-throughput 16S rRNA sequencing and culture-based techniques. There was specific interest in the proventriculus of which there is limited research currently in the literature and the caecum because it contains the highest density of bacterial cells in the gut at 10\(^1\)\(^1\) per gram. The results showed no significant difference in the first stages of the gut which shared a low-diversity microbiota dominated by a few \(Lactobacillus\) species. The microbiota becomes more diverse in the latter pa1ts of the small intestine where \(C/ostridiales\) and \(Enterobacteriaceae\) were present in higher numbers. The caecum was the most diverse organ with the majority of species belonging to Ruminococcaceae, Lachnospiraceae and \(Alistipes\). A number of novel species were isolated from the chicken gut and six of these were whole-genome sequenced.

For most pathogenic bacteria, the adherence to host cells is a crucial step for infection. Therefore, targeting bacterial attachment could be a potential way for treating bacterial infections. This thesis focuses on the interactions between the adhesin Multivalent Adhesion Molecule (MAM7) from the commensal Escherichia coli strain HS with host receptors, aiming to understand the molecular basis of these interactions. Targeting the interactions between MAM7 and host cells was one of this study’s aims towards developing anti-bacterial agents. In this study, HSMAM7 based adhesion inhibitors- beads coupled to HSMAM7 and an engineered bacterium expressing HSMAM7- were designed and their role as anti–adhesion agents against pathogenic bacteria was investigated. This study demonstrates that HSMAM7 based inhibitors have the ability to displace pathogenic bacteria from the host cells. Dissecting the binding of HSMAM7 to host receptors was the second part of this study. The findings revealed that HSMAM7 binds to sulfatide, several components of the extracellular matrix (ECM) and mucin. Further investigation of the interaction between HSMAM and host receptors revealed that HSMAM7 binds to mucin and the host lipid sulfatides via specific recognition of a shared 3-O-sulfo-galactosyl moiety in a sulfation dependent manner. Sulfatase-producing Bacteroides thetaiotaomicron affect the binding of HSMAM7 to mucin. HSMAM7 binding to mucin was inhibited in the presence of B. thetaiotaomicron and that means the commensal E. coli would be free and able to attach to the host epithelial cells, a finding of potential relevance for inflammatory bowel diseases (IBDs).

Genotoxicants are well known and important environmental contaminants. However, current ecotoxicological testing strategies generally focus on apical and reproductive endpoints, with little consideration given to subtle sub-lethal effects such as genotoxicity. Moreover, even less information is available on the responses of lower animals and plants to such substances. In this study, the sub-lethal responses of the water flea Daphnia magna, and the unicellular alga Chlamydomonas reinhardtii were investigated following exposure to known genotoxic substances. These studies were aimed at providing fundamental information on the responses of these two species thus addressing their relevance as potential alternatives to the use of vertebrates (e.g. fish) for the ecotoxicological testing of the effects of such compounds. Methods were developed to allow the Comet assay to be applied to algal and daphnid cells. Statistically significant increases in DNA strand breaks were detected following exposure of algal cells to selected direct- and indirect-acting genotoxicants. An apparent relatively low sensitivity was not elevated by using a wall-free mutant. In contrast, the DNA damage responses in D. magna did not achieve statistical significance. Methods were also developed to allow the xenobiotic biotransformation capability (via 7-ethoxyresorufin-O-deethylase or EROD activity) of both organisms to be measured. C. reinhardtii and D. magna possessed low, but measurable, basal EROD activity (0.03 pmol/minute/106 algal cells and 7 fmol/minute/daphnid). In C. reinhardtii, a 48h exposure to β-naphthoflavone (0.2 and 1nM) did not significantly induce activity, and in D. magna, EROD activity was not significantly affected by the presence of dimethylsulfoxide (up to 0.1%) or methanol (up to 0.05%). In addition to the analysis of effects at the sub-cellular level, gene expression analysis using D. magna oligonucleotide microarrays was also undertaken to assess the effect of these gentoxicants, on DNA repair, at the transcriptomic level. The microarrays revealed unique expression profiles for adults and neonates and significantly higher expression of some DNA repair genes in adults. Following exposure to a mixture of sodium dichromate and benzo[a]pyrene, a greater number of DNA repair genes showed up-regulation in adults compared to neonates. The majority of modulated genes implicated sodium dichromate as the primary stressor (e.g. glutathione-S-transferases, peroxiredoxins, ferritins), and potential reproductive and population level effects were identified. In all this study has highlighted the potential use of C. reinhardtii and D. magna in genotoxicity testing, using the methods developed herein. These studies revealed that both species are able to activate and respond to genotoxicants, although there were some clear differences in terms of sensitivity to the compounds applied. Furthermore, novel gene expression profiling in D. magna also offers a potential complementary measurement of genotoxicant exposure, via the assessment of DNA repair capacity. This methodology could be applied in addition to standard mutation and DNA damage assays to provide a battery approach to non-vertebrate

Fibroblasts are important regulators of inflammatory processes. The phenotype of fibroblasts differ according to anatomical site which may dtermine immune functions such as leukocyte accumulation and predilection for inflammatory disease in certain tissues. This thesis describes the expression profile and explores the function of a family of immunomodulatory proteins (galectins) in fibroblasts from rhematoid arthritis patients. Synovial fibroblasts were found to differ significantly from bone marrow and skin fibroblasts with higher expression of galectin-9 and galectin-12 in synovial fibroblasts. Galectin-9 and galectin-3 expression was also examined in situ in synovial tissue from rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Expression of both galectins were higher in RA synovial tissue compared to OA but not in synovial fibroblasts cultured in vitro. Galectin-3 expression seemed to be controlled by epigenetic factors (methylation) but not cytokine stimulation. Galectin-9 production was up-regulated by interferon-y, interleukin-1b and ligands for Toll-like receptors 3 (TLR3) and 4 (TLR4). It was found that intracellular presence of galectin-9 in RA synovial firboblasts increased their resistance to apoptosis. Galectin-3 level are increased in teh joints of patients with rheumatoid arthritis. Studies on the effect and mechanism of galectin-3 action on fibroblasts revealed that exogenously added galectin-3 induced production of cytokines (IL-6) from synovial and skin fibroblasts but the production of moncyte attracting chemokines (CCL5, CCL2) was induced uniquely in fibroblasts derived from teh synovium. Different signalling pathways mediated the secretion of those mediators. IL-6 release depended on MAP kinases p38, ERK and JNK as well as NFkB transcription factor, whereas CCL5 production required PI3/Akt and NFkB

Ageing, immunity and stress tolerance are inherent characteristics of all organisms. In animals, these traits are regulated, at least in part, by forkhead transcription factors in response to upstream signals from the Insulin/Insulin–like growth factor signalling (IIS) pathway. In the nematode Caenorhabditis elegans, these phenotypes are molecularly linked such that activation of the forkhead transcription factor DAF-16 both extends lifespan and simultaneously increases immunity and stress resistance. It is known that lifespan varies significantly among the Caenorhabditis species but, although DAF-16 signalling is highly conserved, it is unclear whether this phenotypic linkage occurs in other species. In this project we investigate this phenotypic covariance by comparing longevity, stress resistance and immunity in four Caenorhabditis species. We show, using phenotypic analysis of DAF-16 influenced phenotypes, that among four closely related Caenorhabditis nematodes, the gonochoristic species (Caenorhabditis remanei and Caenorhabditis brenneri) have diverged significantly with a longer lifespan, improved stress resistance and higher immunity than the hermaphroditic species (Caenorhabditis elegans and Caenorhabditis briggsae). Interestingly, we also observe significant differences in expression levels between the daf-16 homologues in these species using Quantitative Real-Time PCR, which positively correlate with the observed phenotypes. We also provide additional evidence in support of a role for DAF-16 in regulating phenotypic coupling by using a combination of wildtype isolates, constitutively active daf-16 mutants and bioinformatic analysis. Finally, we take a closer look at the daf-16 gene and its isoforms in C. elegans and their role in driving specific responses to stress. These findings impact upon our understanding of the diversification of the IIS pathway and the evolution of longevity in general, and illustrate how such differences could explain both inter and intra-species differences in ageing, immunity and stress response.

The lymph node (LN) microenvironment in Chronic Lymphocytic Leukaemia (CLL) is the main site of disease progression and maintenance. Whilst isolated components of the LN niche have been studied in vitro, to date, no comprehensive architectural overview of the microenvironment has been attempted. A more holistic view is essential in order to fully understand this disease. LN CLL cells are likely to receive a complex array of survival signals from accessory cells which drive disease and protect against conventional therapeutics. This study embarked upon establishing reliable combinations of primary and secondary antibodies that permit multicolour immunohistochemistry (IHC) interrogation of the CLL LN in formalin fixed paraffin embedded samples (FFPE). This work serves to demonstrate that the architecture of the CLL microenvironment is complex, dynamic and heterogeneous and highlights the advantages multicolour IHC can present to the field for understanding the therapeutic opportunities in this disease.

In eukaryotes, transcription and RNA processing events are spatially separated from translation by the nuclear envelope. Although ribosomal subunits are synthesised and assembled in the nucleus, it is believed that they are kept inactive whilst in the nucleus. Yet, there were observations from this and other laboratories that suggest translation can occur in the nucleus. To further investigate whether translating ribosomes exist in the nucleus, I employed a bimolecular fluorescence complementation (BiFC) 80S reporter based technique previously developed in our laboratory that detects 80S assembly in \(Drosophila\) cells. The initial characterization indicated that the assay reports ribosomal subunit interaction, but other explanations could not be excluded. My first aim was to assess whether this technique is genuinely reporting translation-dependent subunits association. My results were consistent with the assay reporting 80S ribosomes formed as a consequence of translation. Following up from this, I developed a similar technique with Venus, which resulted in a more sensitive 80S reporter. While the 80S signal was more apparent in the cytoplasm, in both cell culture and fly tissue cells, a fraction of the cells showed a signal in the nucleus, particularly concentrated in the nucleolus. This signal was enhanced by translation elongation inhibitors in both cytoplasm and nucleus indicating that the detected 80S are engaged in translation. Notably, the nucleolar signal was prevented by RNA Pol II inhibition, suggesting that the 80S might be associated with mRNA in the nucleolus.