Investigation of the genetic susceptibility to osteoporosis in the Irish population

Osteoporosis is a complex skeletal disorder characterized by compromised bone strength. Variation in bone mineral density (BMD) is a contributing factor. The aim of this research as to select informative single nucleotide polymorphisms (SNPs) in potential candidate genes from loci suggestively linked to BMD variation for fine mapping. The gene regulated by oestrogen in breast cancer 1 (GREB1), located at 2p25.1, was selected. GREB1 transcription is initiated early in the oestrogen receptor alpha regulated pathway. There was significant association between GREB1_03 and BMD variation at the lumbar spine and femoral neck (FN) in the discovery cohort. Significant association was observed between GREB1_04 and FN BMD in the replication cohort. The development and differentiation enhancing factor 2, the integrin cytoplasmic domain associated protein 1 and A-disintegrin and metalloprotease 17 were selected due to their respective roles in cell mobility and adhesion. There was no linkage or association observed between the Chr2 cluster SNPs and BMD. Two factors in bone remodelling are the attraction of bone cell precursors and endocrine regulation of the process, primarily through the action of parathyroid hormone (PTH). The C-C chemokine receptor type 3 (CCR3) encodes a CC chemokine receptor expressed in osteoclast precursors. The PTH receptor type 1 (PTHR1) encodes a G-protein coupled receptor for PTH. Association was observed between CCR3 haplotypes and BMD variation at the FN. There was no linkage or association observed between PTHR1 SNPs and BMD variation. Population genetic studies with complex phenotypes endeavour to elucidate the traits genetic architecture. This study presents evidence of association between GREB1 and BMD variation and as such, introduces GREB1 as a novel gene target for osteoporosis genetics studies. It affirms that common genomic variants in PTHR1 are not associated with BMD variation in Caucasians and supports the evidence that CCR3 may be contributing to BMD variation

The Irish paradigm on the natural progression of hepatitis C virus infection: an investigation in a homogeneous patient population infected with HCV 1b (Review)

The aetiological agent of chronic hepatitis C is the hepatitis C virus. The hepatitis C virus is spread by parenteral transmission of body fluids, primarily blood or blood products. In 1989, after more than a decade of research, HCV was isolated and characterised. The hepatitis C viral genome is a positive-sense, single-stranded RNA molecule approximately 9.4 kb in length, which encodes a polyprotein of about 3100 amino acids. There are 6 main genotypes of HCV, each further stratified by subtype. In 1994, a cohort of women was identified in Ireland as having been iatrogenically exposed to the hepatitis C virus. The women were all young and exposed as a consequence of the receipt of HCV 1b contaminated anti-D immunoglobulin. The source of the infection was identified as an acutely infected female. As part of a voluntary serological screening programme involving 62,667 people, 704 individuals were identified as seropositive for exposure to the hepatitis C virus; 55.4% were found to be positive for the viral genome 17 years after exposure. Of these women 98% had evidence of inflammation, but suprisingly, a remarkable 49% showed no evidence of fibrosis. Clinicopathology and virological analysis has identified associations between viral load and the histological activity index for inflammation, and, between inflammation and levels of the liver enzyme alanine aminotransferase. Infection at a younger age appears to protect individuals from progression to advanced liver disease. Molecular analyses of host immunogenetic elements shows that particular class II human leukocyte associated antigen alleles are associated with clearance of the hepatitis C virus. Additional class II alleles have been identified that are associated with stable viraemia over an extended period of patient follow-up. Although, investigation of large untreated homogeneous cohorts is likely to become more difficult, as the efficacy of anti-viral therapy improves, further investigation of host and viral factors that influence disease progression will help provide an evidence based approach were realistic expectations regarding patient prognosis can be ascertained.

Design, fabrication and characterization of resonant waveguide grating based optical biosensors

The absence of rapid, low cost and highly sensitive biodetection platform has hindered the implementation of next generation cheap and early stage clinical or home based point-of-care diagnostics. Label-free optical biosensing with high sensitivity, throughput, compactness, and low cost, plays an important role to resolve these diagnostic challenges and pushes the detection limit down to single molecule. Optical nanostructures, specifically the resonant waveguide grating (RWG) and nano-ribbon cavity based biodetection are promising in this context. The main element of this dissertation is design, fabrication and characterization of RWG sensors for different spectral regions (e.g. visible, near infrared) for use in label-free optical biosensing and also to explore different RWG parameters to maximize sensitivity and increase detection accuracy. Design and fabrication of the waveguide embedded resonant nano-cavity are also studied. Multi-parametric analyses were done using customized optical simulator to understand the operational principle of these sensors and more important the relationship between the physical design parameters and sensor sensitivities. Silicon nitride (SixNy) is a useful waveguide material because of its wide transparency across the whole infrared, visible and part of UV spectrum, and comparatively higher refractive index than glass substrate. SixNy based RWGs on glass substrate are designed and fabricated applying both electron beam lithography and low cost nano-imprint lithography techniques. A Chromium hard mask aided nano-fabrication technique is developed for making very high aspect ratio optical nano-structure on glass substrate. An aspect ratio of 10 for very narrow (~60 nm wide) grating lines is achieved which is the highest presented so far. The fabricated RWG sensors are characterized for both bulk (183.3 nm/RIU) and surface sensitivity (0.21nm/nm-layer), and then used for successful detection of Immunoglobulin-G (IgG) antibodies and antigen (~1μg/ml) both in buffer and serum. Widely used optical biosensors like surface plasmon resonance and optical microcavities are limited in the separation of bulk response from the surface binding events which is crucial for ultralow biosensing application with thermal or other perturbations. A RWG based dual resonance approach is proposed and verified by controlled experiments for separating the response of bulk and surface sensitivity. The dual resonance approach gives sensitivity ratio of 9.4 whereas the competitive polarization based approach can offer only 2.5. The improved performance of the dual resonance approach would help reducing probability of false reading in precise bio-assay experiments where thermal variations are probable like portable diagnostics.

Investigating the function of PINK1 in cancer development and pathogenesis

PTEN‐induced kinase 1 (PINK1) was identified initially in cancer cells as a gene up‐regulated by overexpression of the central tumour suppressor, PTEN. Loss‐of‐function mutations in PINK1 were discovered subsequently to cause autosomal recessive Parkinsonʹs disease (ARPD). Despite much research focusing on the proposed mechanism(s) through which loss of PINKI function causes neurodegeneration, few studies have focused on a direct role for this serine/threonine kinase in cancer biology. The focus of this thesis was to examine a direct role for PINK1 function in tumourigenesis. Initial studies showed that loss of PINK1 reduces tumour‐associated phenotypes including cell growth, colony formation and invasiveness, in several cell types in vitro, indicating a pro‐tumourigenic role for PINK1 in cancer. Furthermore, results revealed for the first time that PINK1 deletion, examined in mouse embryonic fibroblasts (MEFS) from PINK1 knock‐out animals, causes cell cycle defects, whereby cells arrest at in cytokinesis, giving rise to a highly significant increase in the number of multinucleated cells. This results in several key changes in the expression profile of cell cycle associated protein. In addition, PINK1‐deficient MEFs were found to resist cell cycle exit, with a proportion of cells remaining in proliferative phases upon removal of serum. The ability of cells to progress through mitosis conferred by PINK1 expression was independent of its kinase activity, while the cell cycle exit following serum withdrawal was kinase dependent. Investigations into the mechanism through which loss of PINK1 function gives rise to cell cycle defects revealed that dynamin related protein 1 (Drp1)‐mediated mitochondrial fission is enhanced in PINK1‐ deficient MEFs, and that increased expression of Drp1 on mitochondria and activation of Drp1 is highly significant in PINK1‐deficient multinucleated cells. Deregulated and increased levels and activation of mitochondrial fission via Drp1 was shown to be a major feature of cell cycle defects caused by PINK1 deletion, both during progression through G2/M and cell cycle exit following serum removal. Altered PINK1 localisation was also observed during progression of mitosis, and upon serum deprivation. Thus, PINK1 dissociated from the mitochondria during the mitotic phases and localised to mitochondria upon serum withdrawal. During serum withdrawal deletion of PINK1 disabled the ability of MEFs to increase mitochondrial membrane potential (ΔΨm), and increase autophagy. This was co‐incident with increased mitochondrial fission, and increased localisation of Drp1 to mitochondria following serum deprivation. Together, this indicates an inability of PINK1‐negative cells to respond protectively to this stress‐induced state, primarily via impaired mitochondrial function. In contrast, PINK1 overexpression was found to protect cells from DNA damage following treatment with oxidants. In addition, deletion of PINK1 blocked the ability of cells to re‐enter the cell cycle in response to insulin‐like growth factor‐1 (IGF‐1), a major cancer promoting agonistwhich acts primarily via PI3‐kinase/Akt activation. Furthermore, PINK1 mRNA expression was significantly increased following serum deprivation of MCF‐7 cells, and this was rendered more significant upon additional inhibition of PI3‐kinase. Conversely, IGF‐1 activation of PI3‐kinase/Akt causes a time‐dependent and significant reduction of PINK1 mRNA expression that was PI3‐kinase dependent. Together these results indicate that PINK1 expression is necessary for IGF‐1 signalling and is regulated reciprocally in the absence and presence of IGF‐1, via PI3‐kinase/Akt, a signalling system which has major tumour‐promoting capacity in cancer cell biology. The results of this thesis indicate PINK1 is a candidate tumour-promoting gene which has a significant function in the regulation of the cell cycle, and growth factor responses, at key cell cycle checkpoints, namely, during progression through G2/M and during exit of the cell cycle following removal of serum. Furthermore, the results reveal that the regulation of mitochondrial fission and Drp1 function is mechanistically important in the regulation of cell cycle control by PINK1. As deregulation of the cell cycle is linked to both tumourigenesis and neurodegeneration, the findings of this thesis are of importance not just for understanding cancer biology, but also in the context of PINK1‐associated neurodegeneration.

The role played by angiotensin (1-7) in the regulation of renal haemodynamics and excretory function in anesthetized rats

Initial studies have demonstrated that intra- renal infusion of Ang (1-7) caused a diuresis and natriuresis that was proportional to the degree of activation of the Renin Angiotensin Aldosterone System (RAAS). This raised the question as why the magnitude of this diuresis and natriuresis was compromised in rats receiving a high sodium diet (suppressed RAAS) and enhanced in low sodium fed rats (activated RAAS)? Could the answer lie with changes in intra-renal AT1 or Mas receptor expression? Interestingly, the observed Ang (1-7) induced increases in sodium and water excretion in rats receiving either a low or normal sodium diet were and blocked in the presence of the AT 1 receptor antagonist (Losartan) in the presence of the, 'Mas' receptor antagonist (A-779). These data suggest that both AT1 and 'Mas' receptors need to be functional in order to fully mediate the renal responses to intra-renal Ang (1-7) infusion. Importantly, further experimentation also revealed that there is a proportional relationship between AT 1 receptor expression in the rat renal cortex and the magnitude of the excretory actions of intra renal Ang (1-7) infusion, which is only partially dependent on the level of 'Mas' receptor expression. These observations suggest that although Ang (1-7) induced increases in sodium and water excretion are mediated by the Mas receptor, the magnitude of these excretory responses appear to be dependent upon the level of AT 1 receptor expression and more specifically Ang II/ AT 1 receptor signalling. Thus in rats receiving a low sodium diet, Ang (1-7) acts via the Mas receptor to inhibit Ang II/ AT 1 receptor signalling. In rats receiving a high sodium diet the down regulated AT 1 receptor expression implies a reduction in Ang II/ AT 1 receptor signalling which renders the counter-regulatory effects of intra-renal Ang (1-7) infusion redundant.

Mutational and molecular analyses of lactococcal bacteriophages TP901-1 and Tuc2009

Bacteriophages belonging to the Siphoviridae family represent viruses with a noncontractile tail that function as extremely efficient bacterium-infecting nanomachines. The Siphoviridae phages TP901-1 and Tuc2009 infect Lactococcus lactis, and both belong to the so-called P335 species. As P335 phages are typically capable of a lytic and lysogenic life cycle, a number of molecular tools are available to analyse their virions. This doctoral thesis describes mutational and molecular analyses of TP901-1 and Tuc2009, with emphasis on the role of their tail-associated structural proteins. Several novel and intriguing findings discovered during the course of this study on the nature of Siphoviridae phages furthers a basic molecular understanding of their virions, and the role of their virion proteins, during the initial stages of infection. While Siphoviridae virions represent complex quaternary structures of multiple proteins and subunits thereof, mutagenic analysis represents an efficient mechanism to discretely characterize the function of individual proteins, and constituent amino acids, in the assembly of the phage structure and their biological function. However, as always, more research is required to delve deeper into the mechanisms by which phages commence infection. This is important to advance our understanding of this intricate process and to facilitate application of such findings to manipulate phage infections. On the one hand, we may want to prevent phages from infecting starter cultures used in the dairy industry, while on the other hand it may be desirable to optimize viral infection for the application of phages as bacterial parasites and therapeutic agents.

Pregnancy-specific glycoprotein function, conservation and receptor investigation

Pregnancy-specific glycoproteins (PSGs) are highly glycosylated secreted proteins encoded by multi-gene families in some placental mammals. They are carcinoembryonic antigen (CEA) family and immunoglobulin (Ig) superfamily members. PSGs are immunomodulatory, and have been demonstrated to possess antiplatelet and pro-angiogenic properties. Low serum levels of these proteins have been correlated with adverse pregnancy outcomes. Objectives: Main research goals of this thesis were: 1). To attempt to replicate previously reported cytokine responses to PSG-treatment of immune cells and subsequently to investigate functionally important amino acids within PSG1. 2). To determine whether candidate receptor, integrin αvβ3, was a binding partner for PSG1 and to investigate whether PSG1 possessed functionality in a leukocyte-endothelial interaction assay. 3). To determine whether proteins generated from recently identified putative PSG genes in the horse shared functional properties with PSGs from other species. Outcomes: 1). Sequential domain deletion of PSG1 as well as mutation of conserved residues within the PSG1 Ndomain did not affect PSG1-induced TGF-β1. The investigated response was subsequently found to be the result of latent TGF-β1 contaminating the recombinant protein. Protein further purified by SEC to remove this showed no induction of TGF-β1. The most N-terminal glycosylation site was demonstrated to have an important role in PSG N domain secretion. PSG1 attenuated LPS-induced IL-6 and TNF-α. Investigations into signalling underpinning this proved inconclusive. 2). Integrin αvβ3 was identified as a novel PSG1 receptor mediating an as yet unknown function. Preliminary investigations into a role for PSGs as inhibitors of leukocyte endothelial interactions showed no effect by PSG1. 3). Horse PSG protein, CEACAM49, was shown to be similarly contaminated by latent TGF-β1 particle and once removed did not demonstrate TGF-β1 release. Interestingly horse PSG did show anti-platelet properties through inhibition of the plateletfibrinogen interaction as previously published for mouse and human PSGs.