The main aim of my research, presented here, is to develop proteomic research techniques, for their use in biomarker discovery and identification. This is broken down into three main chapters: • Biomarker Identification in Stroke Brain guided by MALDI-imaging. • Evaluation of the Effectiveness of Heat Treatment for Prevention of Proteomic Sample Degradation using Label Free Relative Quantitation. • Discovery and Identification of Biomarkers for Hypertension Within these chapters special attention is paid to sample preparation, development and assessment of new methods for biomarkers discovery and identification, the importance of experimental design and the application of relevant and useful statistical methods to enable the mining of useful information from rich datasets. The biological changes in stroke induced mouse brain tissue are studied, the prevention of degradation to tissue samples by a novel heat treatment method and changes to plasma samples from hypertensive, wild type and a congenic strain of rat are also studied. The outcomes of this work are multiple, namely: • The identification of a possible marker for stroke from mouse brain tissue • The effect of a new heat treatment device on proteomic data obtained from mouse brain tissue • The novel application of a statistical analysis to a new type of dataset (LC-MS and label free quantitation of biological samples) • The identification of possible biomarkers for hypertension in rat plasma using this method

Living systems exert exquisite control over biomineral production determining mineral type, polymorph, morphology and ultrastructure and ultimately producing biomineral structures that perform a range of functions. In addition to this biological control, the environment also influences aspects of biomineralisation. In marine invertebrates, biominerals record seawater chemistry and temperature at deposition. Past chemistry and temperature information is interpreted via proxies such as oxygen isotope and Mg/Ca ratios in calcium carbonate biominerals and used to help predict future climatic trends. Proxies must be robust and accurate yet the presence of biological or biologically-induced kinetic effects, many of which are a consequence of ontogeny, can make it difficult to isolate the environmental signal. In many cases, proxies are applied without detailed knowledge or assessment of shell microstructures and how this is altered as the animal ages. Interpretation of detailed crystallographic, chemical and isotopic changes in the context of ontogeny is therefore essential. This study considers crystallography, trace element chemistry and stable isotopic composition of six ontogenetic stages of farmed Mytilus edulis collected from a single location at the same time. The shell of Mytilus edulis is comprised of two calcium carbonate polymorphs: an outer layer of prismatic calcite and an inner layer of nacreous aragonite, both with very different morphologies. The crystallographic ultrastructure is analysed through Electron Backscatter Diffraction (EBSD) and Scanning Electron Microscopy (SEM). No significant changes in crystallography are evident between ontogenetic stages. Crystallographic orientation of calcite is more strictly constrained towards the polymorph interface and from anterior to posterior in all stages of ontogeny. Further evidence of ‘mineral bridging’ is observed in nacre. The minor element distribution varies between the polymorphs, through the shell thickness and between ontogenetic stages. Younger specimens have higher Mg, S and Sr concentrations while older specimens have increasing Na concentrations. Mg and S are present in the calcite layer but are virtually absent in aragonite. While Na and Sr occur in both polymorphs, higher concentrations occur in aragonite. Changes in Na and Sr concentrations in the aragonite layer can be linked to prismatic bands of myostracal aragonite in the nacre. A decrease in Na concentration through the calcite layer toward the polymorph interface is observed in all specimens. Increases in Mg and S concentration in the calcite layer near the interface are often observed. Greater trace element concentrations are also observed in the umbo region of the shell. Stable oxygen isotope signatures show small amounts of variation between regions of the shell and between ontogenetic stages. Decreasing δ18O values are observed in older animals and in the posterior edge of the shell. Large variations of δ18O are also observed through the shell thickness which cannot be accounted for by environmental changes. The majority of δ18O data falls within the expected range for equilibrium of calcite and aragonite with ambient seawater. No significant change is observed in δ13C values between ontogenetic stages or through the shell. Values of δ13C and δ18O co-vary in aragonite but not calcite. Refinement in crystallography occurs in conjunction with changes in trace element chemistry and stable oxygen isotope composition moving towards the shell posterior. This indicates a change as the animal grows, resulting in differences in chemical information retained in different parts of the shell. The differences in trace element chemistry between polymorphs and isotopic and chemistry changes across the shell thickness indicate changes that can be attributed to ultrastructure as crystal habit, polymorph and orientation change occur across the shell thickness. These results suggest that greater understanding of factors involved in biomineralising systems is required before undertaking work involving application of climate proxies.

The objectives of the studies presented in this thesis were to identify and quantify the major phenolic components of red wine and to assess the contribution of individual compounds to the total antioxidant activity. Red wines were analysed for their phenolic content and antioxidant activity using a range of complementary techniques including HPLC-tandem mass spectrometry, preparative HPLC and HPLC with an on-line antioxidant detection system. HPLC-MS2 revealed the presence of a number of flavoids and phenolic compounds of which 19 were identified, with gallic acid, the flavin-3-ols and anthocyanins being the most abundant. Preparative HPLC was used in an effort to isolate the antioxidant components in red wine and 60 aliquots were collected. Each wine fraction was analysed for total phenolics, catechins and anthocyanins, while antioxidant activity was determined by electron spin resonance spectroscopy (ESR). The preparative HPLC step did into completely separate the compounds in red wine, nonetheless increasing antioxidant activity was highly and significantly associated with total phenolics (r = 0.816, P < 0.001) and total catechins (r = 0.188, p = 0.151). HPLC with an on-line antioxidant detection system was subsequently used to separate and identify red wine phenolics. The findings from this study indicate that gallic acid, (+)-catechin, (-)-epicatechin, and procyanidin dimersB1 and B2 were the major in vitro antioxidants identified in red wine. Collectively, the flavin-3-ols contributed > 50% of the total antioxidant capacity of each wine, while gallic acid contributed between 24-44%. The flavonols and anthocyanins were minor antioxidant components in red wines. By combining HPLC, MS2 and on-line assessment of antioxidant activity, the major phenolic compounds present in red wine were identified, together with their direct contribution to the total antioxidant activity.

cAMP is a well studied second messenger that is ubiquitously expressed in mammals. It conducts its function by activating its downstream effectors: protein kinase A (PKA), exchange proteins regulated by cAMP (EPAC) and cyclic nucleotide gated ion-channels. The sole mechanism to inactivate cAMP is through degradation via cyclic-phosphodiesterases (PDEs). The PDEs, especially PDE4s, are involved in many diseases including asthma, chronic obstructive pulmonary disease (COPD) and depression. Therefore, PDEs have been a consistently popular research subject for decades and pharmaceutical companies have devoted considerable effort in developing PDE inhibitors. β-arrestin interacts with PDE4D5 and is a multifunctional protein that plays pivotal roles in signal transduction. It functions as an adaptor protein in the c-Raf/MEK/ERK cascade by interacting with c-Raf and ERK. In this study, I have shown that (1) β-arrestin1 can bind MEK1 directly, mediated at least in part by D26D29 of β-arrestin1 and R47K48R49 of MEK1. (2) Disruption of this association by mutagensis or small peptides decreases the phosphorylation of Ser412 on β-arrestin1, and accelerates isoprenaline-stimulated G-protein coupled receptor (GPCR) internalization. Dimerization of PDEs is considered important for their specificity. In this study, yeast two hybrid and co-immunoprecipitation were utilised to demonstrate that (1) PDE4D5 can form stable homodimers in both yeast and mammalian cell lines. (2) The dimerisation requires multiple interaction sites such as R173/N174/N175, E228/T229/L230, L306/M307/H308 and K323/T324/E325 (together named QUAD) in which R173/N174/N175 contributes most toward the dimerisation. (3) Association of an ion-pair R499/D463 also proved to be a necessary condition for dimer formation. (4) PDE4D5 dimerisation can be affected by challenge with the PDE inhibitor, anisomycin, and cAMP elevating agonists forskolin (Fsk) and isobutylmethylxanthine (IBMX). RACK1, another PDE4D5 binding partner, mediates and initiates cell migration in many cell types and affects the activity of the c-Jun NH2-terminal kinase (JNK) signalling pathway, via its interaction with PKC. SUMOylation of proteins is an important method of regulation. In the current study, preliminary investigations were undertaken to determine whether RACK1 is SUMOylated. SUMOylation of K271 of a 25-mer peptide sequence from RACK1 was observed, yet there was no SUMOylation of RACK1 observed in HEK293 cells in the presence or absence of overexpressed E3 ligases.

No description available.

The mitotic cell cycle underlies propagation of eukaryotic cells, continually duplicating and dividing. The past few years have seen major advances in understanding of the regulatory mechanisms that impose on the cell cycle to tightly co-ordinate progression through its individual phases, safeguarding the timing and integrity of its hallmark events, DNA synthesis and mitosis. Transcription is prominent among these processes, manifesting its importance for cell cycle controls by the large number of eukaryotic genes that are expressed at specific cell cycle times. Certain genes are cell cycle regulated in a number of organisms, suggesting that their phase-specific transcription is important for all eukaryotic cells. The budding and fission yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, have been used extensively as model organisms for the study of the eukaryotic cell cycle and cell cycle-regulated transcription, because the cell cycle machinery is conserved among eukaryotes and they are experimentally tractable. Recent microarray analyses have shown that cell cycle-specific expression is a frequent theme in the two yeasts, identifying consecutive, inter-dependent, waves of transcriptional activity, coinciding with the four main cell cycle transitions; G1-S, S, G2-M and M-G1 phases. Each phase-specific transcriptional wave corresponds to at least one group of co-regulated genes, sharing common cis- and trans- acting elements. The work presented in this thesis delves into the regulatory network that drives phase-specific gene expression during late mitosis-early G1 phase in fission yeast. During this late cell cycle stage, fission yeast and, indeed, every eukaryotic cell, undergo major changes; each completes mitosis and cytokinesis, partitioning its duplicated genetic and cytoplasmic material into two progeny cells, which then themselves prepare for a new round of mitotic cell division. Consistent with their periodic pattern of expression, most of the genes transcribed during the M-G1 interval in S. pombe encode proteins that execute important functions during late mitosis and cytokinesis. A DNA sequence promoter motif, the PCB (Pombe cell cycle box), has been identified in fission yeast that confers M-G1 specific transcription, and is bound by the PBF (PCB binding factor) transcription factor complex. PCB promoter motifs are present in several M-G1 transcribed genes, including cdc15+, spo12+, sid2+, fin1+, slp1+, ace2+, mid1+/dmf1+ and plo1+, the latter encoding a Polo-like kinase that also regulates M-G1 gene expression and influences the PCB-dependent binding properties of PBF. Three transcription factors, Sep1p and Fkh2p, both forkhead-like transcription factors, and Mbx1p, a MADS-box protein, have been implicated in M-G1 specific gene expression and are thought to be components of PBF. Consistent with Fkh2p and Sep1p regulating M-G1 specific transcription, forkhead-related sequences are present in the genes’ promoters. Notably, fkh2+ contains both PCB and forkhead promoter sequences and is transcribed during the M-G1 interval, implying that Fkh2p and Plo1p regulate gene transcription during late mitosis and ensuing passage through cytokinesis via feedback loops. This study provides further evidence about transcriptional regulation late in the fission yeast cell cycle, revealing that the PCB sequence is crucial for M-G1 specific transcription, with forkhead-associated DNA motifs playing a parallel but smaller regulatory role. Consistent with this hypothesis, work here and elsewhere shows that both Fkh2p and Sep1p control phase-specific expression of their co-regulated genes through the PCB and forkhead sequences. Notably, data in this thesis reveal that these two forkhead transcription factors associate with each other in vitro and in vivo and bind in vivo to the PCB promoter regions of M-G1 transcribed genes, including cdc15+ and plo1+, in a cell cycle specific manner, consistent with Fkh2p repressing and Sep1p activating transcription. Furthermore, Fkh2p contacts its own promoter, suggesting that it regulates its own expression via a negative feedback mechanism. The Plo1p kinase is shown here to bind in vivo to Mbx1p throughout the cell cycle and in a manner that requires both its kinase and polo-box domains. In agreement with this observation, Plo1p can phosphorylate in vitro Mbx1p, itself known to become phosphorylated during late mitosis. This is the first time that a Polo-like kinase has been shown to bind and phosphorylate a MADS-box protein in any organism. Moreover, in concert with Plo1p binding and phosphorylating Mbx1p, ChIP assays here reveal that this kinase interacts in vivo with the PCB promoter DNA of M-G1 expressed genes, including cdc15+ and fkh2+, in a cell cycle-dependent manner with a timing that coincides with low levels of expression, but follows promoter binding by Fkh2p. Given that Plo1p has previously been shown to positively influence M-G1 dependent transcription, its cell cycle pattern of promoter contact suggests that this Polo-like kinase functions at the genes’ promoters, most-likely via binding and phosphorylation of Mbx1p, to re-stimulate transcription, following repression by Fkh2p. In parallel, these findings suggest that Plo1p regulates its own expression via a positive feedback loop. Overall, the work presented in this thesis unravels crucial regulatory aspects of the transcriptional network that drives M-G1 specific transcription in S. pombe: it suggests an important role for the PCB promoter motif in transcriptional regulation; it proposes that Fkh2p acts as a repressor while Sep1p as an activator of late mitotic transcription; it reveals and proposes novel functions for Plo1p, a conserved Polo-like kinase family member, involving its association with Mbx1p, a MADS box protein, and its cell cycle specific recruitment to PCB promoters of M-G1 transcribed genes. As transcriptional systems, encompassing homologues of most of the components of this S. pombe M-G1 specific transcriptional network operate both in S. cerevisiae and humans, this demonstrates their importance for mitotic cell cycle progression. Thus this work potentially offers new insights into M-G1 specific gene expression in all eukaryotes.

Citrus fruits are of special interest as they accumulate large amounts of flavonoids and are consumed in substantial quantities worldwide. Despite extensive research on citrus flavonoids, many compounds remain unidentified in tropical citrus species. High performance liquid chromatography with mass spectrometry and electrospray ionisation (HPLC-MS–ESI) has proved to be a powerful tool for flavonoid characterisation. This study describes how HPLC-MS-ESI and HPLC with a photodiode array detector (PDA) was used to identify and quantify flavonoids in the tropical species Citrus microcarpa, Citrus hystrix, Citrus medica var. 1 and 2, Citrus suhuiensis and Citrus medica var. sarcodactylis. Among the major compounds detected were apigenin-6-8-di-C-glucopyranoside, apigenin-8-C-glucoside-2′-rhamnoside, phloretin-3′,5′-di-C-glucopyranoside, diosmetin-7-O-neohesperidoside, hesperetin-7-O-rutinoside, diosmetin-7-O-rutinoside and hesperetin-7-O-neohesperidoside. Most of the C-glycosylated flavones and dihydrochalcone have not been found previously in tropical citrus. C. microcarpa contained a high amount of phloretin-3′,5′-di-C-glucopyranoside that was shown to possess a high Trolox Equivalent Antioxidant Ratio (TEAR) value due to its 2,4,6-trihydroxyacetophenone structure. In general, most of tropical citrus flavanones were neohesperidoside conjugates, which are responsible for the bitter taste of the fruits and juices. Tropical citrus essential oils were extracted by steam distillation (SD) and simultaneous distillation extraction (SDE) and analysed using gas chromatography-mass spectrometry (GC-MS). More than 40 compounds were identified and the major component in almost all citrus fruits and leaves was R–(+)-limonene. Citronellal was detected and was highest in C. hystrix leaf (87.8%). The radical-scavenging activity of each oil was assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. Essential oils obtained from all citrus fruits showed very high radical-scavenging activity against the DPPH radical, well in excess that in leaves. The antioxidant activities of standard flavour compounds were also determined. The antioxidant activities of essential oils of selected tropical citrus fruits were attributed mainly to the presence of gamma-terpinene and terpinolene. These compounds showed high free radical-scavenging activity against DPPH radicals and were more active than vitamin E analogue. The colonic bacterial contribution to the catabolism of the flavanoids in a range of beverages was tested by comparing the urine profile of intact adults and adults with ileostomies and also using in vitro fermentation studies. Analysis of catabolic products i.e. free phenolic acids in human urine and faecal slurries following ingestion and supplementation of green tea and green tea catechins was conducted using GC-MS. Pyrogallol, pyrocatechol, 4-hydroxyphenylacetic acid, (-)5-(3',4'-dihydroxyphenyl)-gamma-valerolactone, (-)-5-(3',4',5'-trihydroxyphenyl)-gamma-valerolactone, 3-(3-hydroxyphenyl)propionic acid and 5-(3,4-dihydroxyphenyl)-gamma-valeric acid were among catabolites produced during a 4-48 h fermentation of green tea catechins. In total, 58.4% of (-)-epigallocatechin gallate, 25.2% of (-)-epigallocatechin and 50.1% of (-)-epicatechin were catabolised to phenolic acids in the human faecal slurries in the presence of glucose. Hippuric acid and 4-hydroxyphenylacetic acid were found to be abundant after drinking green tea, but did not appear to be major phenolic acids produced from the catabolism of green tea catechins. Calculation based on the levels of selected phenolic acids excreted in urine of healthy volunteers after drinking green tea indicated excretion was equivalent to 23.8% of flavan-3-ols intake. The mean 0-24 h excretion of 163 ± 48 μmole phenolic acid for healthy volunteers who drank green tea was significantly higher (p<0.05) than the excretion of phenolic acids by volunteers who drank water (20 ± 3 μmole). Analysis of phenolic acids in brewed green tea was also carried out in this study. Gallic acid and p-coumaric acid were by far the most abundant phenolic acids present both in free and conjugate forms, but the amounts were small compared to the quantities of phenolic acids excreted in urine 0-24 h after drinking green tea. This study also describes the pharmacokinetics study of urinary excretion of phenolic acids in human volunteers (with and without a colon) after drinking instant coffee. All volunteers displayed a substantial increase in excretion of 3-(3-hydroxyphenyl)-3-hydroxypropionic acid, dihydroferulic acid, ferulic acid and 3-hydroxyhippuric acid 8-24 h after drinking coffee. The mean amount of urinary phenolic acids excreted after drinking coffee was 134 ± 43 μmole, which was equivalent to 29.4% of chlorogenic acid intake and significantly higher (p<0.05) than phenolic acids excreted when water was consumed instead coffee. The influence of the food matrixes (yoghurt and milk) on the catabolism of polyphenols is described in this study. 3-Methoxy 4-hydroxyphenylhydracrylic acid, 3-(3-hydroxyphenyl)-3-hydroxypropionic acid, hippuric acid, 3-hydroxyhippuric acid, 3-dihydroxyphenylacetic acid and dihydroferulic acid were found in human urine after drinking orange juice enriched with hesperetin-7-O-rutinoside. The total 0-24 h urinary excretion of flavanone-derived phenolic acids increased by a statistically significant nine-fold (p<0.05) following ingestion of orange juice. After consumption of orange juice with yoghurt, only small amounts of phenolic acids were excreted. A study on the effect of milk on the catabolism of cocoa flavan-3-ols showed low concentrations of phenolic acids were found in human urine after drinking hot cocoa and phenolic acid excretion was suppressed in healthy volunteers after drinking cocoa with milk. On the other hand, studies on cocoa polyphenol catabolism using human faecal slurries revealed breakdown to phenolic acids. This may indicate that in vivo cocoa polyphenols may form complexes with proteins, which reduces the extent to which they are degraded to phenolic acids when they reach the colon.