Identifying and correcting non-Markov states in peptide conformational dynamics

Conformational transitions in proteins define their biological activity and can be investigated in detail using the Markov state model. The fundamental assumption on the transitions between the states, their Markov property, is critical in this framework. We test this assumption by analyzing the transitions obtained directly from the dynamics of a molecular dynamics simulated peptide valine-proline-alanine-leucine and states defined phenomenologically using clustering in dihedral space. We find that the transitions are Markovian at the time scale of ˜ 50 ps and longer. However, at the time scale of 30–40 ps the dynamics loses its Markov property. Our methodology reveals the mechanism that leads to non-Markov behavior. It also provides a way of regrouping the conformations into new states that now possess the required Markov property of their dynamics.

Interaction of peptides, peptidomimetics and other drug candidates with the DI/tripeptide transport system in intestinal epithelial cells, using the in vitro caco-2 cell culture system

An uptake system was developed using Caco-2 cell monolayers and the dipeptide, glycyl-[3H]L-proline, as a probe compound. Glycyl-[3H]L-proline uptake was via the di-/tripeptide transport system (DTS) and, exhibited concentration-, pH- and temperature-dependency. Dipeptides inhibited uptake of the probe, and the design of the system allowed competitors to be ranked against one another with respect to affinity for the transporter. The structural features required to ensure or increase interaction with the DTS were defined by studying the effect of a series of glycyl-L-proline and angiotensin-converting enzyme (ACE)-inhibitor (SQ-29852) analogues on the uptake of the probe. The SQ-29852 structure was divided into six domains (A-F) and competitors were grouped into series depending on structural variations within specific regions. Domain A was found to prefer a hydrophobic function, such as a phenyl group, and was intolerant to positive charges and H+ -acceptors and donors. SQ-29852 analogues were more tolerant of substitutions in the C domain, compared to glycyl-L-proline analogues, suggesting that interactions along the length of the SQ-29852 molecule may override the effects of substitutions in the C domain. SQ-29852 analogues showed a preference for a positive function, such as an amine group in this region, but dipeptide structures favoured an uncharged substitution. Lipophilic substituents in domain D increased affinity of SQ-29852 analogues with the DTS. A similar effect was observed for ACE-NEP inhibitor analogues. Domain E, corresponding to the carboxyl group was found to be tolerant of esterification for SQ-29852 analogues but not for dipeptides. Structural features which may increase interaction for one series of compounds, may not have the same effect for another series, indicating that the presence of multiple recognition sites on a molecule may override the deleterious effect of anyone change. Modifying current, poorly absorbed peptidomimetic structures to fit the proposed hypothetical model may improve oral bioavailability by increasing affinity for the DTS. The stereochemical preference of the transporter was explored using four series of compounds (SQ-29852, lysylproline, alanylproline and alanylalanine enantiomers). The L, L stereochemistry was the preferred conformation for all four series, agreeing with previous studies. However, D, D enantiomers were shown in some cases to be substrates for the DTS, although exhibiting a lower affinity than their L, L counterparts. All the ACE-inhibitors and β-lactam antibiotics investigated, produced a degree of inhibition of the probe, and thus show some affinity for the DTS. This contrasts with previous reports that found several ACE inhibitors to be absorbed via a passive process, thus suggesting that compounds are capable of binding to the transporter site and inhibiting the probe without being translocated into the cell. This was also shown to be the case for oligodeoxynucleotide conjugated to a lipophilic group (vitamin E), and highlights the possibility that other orally administered drug candidates may exert non-specific effects on the DTS and possibly have a nutritional impact. Molecular modelling of selected ACE-NEP inhibitors revealed that the three carbonyl functions can be oriented in a similar direction, and this conformation was found to exist in a local energy-minimised state, indicating that the carbonyls may possibly be involved in hydrogen-bond formation with the binding site of the DTS.

Biochemical aspects of Tourette's Syndrome

Kynurenine (KYN) is the first stable metabolite of the kynurenine pathway, the major route of tryptophan. (TRP) metabolIsm. In the liver, cortisol-inducible tIyptophan-2,3-dioxygenase (TDO) is the first enzyme and rate limiting step. In extrahepatic tissues, it is superceded by indoleamine-2,3-dioxygenase (IDO), an enzyme with a wider substrate specificity. Earlier work in this research group has found substantial elevations in plasma KYN in fasting Tourette's Syndrome (TS) patients with normal TRP and neopterin. The aim of our initial pilot study was to confirm this increase in KYN in fasting human TS patients compared with normal controls, and to see how changes in diet :ay influence certain kynurenine pathway variables. However, we failed to detect a change in plasma KYN, TRP, kynurenic acid (KYNA), neopterin or cortisol between the fasting TS and control groups. Moreover, none of the variables was affected by dietary status, and thus candidates selected for the larger cross-sectional study were permitted to eat and drink freely on the day that blood samples were submitted, but were requested to avoid products containing caffeine, aspirin or nicotine. In the cross-sectional study, TS patients exhibited significantly higher plasma KYN concentrations than controls, although the magnitude of the change was much smaller than originally found. This may be due to differences in detection procedure and the seasonal fluctuation of some biochemical variables, notably cortisol. The generalised increase in neopterin in the TS subject group, suggests a difference in the activity of cytokine-inducible IDO as a likely source for this elevated KYN. Other kynurenine pathway metabolites, specifIcally TRP, 3-hydroxykynurenine (HKY), 3-hydroxyanthranilic acid (HAA) and KYNA were unchanged. In view of recent speculation of the potential therapeutic effects of nicotine in TS, the lower KYN concentrations observed in TS smokers, compared with non-smoking TS patients, was another interesting finding. Tic-like movements, such as head-shakes (HS), which occur in rodents both spontaneously and following diverse drug treatments, closely resemble tic behaviours in humans. The animal tic model was used to examine what effects nicotine may have on shaking behaviours and on selected TRP metabolites. Acute systemic administration of nicotine to mice, produced a dose-dependent reduction in HS frequency (induced by the 5-HT2A/2C agonist DOl), which appeared to be mediated via central nicotinic cholinergic receptors, since mecamylamine pretreatment abolished this effect. Conversely, twice daily subcutaneous injections of nicotine for 7 days, led to an increase in spontaneous and DOI-induced HS. Chronic nicotine also caused a significant elevation m plasma and whole brain KYN concentrations, but plasma TRP, HKY, HAA and KYNA were unaltered. In addition, no change in brain 5-HT or 5-HIAA concentrations or 5-HT turnover, was found. Despite contrasting results from human and animal studIes, a role for nicotine in the mediation of tic-like movements is indicated. The relevance of the kynurenine pathway to TS and the potential role played by nicotine in modifying tic-like behaviours is discussed.

Intestinal absorption barriers as modelled by p-glycoprotein and cytochrome p450 a34 in caco2 cells

The passage number and origin of two populations of Caco-2 cells influence their enterocyte-like characteristics. Caco-2 cells of passage number >90 from Novartis pharmaceutical company possess higher levels of expression of alkaline phosphatase and P-glycoprotein and a greater cellular uptake of Gly-1.-Pro than those of passage number <40 from the American Type Tissue Culture collection. High P-gp expressing Caco-2 cells have been developed through stepwise selection of the cells with doxonibicin. This newly-developed cell line (hereafter referred to as Type I) possesses approximately twice as much P-gp protein than non-exposed cells, restricts the transepithelial transport of vincristine in the apical-to-basolateral direction whilst facilitating its transport in the reverse direction and accumulates less vincristine than non-exposed cells. There is no apparent evidence of the co-existence of the multidrug resistance protein (MIT) in Type I cells to account for the above-listed observations. Stopping the exposure for more than 28 days decreases the P-gp protein expression in previously doxorubicin-exposed Type I Caco-2 cells and reduces the magnitude of vincristine transepithelial fluxes in both directions to the levels that are almost similar to those of non-exposed cells. Exposing Caco-2 cells to 0.25 JAM la, 25-dihydroxyvitamin D3 induces their expression of cytochrome P450 3A4 protein to the level that is equivalent to that from isolated human jejunal cells. Under the same treatment, doxorubiein-exposed (Type I) cells metabolise naidazolam poorly and less extensively compared to non-exposed cells, suggesting that there is no such co-regulation of P-gp and CYP3A4 in Caco-2 cells. However, there is evidence which suggests CYP3A metabolises mida_zolam into 1- and 4-hydroxymidazolam, the latter may possibly be a P-gp substrate and is transported extracellularly by P-gp, supporting the hypothesis of P-gp-CYP3A4 synergistic roles in keeping xenobiotics out of the body. Doxoru.bicin-exposed (Type I) cells are less effective in translocating L-proline and glycyl-L-proline across the cell mono layers.

The development of functional in vitro toxicity tests

In vitro toxicity tests which detect evidence of the formation of reactive metabolites have previously relied upon cell death as a toxicity end point. Therefore these tests determine cytotoxicity in terms of quantitative changes in specified cell functions. In the studies involving the CaC0-2 cell model, there was no significant change in the transport of [3H] L-proline by the cell after eo-incubation with either dapsone or cyclophosphamide (50µM) and rat liver microsomal metabolite generating system. The pre incubation of the cells with N-ethylmalemide to inhibit Phase II sulphotransferase activity, prior to the microsomal incubations, resulted in cytotoxcity in all incubation groups. Studies involving the L6 cell model showed that there was no significant effect in the cell signalling pathway producing the second messenger cAMP, after incubation with dapsone or cyclophosphamide (50µM) and the rat microsomal metabolite generating system. There was also no significant affect on the vasopressin stimulated production of the second messenger IP3, after incubation with the hydroxylamine metabolite of dapsone, although there were some morphological changes observed with the cells at the highest concentration of dapsone hydroxylamine (100µM). With the test involving the NG115-401 L-C3 cell model, there was no significant changes in DNA synthesis in terms of [3H] thymidine incorporation, after eo-incubation with either phenytoin or cyclophosphamide (50µM) and the rat microsomal metabolite generating system. In the one compartment erythrocyte studies, there were significant decreases in glutathione with cyclophosphamide (50µM) (0.44 ± 0.04 mM), sulphamethoxazole (50µM) (0.43 ± 0.08mM) and carbamazepine (50µM) (0.47 ± 0.034 mM), when eoincubated with the rat microsomal system, compared to the control (0.52 ± 0.07mM). There was no significant depletion in glutathione when the erythrocytes were eoincubated with phenytoin and the rat microsomal system. In the two compartment erythrocyte studies, there was a significant decrease in the erythrocyte glutathione with cyclophosphamide (50µM) (0.953 ± 0110mM) when co-incubated the rat microsomal system, compared to the control (1.124 ± 0.032mM). Differences were considered statistically significant for p<0.05, using the Student's two tailed 't' test with Bonferroni's correction. There was no significant depletion of glutathione with phenytoin, carbamazepine and sulphamethoxazole when co-incubated with the rat microsomalsystem, compared to the control.

Investigation into the biochemical changes in Tourette Syndrome with a potential for pharmacological manipulation

Kynurenine (KYN) is the first stable metabolite of the kynurenine pathway, which accounts for over 95% of tryptophan metabolism. Two previous studies by this research group reported elevated plasma KYN in Tourette syndrome (TS) patients when compared with age and sex matched controls and another study showed that KYN potentiated 5-HT2A-mediated head-shakes (HS) in rodents. These movements have been suggested to model tics in TS. This raised the questions how KYN acts in eliciting this response and whether it is an action of its own or of a further metabolite along the kynurenine pathway. In the liver, where most of the kynurenine pathway metabolism takes place under physiological conditions, the first and the rate limiting enzyme is tryptophan-dioxygenase (TDO) which can be induced by cortisol. In extrahepatic tissues the same step of the pathway is catalyzed by indoleamine-dioxygenase (IDO), which is induced by cytokines, predominantly interferon-y (INF-y). Plasma neopterin, which shows parallel increase with KYN following immune stimulation, was also found elevated in one of these studies positively correlating with KYN. In the present work animal studies suggested that KYN potentiates and quinolinic acid (QUINA) dose dependently inhibits the 5-HT2A-mediated HS response in mice. The potentiating effect seen with KYN was suggested to be an effect of KYN itself. Radioligand binding and phosphoinositide (PI) hydrolysis studies were done to explore the mechanisms by which kynurenine pathway metabolites could alter a 5-HT2A-receptor mediated response. None of the kynurenine pathway metabolites tested showed direct binding to 5-HT2A-receptors. PI hydrolysis studies with KYN and QUINA showed that KYN did not have any effect while QUINA inhibited 5-HT2A-mediated PI hydrolysis. Plasma cortisol determination in TS patients with elevated plasma KYN did not show elevated plasma cortisol levels, suggesting that the increase of plasma KYN in these TS patients is unlikely to be due to an increased TDO activity induced by increased cortisol. Attention deficit hyperactivity disorder (ADHD) is commonly associated with TS. Salivary cortisol detected in a group of children primarily affected with ADHD showed significantly lower salivary cortisol levels when compared with age and sex matched controls. Plasma tryptophan, KYN, neopterin, INF-y and KYN/tryptophan ratio and night-time urinary 6-sulphatoxymelatonin (aMT6s) excretion measured in a group of TS patients did not show any difference in their levels when compared with age and sex matched controls, but TS patients failed to show the expected positive correlation seen between plasma INF-y, neopterin and KYN and the negative correlation seen between plasma KYN and night-time urinary aMT6s excretion seen in healthy controls. The relevance of the kynurenine pathway, melatonin secretion and cortisol to Tourette Syndrome and associated conditions and the mechanism by which KYN and QUINA alter the 5-HT2A-receptor mediated HS response are discussed.

DNA methylation at cytosine position 5

DNA methylation appears to be involved in the regulation of gene expression. Transcriptionally inactive (silenced) genes normally contain a high proportion of 5-methyl-2'-deoxycytosine residues whereas transcriptionally active genes show much reduced levels. There appears good reason to believe that chemical agents capable of methylating 2'-deoxycytosine might affect gene expression and as a result of hypermethylating promoter regions of cytosine-guanine rich oncogenic sequences, cancer related genes may be silenced. This thesis describes the synthesis of a number of `electrophilic' S-methylsulphonium compounds and assesses their ability to act as molecules capable of methylating cytosine at position 5 and also considers their potential as cytotoxic agents. DNA is methylated in vivo by DNA methyltransferase utilising S-adenoxylmethionine as the methyl donor. This thesis addresses the theory that S-adenoxylmethionine may be replaced as the methyl donor for DNA methytransferase by other sulphonium compounds. S-[3H-methyl]methionine sulphonium iodide was synthesised and experiments to assess the ability of this compounds to transfer methyl groups to cytosine in the presence of DNA methyltransferase were unsuccessful. A proline residue adjacent to a cysteine residue has been identified to a highly conserved feature of the active site region of a large number of prokaryotic DNA methyltransferases. The thesis examines the possibility that short peptides containing the Pro-Cys fragment may be able to facilitate the alkylation of cytosine position 5 by sulphonium compounds. Peptides were synthesised up to 9 amino acids in length but none were shown to exhibit significant activity. Molecular modelling techniques, including Chem-X, Quanta, BIPED and protein structure prediction programs were used to assess any structural similarities that may exist between short peptides containing a Pro-Cys fragment and similar sequences present in proteins. A number of similar structural features were observed.

Amino acid, peptide and drug transport across monolayers of human intestinal (CAC0-2) cells in vitro

The properties of Caco-2 monolayers were compared on aluminium oxide and nitrocellulose permeable-supports. On nitrocellulose, Caco-2 cells displayed a higher rate of taurocholic acid transport than those cultured on aluminium oxide inserts. In addition, Caco-2 cells grown on these two inserts were not comparable with respect to cell morphology, cell numbers and transepithelial electrical resistance. The low adsorption potential of the aluminium oxide inserts, particularly for high molecular weight or lipophilic ligands, offers a distinct advantage over nitrocellulose inserts for drug transport studies. The carrier-mediated uptake and transport of the imino acid (L-proline) and the acidic amino acids (L-aspartate and L-glutamate) have been studied. At pH7.4, L-proline uptake is mediated via an A-system carrier. Elevated uptake and transport under acidic conditions occurs by activation of a distinct carrier population. Acidic amino acid transport is mediated via a X-AG system. The flux of baclofen, CGP40116 andCGP40117 across Caco-2 monolayers was described by passive transport. The transport of three peptides, thyrotrophin-releasing hormone, SQ29852 and cyclosporin were investigated. Thyrotrophin-releasing hormone transport acrossCaco-2 monolayers was characterised by a minor saturable (carrier-mediated,approximately 25%) pathway, superimposed onto a major non-saturable (diffusional)pathway. SQ29852 uptake into Caco-2 monolayers is described by a major saturable mechanism (Km = 0.91 mM) superimposed onto a minor passive component.However, the initial-rate of SQ29852 transport is consistent with a passive transepithelial transport mechanism. These data highlight the possibility that itsbasolateral efflux is severely retarded such that the passive paracellular transportdictates the overall transepithelial transport characteristics. In addition, modelsuitable for investigating the transepithelial transport of cyclosporin A has been developed. A modification of the conventional Caco-2 model has been developed which has a calcium-free Ap donor-solution and a Bl receiver-solution containing the minimumcalcium concentration required to maintain monolayer integrity (100 μM). The influence of calcium and magnesium on the absorption of [14C]pamidronate was evaluated by comparing its transport across the conventional and minimum calciumCaco-2 models. Ap calcium and magnesium ions retard the Ap-to-Bl flux of pamidronate across Caco-2 monolayers. The effect of self-emulsifying oleic acid-Tween 80 formulations on Caco-2monolayer integrity has been investigated. Oleic acid-Tween 80 (1 0:1) formulations produced a dose-dependent disruption of Caco-2 monolayer integrity. This disruption was related to the oleic acid content of the formulation.

Redox signalling and detection of protein oxidation by mass spectrometry

It is now recognised that redox control of proteins plays an important role in many signalling pathways both in health and disease. Proteins can undergo a wide variety of oxidative post-translational modifications (oxPTM); while the reversible modifications are thought to be most important in physiological processes, non-reversible oxPTM may contribute to pathological situations and disease. The oxidant is also important in determining the type of oxPTM (chlorination, nitration, etc.), and the susceptibilities of residues vary depending on their structural location. The best characterized oxPTMs involved in signalling modulation are partial oxidations of cysteine to the disulfide, glutathionylated or sulfenic acid forms, but there is increasing evidence that specific oxidations of methionine and tyrosine may have some biological roles. Well understood examples of oxidative regulation include protein tyrosine phosphatases, e.g. PTP1B/C, and members of the MAPK pathways such as MEKK1 and ASK1. Transcription factors such as NFkB and Nrf-2 are also regulated by redox-active cysteines. Improved methods for analysing specific oxPTMs in biological samples are critical for understanding the physiological and pathological roles of these changes, and tandem or MS3 mass spectrometry techniques interfaced with nano-LC separation are being now used. MS3 fragmentation markers for a variety of oxidized residues including tyrosine, tryptophan and proline have been identified, and a precursor ion scanning method that allows the selective identification of these oxPTMs in complex samples has been developed. Such advances in technology offer potential for biomarker development, disease diagnosis and understanding pathology.

Investigation of formulation and process of lyophilised orally disintegrating tablet (ODT) using novel amino acid combination

Lyophilised orally disintegrating tablets (ODTs) have achieved a great success in overcoming dysphagia associated with conventional solid dosage forms. However, the extensive use of saccharides within the formulation limits their use in treatment of chronic illnesses. The current study demonstrates the feasibility of using combination of proline and serine to formulate zero sacharide ODTs and investigates the effect of freezing protocol on sublimation rate and tablets characteristics. The results showed that inclusion of proline and serine improved ODT properties when compared to individual counterparts. Additionally, annealing the ODTs facilitated the sublimation process and shortened the disintegration time. © 2010 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.

Biosynthesis and analysis of plant oxylipins

The term oxylipin is applied to the generation of oxygenated products of polyunsaturated fatty acids that can arise either through non-enzymatic or enzymatic processes generating a complex array of products, including alcohols, aldehydes, ketones, acids and hydrocarbon gases. The biosynthetic origin of these products has revealed an array of enzymes involved in their formation and more recently a radical pathway. These include lipoxygenases and α-dioxygenase that insert both oxygen atoms in to the acyl chain to initiate the pathways, to specialised P450 monooxygenases that are responsible for their downstream processing. This latter group include enzymes at the branch points such as allene oxide synthase, leading to jasmonate signalling, hydroperoxide lyase, responsible for generating pathogen/pest defensive volatiles and divinyl ether synthases and peroxygenases involved in the formation of antimicrobial compounds. The complexity of the products generated raises significant challenges for their rapid identification and quantification using metabolic screening methods. Here the current developments in oxylipin metabolism are reviewed together with the emerging technologies required to expand this important field of research that underpins advances in plant-pest/pathogen interactions.

Residue-specific investigation of the relationship between oxidation and activity of a dual specificity phosphatase by mass spectrometry

Redox regulation of signalling pathways is critical in proliferation and apoptosis; redox imbalance can lead to pathologies such as inflammation and cancer. Vaccinia H1-related protein (VHR; DUSP3) is a dual-specificity phosphatase important in controlling MAP kinase activity during cell cycle. the active-site motif contains a cysteine that acts as a nucleophile during catalysis. We used VHR to investigate the effect of oxidation in vitro on phosphatase activity, with the aim of determining how the profile of site-specific modification related to catalytic activity. Recombinant human VHR was expressed in E. coli and purified using a GST-tag. Protein was subjected to oxidation with various concentrations of SIN-1 or tetranitromethane (TNM) as nitrating agents, or HOCl. the activity was assayed using either 3-O-methylfluorescein phosphate with fluorescence detection or PIP3 by phosphate release with malachite green. the sites of oxidation were mapped using HPLC coupled to tandem mass spectrometry on an ABSciex 5600TripleTOF following in-gel digestion. More than 25 different concentration-dependent oxidative modifications to the protein were detected, including oxidations of methionine, cysteine, histidine, lysine, proline and tyrosine, and the % oxidized peptide (versus unmodified peptide) was determined from the extracted ion chromatograms. Unsurprisingly, methionine residues were very susceptible to oxidation, but there was a significant different in the extent of their oxidation. Similarly, tyrosine residues varied greatly in their modifications: Y85 and Y138 were readily nitrated, whereas Y38, Y78 and Y101 showed little modification. Y138 must be phosphorylated for MAPK phosphatase activity, so this susceptibility impacts on signalling pathways. Di- and tri- oxidations of cysteine residues were observed, but did not correlate directly with loss of activity. Overall, the catalytic activity did not correlate with redox state of any individual residue, but the total oxidative load correlated with treatment concentration and activity. This study provides the first comprehensive analysis of oxidation modifications of VHR, and demonstrates both heterogenous oxidant effects and differential residue susceptibility in a signalling phosphatase.

The role of splicing factors and small nuclear RNAs in spliceosomal formation

Protein coding genes are comprised of protein-coding exons and non-protein-coding introns. The process of splicing involves removal of the introns and joining of the exons to form a mature messenger RNA, which subsequently undergoes translation into polypeptide. The spliceosome is a large, RNA/protein assembly of five small nuclear RNAs as well as over 300 proteins, which catalyzes intron removal and exon ligation. The selection of specific exons for inclusion in the mature messenger RNA is spatiotemporally regulated and results in production of an enormous diversity of polypeptides from a single gene locus. This phenomenon, known as alternative splicing, is regulated, in part, by protein splicing factors, which target the spliceosome to exon/intron boundaries. The first part of my dissertation (Chapters II and III) focuses on the discovery and characterization of the 45 kilodalton FK506 binding protein (FKBP45), which I discovered in the silk moth, Bombyx mori, as a U1 small nuclear RNA binding protein. This protein family binds the immunosuppressants FK506 and rapamycin and contains peptidyl-prolyl cis-trans isomerase activity, which converts polypeptides from cis to trans about a proline residue. This is the first time that an FKBP has been identified in the spliceosome. The second section of my dissertation (Chapters IV, V, VI and VII) is an investigation of the potential role of small nuclear RNA sequence variants in the control of splicing. I identified 46 copies of small nuclear RNAs in the 6X whole genome shotgun of the Bombyx mori p50T strain. These variants may play a role in differential binding of specific proteins that mediate alternative splicing. Along these lines, further investigation of U2 snRNA sequence variants in Bombyx mori demonstrated that some U2 snRNAs preferentially assemble into high molecular weight spliceosomal complexes over others. Expression of snRNA variants may represent another mechanism by which the cell is able to fine tune the splicing process.

The Role of Splicing Factors and Small Nuclear RNAS in Spliceosomal Formation

Protein coding genes are comprised of protein-coding exons and non-protein-coding introns. The process of splicing involves removal of the introns and joining of the exons to form a mature messenger RNA, which subsequently undergoes translation into polypeptide. The spliceosome is a large, RNA/protein assembly of five small nuclear RNAs as well as over 300 proteins, which catalyzes intron removal and exon ligation. The selection of specific exons for inclusion in the mature messenger RNA is spatio-temporally regulated and results in production of an enormous diversity of polypeptides from a single gene locus. This phenomenon, known as alternative splicing, is regulated, in part, by protein splicing factors, which target the spliceosome to exon/intron boundaries. The first part of my dissertation (Chapters II and III) focuses on the discovery and characterization of the 45 kilodalton FK506 binding protein (FKBP45), which I discovered in the silk moth, Bombyx mori, as a U1 small nuclear RNA binding protein. This protein family binds the immunosuppressants FK506 and rapamycin and contains peptidyl-prolyl cis-trans isomerase activity, which converts polypeptides from cis to trans about a proline residue. This is the first time that an FKBP has been identified in the spliceosome. The second section of my dissertation (Chapters IV, V, VI and VII) is an investigation of the potential role of small nuclear RNA sequence variants in the control of splicing. I identified 46 copies of small nuclear RNAs in the 6X whole genome shotgun of the Bombyx mori p50T strain. These variants may play a role in differential binding of specific proteins that mediate alternative splicing. Along these lines, further investigation of U2 snRNA sequence variants in Bombyx mori demonstrated that some U2 snRNAs preferentially assemble into high molecular weight spliceosomal complexes over others. Expression of snRNA variants may represent another mechanism by which the cell is able to fine tune the splicing process.

The role of protein oxidation in ageing & disease

Ageing is a natural phenomenon of the human lifecycle, yet it is still not understood what causes the deterioration of the human body near the end of the lifespan. One popular theory is the Free Radical Theory of Ageing, which proposes that oxidative damage to biomolecules causes ageing of tissues. The ageing population is affected by many chronic diseases. This study focused on sarcopenia (muscle loss in ageing) and obesity as two models for comparison of oxidative damage in muscle proteins in mice. The aim of the study was to develop advanced mass spectrometry methods to detect specific oxidative modifications to mouse muscle proteins, including oxidation, nitration, chlorination, and carbonyl group formation, but western blotting was also used to provide complementary information on the oxidative state of proteins from aged and obese muscle. Mass spectrometry proved to be a powerful tool, enabling identification of the types of modifications present, the sites at which they were present and percentage of the peptide populations that were modified. Targeted and semi-targeted mass spectrometry methods were optimised for the identification and quantitation of the oxidised residues in muscle proteins. The development of the quantitative methods enabled comparisons of mass spectrometry instruments. Both the Time of Flight and QTRAP systems showed advantages of using the different mass analysers to quantify oxidative modifications. Several oxidised residues were characterised and quantified in both the obese and sarcopenic models, and higher levels of oxidation were found compared to their control counterparts. Residues found to be oxidised were oxidation of proline, tyrosine and tryptophan, dioxidation of methionine, allysine and nitration of tyrosine. However quantification was performed on methionine dioxidation and cysteine trioxidation containing residues in SERCA. The combination of measuring residue susceptibility and functional studies could contribute to understanding the overall role of oxidation in ageing and obesity.