No description available.

The body's carnitine pool is almost entirely confined to skeletal muscle where it plays a dual role in cellular energy metabolism. At rest and during moderate intensity exercise, carnitine is an obligatory cofactor in long chain fatty acid metabolism, whereas during intense contraction, carnitine plays a central role in the maintenance of the mitochondrial free Co enzyme A (CoASH) pool. Although carnitine supplementation has been touted as a means to alter skeletal muscle fuel metabolism for several decades, only recently has it been shown that skeletal muscle carnitine availability can be elevated in humans, and that this leads to alterations in muscle fuel metabolism, a finding which has led to renewed interest in carnitine as a regulator of skeletal muscle fuel metabolism. Despite recent developments in our understanding of the physiological impact of skeletal muscle carnitine loading, little is known regarding the metabolic impact of carnitine depletion on skeletal muscle fuel metabolism. The first objective of the work presented in this thesis was to establish a rodent model of skeletal muscle carnitine depletion. This was achieved via oral supplementation with mildronate, a compound which has been shown to attenuate carnitine biogenesis, while also accelerating its renal clearance of carnitine in vivo. Thereafter, the impact of skeletal muscle carnitine depletion on whole body and skeletal muscle fuel metabolism was investigated in non-obese and obese, insulin resistant rodents. Collectively, the experiments detailed in this thesis offer a novel insight regarding the metabolic consequences of skeletal muscle carnitine depletion. More specifically, mildronate administration resulted in a significant reduction in skeletal muscle total carnitine content, which was largely attributable to a near complete depletion of the muscle free carnitine pool. This resulted in a reduction in muscle long chain acylcarnitine content, indicative of impaired carnitine palmitoyl transferase 1 (CPTl) flux and mitochondrial long chain fatty acid transport. Indeed, skeletal muscle carnitine depletion attenuated fat oxidation in both non-obese and obese insulin resistant rodents. In addition to a marked reduction in fat oxidation, carnitine depletion also resulted in an increase in skeletal muscle glycogen utilisation, an effect which was more apparent in obese insulin resistant rodents when compared to non-obese rodents. Interestingly, an additional novel finding of this work was the fact that despite driving skeletal muscle glycogenolysis, skeletal muscle carnitine depletion impaired glucose tolerance in obese insulin resistant rodents. This finding is most likely a result of hepatic lipid accumulation and a subsequent reduction in hepatic insulin sensitivity. Taken together, the data presented in this thesis clearly demonstrates that skeletal muscle carnitine depletion attenuates CPTl flux and fat oxidation while driving skeletal muscle CHO oxidation. However, despite increased glycogenolysis in carnitine depleted skeletal muscle, carnitine depletion does not improve glucose tolerance in obese insulin resistant rodents, and therefore would not be a suitable intervention to manage hyperglycaemia in diabetic subjects.

Family B DNA polymerases from archaea, such as Pyrococcus furiosus (Pfli-Pol), stall replication upon encountering template strand uracil and hypoxanthine (the deamination products of cytosine and adenine spectively) four base pairs ahead of the primer template junction. The deaminated bases bind to a specialized pocket within the amino terminal domain of the polymerase. This read-ahead mechanism could be a final attempt to detect deaminated bases and repair them by an as yet undetermined pathway, preventing 50% of the progeny inheriting a transition mutation.

No description available.

No description available.

Poly(ADP-ribose)polymerase (PARP) is a nuclear enzyme involved in the repair of DNA single strand breaks via the Base Excision Repair pathway (BER). Temozolomide, a DNA alkylating agent recently licensed for the treatment of gliomas and melanoma, produces DNA lesions which are targets for BER. Preclinical studies demonstrate that PARP inhibitors increase the cytotoxicity and antitumour activity of temozolomide suggesting that PARP inhibitors may have a clinical role as chemopotentiating agents. This thesis describes the protocol development of a First-in-Human phase I clinical trial of AGO14699, a potent PARP inhibitor, in combination with temozolomide in patients with advanced solid malignancies discussing the rationale for the study design, definition of pharmacodynamic (PD) endpoints and starting dose. A two part trial escalating first the dose of AG014699 then that oftemozolomide was designed with PD endpoints for part 1 and classical toxicity endpoints for part 2. The development and validation of two PARP activity assays to measure enzyme activity and inhibition in human peripheral blood lymphocytes (PBLs) and homogenised tumour biopsies are discussed. An established tumour cell line (L1210) was evaluated to provide Quality Assurance, and preparation, storage and transport stability of samples investigated. The first assay developed relied upon measuring incorporation ofe2p] NAD+ into poly(ADP-ribose) (PAR), this assay proved robust but depended upon the availability of large numbers of PBLs, limiting its clinical application. An alternative assay based on detection of PAR with a monoclonal antibody and electronic digitisation of the chemiluminescence signal was validated and then assessed in a phase II mechanistic study of temozolomide alone in patients with advanced malignant melanoma. This small study provided additional validation for the assay and also data on DNA damage and repair after temozolomide which can be used as control data to interpret the pharmacodynamic results of the First-inHuman study.

Caleosins were originally described as one of the two major protein components of storage lipid bodies in the seeds of higher plants, the other being oleosins. In contrast with oleosins, caleosins have a single calcium-binding EF-hand domain plus several potential phosphorylation sites and have been hypothesised as playing a role in lipid-body formation and possibly mobilisation in seeds. In Arabidopsis, there are six functional caleosin genes, two of which encode seed-specific proteins while the other isoforms are expressed in a variety of vegetative and reproductive tissues. More recently, seed caleosins have been shown to have a peroxygenase activity but the function of this was uncertain. This study describes the characterisation of a specific membrane-bound caleosin isoform, termed Clo-3 in Arabidopsis and Brassica spp, that appears to be present in all plant tissues and is responsive to a variety of biotic and abiotic stresses. Bioinformatic analysis reveals that similar caleosin-like genes/proteins are present in all vascular plants, as well as in nonvascular plants such as mosses, and even in single-celled algae. Intriguingly, caleosin-like genes are also present in the genomes of most fungi described to date, with the surprising exception of the yeasts. In order to understand the function of caleosins in plants, a detailed structural and functional analysis of this novel class of protein is reported here. Biochemical studies demonstrate that the Clo-3 isoform binds calcium (one atom per molecule), can be phosphorylated most likely, by a casein kinase 2 (CK2) protein kinase, and has putative peroxygenase activity. In addition to biochemical data, microscopy analysis shows that Clo-3 may be located both on the endoplasmic reticulum and chloroplastid envelope membranes. specifically the chloroplast envelope. Biochemical evidence of cell membrane localisation is also presented. Protease digestion experiments show that the membrane bound Clo-3 has a Type I transmembrane orientation, where its N-terminal domain faces the lumen of microsomes while the C-terminal is on the cytosolic face. Such an orientation is common for receptors or proteins that may be activated by signalling molecules. The Clo-3 gene and its encoded protein are each upregulated by salt and drought stresses and by abscisic acid (ABA) treatment. Reverse genetics using RNAi knockdown mutants demonstrate specific transcription factors involved in regulating Clo-3 during different stresses. Peroxygenase activities of Clo-3 enriched microsomes were higher following salt stress. Although the data is representative of potentially many peroxygenases, it does provide indirect evidence that Clo-3 abundance increases and/or catalytic activity is induced during stress. The study also presents evidence of the response of Clo-3 to biotic stress and related signalling molecules. Arabidopsis Clo-3 is highly responsive to the phytohormone salicylic acid, to the salicylic acid synthetic analogue DCINA, the biotic signalling molecule hydrogen peroxide, and to infection by the common fungal pathogen of Brassicas, Leptosphaeria maculans (Phoma), while experiments utilising the non-expressor of pathogenesis related protein 1 (npr1) knockout mutant plant demonstrates Clo-3 response to salicylic acid (SA) is chiefly via npr1 translocation to the nucleus. The type of peroxygenase epitomised by Clo-3 is similar to those involved in the formation of epoxy alcohols from fatty acid hydroperoxides. The latter are a class of oxylipins that are seen in fungal infection, and also play a role in various aspects of fungal spore development including sporulation and a role in cuticle synthesis. As such, Clo-3 in Arabidopsis and possibly similar caleosins in other species might play roles in oxylipin signalling pathways that are involved in a protective role during both biotic and abiotic stress responses.

DNA replication is a key process required by organisms during cell division, with a concomitant requirement for genome synthesis by DNA polymerases. Biotechnological exploitation of thermostable DNA polymerases for DNA amplification by the Polymerase Chain Reaction (PCR), provides a significant market for novel enzymes or those with improved properties. An approach was taken to isolate alternative thermostable DNA polymerases, by enriching thermophilic bacteria from a novel thermal environment, aerobically spoiling silage. In addition, a novel DNA polymerase (Abr polBl) was cloned from the thermoacidophilic archaeon, Acidianus brierleyi, with the intention of characterising its in vivo role and application to PCR. Protein sequence analysis suggested a proofreading (high fidelity) DNA synthesis activity most related to polBl DNA polymerases from Crenarchaeota. Abr polBl was heterologously expressed in bacteria and protein purified to homogeneity. Biochemical assays confirmed high-temperature DNA polymerase and 3'-5'exonuclease activities of Abr polBl, with an accompanying proofreading ability. Sequence analysis, processivity, strand displacement and lesion bypass activities indicated potential roles in genome replication and DNA repair. Abr polBl could not amplify DNA under a range of PCR conditions, presumably following its low intrinsic thermostability. Biophysical analyses confirmed irreversible unfolding of Abr polBl at temperatures required for PCR. Supplementation with organic compounds and ionic salts stabilised Abr polBl, promoting retention of conformational stability and DNA synthesis activity following thermal incubation, but could not promote DNA amplification with Abr polB 1.

Molecular genetics use molecular methods that amplify specific fragments of DNA. Today, the molecular techniques which were developed for amplification and detection of specific sequences of nucleic acids helped in a great deal to understand the structure of many diseases. Polymerase chain reaction or PCR is a technique that is used for isolation and amplification of a specific sequence of DNA. PCR is an in vitro method that exploits the in vivo procedure of replication of DNA. DNA polymerase is used to amplify a piece of DNA by in vitro enzymatic replication. As PCR progresses, the DNA thus generated is itself used as a template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. PCR was designed and presented by Dr Kary Mullis (1983).Today PCR has a great range of implementations related to 1) the cloning and the study of gene’s expression 2) the detection of mutations that are responsible for hereditary diseases 3) Criminology, Toxicology and Forensics. / H Μοριακή Γενετική χρησιμοποιεί μοριακές μεθόδους οι οποίες ενισχύουν συγκεκριμένα τμήματα DNA. Σήμερα, οι μοριακές τεχνικές οι οποίες αναπτύχθηκαν για ενίσχυση και ανίχνευση συγκεκριμένων ακολουθιών νουκλεϊνικών οξέων βοήθησαν σε μεγάλο βαθμό στην κατανόηση της δομής πολλών ασθενειών. Η αλυσιδωτή αντίδραση πολυμεράσης είναι μια τεχνική η οποία χρησιμοποιείται για απομόνωση και ενίσχυση μιας συγκεκριμένης ακολουθίας DNA. Η αλυσιδωτή αντίδραση της πολυμεράσης είναι μια διαδικασία που πραγματοποιείται σε ελεγχόμενες συνθήκες έξω απο ζωντανούς οργανισμούς, η οποία εκμεταλλεύεται την αντιγραφή του DNA που πραγματοποιείται μεσα στον ζωντανό οργανισμό. Όσο η PCR εξελίσσεται δημιουργούνται αντίγραφα DNA χρησιμοποιώντας ως πρότυπο το αρχικό DNA. Αυτό ενεργοποιεί μια διαδικασία αλυσιδωτής αντίδρασης ώπου το DNA αναπαράγεται εκθετικά. Με την PCR μπορείς να δημιουργήσεις εκατομμύρια αντίγραφα DNA από μία συγκεκριμένη ακολουθία. Η PCR σχεδιάστηκε και παρουσιάστηκε απο τον Dr Kary Mullis. Σήμερα η PCR έχει μέγαλο εύρος εφαρμογής σε πεδία σχετικά με τη μελέτη της γενετικής έκφρασης την ανίχνευση μεταλλάξεων οι οποίες είναι υπεύθυνες για κληρονομικές ασθένειες, Εγκληματολογία, Τοξικολογία και Ιατροδικαστική.

L’optimisation de l’incorporation et de la libération de principes actifs dans les produits formulés constitue un des enjeux majeurs des industries pharmaceutiques et cosmétiques. L'objectif principal de notre étude est de proposer un modèle prédictif de la diffusion de petites sondes au sein des émulsions concentrées. Pour cela, il a fallu considérer à la fois la formulation d’émulsions concentrées stables et leur caractérisation rhéologique et structurelle ainsi que la prédiction des paramètres de transfert des sondes au sein des émulsions concentrées. On entend par paramètres de transfert, tous les paramètres permettant de caractériser les différents mécanismes de transfert de sondes dans les émulsions concentrées pris en compte dans notre système, à savoir le coefficient de diffusion dans les phases continue et dispersée, le coefficient de transfert à l’interface eau/huile, le coefficient de partage à l’équilibre de la sonde entre les deux phases de l’émulsion. Une nouvelle approche de caractérisation de la structure des émulsions concentrée a permis l’obtention des paramètres importants de structure (taille des gouttes et épaisseur du film de la phase continue). L’étude détaillée des mécanismes et processus diffusionnels est réalisée avec la prise en compte des résultats liés à la caractérisation structurelle du système d’étude. Ainsi, un modèle de diffusion fondé sur une approche phénoménologique est proposé pour prédire l'évolution des profils de concentration de la sonde dans les émulsions concentrées. Les cinétiques expérimentales de libération des sondes sont comparables à celles simulées par le modèle sans paramètres ajustables. Cette comparaison montre une bonne adéquation entre le modèle de diffusion et l’expérience / In the field of controlled release technology for new drugs, models that can predict its delivery during application are important for device design. The main objective of this work is to develop a predictive model able to describe the drug delivery from highly concentrated water-in-oil emulsions. These systems consist of deformed droplets dispersed in a continuous film. Their structure’s characteristics make them favourable for their use as releasing devices. A combination of different transfer mechanisms has been implemented in a mathematical model in order to simulate release experiments under different operating conditions (volume fraction, oil/surfactant ratio). A sensitivity analysis has been performed to point out the most relevant parameters affecting the drug’s release: drug partition and diffusion coefficients. Partition coefficient of the drug for different surfactant concentrations has been obtained through a predictive thermodynamic model UNIFAC, and the diffusion coefficient using Chang and Wilke equations in addition to the Stefan- Maxwell development. An original and simple technique has been used to determine indirectly the mean droplet size of the concentrated emulsions, through measurements of continuous phase’s thickness by analysis of incoherent polarized steady light transport through emulsion samples. In a general view, the diffusion model proposed for small drug diffusion in concentrated emulsions, which was first proposed for diluted emulsions, predicts successfully the evolution of mandelic acid concentrations during release experiments undertaken in perfect sink conditions