Association between Frequency of Chromosomal Aberrations and Cancer Risk Is Not Influenced by Genetic Polymorphisms in GSTM1 and GSTT1

To evaluate the role of polymorphisms in glutathione S-transferase M1 (GSTM1) and theta 1 (GSTT1) as effect modifiers of the association between CA and cancer risk. A case-control study was performed pooling data from cytogenetic studies carried out in 1974-1995 in three laboratories in Italy, Norway, and Denmark. The subjects were classified as low, medium, and high by tertile of CA frequency. The data were analysed by setting up a Bayesian model which included prior information about cancer risk by CA frequency

Exploring the photochemistry and photosignalling mechanisms in cryptochromes and related model systems

The ability of migratory birds to sense and use the Earth’s magnetic field during migration is hypothesised to involve cryptochrome proteins located in their retinae. It is proposed that photo-initiated reactions within these proteins generate radical pairs (RPs) as transient intermediates, which have product yields that are sensitive to the magnitude and direction of the external magnetic field, and ultimately allow birds to visualize the geomagnetic field. Cryptochromes are currently the only candidate chemical compass magnetoreceptor for this radical pair mechanism (RPM)- based hypothesis (Chapter 1) and thus it is important to take steps towards more fully understanding the photochemistry and photosignalling mechanisms occurring. This thesis has two main themes. Firstly, a highly sensitive fluorescence-based spectroscopic technique has been developed to further increase sensitivity and make it more suitable for studying precious biological samples. Whilst many (absorption- based) spectroscopic techniques have investigated magnetic field effects (MFEs) on isolated cryptochrome proteins, these experiments have typically been performed in vitro and thus are still very far removed from biological conditions. This thesis describes the first attempts towards studying cryptochromes in living cells and thus observing an MFE in vivo (Chapter 2). A very small, negative MFE is observed, however this was attributed to the RP chemistry occurring in the growing medium rather than the cryptochromes. The exquisite sensitivity of the experiment is still highlighted, however, and the re-designed fluorescence technique is further used to explore the photochemistry of RPs in restricted environments (micelles) to investigate how altering the RP environment can affect the photochemistry of a system (Chapter 3). A negative MFE is observed in a thionine/DABCO system which is vastly increased when the system is constricted to an SDS micelle. The relationship between quencher concentration and MFE was shown to be biphasic, with dynamic quenching changing to static quenching with increasing DABCO concentration, and the importance of the micelle environment was demonstrated as altering the size or charge of the micelle affected the MFE. Secondly, the relationship between the photochemistry and signal transduction mechanism in cryptochromes has been explored. Currently, very little is known about the photosignalling pathway in cryptochromes (specifically animal cryptochromes), however one widely held model proposes the involvement of a light-induced conformational change which initiates the signalling cascade. By using a technique known as Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS), a blue-light induced conformational change has been observed on DmCry WT and been localised to a region of the protein known as the C-terminal tail (CTT). Further, by comparing the behaviour of DmCry WT with that of its mutants, DmCry W342F and W394F, in which the electron transfer (ET) chain is interrupted at the third and fourth tryptophan position respectively (by replacing the tryptophan with a redox inert phenylalanine), altering the photochemistry has been shown to have a direct effect on the magnitude of the conformational change, highlighting the importance of these tryptophans for the signalling state of the protein (Chapter 4).</p

Investigation of preparation methods on surface/bulk structural relaxation and glass fragility of amorphous solid dispersions

The objective of this study was to investigate the effect of preparation methods on the surface/bulk molecular mobility and glass fragility of solid dispersions. Solid dispersions containing indomethacin and PVP K30 were chosen as the model system. An inverse gas chromatography method was used to determine the surface structural relaxation of the solid dispersions and these data were compared to those for bulk relaxation obtained by DSC. The values of τ(β) for the surface relaxation were 4.6, 7.1 and 1.8h for melt quenched, ball milled and spray dried solid dispersions respectively, compared to 15.6, 7.9 and 9.8h of the bulk. In all systems, the surface had higher molecular mobility than the bulk. The glass fragility of the solid dispersions was also influenced by the preparation methods with the most fragile system showing the best stability. The zero mobility temperature (T(0)) was used to correlate with the physical stability of the solid dispersions. Despite having similar T(g) (65°C), the T(0) of the melt quenched, ball milled and spray dried samples were 21.6, -4.2 and 16.7°C respectively which correlated well with their physical stability results. Therefore, T(0) appears to be a better indicator than T(g) for predicting stability of amorphous materials

Comparison of cytologic and genetic distances between long arm subtelomeric markers of human autosome 14 suggests uneven distribution of crossing-over

The analysis of two rodent X human somatic cell hybrids, carrying different inborn translocations of the human chromosome 14 long arm, has permitted us to narrow down the localization of the structural locus for alpha-1-antitrypsin (PI) to band 14q32.1, proximally to the highly polymorphic DNA locus D14S1 which has been localized by previous studies between 14q32.1 and 14q32.2. These data, evaluated in conjunction with other published information, suggest that the D14S1 locus is cytologically equidistant from both the PI locus and the complex locus for the immunoglobulin heavy chains (IGH) but, genetically, it appears much closer to the latter since the recombination frequency reported between the IGH complex and PI is six times greater than that between the IGH complex and D14S1 (lod score peaks respectively at 26% and 4% with narrow fiducial limits). The present report adds further strength to the frequently proposed hypothesis of a nonlinear relationship between cytologic and genetic distances of human genes. The possibility that this phenomenon may be a feature of frequent occurrence throughout the entire human genome is discusse

Lifespan of human lymphocyte subsets defined by CD45 isoforms.

The lifespan of thymic-derived or T lymphocytes is of particular interest because of their central role in immunological memory. Is the recall of a vaccination or early infection, which may be demonstrated clinically up to 50 years after antigen exposure, retained by a long-lived cell, or by its progeny? Using the observation that T lymphocyte expression of isoforms of CD45 corresponds with their ability to respond to recall antigens, we have investigated the lifespan of both CD45R0 (the subset containing responders, or 'memory' cells) and CD45RA (the unresponsive, or 'naive' subset) lymphocytes in a group of patients after radiotherapy. Here we report rapid loss of unstable chromosomes from the CD45R0 but not the CD45RA pool. Immunological memory therefore apparently resides in a population with a more rapid rate of division. Differing survival curves for the two subsets are best described by a model in which there is also reversion in vivo from the CD45R0 to the CD45RA phenotype. Expression of CD45R0 in T cells may therefore be reversible