Hyperspectral image representation and processing with binary partition trees

The optimal exploitation of the information provided by hyperspectral images requires the development of advanced image processing tools. Therefore, under the title Hyperspectral image representation and Processing with Binary Partition Trees, this PhD thesis proposes the construction and the processing of a new region-based hierarchical hyperspectral image representation: the Binary Partition Tree (BPT). This hierarchical region-based representation can be interpreted as a set of hierarchical regions stored in a tree structure. Hence, the Binary Partition Tree succeeds in presenting: (i) the decomposition of the image in terms of coherent regions and (ii) the inclusion relations of the regions in the scene. Based on region-merging techniques, the construction of BPT is investigated in this work by studying hyperspectral region models and the associated similarity metrics. As a matter of fact, the very high dimensionality and the complexity of the data require the definition of specific region models and similarity measures. Once the BPT is constructed, the fixed tree structure allows implementing efficient and advanced application-dependent techniques on it. The application-dependent processing of BPT is generally implemented through a specific pruning of the tree. Accordingly, some pruning techniques are proposed and discussed according to different applications. This Ph.D is focused in particular on segmentation, object detection and classification of hyperspectral imagery. Experimental results on various hyperspectral data sets demonstrate the interest and the good performances of the BPT representatio

Aspects of the Regulation and Role of Focal Adhesion Kinase and Src in Oncogenic Transformation

Focal adhesion kinase (FAK) is a non-receptor tyosine kinase present in cell-ECM focal adhesions. As a part of the integrin signalling complex, FAK is implicated to play a role in the mediation of integrin induced cell growth, motility and survival- processes that are also required for tumour development and progression. A role for FAK has thus been proposed in the process of tumorigenesis. In support of this, FAK is found to be upregulated in various different tumours and tumour cell lines. However, the mechanisms that lead to the upregulation of FAK in cancer cells have not been investigated thus far. The first part of this study focused on establishing some of the genetic alterations adopted by cancer cells to elevate the level of FAK protein. We used fluorescent in situ hybridisation ((FISH) to first confirm that fak localised to human chromosome 8q in normal cells. Elevated FAK protein levels in cell lines derived from invasive squamous cell carcinomas of the head and neck were accompanied by gain in fak gene copy number in all the cases examined. Moreover, increased fak gene dosage, including genetic alterations like amplification and isochromosome formation, were observed in cell lines derived from human tumours of lung, breast and colon. In addition, conversion from adenoma to carcinoma, in an in vitro model for human colon cancer progression, was accompanied by an elevation in FAK protein level and gain in fak gene copy number. Although, other genes like c-myc, lying near the fak locus, were also found to be co-amplified or increased in copy number, given the biological functions FAK is proposed to play a role, it may contribute in exerting the selective pressure for the re retention of the whole region in increased number of copies. In order to further investigate the role of FAK in tumour development, we studied its importance in cell survival. For this we used an in vitro model of apoptosis, wherein, apoptosis was induced in v-Src transformed Rat-1 cells after serum-deprivation and inhibition of v-Src activity. In particular, we examined the importance of FAK proteolysis in the induction of apoptosis. We found that although induction of apoptosis was accompanied by FAK cleavage, inhibition of FAK proteolysis was unable to promote cell survival. The in vitro model of apoptosis using v-Src transformed Rat-1 cells was also characterised further in this thesis. Like FAK, the v-Src oncoprotein also resides in the focal adhesions where its constitutive kinase activity induces disruption of focal adhesions and cell transformation. Unlike other oncoproteins like c-Myc, and v-Jun, v-Src does not induce cell death under low serum conditions. However, the data described in this thesis suggested that v-Src primes the cells to undergo apoptosis, partly through disruption of integrin signalling, while providing them with a survival signal at the same time. Removal of this survival signal, thus, induced the cells to undergo apoptosis. Further investigations indicated that the surrogate integrin survival signal provided by v-Src required the activation of the PI 3-kinase/Akt pathway, while the MAP kinase pathway did not play any role in the mediation of the survival signalling. In addition, the apoptotic response induced in v-Src transformed Rat-1 cells was shown to be accompanied by activation of caspases, and stress-activated kinase, p38. Cell death was inhibited by overexpression of the anti-apoptotic protein Bcl-2 or by the inhibtion of caspase and p38 activity simultaneously

Studies on catecholamines and 5-hydroxytryptamine and the significance of their metabolites in animal tissues and body fluids

The papers presented in this thesis describe the development of methods for the estimation of adrenaline, noradrenaline, dopamine, 5-hydroxytryptamine and some of their metabolites and the application of such estimations to some problems of biological interest. The major part of the thesis is concerned with the metabolism of these amines in the mammalian central nervous system. The papers are presented in three groups.The first group is made up of papers in which the estimation of 5-hydroxytryptamine (5-HT) or its metabolite 5-hydroxy-indol-3-yl acetic acid (5-HIAA) was measured.1. On the question of the occurrence and metabolism of 5-hydroxy-tryptamine and related indole compounds in mammalian semen. By T. Mann, R. P. Seamark and D. F. Sharman. Br. J. Pharmac. Chemother. 17, 208 - 217, 1961.In this paper it was shown conclusively that the semen of man, bull, boar, ram and dog contains little or no 5-hydroxytryptamine.2. Drug-induced changes in the concentration of 5-0R indolyl compounds in cerebrospinal fluid and caudate nucleus. By G. V. Ashcroft and D. P. Sharman. Br. J. Pharmac. Chemother. 19. 155 - 160, 1962.Because of an earlier observation by Ashcroft and Sharman (Nature, Lond.,186, 1050 - 1051, i960) that the cerebrospinal fluid of depressed patients contained a lower concentration of 5-hydroxyindolyl compounds than normal, the effect of reserpine, a drug known to reduce the concentration of 5-hydroxytryptamine in the brain, on the concentration of such compounds in the cerebrospinal fluid of the dog was examined. It was found that the concentration of 5-hydroxyindolyl compounds in the cerebrospinal fluid was increased after reserpine.3. The effect of a-methyldopa on the metabolism of 5-hydroxy-tryptamine in rat brain. By D. P. Sharman and S. E. Smith. J. Neurochem. 403 - 406, 1962.In this paper, the concentration of 5-HIAA in the brain was used as an index of the rate at which 5-HT was released in this tissue after a-methyldopa, an inhibitor of the formation of 5-HT,was given to rats.4. The action of 2-aminotetralin ({3-tetrahydronaphthylamine) on the metabolism of 5-hydroxytryptamine in the brain of the mouse. By D. Robinson and D. P. Sharman. Br. J. Pharmao. Chemother. 29. 535 - 341, 1967.2-Aminotetralin causes a reduction in the concentration of 5- hydroxyindol-3-ylacetic acid in the brain of the mouse (paper 12). The possible causes of this effect were' examined.The second group consists mainly of papers which describe the development of methods of estimating dopamine and its acid metabolites 3,4-dihydroxyphenylacetio acid (DOPAC) and 4-hydroxy-3-methoxyphenyl acetic acid (homovanillic acidj HVA), their application to several problems, chiefly to a study of the rate of utilisation of dopamine in the brain and also the effect of drugs on the metabolism of this amine.5. Chemical and physiological changes produced by arterial infusion of dihydroxyphenylalanine into one cerebral hemisphere of the cat. By R. Dagirmanjian, R. Laverty, P. Mantegazzini, D. P. Sharman and M. Vogt. J. Neurochem. 10, 177 - 182, 1965.The infusion of 3,4-dihydroxyphenylalanine (DOPA) into one carotid artery of the cat can cause arousal of the brain on the side of the infusion. It was shown that this unilateral arousal is accompanied by an increase in the concentration of dopamine in the caudate nucleus, hypothalamus and midbrain reticular formation on the same side of the brain.6. The subcellular localisation of dopamine and acetylcholine in the dog caudate nucleus. By R. Laverty, I. A. Michaelson, D.P. Sharman and V. P. Whittaker. Br. J. Pharmac. Chemother. 21, 482 - 490, 1963.7. Localisation of acetylcholine, 5-hydroxytryptamine and noradrenaline within subcellular particles derived from guinea-pig subcortical brain tissue. By I. A. Michaelson, V. P. Whittaker, R. Laverty and D. P. Sharman. Biochem. Pharmacol. 12. 1450-1453, 1963.8. A fluorimetric method for the estimation ti 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid) and its identification in brain tissue. By D. F. Sharman. Br. J. Pharinac. Chemother. 20. 204 - 213, 1963.9. The estimation of small quantities of 3,4-dihydroxyphenylethylamine in tissues. By R. Laverty and D. F. Sharman. Br. J. Pharmac. Chemother. 24, 538 - 548, 1965.10. Modification by drugs of the metabolism of 3,4-dihydroxyphenylethylamine, noradrenaline and 5-hydroxytryptamine in the brain. By R. Laverty and D. F. Sharraan. Br. J. Pharmac. Chemother. 24. 759 - 772, 1965.11. The effect of drugs on the homovanillie aoid content of the corpus striatum of some rodents. By A. V. Juorio, D. F. Sharman and T. Trajkov. Br. J. Pharmac. Chemother. 26. 385 - 392, 1966.12. Changes in the metabolism of 3,4-dihydroxyphenylethylamine (dopamine) in the striatum of the mouse induced by drugs. By D. F. Sharman, Br. J. Pharmac. Chemother. 28. 153 - 163, 1966.13. A discussion of the modes of action of drugs which increase the concentration of 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid) in the striatum of the mouse. By D. P. Sharman. Br. J. Pharmac. Chemother. 30, 620 - 626, 1967.14. Homovanillic acid and dihydroxyphenylacetic acid in the striatum of monkeys with brain lesions. By D. P. Sharman, L. J. Poirier, G. P. Murphy and T. L. Sourkes. Can. J. Physiol. Pharmacol. 49. 57 - 62, 1967.15. Release by tubocurarine of dopamine and homovanillic acid from the superfused caudate nucleus. By P. J. Portig, D. P. Sharman and Marthe Vogt. J. Physiol. Lond. 194. 565 - 572, 1968.16. The effect of tropolone on the formation of 3,4-dihydroxyphenylacetic acid and 4-hydroxy-3-methoxyphenylacetic acid in the brain of the mouse. By G. P. Murphy, D. Robinson and D. P. Sharman. Br. J. Pharmac. Chemother. j>6, 107 - 115, 1969.17. Turnover of amines using probenecid to block the egress of metabolites. By D. P. Sharman. Metabolism of brain amines. Edited by G. Hooper, Macmillan, London, pp. 34 - 37, 1969.These papers form the main part of the thesis and attempt to relate the concentration of HVA in the central nervous system to the rate at which dopamine is utilised in this tissue. The locus of the metabolism of dopamine in the central nervous system is discussed.The papers in the third group are concerned with noradrenaline or its glycol metabolites.18. Noradrenaline content in the heart and spleen of the mouse tinder normal conditions and after administration of some drugs. By D. P. Sharman, S. Vanov and Marthe Vogt. Br. J. Pharmac. Chemother. 11, 527 - 533, 1962.This study was made to investigate a report of what appeared to he unusual behaviour of the tissue catecholamines in the mouse. The method used to estimate the noradrenaline was designed to incorporate as many controls as was possible to ensure that correct estimates were obtained. The earlier report could not be confirmed.19. Iontophoretic release of adrenaline noradrenaline and 5-hydroxytryptamine from raicropipettes. By K. Krnjevic, R. Laverty and D. P. Sharman. Br. J. Pharmac. Chemother. 20, 491 - 496, 1963.Fluorimetric methods were used to measure the relation between the release of these amines and the electrical charge applied to micropipettes used for the iontophoretic application of drugs to single neurones.20. The action of 2,4,5-trihydroxyphenylethylamine on the storage and release of noradrenaline. By R. Laverty, D. P. Sharman and Marthe Vogt. Br. J. Pharmac. Chemother. 24, 549 - 560, 1965.2,4,5-Trihydroxyphenylethylamine causes a rapid and long lasting depletion of noradrenaline from the mouse heart. A new method was developed for the estimation of the former amine and was used to show that it did not persist in the tissue or was tightly bound in the tissue.21. The noradrenaline content of the caudate nucleus of the rabbit. By D. P. Sharman and Marthe Vogt. J. Neurochem. 12. 62, 1965.This short paper illustrates a frequently reported erroneous result when a commonly used fluorimetric method for the estimation of nor-adrenaline is applied to those brain tissues which contain very little of this amine.22. Gas chromatographic evidence for the presence of glycol metabolites of catecholamines in brain tissue. By B. F. Sharman. J. Physiol. Lond. 200. 35 - 35P, 1969.23. Glycol metabolites of noradrenaline in brain tissue. By D. P. Sharman. Accepted for publication by Br. J. Pharmac. Chemother. 1969.The application of gas liquid chromatography and electron capture detection to the estimation of glycol metabolites of noradrenaline in the brain is described. The possibility of using a similar technique for the estimation of noradrenaline and normetanephrine is discussed.These two papers describe the estimation of noradrenaline, dopamine, 5-hydroxytryptamine and acetylcholine in particles obtained by sub-cellular fractionation of brain tissues. The first shows that dopamine is associated with a particle that is similar to,but distinguish¬ able from, that with which acetylcholine is associated. The second paper demonstrates that the storage sites within subcellular particles for acetylcholine and those for noradrenaline and 5-hydroxytryptamine are different

Flow cytometric observations in quantitative and qualitative platelet disorders and in humans receiving infusions of a chimaeric monoclonal antibody to glycoprotein IIb-IIIa.

A flow cytometric method was developed for quantitation of platelet surface-bound immunoglobulin G (IgG). A novel approach was used to calibrate the assay; this was based on the creation of reference curves by measuring PSIgO in platelets coated with known amounts of a chimaeric construct (supplied by Centocor Inc.) consisting of human IgG whose hypervariable region (HVR) had been replaced by the HVR of the murine monoclonal antibody (MoAb) 7E3, which binds with high affinity to human platelet glycoprotein (gp) Ilb-IIIa. By using a fluorescein- conjugated polyclonal anti-IgG it was calculated that platelets of healthy subjects (n=71) bear 1463 (SD=927) IgG molecules, whereas PSIgG elevations of up to 39,000 molecules/platelet were often detected in a cohort (n=87) of thrombocytopenic individuals. FC was also used to monitor platelet-bound 7E3, residual 7E3 binding sites and PSIgG instable angina patients receiving infusions of Fab fragments of chimaeric MoAb 7E3 (c7E3-Fab), which is currently under evaluation as an anti-platelet agent. The distribution of c7E3-Fab in the platelet population was unimodal at all time points following the infusion, demonstrating in vivo transfer of antibody to newly released platelets; clearance of platelet-bound antibody followed an exponential model but all circulating platelets were bearing c7E3-Fab at time points beyond the lifespan of the platelets exposed to c7E3-Fab during the infusion. Ex vivo and in vitro mixing experiments suggested that c7E3- Fab transfer between platelets was possible. TurbidometricaUy measured platelet aggregation response to ADP was shown to be a linear function of the logarithm of residual binding sites for 7E3. Infusion of c7E3-Fab was frequently associated with recruitment of IgG onto the platelet surface; in vitro evidence suggested that this was caused by naturally occurring IgG bindable to proteolytically derived Fab fragments of chimaeric but not murine 7E3-IgG. A new type of pseudothrombocytopenia, seen upon EDTA exposure of c7E3-Fab-bearing platelets was observed; this also appeared to be immune-mediated. The physiological, pathophysiological and therapeutic implications of these findings are discussed

The effects of isolation technique, substrata and pyrazole on cytochrome P450 enzymes in cultured male rat hepatocytes.

The cytochrome P450 enzymes are of particular interest in the study of drug metabolism since they play a key role in the transformation of xenobiotics. The recent explosion of in vitro techniques has facilitated detailed examination of drug metabolism in isolated systems such as primary hepatocyte culture. However, the expression of cytochrome P450 enzymes declines under culture conditions, limiting the use of this method. These studies examined the effects of combinations of isolation technique and substrata on the activity and amount of immunodetectable protein of several cytochrome P450 enzymes in male rat hepatocytes cultured for up to 96 hours. Two techniques, collagenase and EDTA, were used to isolate hepatocytes subsequently cultured on Matrigel®, Vitrogen®, fibronectin or uncoated plastic. Collagenase digestion was a significantly superior method of isolating hepatocytes compared to EDTA, in terms of cell yield and viability. The low cell yield and viability following EDTA isolation suggest that this technique is unsuitable for routine use. The data generated show that both substrata and, to a greater degree, isolation technique significantly affected the activity and immunodetectable amounts of several cytochrome P450 enzymes in rat hepatocytes throughout the culture period examined. The rates of decline of these enzymes, and the effects of isolation technique and substrata, were variable. No combination of isolation technique and substratum was found to halt the decline in cytochrome P450 enzymes and it is likely that a number of factors involved in their expression are lacking in the culture system The expression of CYP2E1 in cultured rat hepatocytes was also examined following exposure to the CYP2E1 inducing agent pyrazole. Increases in both CYP2E1 activity and amount of immunodetectable protein show that the hepatocytes have retained the ability to respond to pyrazole, demonstrating that the decline in cytochrome P450 expression is not irreversible