Integration of biological data: systems, infrastructures and programmable tools

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid. Escuela Politécnica Superior, Departamento de Ingeniería informática. Fecha de lectura: 19-05-200

Determinants of survival and virulence of Campylobacter

The pathogenesis of Campylobacter enteritis is not well understood including the mechanisms involved in invasion and translocation across intestinal epithelial cells. The genetic make-up of the pathogen and its responses to different environmental cues are thought to contribute to the organism's ability to survive and cause disease. The extremes of environment which Campylobacter can with-stand, and the effect that this has on virulence and invasive ability remains undefined. For the first time, several isolates were compared quantitatively to determine the extent to which intracellular invasion contributes to translocation across epithelial cell mono layers. Translocation ability did not correlate with intracellular invasiveness, suggesting that different "invasion" phenotypes exist among Campylobacter isolates. Repeated exposure of Campylobacter isolates to Caco-2 cells caused an increase in their ability to invade and survive, which was associated with changes in protein expression. Campylobacter was grown in continuous culture under conditions of iron sufficiency, iron limitation, oxidative stress and low pH. Uniquely, growth under oxidative stress and iron replete conditions caused an increase in the invasive ability of C. jejuni 81116, which was correlated with the up-regulation of specific proteins. The role of three proteins, HtrB, Tpx and PEB-4, was investigated at the molecular level. Two of the encoding genes, peb4A and htrB, were found to be essential for viability. Homologous recombination of an inactivated Ipx gene into the genome of C. jejuni caused increased sensitivity to H20 2, but did not affect the ability of C. jejuni to invade and survive within Caco-2 cell monolayers. This study demonstrated that isolates of Campylobacter differ significantly in their virulence potential with respect to their invasive phenotypes. In addition Campylobacter grown in well defined continuous culture conditions demonstrated for the first time the importance of iron and oxidative stress as acting as potenital cues for the expression of survival and invasion determinants

Biochemical and molecular studies of some aspects of disease resistance in potato (solanum tuberosum L.)

Disease problems in crop plants are still a major threat to the agro-industry worldwide. Various strategies have been developed and evaluated in recent years. One strategy is to use naturally-occurring antipathogen factors such as lysozymes and chitinases in transgenic plants. In the present study, transgenic plants containing chick-egg white lysozyme (Lys 75) have been evaluated for lysozyme production in planta, subcellular localisation, and resistance to some potato pathogens, including Phytophthora infestans and Erwinia carotovora subsp. atroseptica, the two major potato pathogens worldwide. In addition, the evaluation of resistance was also undertaken for transgenic plants carrying other naturally-occurring antipathogen factors including a bean chitinase gene (BCH 35) and a snowdrop lectin gene (GNA 74). In order to accurately quantify the lysozyme production in Lys 75 plants, the turbidimetric lysozyme enzyme assay was optimised. Also, a modified substrate for the enzyme has been developed by covalently linked the Micrococcus lysodeikticus cell wall with a dye, remazol brilliant violet 5R to enable a colorimetric assay of the enzyme. In order to quantitatively assess resistance levels of the transgenic plant, a new method (leaf-bridge bioassay) for conducting and evaluating resistance in planta has been developed. All transgenic plants in tissue culture were tested for resistance using this technique. Evaluation of the progress of infection in detached leaves of Lys 75 showed that lysozyme gave some degree of protection against the bacterial pathogen, Erwinia carotovora subsp. atroseptica and the fungal pathogen, Fusarium sulphureum. Analysis of intercellular fluid from the Lys 75 leaves showed that more than 80% of the total lysozyme expressed in the leaf was located in the intercellular space which is a strategic place to combat pathogen attack. In contrast, the levels of protection in BCH 35 plants were relatively low compared with Lys 75. The progress of infection was delayed in BCH 35 leaves challenged with F. sulphureum only. No resistance at all was observed in GNA 74 to all the pathogens used. All the transgenic potato lines were susceptible to P. infestans. Recently, a new strategy to combat disease problems has been suggested based on a 'durable resistance'. Potato variety Stirling' which shows durable resistance in the field has been used to study the early biochemical and molecular events during elicitation of 'Stirling' cell suspension cultures with an elicitor mix derived from infective units of a compatible strain of P. infestans. For comparison, an elicitor mix from an incompatible strain of P. infestans was also prepared and used. The mixed elicitor comprising zoospore extract, culture filtrate and mycelium homogenate induced defence responses in 'Stirling' cell suspension cultures as judged by the increase in PAL enzyme activity. PAL activity in 'Stirling' ceUs elicited with an elicitor mix derived from an incompatible strain of P. infestans was twice the activity in the compatible interaction. The peak levels in both types of interaction were at 6 h post-elicitation. An oxidative burst was demonstrated also in both types of interactions indicated by rapid release of H(_2)O(_2) into the culture medium. The H(_2)O(_2) level peaked at 2 h post-elicitation in both interactions before being reduced to its normal level at 4 h. The H(_2)O(_2) released during incompatible interaction was twice the levels monitored in the compatible. A subtracted cDNA library of differentially expressed mRNAs during elicitation of 'Stirling' cell suspension cultures with the elicitor mix from a compatible strain of P. infestans was constructed using suppression subtractive hybridisation. Two cDNA clones, STS 42 and STS 52, relevant to the present study were identified and characterised. STS 42 showed high degree of similarity to potato leucine aminopeptidase gene which is induced in response to wounding. Gene expression studies using RT-PCR showed that the mRNA levels of STS 42 increased gradually throughout the 18 h elicitation. STS 51 was identified as a member of the ribonuclease T2 histidine proteins. It showed some degree of similarity to plant ribonucleases involved in self-incompatibility reactions during pollination. It has a site for tyrosine kinase phosphorylation at the hydrophilic region of the sequence and could possibly be involved in phosphorylation during signal transduction. mRNA levels of STS 51 were increased during the first 12 h of elicitation

Novel bioinformatics approach for encoding and interrogating the progression and modulation of the mammalian cell cycle

The cell cycle, with its highly conserved features, is a fundamental driver for the temporal control of cell growth and proliferation in tissues - while abnormal control and modulation of the cell cycle are characteristic of cancer cells, particularly in response to therapy. A central theme in cancer biology is to resolve and understand the origin and nature of innate and induced heterogeneity at the cell population level. Cellular heterogeneity - comprising structural, temporal and functional dimensions - is a confounding factor in the analysis of cell population dynamics and has implications at physiological, pathological and therapeutic levels. There is an exceptional advancement in the applications of imaging and cell tracking technologies dedicated to the area of cytometric research, that demand an integrated bioinformatics environment for high-content data extraction and interrogation. Image-derived cell-based analyses, where time is the quality parameter also demand unique solutions with the aim of enabling image encoding of spatiotemporal cellular events within complex cell populations. The perspective for this thesis is the complex yet poorly understood nature of cancer and the opportunities offered by rapidly evolving cytometric technologies. The research addresses the intellectual aspects of a bioinformatics framework for cellular informatics that encompass integrated data encoding, archiving, mining and analysis tools and methods capable of producing in silico cellular fingerprints for the responses of cell populations to perturbing influences. The overall goal is to understand the effects of anti-cancer drugs in complex and potentially heterogeneous neoplastic cellular systems by providing hypothesis testing opportunities. Cell lineage maps encoded from timelapse microscopy image sequences sit at the core of the proposed bioinformatics infrastructure developed in the current work. Through a number of data mining, analysis and visualisation tools the interactions and relationships within and between lineages have provided dynamic patterns for the modulation of the cell cycle in disease and under stress. The lineage data, accessible through databases implemented during the current study, has provided a rich repository for pharmacodynamic (PD) modelling and validation and has thus laid the foundation for fabricating a comprehensive knowledge base for linking both cellular and molecular behaviour patterns. These provide the foundation for meeting the aspirations of systems biology and drug discovery.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

A molecular approach to questions in the phylogeny of planktonic sarcodines

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 1996.Includes bibliographical references.by Linda Angela Amaral Zettler.Ph.D

Novel bioinformatics approach for encoding and interrogating the progression and modulation of the mammalian cell cycle

The cell cycle, with its highly conserved features, is a fundamental driver for the temporal control of cell growth and proliferation in tissues - while abnormal control and modulation of the cell cycle are characteristic of cancer cells, particularly in response to therapy. A central theme in cancer biology is to resolve and understand the origin and nature of innate and induced heterogeneity at the cell population level. Cellular heterogeneity - comprising structural, temporal and functional dimensions - is a confounding factor in the analysis of cell population dynamics and has implications at physiological, pathological and therapeutic levels. There is an exceptional advancement in the applications of imaging and cell tracking technologies dedicated to the area of cytometric research, that demand an integrated bioinformatics environment for high-content data extraction and interrogation. Image-derived cell-based analyses, where time is the quality parameter also demand unique solutions with the aim of enabling image encoding of spatiotemporal cellular events within complex cell populations. The perspective for this thesis is the complex yet poorly understood nature of cancer and the opportunities offered by rapidly evolving cytometric technologies. The research addresses the intellectual aspects of a bioinformatics framework for cellular informatics that encompass integrated data encoding, archiving, mining and analysis tools and methods capable of producing in silico cellular fingerprints for the responses of cell populations to perturbing influences. The overall goal is to understand the effects of anti-cancer drugs in complex and potentially heterogeneous neoplastic cellular systems by providing hypothesis testing opportunities. Cell lineage maps encoded from timelapse microscopy image sequences sit at the core of the proposed bioinformatics infrastructure developed in the current work. Through a number of data mining, analysis and visualisation tools the interactions and relationships within and between lineages have provided dynamic patterns for the modulation of the cell cycle in disease and under stress. The lineage data, accessible through databases implemented during the current study, has provided a rich repository for pharmacodynamic (PD) modelling and validation and has thus laid the foundation for fabricating a comprehensive knowledge base for linking both cellular and molecular behaviour patterns. These provide the foundation for meeting the aspirations of systems biology and drug discovery

The Prevention and Treatment of Atherosclerosis

This special issue of JCM ‘The Prevention and Treatment of Atherosclerosis’ will walk the Readers across novel diagnostic achievements in atherosclerosis and contemporary actions towards optimizing therapy.Everything begins with diagnosis. Accurate diagnostic tools and tests are of utmost importance. Contemporary research on microparticles, non-coding RNAs, proteomic characterization, …, offers detailed molecular characteristics of athero-thrombosis.Prevention is equally important as treatment. The impact of eating habits in prophylaxis of many pathologies, including cardiovascular disease has been documented.Then new pharmacological agents. Managing hypercholesterolemia with PCSK9 inhibitors, shown great potential in efficient lipid lowering to achieve LDL-C treatment goals, as well as reduction in cardiovascular mortality and morbidity.However, therapeutic goals accomplishment requires supervision. Arising number of data support that cardiovascular risk prediction can be improved with imaging modalities displaying atheroma: carotid plaque ultrasonography, coronary calcium score, intravascular ultrasonography, and optical coherent tomography or many others.As atherosclerosis is a progressive disease, it comes the time for more radical management, including endovascular and surgical intervention. There is field for new stent and equipment technologies, new surgical and endovascular techniques, supervision of endovascular procedures with IVUS, OCT, functional flow assessment or cell therapy.From diagnosis to risk stratification, elaborated prevention models, finally to modern and optimized therapeutic intervention

Lysosomal neuraminidase : a unique member of the sialidase superfamily

Lysosomal neuraminidase initiates the hydrolysis of oligosaccharldes, gangliosides, glycolipids and glycoprotelns by removing their terminal sialic acid residues. The enzyme functions exclusively In a multi-enzyme complex, together with flgalactosidase and protective protein/cathepsin A (PPCA) and Is dependent for Its activity and stability on the latter protein. Mutations In the neuraminidase gene are the basis for the lysosomal storage disorder sialidosls, while mutations In the PPCA gene Indirectly results In the Impairment of neuraminidase and p·galactosidase activity, causing the lysosomal storage disorder galactoslalldosls. The experimental work that is described in this thesis involves the Isolation and characterization of the cDNA's encoding human and mouse lysosomal neuraminidase. Structural and functional analysis of this enzyme has focused on the Interaction with PPCA in relation to Its transport to the Iysosomes and the mechanism of catalytic activation. These studies, In combination with the Identification and functional analysis of a large number of novel mutations In slalidosls patients, have given new Insights Into the biochemical functions and properties of neuraminidase and provided reliable phenotype-genotype correlations. The results presented In this thesis may form the basis for understanding the structural characteristics of this unusual lysosomal protein and for the future development of strategies for therapy of both slalldosis and galactoslalldosis patients