New Methods to Improve Protein Structure Modeling

Proteins are considered the central compound necessary for life, as they play a crucial role in governing several life processes by performing the most essential biological and chemical functions in every living cell. Understanding protein structures and functions will lead to a significant advance in life science and biology. Such knowledge is vital for various fields such as drug development and synthetic biofuels production. Most proteins have definite shapes that they fold into, which are the most stable state they can adopt. Due to the fact that the protein structure information provides important insight into its functions, many research efforts have been conducted to determine the protein 3-dimensional structure from its sequence. The experimental methods for protein 3-dimensional structure determination are often time-consuming, costly, and even not feasible for some proteins. Accordingly, recent research efforts focus more and more on computational approaches to predict protein 3-dimensional structures. Template-based modeling is considered one of the most accurate protein structure prediction methods. The success of template-based modeling relies on correctly identifying one or a few experimentally determined protein structures as structural templates that are likely to resemble the structure of the target sequence as well as accurately producing a sequence alignment that maps the residues in the target sequence to those in the template. In this work, we aim at improving the template-based protein structure modeling by enhancing the correctness of identifying the most appropriate templates and precisely aligning the target and template sequences. Firstly, we investigate employing inter-residue contact score to measure the favorability of a target sequence fitting in the folding topology of a certain template. Secondly, we design a multi-objective alignment algorithm extending the famous Needleman-Wunsch algorithm to obtain a complete set of alignments yielding Pareto optimality. Then, we use protein sequence and structural information as objectives and generate the complete Pareto optimal front of alignments between target sequence and template. The alignments obtained enable one to analyze the trade-offs between the potentially conflicting objectives. These approaches lead to accuracy enhancement in template-based protein structure modeling

Aerospace Medicine and Biology: A Continuing Bibliography with Indexes

This bibliography lists 237 reports, articles, and other documents introduced into the NASA scientific and technical information system in November 1985

Identification and characterization of the activated defence response in the commercially important Agarophyte, Gracilaria Gracilis, following exposure to disease elicitors

To our knowledge, this study represents the first analysis of gene expression using cDNA microarrays in the red macroalga G. gracilis. Western hybridization analysis was used to establish whether the observed changes in gene expression following exposure to disease elicitors positively correlated to changes at the protein level

Genetic modification of heavy metal resistant Streptomyces sp. strains

Two heavy metal resistant Streptomyces sp. strains, S. mirabilis P16 B-1 and S. acidiscabies E13, isolated from a former uranium mining site are investigated for genetic metal resistance determinants. After establishing a transformation system for Streptomyces sp. using conjugation with E. coli, the streptomycetes are transformed by random transposon mutagenesis yielding mutants with reduced metal resistance. The integration sites serve as targets for directed deletion via PCR-targeted gene replacement. It is demonstrated that a large linear S. mirabilis plasmid plays a major role in conferring nickel resistance and is transferrable to S. lividans. An NreB-like transporter encoded on this plasmid is identified as a nickel resistance factor, which could also be expressed in E. coli. Additionally, the role of plasmid-borne copper homeostasis system components, i.e. a transcriptional CsoR-type repressor and a CopZ-like chaperone, is surveyed. The tyrosinase-encoding gene melC2, located on a second linear S. mirabilis plasmid, is shown to hamper nickel resistance on complex medium, providing one reason for the strains higher nickel resistance on minimal medium. Furthermore, genes encoding means for coping with different environmental stressors are investigated, e.g. an HAD-like hydrolase, a Na+,Li+/H+ antiporter, a phosphinothricin N-acetyltransferase. Besides, the composition of extracellular matrix around the streptomycete hyphae and cell membrane composition are shown to impact metal resistance, i.e. loss of CslA function increases nickel resistance. Taken together, by demonstrating the applicability of molecular transformation methods for heavy metal resistant Streptomyces sp. strains this study provides the basis for further investigations of the metal resistome. The results indicate that metal resistance of both investigated strains is a result of multiple genetic determinants, several of which are plasmid-encoded

Studies on nucleotide levels and electron transport genes of Clostridium acetobutylicum P262

Clostridium acetobutylicum P262 is an endospore-forming Gram positive anaerobic bacterium, and for many years this organism has been used in the industrial fermentation for the production of acetone and butanol from carbohydrate substrates. The aims of this thesis included studies on small phosphorylated molecules involved in energy metabolism and cell differentiation, and an investigation into the genetics and molecular biology of C. acetobutylicum electron transport genes. To facilitate quantitation of nucleoside triphosphates in extracts of C. acetobutylicum, a chromatographic data acquisition and analysis system was constructed. Samples were prepared from C. acetobutylicum cultures by treatment with formic acid, and nucleotides contained in these extracts were separated by strong anion exchange HPLC. The developed manual integration system features the ability to collect and store chromatographic data, allowing for multiple integration using different calibration curves. Nucleoside triphosphate profiles were obtained from batch fermentations of the C. acetobutylicum P262 wild type, sporulation deficient (spo-1) and solvent deficient (ds-1) strains. The nucleoside triphosphate profiles of the wild type and spo-1 mutant were similar and were characterized by a trough in nucleotide levels which occurred just prior to the pH break point, the onset of the stationary growth phase, clostridial stage formation and the transition from the acidogenic to the solventogenic phase. The nucleoside triphosphate concentrations during the exponential growth phase were much lower than those found during the stationary phase. Exponential phase nucleotide levels in the cls-1 mutant were comparable to those observed in the wild type and spo-1 mutant. Unlike the wild type and spo-1 strains, the cls-1 mutant, which does not switch to solventogenesis, did not demonstrate an increase in nucleotide levels after the cessation of cell division. The involvement of nucleotide levels, particularly that of GTP, in the differentiation of C. acetobutylicum was indicated by the effect of inhibitors, which have been shown to decrease ribonucleotide levels in other organisms and cause an increase in sporulation. The antibacterial agent metronidazole, was used as a tool for the isolation of C. acetobutylicum electron transport genes. Since it was desired to clone these genes in Escherichia coli, and investigation into the activation of metronidazole by E. coli strains was necessary. E. coli strains with lesions in their DNA repair systems were more susceptible to metronidazole than wild type strains. However, it has been reported that DNA repair deficient strains of E. coli that also had a diminished ability to reduce chlorates and nitrates were no more susceptible to metronidazole than their wild type parents (Jackson et al., 1984; Yeung et al., 1984). To isolate a suitable E. coli cloning host for the selection of C. acetobutylicum electron transport genes which activated metronidazole, transposon mutagenesis of the recA E. coli strain CC118 with TnphoA, was used to construct the recA, metronidazole resistant E. coli strain Fl9. F19 was shown to have diminished nitroreductase activity, which was presumed to be responsible for the metronidazole resistant phenotype. However, the recA mutation renders E. coli F19 highly susceptible to the reduced toxic intermediates of metronidazole. The E. coli F19 recA, nitroreductase deficient mutant was used for the isolation of C. acetobutylicum genes on recombinant plasmids which activated metronidazole. Twenty-five E. coli F19 clones which contained different recombinant plasmids were isolated. The clones were tested for nitroreductase, pyruvate-Fd­oxidoreductase and hydrogenase activities. Nitroreductase and pyruvate-Fd­oxidoreductase activity was not demonstrated in any of the isolated clones, and only one clone tested positive for hydrogenase activity. DNA hybridization and restriction endonuclease mapping revealed that four of the C. acetobutylicum insert DNA fragments on recombinant plasmids were linked in an 11.1 kb chromosomal fragment. It was determined that this 11.1 kb fragment contained at least two regions responsible for activating metronidazole. The one gene responsible for making E. coli F19 extremely sensitive to metronidazole was localized to a 2 kb region. The nucleotide sequence of this 2 kb region was determined and two truncated open reading frames and one complete open reading were present. The complete open reading frame was shown to be responsible for activating metronidazole. The deduced amino acid sequence of this open reading frame was determined to be 160 amino acids in length, and database searches showed good similarity to flavodoxin proteins from many organisms. Based on alignments to the amino acid sequences of these flavodoxins, as well as the fact that Chen and Blanchard (1979) reported that reduced flavodoxin can transfer electrons to metronidazole, the sequence corresponding to this C. acetobutylicum metronidazole activating gene was identified as coding for a flavodoxin gene. The role of flavodoxin in C. acetobutylicum and other organisms is presented. Possible relationships between the cloned C. acetobutylicum flavodoxin gene and metronidazole sensitivity are discussed

Flow-Through Microchannel DNA Chips

This work represents a contribution to the rapidly expanding field of microarray technology, which consists in the pooling of a large number of miniaturised biorecognition elements on a single solid substrate so as to allow heterogeneous bioassays to be performed in a highly multiplexed fashion. Numerous applications of life science research and pharmaceutical development, such as diagnostics and molecular medicine, are expected to greatly benefit from the increased levels of throughput provided by the microarray approach. However, most microarray platforms developed as of this writing have merely been based on a two-dimensional configuration in which the biorecognition sites are distributed on the surface of a planar, unpenetrable substrate; this format does not adress the mass-transfer limitations inherent to heterogeneous ligand binding kinetics and thus does not provide optimal assay performance in terms of speed and sensitivity. In this thesis, a novel concept is presented that has been devised to increase the intrinsic analytical performance of microarrays. The approach is based on the use of three-dimensional, uniformly porous substrates featuring a regular array of discrete, ordered, high aspect ratio microchannels. The fabrication of such microstructures, with feature sizes in the micrometre range and aspect ratios in excess of 30:1, was achieved using two subtractive silicon processing techniques, electrochemical etching and deep dry etching, whose relative merits have been assessed. Commercially available microchannel glass substrates were also used. Nucleic acid molecules were immobilised as capture probes on the sidewalls of the microchannels, in a conformation allowing biorecognition through heterogeneous hybridisation. To that end, a chemical strategy was developed for the functional immobilisation of oligonucleotide probes onto silica-rich substrates such as glass and oxidised silicon. Nucleic acid hybridisation was chosen as the model ligand binding process due not only to the relative ease of handling of DNA (including the availability of chemically pure, synthetic oligonucleotides) but also to its relevance, through genomics, to a number of clinical, medical and pharmaceutical applications. The particular wetting properties of microchannel substrates were characterised and subsequently taken into account in the development of a microarraying procedure based on non-contact, ink-jet dispensing technology. In this respect, the physical-chemical processes that control the formation of spots on microchannel substrates, i.e. the wetting properties of the substrates, as well as the main parameters involved in the arraying process, such as the concentration of the capture probe solution and volume of the aliquots dispensed, have been investigated. Medium-density microchannel oligoarrays were produced that comprised as many as 256 probe spots over a 1 cm2 area. Since the effective surface area available for probe immobilisation was increased by expansion into the third dimension, a higher spatial density of probes could be achieved as compared to planar substrates with the same lateral dimensions. Biorecognition events between the immobilised capture probes and solution-borne target molecules were allowed to take place, in a dynamic mode, by convectively pumping sample through the microchannel array. As each microchannel acted as a miniature analytical chamber, probe-target hybridisation kinetics were enhanced by spatial confinement, while recirculation of the sample provided enhanced mass transfer. The feasibility of using electrokinetic transport of the targets was also assessed. Quantitative detection of heterogeneous hybridisation events taking place along the microchannel sidewalls was achieved through a CCD camera-based epifluorescence detection scheme, in a first instance, conventional low magnification microscope objective lenses were used as imaging optics whose depth-of-field characteristics matched the thickness of the microchannel substrate. The suitability of the approach was illustrated by the achievement of detection limits as low as a few attomoles of fluorescent dye per spot. This level of performance was made possible by the remarkable optical properties of the substrate, which were characterised both experimentally and through simulations based on ray-tracing procedures. Since the use of microchannel chips of increased thickness provided a way of increasing the number of capture probe molecules immobilised within each spot without increasing the lateral dimensions of the spots, a range of chip thicknesses were considered in a first step towards optimisation of the substrate geometry. Potential limitations to the benefits associated with an increase in chip thickness were discussed, in terms of the effectiveness of both the fluorescence detection scheme and the process of probe immobilisation on the microchannel sidewalls. Comparative hybridisation experiments were conducted which demonstrated the improved performance of the flow-through microchannel configuration over the conventional planar configuration, with an observed circa 5 times enhancement in hybridisation kinetics and a one to two orders of magnitude higher detectability. A few routes for further improving the performance of flow-through microchannel DNA chips are suggested, along with other possible bioanalytical applications of the microchannel biochip platform

Identification and characterisation of the Cdx1 and Apc1 cis-regulatory elements in mouse and Fugu rubripes

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Fluorescent molecular cytogenetics: Preimplantation diagnosis, colorectal cancer and mapping chromosome 9q.

The technique of fluorescent in-situ hybridisation (FISH) on to metaphase and interphase nuclei has many applications. In this thesis, FISH has been used in three areas of research:- Preimplantation diagnosis, genetics of colorectal cancer and early stages in mapping the terminal region of chromosomal 9q to search for the tuberous sclerosis locus (TSC1). Linkage data has suggested possible tuberous sclerosis loci on chromosomes 9, 11, and/or 12. In this study a hybrid was screened by FISH and found to contain only chromosome 9q. Fragment hybrids made from this were also screened. Phage clones isolated from a hybrid containing three small human pieces and clones linked to TSC1 were mapped to chromosome 9q. Cosmid clones isolated from the original hybrid were also mapped

On Musical Self-Similarity : Intersemiosis as Synecdoche and Analogy

Self-similarity, a concept borrowed from mathematics, is gradually becoming a keyword in musicology. Although a polysemic term, self-similarity often refers to the multi-scalar feature repetition in a set of relationships, and it is commonly valued as an indication for musical ‘coherence’ and ‘consistency’. In this study, Gabriel Pareyon presents a theory of musical meaning formation in the context of intersemiosis, that is, the translation of meaning from one cognitive domain to another cognitive domain (e.g. from mathematics to music, or to speech or graphic forms). From this perspective, the degree of coherence of a musical system relies on a synecdochic intersemiosis: a system of related signs within other comparable and correlated systems. The author analyzes the modalities of such correlations, exploring their general and particular traits, and their operational bounds. Accordingly, the notion of analogy is used as a rich concept through its two definitions quoted by the Classical literature—proportion and paradigm, enormously valuable in establishing measurement, likeness and affinity criteria. At the same time, original arguments by Benoît B. Mandelbrot (1924–2010) are revised, alongside a systematic critique of the literature on the subject. In fact, connecting Charles S. Peirce’s ‘synechism’ with Mandelbrot’s ‘fractality’ is one of the main developments of the present study