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1	RECONSTRUCTION OF MOLECULAR REGULATORY NETWORKS IN <i>Arabidopsis thaliana</i> Fitzek, Elisabeth 01 May 2012 (has links) Bioinformatics is a valuable tool to understand gene regulatory networks. Cis-regulatory elements (CREs) previously found in promoter regions are known to recruit transcription in signaling pathways. In this work it has been hypothesized to consider CREs as a family of related words that interact/bind to a family of related transcription factors, and thus have similar but distinct regulation patterns. A 1460 microarray gene expression collection was obtained via online databases to create a transcriptomic meta-dataset. A novel bioinformatic algorithm was applied to annotate all 65536 (64k) potential 8-letter CREs in the 500 bp upstream promoter region of all A. thaliana genes across the transcriptomic meta-dataset. Of the possible words, only 2,498 were significantly associated with a pattern of regulation in any of the 1,460 microarrays tested whereas the remaining motifs appeared not to be regulatory. Unique CREs were categorized into 4 regulatory types: inducer, suppressor, biregulator and insulator. A predicted protein protein interactome was created for an economically important plant Coffea canephora. Here, it has been hypothesized that evolutionary conservation of many core biological processes enable generation of predicted protein interactome for species with few resources other than sequenced genome. Of over 12,000 genes identified, 939 were predicted to have 4,587 interactions. Gene Ontology analysis revealed enrichment of processes conserved in all eukaryotes but depletion in unique plant processes. A third study was conducted to determine if homology modeling, evolutionary analysis, and structural evolution could determine key factors involved in function, and interaction specificity in Pus10 (EC 5.4.99.25) found in Archaea and Eukaryotes. Redundancy of Pus10 and the bacterial TrmA and TruB orthologs appear to have resulted in significant molecular evolution of Pus10 function. Neofunctionalization was identified in animal kingdom where thiouridine synthase, methylases and PSUSs (THUMP)-domain modification in early animal evolution coincides with appearance of TNF-related apoptosis-inducing ligand (TRAIL) apoptosis components. Subfunctionalization was identified for Thermococcales lineage of Archaea where a shorter forefinger-loop coincides with the loss of Ψ54 specificity as experimentally verified in P. furiosus. Absence of Pus10 was observed in Sulfolobus and higher fungi whereas in plant kingdom Pus10 function remains unknown with possible pseudogene in some lineages Arabidopsis thaliana Archaea Bioinformatics cis-regulatory elements protein-protein interactome pseudouridinesynthase
2	The Development and Application of Mass Spectrometry-based Structural Proteomic Approaches to Study Protein Structure and Interactions Makepeace, Karl A.T. 26 August 2022 (has links) Proteins and their intricate network of interactions are fundamental to many molecular processes that govern life. Mass spectrometry-based structural proteomics represents a powerful set of techniques for characterizing protein structures and interactions. The last decade has witnessed a large-scale adoption in the application of these techniques toward solving a variety of biological questions. Addressing these questions has often been coincident with the further development of these techniques. Insight into the structures of individual proteins and their interactions with other proteins in a proteome-wide context has been made possible by recent developments in the relatively new field of chemical crosslinking combined with mass spectrometry. In these experiments crosslinking reagents are used to capture protein-protein interactions by forming covalent linkages between proximal amino acid residues. The crosslinked proteins are then enzymatically digested into peptides, and the covalently-coupled crosslinked peptides are identified by mass spectrometry. These identified crosslinked peptides thus provide evidence of interacting regions within or between proteins. In this dissertation the development of tools and methods that facilitate this powerful technique are described. The primary arc of this work follows the development and application of mass spectrometry-based approaches for the identification of protein crosslinks ranging from those which exist endogenously to those which are introduced synthetically. Firstly, the development of a novel strategy for comprehensive determination of naturally occurring protein crosslinks in the form of disulfide bonds is described. Secondly, the application of crosslinking reagents to create synthetic crosslinks in proteins coupled with molecular dynamics simulations is explored in order to structurally characterize the intrinsically disordered tau protein. Thirdly, improvements to a crosslinking-mass spectrometry method for defining a protein-protein interactome in a complex sample is developed. Altogether, these described approaches represent a toolset to allow researchers to access information about protein structure and interactions. / Graduate Mass Spectrometry Structural Proteomics Crosslinking Cross-linking Disulfide bonds Proteomics Tau Mitochondria Method development Molecular dynamics Protein chemistry Protein crosslinking Protein cross-linking Shotgun proteomics Bottom-up proteomics Data-dependent acquisition DDA Protein-protein interactome Interactome Interactomics Proteinase K Trypsin Higher-order crosslinking Machine learning Feature engineering Yeast Yeast mitochondria Algorithms Data analysis Surface modification Molecular modelling Non-specific digest Structural mass spectrometry Structural biology Biochemistry LC-MS

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RECONSTRUCTION OF MOLECULAR REGULATORY NETWORKS IN <i>Arabidopsis thaliana</i>

The Development and Application of Mass Spectrometry-based Structural Proteomic Approaches to Study Protein Structure and Interactions