Molecular interactions enable us to understand the complexity of the human living system and how it can be exploited or malfunction to cause disease. The biomedical literature presents detailed knowledge of molecular functions and therefore represents a valuable reservoir of data for studying disease. However, extracting this data efficiently is difficult as it is spread over millions of publications in text that is not machine-readable. In this thesis we investigate how text mining can be used to automatically extract data for molecular interactions and their context relevant to disease. We focus on two globally relevant classes of diseases of which manifest from contrasting mechanisms: pain-related diseases and diseases caused by pathogenic organisms. Using HIV-1 as a case study, we first show that text mining can be used to partially recreate a large, manually curated database of HIV-1-human molecular interactions derived from the literature. We highlight both weaknesses in the quality of the data produced by the text-mining approach and strengths in it being able to extract this data rapidly, identifying instances missed in the manual curation and its potential as a support tool. We then expand on this approach by showing how an entirely new database of protein interactions relevant to pain can be created efficiently and accurately using text mining to generate the data and manual curation to validate the data quality. The following chapter then presents an analysis of 1,002 unique pain-related protein-protein interactions derived from this database, showing that it is of greater relevance to pain research than databases of pain interactions created from other common starting points. We highlight its value by, for example, identifying new drug repurposing opportunities and exploring differences in specific pain diseases using the contextual detail afforded by the text mining. Finally, we expand further on our approach to extracting molecular interactions from the literature, by showing how interactions between human proteins and pathogens can be curated across pathogenic organisms. We demonstrate how these techniques can be used to expand our knowledge of human pathogen related interaction data already stored in public databases, by identifying 42 new HIV-1-human molecular interactions, 108 new interactions between pathogen species and human proteins and 33 new human proteins that were found to interact with pathogens. Together, the results show that contexualised text mining, when supported by manual curation, can be used to extract molecular interactions for contrasting disease types in an efficient and accurate manner.

Control of Rhipicephalus microplus is predominantly mediated by the application of acaricides, but the rapid acquisition of resistance by this species and environmental pollution resulting from discarded acaricides, necessitates the discovery of new control measures. Due to the fact that Rhipicephalus spp. are genetically diverse and often have more than one host, it has been difficult to identify a common protective vaccine candidate able to target all species of this genus. Only one anti-tick antigen, Bm86, has been commercialized to date and is sold as GAVAC® and GAVACPlus® in South America. In an attempt to identify protective antigens, a protein termed subolesin was identified using expression library immunisation. RNAi studies showed that subolesin knockdown causes the degeneration of tick guts, salivary glands, reproductive tissues and embryos. Subolesin additionally mediates tick gene expression, impacts the innate immune response and affects tick infection by Anaplasma, Ehrlichia, Rickettsia, Babesia or Theileria spp. The R. microplus EF-1α homolog was identified as a subolesin-interacting protein via yeast two-hybrid and co-affinity purification experiments. RNAi experiments have suggested that EF-1α is another possible anti-tick vaccine candidate since it exhibits a similar phenotype as subolesin upon knockdown. The aim of the present research was to express R. microplus EF-1α in the yeast, Pichia pastoris and to exploit the yeast two-hybrid system in an attempt to identify its protein-binding partners. This will provide insight into understanding the translational machinery of this species and of ixodid ticks. Recombinant EF-1α was expressed as a 24 kDa protein, validated by western blotting. A highly representative cDNA library was produced from R. microplus mixed lifestages mRNA, fractionated and cloned into a two-hybrid prey vector. No definitive hits were obtained during the two-hybrid screen of reporter genes, as E-values attained after tblastx and PSI-BLAST analysis were higher than the required limit of 1 x 10-4. / Dissertation (MSc)--University of Pretoria, 2013. / Biochemistry / unrestricted

Orientadores: Marcelo Menossi Teixeira, Ricardo Aparicio / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T14:13:51Z (GMT). No. of bitstreams: 1 Soares_JoseSergiodeMacedo_D.pdf: 4995080 bytes, checksum: ad3458f447f7044749e0ee6cb95f4315 (MD5) Previous issue date: 2013 / Resumo: O agronegócio da cana de açúcar movimenta cerca de R$ 40 bilhões por ano no Brasil. A cadeia produtiva da cana de açúcar como atividade na economia é responsável por 1,5% do produto interno bruto (PIB) nacional e um dos principais componentes econômicos é a quantidade de sacarose acumulada nos colmos. No entanto, a síntese de sacarose e sua acumulação em plantas superiores é o resultado do produto de uma extensa rede de interações. Quando descarregada nas células do parênquima de armazenamento, a sacarose é metabolizada por diferentes enzimas, sendo a UDP-glucose pirofosforilase (UGPase) uma das enzimas responsáveis pela síntese de sacarose em cana de açúcar. O objetivo deste trabalho foi avaliar o padrão de expressão do gene ScUGPase-1 e os mecanismos regulatórios que controlam a atividade da proteína UGPase de cana de açúcar. Análises por RT-qPCR revelaram que a expressão do gene ScUGPase-1 diminui ao longo da maturação dos colmos e o gene é mais expresso nos entrenós em comparação com o tecido de folha. Porém, nenhuma diferença de expressão significativa foi observada entre dois cultivares contrastantes em teor de sacarose. In vivo, a localização subcelular da proteína ScUGPase-1 indicou uma associação à membrana nos tecidos de folha e colmo. Utilizando anticorpo primário fosfo-específico, observamos a fosforilação da proteína ScUGPase-1 apenas na fração solúvel e microssomal do tecido de folha. In vitro, a proteína ScUGPase-1 formou um complexo com a proteína recombinante caseína quinase 1 (CK1) e sua atividade foi afetada por agentes óxido-redutores. Para complementar os dados de óxido-redução, análises de espalhamento de luz a baixo ângulo (SAXS) forneceram o primeiro modelo estrutural do dímero da proteína ScUGPase-1 em solução, destacando que a interface de dimerização está localizada na região C-terminal. Os dados indicam que a fosforilação, interação protéica e oligomerização podem exercem um papel importante na regulação da proteína ScUGPase-1 durante a síntese de sacarose em cana de açúcar. / Abstract:The sugarcane agribusiness generates around R$ 40 billion per year in Brazil, while the entire supply chain of sugarcane is responsible for 1.5% of the gross domestic product (GDP). Sugarcane productivity is mainly determined by the accumulation of sucrose in the culms. However, the synthesis and accumulation of sucrose in plants is the result of an extensive network. When sucrose is unloaded in the storage parenchyma cells, it is metabolized by different enzymes, and UDP-glucose pyrophosphorylase (UGPase) is one of the enzymes responsible for the synthesis of sucrose in sugarcane. The objective of this work was to gain insights on the ScUGPase-1 expression pattern and the regulatory mechanisms that control protein activity. ScUGPase-1 transcript levels were negatively correlated with sucrose content in the internodes and only a slight difference in the expression pattern was observed between two cultivars that differ in their sucrose content. The intracellular localization of ScUGPase-1 indicated association with membranes in both leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo in the soluble and membrane fractions from leaves, but not from internodes. In vitro, the purified recombinant enzyme interacted with recombinant protein casein kinase 1 and its activity was affected by redox modification. To complement the redox data, Small-Angle X-ray Scattering provided the first structural model of the dimer of sugarcane UGPase in solution, highlighting that the dimer interface is located at the C-terminal. The data indicated that phosphorylation, protein interaction and oligomerization may play an important role in the regulation of ScUGPase-1 activity / Doutorado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

Plant natriuretic peptides (PNPs) comprise a novel class of hormones that share some sequence similarity in the active site with their animal analogues that function as regulators of salt and water balance. A PNP present in Arabidopsis thaliana (AtPNP-A) has been assigned a role in abiotic and biotic stress responses, and the recombinant protein has been demonstrated to elicit cyclic guanosine monophosphate (cGMP)-dependent stomatal guard cell opening, regulate ion movements, and induce osmoticum-dependent water uptake. Although the importance of the hormone in maintaining ion and fluid homeostasis has been established, key components of the AtPNP-A-dependent signal transduction pathway remain unknown. Since identification of the binding partners of AtPNP-A, including its receptor(s), is fundamental to understanding the mode of its action at the molecular level, comprehensive protein-protein interaction studies, involving yeast two-hybrid screening, affinity-based assays, protein cross-linking and co-immunoprecipitation followed by mass spectrometric (MS) analyses have been performed. Several candidate binding partners of AtPNP-A identified with at least two independent methods were subsequently expressed as recombinant proteins, purified, and the specificity of their interactions with the recombinant AtPNP-A was verified using surface plasmon resonance. Several specific binary interactants of AtPNP-A were subjected to functional assays aimed at unraveling the consequences of the interactions in planta. These experiments have revealed that reactive oxygen species (ROS) are novel secondary messengers involved in the transduction of AtPNP-A signal in suspension-cultured cells of A. thaliana (Col-0). Further insight into the AtPNP-A dependent signalling events occurring in suspension-cultured cells in ROS-dependent or ROS-independent manner have been obtained from the large-scale proteomics study employing tandem mass tag (TMT) labelling followed by MS analysis to identify and relatively quantify proteins that are differentially expressed upon the treatment with nano- and picomolar concentrations of the biologically active AtPNP-A peptide at different time-points post-treatment. Characterization of both the AtPNP-A interactome and AtPNP-A dependent proteome afforded novel insights into the signal transduction pathways altered by PNPs and shed new light on the mechanisms by which these candidate interactants operate. Taken together, indications are that PNP dependent mechanisms can be harnessed for possible biotechnological applications.

Capillary stamping is a versatile patterning platform to create micron/sub-micron features on surfaces. When used in combination with mesoporous silica stamps, dot arrays with length scale characteristics matching those of various biomolecular organizations on living cells can be printed. Therefore, different types of ink with functional molecules were printed onto a glass surface and assessed toward their capability to enable an analysis of cellular interactions. Among the evaluated patterned surfaces were dot arrays generated with heterocyclic silanes, which react in a ring-opening reaction upon contact with hydroxyl-terminated surfaces and allow post-modifications of the stamped dot array. Similarly, functionalized proteins were stamped from an aqueous solution, analyzed in regards to specific geometric descriptors and overall contrast between dot and background. After the establishment of a robust patterning system, the stamped substrates were used to spatially manipulate protein-protein interactions in live cells. With the introduction of optogenetics, namely the photoactivatable iLID-system into HeLa cells, protein recruitment from the cytosol to the membrane-bound domains upon irradiation with light was investigated. The technique was also utilized to explore the determinants of Wnt signalosome formation. Wnt co-receptor Lrp6 expressed at the surface of living cells was successfully assembled into nanodot arrays. Strikingly, the co-receptor Fzd8 and the cytosolic scaffold proteins Axin1 and Disheveled2 were spontaneously recruited into the nanodot array to form spatially defined signalosomes in the absence of ligand pointing toward Liquid-Liquid Phase Separation driven signalosome assembly. Immunofluorescence staining confirmed ligand-independent Wnt/β-catenin signaling activated the nanodot arrays.

The synthesis of iron-sulfur clusters is an essential cellular process, which depends on complex biosynthetic pathways. In model eukaryotes, these pathways are the ISC pathway in the mitochondria and the CIA pathway in the cytosol. A recent genome and transcriptome analysis showed, that an amitochondriate protist Monocercomonoides exilis lacks the canonical ISC pathway, which has been replaced by a bacterial SUF pathway. A close free-living relative of M. exilis, Paratrimastix pyriformis possesses a mitochondrion-related organelle, yet also possesses a SUF pathway instead of ISC. The acquisition of the SUF pathway has been suggested as the primordial cause for mitochondrial loss in M. exilis, which is the first documented eukaryotic organism without a mitochondrion. The SUF pathway has been the subject of numerous studies in bacteria, however, its role as the core provider of iron-sulfur clusters for eukaryotic cells has been reported in merely a handful of eukaryotes and was based predominantly on genomic data. This thesis focuses on the putative ATPase SufC and the putative scaffold protein SufB. Both proteins were successfully produced in recombinant forms. SufC has been found to possess ATPase activity in vitro, which was increased upon interaction with SufB. The conditions for theATPase...

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>Magister Scientiae - MSc / This thesis describes the cloning and recombinant expression of domains from the human RBBP6 protein for future in vitro binding studies with pRb and p53. RBBP6 is a splicing-associated protein that is known to interact with both p53 and the Retinoblastoma gene product (pRb), and has recently been shown to be highly upregulated in oesophageal cancer. The pRb binding domain (RbBD) and the p53 binding domain (p53BD) were each expressed using the glutathione-S-transferase (GST) tag affinity system, and affinity purified using a glutathione-linked agarose column. Purified fusion proteins were cleaved to separate the target protein from GST using PreScission ™ Protease, for which there is a recognition sequence located immediately upstream of the multiple cloning site on the pGEX-6P series of plasmids. The pRb binding and p53 binding domains were further purified using cation exchange chromatography. Mass spectrometry confirmed that the RbBD was expressed as a single species of the expected molecular weight. However preliminary NMR analysis suggested that the domain was not fully folded. A total yield of 8 mg of protein was achieved from 11 of culture, which make it feasible to express 15Nand 12Clabelled samples for NMR. The p53BD was found to be expressed at lower levels and subject to C-terminal degradation, which suggest that the C-terminus is unstructured most likely due to the presence of polylysine tail. Human pRb protein was also successfully expressed and purified using the GST affinity system. Human p53 protein was expressed but was found to be insoluble and attempts to purify it were not pursued. Attempts to confirm the interactions between human RBBP6 and p53 and pRb proteins are on-going but fall outside the scope of this thesis. Expression constructs for the RING and zinc finger domains from human RBBP6 were also cloned into the pGEX system for future structural studies using NMR. Both domains were found to be expressed as soluble fusion proteins in preliminary expression studies.

Toll-like receptors (TLRs) play important role in the innate immune system. TLR15 is reported to have a unique role in defense against pathogens, but its structural and evolution characterizations are still poorly understood. In this study, we identified 57 completed TLR15 genes from avian and reptilian genomes. TLR15 clustered into an individual clade and was closely related to family 1 on the phylogenetic tree. Unlike the TLRs in family 1 with the broken asparagine ladders in the middle, TLR15 ectodomain had an intact asparagine ladder that is critical to maintain the overall shape of ectodomain. The conservation analysis found that TLR15 ectodomain had a highly evolutionarily conserved region on the convex surface of LRR11 module, which is probably involved in TLR15 activation process. Furthermore, the protein-protein docking analysis indicated that TLR15 TIR domains have the potential to form homodimers, the predicted interaction interface of TIR dimer was formed mainly by residues from the BB-loops and αC-helixes. Although TLR15 mainly underwent purifying selection, we detected 27 sites under positive selection for TLR15, 24 of which are located on its ectodomain. Our observations suggest the structural features of TLR15 which may be relevant to its function, but which requires further experimental validation.