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Dissertations / Theses on the topic 'Protein variants'
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Sadler, David Paul. "Mechanically unfolding variants of protein L." Thesis, University of Leeds, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.444035.
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Jones, Kerrie Margaret. "Mutational variants of E. coli glutamate dehydrogenase." Thesis, University of Leeds, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.278229.
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Lee, Seung-Joo. "Structural and functional consequences of disease-related protein variants." Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1269545015.
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Veltman, Oene Robert. "Engineering high performance variants of Bacillusthermolysin-like proteases." [S.l. : [Groningen] : s.n.] ; [University Library Groningen] [Host], 1997. http://irs.ub.rug.nl/ppn/164267484.
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Bruce, Lesley J. "A study of human erythrocyte band 3 variants." Thesis, University of Bristol, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.240590.
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Waterhouse, Mark Peter. "Specific targeting of FcγRIIIa using artificial scaffold protein variants." Thesis, University of Leeds, 2018. http://etheses.whiterose.ac.uk/20522/.
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Fcγ Receptors (FcγRs) are cell-surface receptors for IgG that are expressed on immune cells including macrophages, monocytes and natural killer (NK) cells. Upon binding to IgG-containing immune complexes, FcγRs trigger cell-mediated effector functions that lead to the clearance of pathogenic material and immune homeostasis. However, aberrant activation of these pathways can result in autoimmune susceptibility and, as such, FcγRs are implicated in the pathogenesis of several autoimmune disorders. For example, FcγRIIIa, expressed on macrophages and NK cells, has been functionally and genetically linked to susceptibility for rheumatoid arthritis and is a valid target for several FcγR-mediated autoimmune disorders. Despite this, FcγRIIIa is currently under-exploited due to a lack of FcγRIIIa-specific agents, which results from the expression of the near identical FcγRIIIb on neutrophils. However, several FcγRIIIa-specific Adhirons (an engineered protein scaffold) have recently been described, which inhibit IgG binding and effector functions (phagocytosis and cytokine release) of THP-1 cells. This thesis aimed to fully characterise and enhance the properties of one of these recently described Adhirons (known as AdG3) through site-directed mutagenesis (SDM); Molecular Dynamics (MD) simulations; and fusion to the IgG1- or IgG2-Fc. SDM identified several key binding residues in the AdG3 variable regions through screening of AdG3 mutants by surface plasmon resonance (SPR). SPR also identified three mutants with enhanced affinity for FcγRIIIa and showed that AdG3 possesses background binding to the highly homologous FcγRIIIbNA1, but not FcγRIIIbNA2, which was supported by MD simulations that revealed the exquisite mode of AdG3 specificity for these receptors, which was mediated through the Arg18Ser polymorphism in the AdG3 binding site. Fusion to the IgG-Fc was not detrimental to the FcγRIIIa-AdG3 interaction, and fusion to the IgG2-Fc was observed to potentially enhance the affinity and specificity of AdG3 for FcγRIIIa. These findings could subsequently be utilised for further improvement of AdG3 affinity and specificity, as well as other clinically relevant parameters such as serum half-life.
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Rivas, Cruz Manuel A. "Medical relevance and functional consequences of protein truncating variants." Thesis, University of Oxford, 2015. http://ora.ox.ac.uk/objects/uuid:a042ca18-7b35-4a62-aef0-e3ba2e8795f7.
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Genome-wide association studies have greatly improved our understanding of the contribution of common variants to the genetic architecture of complex traits. However, two major limitations have been highlighted. First, common variant associations typically do not identify the causal variant and/or the gene that it is exerting its effect on to influence a trait. Second, common variant associations usually consist of variants with small effects. As a consequence, it is more challenging to harness their translational impact. Association studies of rare variants and complex traits may be able to help address these limitations. Empirical population genetic data shows that deleterious variants are rare. More specifically, there is a very strong depletion of common protein truncating variants (PTVs, commonly referred to as loss-of-function variants) in the genome, a group of variants that have been shown to have large effect on gene function, are enriched for severe disease-causing mutations, but in other instances may actually be protective against disease. This thesis is divided into three parts dedicated to the study of protein truncating variants, their medical relevance, and their functional consequences. First, I present statistical, bioinformatic, and computational methods developed for the study of protein truncating variants and their association to complex traits, and their functional consequences. Second, I present application of the methods to a number of case-control and quantitative trait studies discovering new variants and genes associated to breast and ovarian cancer, type 1 diabetes, lipids, and metabolic traits measured with NMR spectroscopy. Third, I present work on improving annotation of protein truncating variants by studying their functional consequences. Taken together, these results highlight the utility of interrogating protein truncating variants in medical and functional genomic studies.
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Baldan, Nikita <1996>. "Computational analysis of NaV1.7 protein variants and tool for 3D visualization of protein structures." Master's Degree Thesis, Università Ca' Foscari Venezia, 2020. http://hdl.handle.net/10579/17572.
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This thesis is composed of two parts. The first part explores the possibility to use Graph Kernels to discriminate pathogenic versus non-pathogenic variants of a specific protein.
All variants are represented as Residue Interaction Networks (RIN), where nodes are amino acids and edges represent non-covalent bonds between atoms of the two involved amino acids.
This part is guided by a previous Master degree thesis that considered protein NaV1.7, which is responsible for the transmission of the pain signal from the peripheral nervous system to the brain. The thesis considered 85 genetic variants of the human NaV1.7, among which 30 are known to cause neuropathic diseases and 55 are instead neutral.
The results of the first part highlight that some Graph Kernels are actually able to discriminate between pathogenic and neutral variants.
This prompted the idea of realizing a 3D viewer able to show the three-dimensional structure of a protein and also its non-covalent bonds.
The second part of the thesis describes Spheremole, an application for the visualization of the three-dimensional structure of a protein. In particular, Spheremole allows the visualization of two proteins structures and their visual comparison, also based on their non-covalent bonds.
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Norman, Jane Eleanor. "Molecular mechanisms of platelet G protein-coupled receptor gene variants." Thesis, University of Bristol, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.687283.
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G protein-coupled receptors (GPCRs) are critical mediators of platelet responses to regulatory agonists and are essential drug targets. This project aimed to identify informative platelet GPCR gene variants and to characterise the mechanism of loss of receptor function for selected variants. Variants were sought in 2400 cardiac surgery patients in which preoperative platelet function test results were used to select subgroups with GPCR dysfunction potentially explained by loss of function P2Y12 receptor, thromboxane A2 receptor and protease-activated receptor 1 gene variants. This approach did not identify variants likely to affect GPCR function. Re-sequencing the protease-activated receptor 4 (PAR4) gene yielded seven different missense variants. After evaluation using computational prediction and homology modelling, the predicted tyrosine 157 to cysteine (Y157C) substitution was demonstrated to reduce PAR4 reactivity and was studied further. Compared to controls, Y157C platelets showed reduced functional responses to PAR4 activating peptide and a greater reduction in responses to a-thrombin in the presence of a PAR1 antagonist, vorapaxar, together consistent with a PAR4 defect. Y157C platelets, showed similar total PAR4 expression levels to controls but reduced surface expression, accounting at least in part for the reduced of PAR4 reactivity. HEK293 cells transfected with a PAR4 Y157C expression construct also showed reduced PAR4 surface expression and functional responses. PAR4 Y157C co-localised with an ER marker in the cell cytoplasm and had an expression pattern consistent with reduced N-glycosylation. Mutagenesis of the putative hydrogen bond partner for the substituted Y157 residue caused a similar phenotype. These findings suggest the Y157C substitution results in receptor mis-trafficking due to a disruption of an intra-molecular hydrogen bond. This first reported characterisation of a variant affecting PAR4 demonstrates that rare variants in the PAR4 gene are a potential source of inter-individual variation in the platelet haemostatic response and the effect of anti-platelet drugs that target the PAR system.
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Yang, Su jeong. "Biochemical and biophysical characterisation of allelic variants of ovine prion protein." Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611255.
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Turk, Casey M. "Paralemmin splice variants and mRNA and protein expression in breast cancers." Connect to this title, 2008. http://scholarworks.umass.edu/theses/194/.
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Chivers, Claire Elizabeth. "Investigating high-affinity non-covalent protein-ligand interaction via variants of streptavidin." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:631c65ed-08d9-484e-a8df-309a4c95df45.
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The Streptomyces avidinii protein streptavidin binds the small molecule biotin (vitamin H / B₇) with extraordinary stability, resulting in the streptavidin-biotin interaction being one of the strongest non-covalent interactions known in nature (Kd ~ 10-14 M). The stable and rapid biotin-binding, together with high resistance to heat, pH and proteolysis, has given streptavidin huge utility, both in vivo and in vitro. Accordingly, streptavidin has become a widely used tool in many different biotechnological applications. Streptavidin has also been the subject of extensive research efforts to glean insights into this paradigm for a high-affinity interaction, with over 200 mutants of the protein reported to date. Despite the high stability of the streptavidin-biotin interaction, it can and does fail under certain experimental conditions. For example, streptavidin-biotin dissociation is accelerated by an increased temperature or lower pH (conditions often encountered in cellular imaging experiments), and by mechanical stress, such as the shear force arising from fluid flow (encountered when streptavidin is used as a molecular anchor in biosensor chips and arrays). This study details efforts made at increasing further the utility of streptavidin, by increasing the stability of biotin and biotin-conjugate binding. A rational site-directed mutagenesis approach was used to create 27 mutants, with eight of these mutants possessing higher-stability biotin-binding. The most stable biotin-binding mutant was named traptavidin and was extensively characterised. Kinetic characterisation revealed traptavidin had a decreased dissociation rate from biotin and biotin-conjugates when compared to wildtype streptavidin, at both neutral pH and pH 5. Atomic force microscopy and molecular motor displacement assays revealed the traptavidin-biotin interaction possessed higher mechanical stability than the streptavidin-biotin interaction. Cellular imaging experiments revealed the non-specific cell binding properties of streptavidin were unchanged in traptavidin. X-ray crystallography was also used to generate structures of both apo- and biotinbound traptavidin at 1.5 Å resolution. The structures were analysed in detail and compared to the published structures of streptavidin, revealing the characteristics of traptavidin arose from the mutations stabilising a flexible loop over the biotin-binding pocket, as well as reducing the conformational change on biotin-binding to traptavidin. Traptavidin has the potential to replace streptavidin in many of its diverse applications, as well as providing an insight into the nature of ultra-stable noncovalent interactions.
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Efthymiou, Maria A. "Activated protein C and severe sepsis : Generation and characterisation of novel variants." Thesis, Imperial College London, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.534976.
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Tong, Shuping. "Molecular characterization of hepatitis B virus variants unable to express HBe protein." Lyon 1, 1992. http://www.theses.fr/1992LYO1T109.
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Fletcher, Jessica Frances. "Novel variants of the DNA damage checkpoint protein Cds1 in Schizosaccharomyces pombe." Thesis, Bangor University, 2017. https://research.bangor.ac.uk/portal/en/theses/novel-variants-of-the-dna-damage-checkpoint-protein-cds1-in-schizosaccharomyces-pombe(9df1851b-ca3f-449f-880f-c8eb627cb786).html.
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In the model organism Schizosaccharomyces pombe, the Cds1 (checking DNA synthesis 1) kinase is activated at the S-phase checkpoint upon stalling of the replication fork during DNA synthesis. Under normal conditions (300C), the role of the full-length protein kinase is to activate downstream processes resulting in mitotic arrest,protection of the stalled replication fork, and prevention of continued DNA replication in an unfavourable environment. In this way, Cds1 acts to ensure the reversible arrest of DNA synthesis. However, under stress conditions such as raised temperature or specific DNA damaging drugs, shorter variants of this protein kinase are rapidly expressed. Initial experimentation revealed the origin of the predominant variant, named Cds1-B, as the internal translation initiation site Methionine 159. The expressed protein is N-terminally truncated, missing amino acids residues 1-158, and therefore lacking the regulatory SQ/TQ and FHA domains whilst retaining the kinase domain. Absence of these regulatory regions suggests that this variant is free to act outside of the chromatin environment to fulfil roles different to that of its full-length counterpart, however it is unlikely to be active as a kinase as it is unable to participate in the model of activation currently proposed in the literature. Experimentation in this project was aimed at elucidating the role of this Cds1-B variant through analysis of drug sensitivity and checkpoint control efficacy, and evaluation of kinase activity. Current literature and results discussed here suggest an interesting hypothesis in which variants are expressed in response to specific genotoxic stresses in order to self-regulate kinase activity and selectively mediate cellular response either by the DNA replication checkpoint effector Cds1 or the DNA damage checkpoint effector Chk1. Mediation of effector kinase action is a promising avenue for cancer treatment, and with a potential Cds1-B homologue for human Chk2 identified (splice variant isoform 13), gaining a greater nderstanding of these variant mechanisms in yeast will aid the development of new therapeutic interventions.
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Adhikari, Sandeep. "FUNCTIONAL CHARACTERIZATION OF IDENTIFIED DEAF1 VARIANTS AND SIGNIFICANCE OF HDAC1 INTERACTIONS ON DEAF1-MEDIATED TRANSCRIPTIONAL REPRESSION." OpenSIUC, 2021. https://opensiuc.lib.siu.edu/theses/2838.
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Deformed epidermal autoregulatory factor 1 (DEAF1) encodes a transcription factor essential in early embryonic and neuronal development. In humans, mutations in the DNA binding domain of DEAF1 cause intellectual disability together with clinical characteristics collectively termed DEAF1-associated neurodevelopmental disorders (DAND). The objective of this study is to 1) assess the pathogenicity of newly identified variants using established functional assays, and 2) confirm and map the interaction domain of DEAF1 with HDAC1 and evaluate the importance of DEAF1-HDAC1 interaction on DEAF1-mediated transcriptional repression. Exome sequencing analysis identified six de novo DEAF1 mutations (p.D200Y, p.S201R, p.K250E, p.D251N, p.K253E, and p.F297S). Promoter activity experiments indicate DEAF1 transcriptional repression activity was altered by p.K250E, p.K253E, and p.F297S. Transcriptional activation activity was altered by p.K250E, p.K253E, p.F297S, and p.D251N. Combined with clinical phenotype of the patients, this work establishes the pathogenicity of new DEAF1 variants. Previous studies identified a potential protein interaction between DEAF1 and several proteins of the nucleosome remodeling and deacetylating (NuRD) complex including Histone Deacetylase 1 (HDAC1), Retinoblastoma Binding Protein 4 (RBBP4), Methyl CpG Binding Domain Protein 3 (MBD3). GST pull-down and co-immunoprecipitation (CoIP) assays confirmed and mapped the interaction with HDAC1 between amino acids 113 – 176 of DEAF1. To determine whether DEAF1-mediated repression requires HDAC1 activity, HEK293t wild type or CRISPR/Cas9-mediated DEAF1 knockout cells were treated with the HDAC inhibitor Trichostatin A (TSA). Interestingly, this study demonstrates that the requirement of HDAC1 activity on DEAF1-mediated transcriptional repression activity is target gene specific and expands our understanding of DEAF1 mediated transcriptional repression.
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Kuthiala, Amrita. "In-vitro Studies on Aptamer - Induced FRET Between λN22 Tagged Fluorescent Protein Variants." Diss., lmu, 2010. http://nbn-resolving.de/urn:nbn:de:bvb:19-124642.
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Panicco, Paola. "Protein engineering of human cytochromes P450 and their allelic variants for nanobiotechnological applications." Thesis, Imperial College London, 2010. http://hdl.handle.net/10044/1/11739.
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Human cytochromes P450 (CYP) constitute one of the most important and studied classes of phase I drug metabolizing enzymes. A small group of 6-7 isoforms of the 57 identified until now accounts for 90-95% of the metabolism of clinically used drugs and can contain mutations (single nucleotide polymorphisms) that, when located in the coding regions, can lead to absent, deficient or enhanced enzyme activity. Thanks to the development of pharmacogenetics and later on pharmacogenomics, the presence of these single nucleotide polymorphisms (SNP) has been associated to inter-individual and inter-ethnic variability in the response to several important therapeutic agents. Due to this direct correlation between polymorphism and efficacy of drug treatment, the pharmaceutical industry is particularly interested in developing new analytical tools for the determination of the cytochrome P450 in vitro metabolism to correlate to the in vivo situation. The use of amperometric sensing systems represents an interesting alternative to the traditional methods. In the present study an electrochemical characterisation of the human cytochrome P450 2C9 and its two main allelic variants the CYP2C9*2 and CYP2C9*3 have been performed. Different methods of immobilisation and electrodes have been used to investigate the conditions that are more suitable to maintain and guarantee the active state and biocatalytic response of an immobilised cytochrome P450. The CYP2C9 and its two main allelic variants CYP2C9*2 and CYP2C9*3 have been inserted into engineered constructs where the human cytochrome P450 gene is linked to artificial redox chains to regulate the electron flow from the electrode surface to the haem. All the constructs have been successfully expressed and purified in heterologous E.Coli cells, with the CYP2C9FLD chimeras showing higher yields. Preliminary spectroelectrochemical studies on semi-conductive and optically transparent tin-dioxide allowed the combination of absorption spectroscopy and electrochemistry (cyclic voltammetry) to ascertain the native state of these P450 enzymes once immobilised. The results showed how the conversion from the active P450 form to the inactive P420 one can be achieved by modifying the surface with polycations with slightly different chemical properties. A proper characterisation of the two species was performed for the first time and, under certain conditions, the inactive P420 species appeared to dominate the cyclic voltammogram (CV). Similar findings have been observed when the CYP2C9 wild type have been electrochemically characterised on DDAB and PDDA modified glassy carbon electrodes. Electrocatalysis in presence of S-warfarin and FT-IR spectra of the CYP2C9/DDAB/GC electrodes revealed higher catalytic activity and maintenance of the native secondary structure of the enzyme. Immobilisation of the CYP2C9FLD, CYP2C9*2FLD and CYP2C9*3FLD on DDAB modified glassy carbon electrodes showed well defined redox couples on the oxygen-free cyclic voltammograms (CVs) and mid point potentials of all enzymes were calculated. Electrocatalysis in presence of substrate and quantification of the product formed showed lower catalytic activities for the CYP2C9*3FLD and CYP2C9*2FLD compared to the wild type CYP2C9FLD as it was expected from literature data. When the CYP2C9FLD, CYP2C9*2FLD and CYP2C9*3FLD were immobilised on alkanethiol modified gold electrodes, the system was tested as both amperometric sensor and catalyser. Parameters such as the apparent Michaelis– Menten constant Km and the Vmax were determined and the metabolic profile of S-warfarin was confirmed to be in line with literature findings. The fundamental knowledge acquired was then transferred to a commercial prototype sensor for testing the metabolic profile of known drugs. Encouraging results in line with the previous findings were achieved.
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Wilson, Michael Christopher. "Comparing the thermal, chemical and mechanical stabilities of extremophilic cold shock protein variants." Thesis, University of Leeds, 2016. http://etheses.whiterose.ac.uk/15982/.
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Proteins conduct a vast array of chemical processes and achieve this through a balance of flexibility and function. Proteins from extremophilic organisms have evolved to adjust this balance to function in extreme conditions. Research into hot-adapted proteins has been encouraged through successes in improving enzyme thermostability, though cold-adapted proteins also offer substantial potential as they can function effectively at lower temperatures. Research into cold-adapted proteins remains limited despite cold climates comprising over 80% of Earth’s biosphere. This study compares the stabilities of cold shock protein (Csp) homologues from bacteria growing at vastly different temperatures. The cold-adapted Csps show reduced thermal denaturation mid-points (Tm values) compared to a temperate Csp with PB6 Csp displaying the lowest Tm of 42.8 ⁰C contrasting with 52.3 ⁰C shown by BsCsp. Thermostability projections using the Gibbs-Helmholtz equation show cold-adapted Csps display slightly reduced thermostabilities and remain folded over a narrower range of temperatures. Csp thermostabilities were found to be very similar at around 8 degrees below the optimal growth temperature of the bacteria each Csp was derived from, suggesting thermostabilities evolved relating to Csp operating temperatures. The roles of electrostatics and hydrophobic packing in stabilizing a hyperthermophilic Csp are evaluated using mutants that highlight how a single side chain length reduction dramatically decreases Csp thermostability. Kinetic studies showed that the Csps exhibit similar folding rate constants but different unfolding rate constants. Initial flexibility studies suggest that DNA binding increases Csp rigidity and triggers a conformational change in a psychrotrophic Csp. Csp mechanical stabilities showed the same hierarchy as thermostabilities and similar sensitivity to temperature changes. Energy landscape projections constructed using Monte Carlo simulations showed the mechanical energy barrier height of Csp unfolding was temperature independent. At 5 ⁰C the Csps showed similar mechanical softness however the hyperthermophilic TmCsp shows greater mechanical softness at higher temperatures.
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Burns, Joyce Nicole. "Development of a quantitative assay to distinguish glaucoma-causing and benign olfactomedin variants." Thesis, Georgia Institute of Technology, 2010. http://hdl.handle.net/1853/42931.
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Myocilin, expressed in the trabecular meshwork of the eye, has been linked to inherited primary open-angle glaucoma (POAG). The biological function of myocilin is unknown, but mutant myocilin exhibits a gain-of-function mechanism, aggregating within the endoplasmic reticulum of human trabecular meshwork cells, causing cell stress and eventually apoptosis. After apoptosis occurs, the trabecular meshwork is compromised, leading to an increase in intraocular pressure, a symptom of glaucoma. In this thesis, I have expressed and purified the wild-type olfactomedin (OLF) domain and 24 reported disease-causing variants. I developed a facile thermal stability assay using differential scanning fluorimetry, which follows the unfolding of a protein through the fluorescence of a dye sensitive to hydrophobic regions of a protein. Also in this thesis I have determined melting temperatures for the wild-type and for each of the disease-causing mutants. I have tested the stability of the mutants in the presence of seven osmolytes, with sarcosine and trimethylamine-N-oxide restoring the melting temperature closest to wild-type. Additionally, I expressed and purified three reported single nucleotide polymorphisms (SNPs) (E352Q, E396D, K398R), which are considered benign variants. Variants were also compared by circular dichroism, revealing high b-sheet content and wild-type structure. When compared to previous studies, there is a positive correlation between the melting temperature, and previously reported qualitative assays, which measure the mutant myocilin solubility in detergent, secretion from mammalian cells, and aggregation propensity. Taken together, these data give insight into the relationship between glaucoma genotypes and phenotypes.
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Lamlum, Hanan. "Variation in human tissue inhibitor of metalloproteinase 1 gene and its effect on the control of connective tissue remodelling in cardiovascular disease." Thesis, University of Oxford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365809.
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García, Alonso Luz María. "Functional profiling of human genomic data using the protein interactome." Doctoral thesis, Universitat Politècnica de València, 2015. http://hdl.handle.net/10251/55848.
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[EN] Our understanding of the biological mechanisms for most common human diseases is far from complete. Even with well established genetic landscapes, our capacity to make accurate phenotypical predictions or determine personalised disease risk using genetics alone is not possible for most diseases due to our lack of understanding of the mechanisms by which genetic alterations cause disease. Several suggestions have been proposed to explain this manifested lack of direct relation between genotype and phenotype, including interactions with other molecules, pleiotropy and environmental perturbations. Due to their essential role in carrying cellular functions, proteins and its interactions seem crucial to translate genomic data to phenotypic states. In this thesis I present three different and independent approaches to integrate human genomic data with prior knowledge in terms of protein-protein interactions (PPIs). The overall objective is, by making use of the interactome structure, to propose functional hypotheses that help to interpret the genetic variability observed in different human phenotypes. First I developed a methodology to extract the network component associated to any gene list ranked by any experimental parameter, as the one coming from case-control genome-wide associations studies. Second I performed a systematic analysis of human variants in the context of the protein interactome. There I study how the interactome structure can help us to explain the amount of apparently deleterious variation observed in actual populations and, therefore, give insight in its role in shaping the patterns of variability. Results are compared against somatic mutation found in Leukemia patients. Finally, I structurally resolved the protein interactome and used it to study how somatic mutations found in primary tumours distribute across the interacting interfaces and identify those with a potential role in driving oncogenesis. Although each chapter covers a different question, all of them demonstrate the potential of the interactome in helping to interpret genomic variation observed under diverse research scenarios.[ES] Nuestro conocimiento acerca de los mecanismos biológicos causantes de la mayoría de enfermedades humanas comunes es aun pobre. Incluso con mapas genéticos de alta resolución, nuestra capacidad para hacer predicciones fenotípicas certeras o determinar el riesgo de una persona a padecer una enfermedad utilizando solamente marcadores genéticos es muy baja. Entre las principales causas de esta aparente falta de relación directa entre genotipo y fenotipo están las interacciones moleculares, los fenómenos de pleiotropía y la influencia de los factores externos. Debido al papel esencial que ejercen en llevar a cabo las funciones celulares, las proteínas y sus interacciones han adquirido una atención especial en la traducción de los datos genotípicos a estados fenotípicos. En esta tesis se presentan tres estrategias diferentes para la integración de datos genómicos humanos con la red de interacciones proteicas (interactoma). El objetivo común de todas ellas es, haciendo uso de la estructura del interactoma, proponer hipótesis funcionales que ayuden a interpretar los patrones de variabilidad observados en diferentes estados fenotípicos humanos. Primero, se propone una metodología para extraer el componente del interactoma asociado a los genes relevantes en una lista ranqueada por cualquier parámetro experimental, como el estadístico derivado de los estudios de asociación genómicos. Es segundo lugar se describe un análisis sistemático de las variantes genéticas observadas en humanos sanos en el contexto del interactoma. En él se estudia cómo la estructura del interactoma puede ayudar en explicar la aparentemente elevada cantidad de variantes deletéreas observadas en los últimos estudios poblacionales de secuenciación de genomas. Los resultados son comparados con las mutaciones somáticas observadas en pacientes de Leucemia. Finalmente, se presenta un estudio de las mutaciones somáticas observadas en tumores primarios utilizando una versión del interactoma que incluye la estructura tridimensional de las proteínas. Aunque cada estudio presentado en la tesis pretende resolver preguntas diferentes, todos ellos demuestran el potencial del interactoma de proteínas en ayudar a interpretar la variación genómica humana observada en un contexto tanto evolutivo como de enfermedad.
[CAT] El nostre coneixement sobre els mecanismes biològics causants de la majoria de malalties humanes comuns es encara pobre. Tot i que en l'actualitat tenim mapes genètics d'alta resolució, la nostra capacitat per a fer prediccions fenotípiques certeres utilitzant únicament marcadors genètics es encara molt baixa degut a que no entenem les bases moleculars a traves de les quals les alteracions genètiques condicionen un fenotip de malaltia. Entre les principals causes d'aquesta aparent falta de relació directa entre genotip i fenotip estan la complexitat introduïda per les interacciones moleculars, els fenòmens de peleiotropia i la influencia dels factors externs. Degut al paper clau en dur a terme la majoria de funcions cel·lulars, les proteïnes i les seues interaccions han adquirit una especial atenció en la traducció de les dades genotípiques en estats fenotípics. Aquesta tesi presenta tres estartègies diferents per a la integració de dades genòmiques humanes amb la xarxa d'interaccions proteiques (interactoma). L'objectiu comú es, fent ús de l'estructura del interactoma, proposar hipòtesis funcionals que ajuden a interpretar els patrons de variabilitat genètica observats en diferents estats fenotípics. En primer lloc, es proposa una metodologia per a extraure el component de l'interactoma associat als gens rellevants en una llista ranquejada per qualsevol paràmetre experimental, com l'estadístic derivat d'estudis d'assocaició de genoma. En segon lloc, es descriu un anàlisi sistemàtic de les variants genètiques observades en humans sans en el context del interactoma. Ací s'analitza com l'estructura del interactoma pot ajudar a explicar l'aparent elevada quantitat de variants deletèries observades en els últims estudis poblacionals de sequenciació de genomes. Els resultats son comparats amb les mutacions somàtiques observades en pacients de Leucèmia. Finalment, es presenta un estudi de les mutacions somàtiques observades en tumors primaris de més de 20 tipus utilitzant una versió del interactoma més resolutiva, que inclou l'estructura tridimensional de les proteïnes. Encara que cada estudi presentat en la tesi planteja resoldre qüestions diferents, tots ells demostren el potencial del interactoma de proteïnes en ajudar a interpretar la variació genòmica humana observada en un context tant poblacional com de malaltia.
García Alonso, LM. (2015). Functional profiling of human genomic data using the protein interactome [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/55848
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Kalita, Ann Marie. "Comparison of the activities of two allelic variants of the human wildtype p53 protein." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/mq29729.pdf.
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Rees, Matthew Geoffrey. "Genetic, functional, and phenotypic analysis of human variants in the glucokinase regulatory protein gene." Thesis, University of Oxford, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.589604.
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Genome-wide association (GWA) studies have provided significant insight into the underlying genetic components of common human diseases such as type 2 diabetes (T2D). However, the translation of such genetic findings into biological and clinical insight remains a major challenge. One of the genes implicated in T2D pathogenesis and effects on related glycaemic and lipidaemic traits by the GWA approach is GCKR, encoding glucokinase regulatory protein (GKRP). GKRP inhibits the glycolytic enzyme glucokinase (GCK) in the liver, sequestering it in an inactive form in the nucleus. Together, GCK and GKRP exert a significant proportion of control of hepatic glycolytic flux, facilitating glucose uptake and disposal in the fed state and inhibiting glycolysis in the fasting state. A common nonsynonymous variant in GCKR, p.P446L, was identified by a combination of genetic and functional approaches as the likely causative variant underlying the GWA findings. The P446L variant protein has been shown to have diminished capacity to inhibit GCK relative to wild-type (WT) GKRP, resulting in enhanced hepatic glycolytic flux and activation of liver synthetic pathways, including those generating triglycerides. Genes such as GCKR harbouring common variants of discernible functional effect may also contain rare variants of large effect. Accordingly, I aimed to comprehensively identify and functionally characterise nonsynonymous variants across the allelic frequency spectrum in GCKR, both by applying and adapting existing kinetic techniques and by developing methodologies to assess the effects of variants on the subcellular localisation of GKRP and GCK. Additionally, I aimed to relate these findings back to clinically important phenotypes, and to further characterise the binding of GCK and GKRP by searching for novel small- molecule modulators of this interaction. Using fluorescent fusion proteins transiently transfected into HeLa cells, I showed that human GKRP localises to the nucleus and sequesters human GCK. I also demonstrated that the common P446L variant significantly reduces the ability of G KRP to localise to the nucleus and sequester GCK. I then investigated the cellular and kinetic characteristics of 18 additional nonsynonymous GCKR variants identified in the National Institutes of Health's ClinSeqTM cohort, determining that the majority of variants affected protein function, and that these effects could be divided into distinct sub-classes. Variants causing a significant loss of function were associated with increased lipid levels in the cohort. Finally, I developed robust assays capable of measuring the interaction of re comb in ant GKRP and GCK in a format suitable for quantitative high-throughput screening of up to 400,000 small-molecule compounds. While no compounds that specifically affected this interaction have been identified to date, the assays developed could be useful in future studies of GCK and GKRP. These data provide further insight into the critical regulatory role of GCKR in metabolism, the structure and function of the GKRP protein, and the potential pathogenic consequences of human variants within GCKR.
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Atkins, Elizabeth Rose. "Functional characterisation of natural variants of the hepatitis C virus p7 ion channel protein." Thesis, University of Leeds, 2013. http://etheses.whiterose.ac.uk/6911/.
Full textAbstract:
The HCV p7 protein is a viroporin that acts to increase endosomal pH to preserve the infectivity of nascent virions. Previous work has identified several key residues in p7 that are critical for its function. A number of compounds have been found to inhibit p7 activity in vitro and genotype variation in the p7 sequence is known to have significant effects on p7 inhibitor sensitivity. This study aimed to further our understanding of the role of p7 during HCV infection. The effects of six naturally-occurring p7 variants, within a single genotype, isolated from 5 patients of varying disease severity were investigated. It was found in in vitro liposome assays that the patient polymorphisms caused a wide variation in p7 channel activity. The previously-observed low-pH activation in J4 p7 was also observed in JFH1 p7, but not in H77 p7; this showed a ‘V’ shaped activation profile, with its lowest activity at pH 6.7 and peak activity at pH 6.2 and 7.4. Four of the patient isolates shared the same activation pattern as H77; two with non-synonymous mutations of S21P and Y31H displaying low-pH activation. In virus, the Y31H mutant was the only Seattle variant to show a significant reduction in the production of infectious virus. JFH1 intracellular virions have previously been shown to be sensitive to transient exposure to reduced pH, while secreted virions were insensitive to such changes. In this study, it is shown that H77 secreted virions are sensitive to transient reductions in pH, while the Seattle isolate viruses showed reduced pH sensitivity. The Y31H isolate also showed increased sensitivity to the p7 inhibitor rimantadine. In conclusion, this study found that natural polymorphisms in p7 within a single genotype can cause significant changes in p7 activity. These changes did not show any correlation with the severity of disease in the original patients.
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Rossetti, Giulia. "Molecular simulation studies of the prion protein: from disease-linked variants to ligand binding." Doctoral thesis, SISSA, 2010. http://hdl.handle.net/20.500.11767/4158.
Full textAbstract:
Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal neu-rodegenerative disorders (198). The crucial event in the development of these diseases is the conformational change of a membrane bound protein, the cellular PrPC in Figure 3.1, into a disease associated, bril-forming isoform (199). Despite their rare incidence, TSEs have captured very large attention from the scienti c community due to the unorthodox mechanism by which prion diseases are transmitted...
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Bronicki, Lucas M. "Characterization of Multiple Exon 1 Variants and Neuron-specific Transcriptional Control of Mammalian HuD." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/23682.
Full textAbstract:
The RNA-binding protein (RBP) and Hu/ELAV family member HuD regulates mRNA metabolism of genes that encode proteins involved in neuronal differentiation, learning and memory, and certain neurological diseases. Given the important functions of HuD in a variety of processes, we set out to characterize the 5’ genomic region of the mammalian HuD gene and determine the mechanisms that regulate its mRNA expression in neurons using P19 cells and mouse brain as models.
Bioinformatic and 5’RACE (rapid amplification of cDNA ends) analyses of the HuD 5’ genomic flanking region identified eight conserved leader exons (E1s), two of which are novel. Expression of all E1 variants was established in differentiating P19 cells, mouse embryonic (E14.5) and adult brains. Through several complementary approaches, we determined that the abundance of HuD mRNA is predominantly under transcriptional control in differentiating neurons. Sequential deletion of the 5’ regulatory region upstream of the predominantly expressed E1c variant revealed a well-conserved 400 bp DNA region that contains five E-boxes and is capable of directing expression of HuD specifically in neurons. Using electrophoretic mobility shift assays (EMSAs), chromatin immunoprecipitations (ChIPs), and E1c 5’ regulatory region (RR) deletion and mutation analysis, we found that two of these E-boxes are targeted by neurogenin 2 (NGN2/NEUROG2) and that this mechanism is important for induction of HuD mRNA in neurons. Additional deletion and mutation of the E1c 5’ RR revealed that putative cis-acting elements for Kruppel-like factors (KLFs) and nuclear DEAF-1-related (NuDR) transcription factors also positively regulate transcription of HuD.
Together, our findings reveal that the intricate transcriptional regulation of mammalian HuD involves eight leader exons and potentially alternate promoters. We further demonstrate that transcription of HuD requires neuron-specific control by NGN2 and possibly KLF and NuDR transcription factors. To our knowledge, this is the first study to identify transcriptional events that positively regulate expression of HuD.
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MARRANCI, ANDREA. "Analysis of the expression of all BRAF transcript variants and of their implication in post-transcriptional regulation mediated by miRNAs in melanoma." Doctoral thesis, Università di Siena, 2017. http://hdl.handle.net/11365/1005876.
Full textAbstract:
BRAF is a widely studied oncogene and its functions are well characterized in several cellular contests and diseases. However the regulation of the expression of BRAF is mostly unknown.
With the aim to understand the post-transcriptional regulation of BRAF, we performed 3’RACE in A375 melanoma cells and we found 2 different 3’UTRs: the one commonly reported in many data bases (Reference) and a new one that is only predicted (X1). The two 3’UTRs are completely different in sequence and length (120nt vs 1350nt). Furthermore, they are transcribed from exon 18 or thanks to an alternative splicing event occurring between exon 18 and a newly discovered exon 19 (X1). By using Real Time PCR, we confirmed the expression of both transcripts in melanoma and non-melanoma cell lines. Moreover, using RNA-SEQ data available at TCGA, we showed the co-expression of Reference and X1 BRAF transcripts also in human biopsies.
Due to our discovery that BRAF exists in at least 2 different transcript variants, we decided to investigate further the expression of all the BRAF isoforms reported in NCBI and Ensembl. To do so, we took advantage of the RNA-seq data of more than 4,800 patients belonging to 9 different cancer types. We show that BRAF mRNA exists as a pool of 3 isoforms (reference BRAF, BRAF-X1 and BRAF-X2) that differ in the last part of their open reading frames, as well as in the length (BRAF-ref: 76nt; BRAF-X1 and X2: up to 7kb) and in the sequence of their 3’UTRs. In melanoma cells, the X1 isoform is expressed at the highest level, while the most prevalent among the three isoforms varies from one cancer type to another. Moreover, the relative abundance among the three BRAF isoforms is maintained in melanoma cells with acquired resistance to BRAF and MEK inhibitors driven by BRAF gene amplification or expression of the Δ[3-10] splicing variant.
Besides their 3’UTRs, also the very last part of the coding sequences differ among the three isoforms. By immunoprecipitation of BRAF in A375 cells and subsequently Mass-SPEC analysis, we revealed the existence of Reference and X1 proteins which are expressed at similar levels, while X2 is not detectable because quicky degraded by the proteasome. Furthermore functional studies show that the two proteins account together for BRAF activities both in vitro and in vivo.
Given the differences in length and sequence between the reference and the X1 3’UTR, we hypothesized that the two isoforms undergo different regulation mediated by RNA-binding proteins or non-coding RNAs. We focused on post-transcriptional regulation by microRNAs. MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate the expression of target messenger RNAs (mRNAs) and for this reason play a key role in virtually all cellular processes. In spite of the availability of several prediction algorithms, the identification of specific miRNA-target interactions remains a challenge.
In order to overcome this problem we developed an innovative method, called miR-CATCH v2.0, for the high-throughput identification of microRNAs that bind a target transcript. The protocol is based on the affinity purification of the target mRNA and bound miRNAs by using two different pools of 3’biotinylated anti-sense DNA probes (ODD and EVEN). We designed 12 probes (6 ODD probes and 6 EVEN probes) for the purification of X1-3’UTR-miRNAs complexes and we performed three separate and independent captures in A375 metastatic melanoma cells. MicroRNAs were identified through small RNA-sequencing and the top-scoring miRNAs that resulted consistently enriched in all the captures will be validated in vitro and in vivo experiments.
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Boopathy, Sivakumar. "Investigating Structural and Functional Defects in ALS-causing Profilin 1 Variants." eScholarship@UMMS, 2009. http://escholarship.umassmed.edu/gsbs_diss/923.
Full textAbstract:
Mutations in profilin 1 (PFN1) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease that targets motor neurons. PFN1 is a 15 kDa protein that is best known for its role in actin dynamics. However, little is known about the pathological mechanisms of PFN1 in ALS. In this dissertation, it is demonstrated that certain familial ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in neuronal cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported functional defects in cell-based assays. The source of this destabilization is illuminated by the crystal structures of several PFN1 proteins, revealing an expanded cavity near the protein core of one ALS variant and predicting a non-surface exposed cavity in another. Functional biochemical experiments point to abnormalities in actin filament nucleation and elongation caused by PFN1 mutants. In HeLa cells, PFN1 is essential for the generation of actin-rich filopodia and expression of mutant PFN1 alters filopodia density further supporting a pathogenesis mechanism involving actin cytoskeleton. Taken together, this dissertation infers that the pathogenesis of ALS due to mutations in PFN1 can be mediated at least by two possibly related mechanisms, a destabilization of the native PFN1 structure and an impact on the actin assembly processes.
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Boopathy, Sivakumar. "Investigating Structural and Functional Defects in ALS-causing Profilin 1 Variants." eScholarship@UMMS, 2017. https://escholarship.umassmed.edu/gsbs_diss/923.
Full textAbstract:
Mutations in profilin 1 (PFN1) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease that targets motor neurons. PFN1 is a 15 kDa protein that is best known for its role in actin dynamics. However, little is known about the pathological mechanisms of PFN1 in ALS. In this dissertation, it is demonstrated that certain familial ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in neuronal cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported functional defects in cell-based assays. The source of this destabilization is illuminated by the crystal structures of several PFN1 proteins, revealing an expanded cavity near the protein core of one ALS variant and predicting a non-surface exposed cavity in another. Functional biochemical experiments point to abnormalities in actin filament nucleation and elongation caused by PFN1 mutants. In HeLa cells, PFN1 is essential for the generation of actin-rich filopodia and expression of mutant PFN1 alters filopodia density further supporting a pathogenesis mechanism involving actin cytoskeleton. Taken together, this dissertation infers that the pathogenesis of ALS due to mutations in PFN1 can be mediated at least by two possibly related mechanisms, a destabilization of the native PFN1 structure and an impact on the actin assembly processes.
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Lackman, J. (Jarkko). "Glycosylation and dimerization of the human δ-opioid receptor polymorphic variants." Doctoral thesis, Oulun yliopisto, 2018. http://urn.fi/urn:isbn:9789526221342.
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Abstract Cellular signaling by G protein-coupled receptors (GPCRs) governs a wide array of physiological functions throughout the body. The human δ-opioid receptor (hδOR) is a GPCR that modulates the sensation of pain and mood and has great potential for the treatment of pain and a variety of neurological disorders. A common single-nucleotide polymorphism (SNP) in the extracellular N-terminal tail of hδOR changes Phe to Cys at position 27. Using various biochemical and cell biological methods, the study demonstrates that several events during receptor biosynthesis and cell surface delivery are affected by the SNP. These events participate in the multifaceted regulation of the receptor and modulate receptor behavior at the cell surface. Two distinct pathways were shown to scrutinize the quality of the synthesized hδOR in the endoplasmic reticulum (ER) and target some for degradation in N-glycan-dependent and -independent ways. The hδORCys27 that matures inefficiently required N-glycan-mediated interactions with the lectin-chaperone calnexin to be expressed in a fully functional form at the cell surface, whereas the N-glycan-independent pathway was sufficient for hδORPhe27. For both variants, the N-glycan-independent quality control, which is likely to operate as a back-up pathway, led to a more rapid export from the ER and receptors at the cell surface that were less stable. Receptor dimerization emerged as an important regulatory step for receptor cell surface delivery. In co-transfected cells, interactions between the newly-synthesized variants led to the retention and subsequent ER-associated degradation of hδORPhe27. This dominant-negative attenuation of hδORPhe27 cell surface expression by hδORCys27 may have unpredictable consequences for opioid signaling in heterozygous individuals. Finally, the study shows that N-acetylgalactosamine (GalNAc)-type O-glycosylation catalyzed in the Golgi modulates hδOR expression at the cell surface by enhancing receptor stability and inhibiting constitutive downregulation. The modification of Ser residues in the receptor N-terminus by GalNAc-transferase 2 was affected by the SNP, which presents another distinction in the cellular processing of the two variants. The findings highlight the importance of the biosynthetic pathway in the regulation of GPCR behavior and pave way for strategies for treatments targeting GPCRs at this levelTiivistelmä Solujenvälisellä viestinnällä on keskeinen tehtävä kehon kaikissa toiminnoissa. δ-opioidireseptori (δOR) on solusignalointiin erikoistuneen kalvoproteiiniperheen (G-proteiiniin kytketyt reseptorit) jäsen, joka ohjaa kivuntuntemusta ja mielialoja. Sitä pidetään mahdollisena lääkekehityksen kohteena paitsi kivunlievityksen, myös useiden neurologisten häiriöiden hoidossa. δOR ilmenee kahtena polymorfisena muotona sen solunulkoisessa osassa tapahtuneen aminohappomuutoksen vuoksi (Phe27Cys). Työssä tutkittiin reseptorin glykosylaatiota ja dimerisaatiota, jotka säätelevät sen prosessointia, käyttäytymistä ja toimintaa. Käyttäen useita biokemiallisia ja solubiologisia menetelmiä työssä osoitettiin polymorfian vaikuttavan useisiin prosessointivaiheisiin ja muokkaavan siten reseptorin viestintää. Proteiinien laadunvalvontakoneiston havaittiin säätelevän reseptorin siirtymistä endoplasmakalvostolta solun pinnalle kahdella eri mekanismilla ohjaten osan reseptoreista hajotukseen. Toisin kuin Phe27-variantin, tehottomasti kypsyvän Cys27-variantin laadunvalvonta on riippuvainen reseptoriin liittyvistä N-glykaaneista ja näihin sitoutuvasta kaitsijaproteiinista, kalneksiinista. Reseptorivariantit, joista N-glykaanit puuttuvat, siirtyvät nopeammin solukalvolle, mutta ne ovat epästabiileja ja häviävät nopeasti solun pinnalta. Vaihtoehtoinen N-glykaaneista riippumaton laadunvalvontamekanismi sallii myös inaktiivisen Cys27-variantin pääsyn solun pinnalle. Varianttien dimerisoitumisen osoitettiin säätelevän niiden kuljetusta soluissa. Cys27-variantin havaittiin sitoutuvan Phe27-varianttiin aikaisessa biosynteesivaiheessa ja ohjaavan osan siitä hajotukseen. Tällä voi olla suuri merkitys opioidiviestinnässä molempia alleeleja kantavilla henkilöillä. Työssä havaittiin myös GalNAc-transferaasi-2-entsyymin ohjaavan Golgin laitteessa tapahtuvaa reseptorin O-glykosylaatiota. Se glykosyloi reseptorin solunulkoisen osan seriinitähteitä (Ser6, Ser25, Ser29), stabiloiden siten solun pinnan reseptoreita ja tehostaen niiden viestintää. Lisäksi havaittiin eroja varianttien O-glykosylaatiossa, mikä voi osaltaan selittää varianttien ilmentymisessä todettuja eroja. Tutkimus luo uutta tietoa biosynteesireitin merkityksestä G-proteiiniin kytkettyjen reseptorien säätelyssä sekä antaa pohjaa keinoille, joilla tätä voitaisiin hyödyntää farmakologisesti
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Gasparini, Alessandra. "From High-Throughput Analysis of Genetic Variants to the Experimental Validation of Putative Protein Function." Doctoral thesis, Università degli studi di Padova, 2018. http://hdl.handle.net/11577/3426808.
Full textAbstract:
The state-of-the-art approach for the genetic molecular cause research relies on massively parallel gene sequencing, which represents a challenge both in data handling and variant prioritization. The univocal assignment of disease pathogenicity to the sequence variants is often difficult, and requires the integration of different lines of evidence for a comprehensive interpretation. During my thesis, I contributed to the development of novel approaches to evaluate rare variant contribution to the clinical phenotype. These methods were presented and evaluated at the Critical Assessment of Genome Interpretation, ranking among top programs considering either performance or the number of correct assigned disease predictions. A similar strategy was employed for identification of disease genes linked to neurodevelopmental disorders (NDDs) comorbidity. In this case, computational methods were applied to select the most promising candidate genes for the design of diagnostic panel, which is currently used for patient screening at Pediatrics Clinic of the University of Padova. The variants found within the panel genes have been selected according frequency, pathogenicity prediction and variant segregation analysis within the family. Furthermore, I took advantage of different computational tools to investigate the mutated gene function, and used this information to demonstrate the impact of likely pathogenic variant on clinical phenotype. In several cases, likely pathogenic mutations mapped to intrinsically disordered regions (IDRs), which lack a fixed three-dimensional structure. Coherently, several studies demonstrate that mutations in IDRs are often associated with the pathogenesis of various human diseases. Thus, IDRs classification could represent a critical step for understanding the impact of possibly disease-causative variants mapping in these regions. Due to the influence of intrinsically disordered proteins (IDPs) in diseases, I participated to the manual curation and update of entries in the DisProt database, the primary repository of disorder-related data on sequence. Interestingly, increasing evidence from literature highlights the IDPs involvement in neuronal signal transduction. Among the proteins encoded by diagnostic panel genes, TANC2 especially emerged as intrinsically disordered protein with a possible role in synaptic signal transduction. As TANC2 and its protein family function was poorly characterized, I performed an in silico analysis to characterize the TANC protein activity, and the implicated biological processes. The functional hypothesis emerged from the bioinformatics analysis was used to drive further experimental investigations. In vitro validation of predicted TANC2-CDKL5 interaction highlighted the relevance of the IDRs in regulating degradation of CDKL5, whose mutations are associated with a heterogeneous set of NDD phenotypes. Furthermore, I demonstrated that TANC2 contributes to downregulate CDKL5 expression levels. For this reason, TANC2 protein could represent a novel therapeutic target to design new drugs for the treatment of CDKL5 over-expression associated diseases.La strategia di elezione per l'identificazione di varianti causative di malattie genetiche consiste nell’utilizzo di piattaforme di Next Generation Sequencing. Questo tipo di approccio rappresenta una sfida, sia per quanto riguarda la gestione della mole di dati da sequenziamento, che per l’interpretazione clinica dei risultati. L’identificazione di varianti chiaramente implicate nella determinazione della patologia è un processo complesso, che richiede l'integrazione di diversi tipi di informazione. Durante il mio dottorato, ho contributo all’implementazione di metodi computazionali per predire la probabilità che un determinato genotipo sia associato al fenotipo clinico di interesse. Questi metodi sono stati presentati, e valutati, in occasione del Critical Assessment of Genome Interpretation (CAGI), dove si sono posizionati tra i migliori classificati sia per prestazioni che numero di predizioni corrette. Una strategia analoga è stata applicata all’identificazione di geni implicati nella comorbidità tra disordini del neurosviluppo. Anche in questo caso, l’utilizzo di tecniche bioinformatiche si è reso fondamentale per la selezione di geni candidati, che sono stati poi utilizzati nella progettazione di un pannello genico diagnostico attualmente in uso presso la Clinica Pediatrica dell’Università di Padova. Data la gran quantità di dati prodotti per esperimento, le varianti trovate nei geni inclusi nel pannello sono state filtrate in base alla frequenza, alla predizione di patogenicità e all'analisi di segregazione all'interno della famiglia. In alcuni casi, un ulteriore contributo a supporto dell’effettiva patogenicità della variante è stato dato dall’analisi bioinformatica della proteina mutata. Frequentemente, la variante candidata provoca alterazioni a livello di regioni intrinsecamente disordinate (IDR), caratterizzate dall’assenza di una conformazione tridimensionale stabile. Questo dato è coerente con la più recente letteratura: diversi studi, infatti, dimostrano l’implicazione di mutazioni nelle IDR in diverse patologie umane. La classificazione delle IDR, quindi, può rappresentare un primo passo per comprendere l'impatto di eventuali varianti causative all'interno di queste regioni. Data la rilevanza delle IDR a livello biologico e clinico, ho partecipato alla curazione manuale e all'aggiornamento delle voci presenti nel database DisProt, la principale banca dati relativa al disordine nelle proteine. È interessante notare che, tra i vari processi biologici in cui le IDR sono coinvolte, queste regioni svolgono un ruolo molto importante nel signaling neuronale. Tra le proteine codificate dai geni inclusi nel pannello genico, TANC2 si è distinta per essere una proteina disordinata, probabilmente implicata alla trasduzione del segnale a livello delle sinapsi neuronali. Dato che la funzione di TANC2 e della rispettiva famiglia proteica risultava ancora poco chiara, ho eseguito un’analisi in silico delle proteine TANC, grazie alla quale è stato possibile caratterizzare le funzioni e i diversi processi cellulari in cui queste sono coinvolte. Le ipotesi funzionali emerse dall'analisi bioinformatica sono state utilizzate per condurre ulteriori indagini sperimentali. In particolare, la validazione in vitro dell'interazione TANC2-CDKL5 ha evidenziato l’estrema importanza di regioni intrinsecamente disordinate nella regolazione della degradazione di CDKL5, le cui mutazioni sono associate con manifestazioni cliniche legate a disordini del neurosviluppo. Inoltre, gli esperimenti hanno dimostrato che TANC2 contribuisce alla down-regolazione dei livelli di espressione di CDKL5. Per questo motivo, TANC2 si candida a rappresentare un nuovo target terapeutico per lo sviluppo di nuovi composti per il trattamento di condizioni cliniche associate all’over-espressione di CDKL5.
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Wright, Amy Joy. "Purification and characterization of beta-protein variants of 20S proteasomes of the haloarchaeon Haloferax volcanii." [Gainesville, Fla.] : University of Florida, 2006. http://purl.fcla.edu/fcla/etd/UFE0014370.
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Neueder, Andreas [Verfasser], Herbert [Akademischer Betreuer] Tschochner, and Reinhard [Akademischer Betreuer] Sterner. "Characterization of r-protein variants in Saccharomyces cerevisiae / Andreas Neueder. Betreuer: Herbert Tschochner ; Reinhard Sterner." Regensburg : Universitätsbibliothek Regensburg, 2010. http://d-nb.info/1048724115/34.
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Al-Mahmoud, Widad Abdulsamad Mansour. "Novel variants of the DNA damage checkpoint protein Hus1 in fission yeast and human cells." Thesis, Bangor University, 2014. https://research.bangor.ac.uk/portal/en/theses/novel-variants-of-the-dna-damage-checkpoint-protein-hus1-in-fission-yeast-and-human-cells(dee8a56b-687f-4f24-9c6d-f81d73edc877).html.
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Hollar, Carol M. "Estimation of Selected Milk Protein Genetic Variants by Multi-Component Analysis of Amino Acid Profiles." DigitalCommons@USU, 1992. https://digitalcommons.usu.edu/etd/5390.
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Cation-exchange fast protein liquid chromatography separated whole casein into β-casein A2, A1, and B, K-casein, αs1-casein, and αs2-casein fractions as well as γ-caseins and several unidentified peaks using a urea-acetate buffer at pH 5 and a NaCl gradient. The whole casein fractions eluted in the following order: breakdown products of β-casein and unidentified peaks; β-casein A2, Al, and B; additional breakdown products of β-casein and unidentified peaks; K-casein; αs1-casein; and αs2-casein. The calculated composition of the four major caseins correlated well with values obtained using anion-exchange fast protein liquid chromatography at pH 7. An acid-PAGE gel confirmed that the three β-casein peaks were variants of β-casein.
Incubating herd bulk whole casein with neuraminidase (EC 3.2.1.18) removed carbohydrate from K-casein. Anion-exchange fast protein liquid chromatography separated whole casein into β-casein breakdown products, K-casein A and B, β-casein, αs2-casein, and αs1-casein peaks as well as three unidentified fractions using bis-Tris-propane-urea buffer at pH 7 and a NaCl gradient. Fractions of whole casein eluted in the following order: breakdown products of β-casein and unidentified fractions A and B; K-casein fraction; unidentified C fraction; β-casein; αs2-casein; and αs1-casein. Following treatment with neuraminidase, K-casein eluted as K-casein B and A rather than a series of peaks. Casein samples from individual cows containing known combinations of K-casein A and B confirmed that the peaks were K-casein variants.
Isoelectric focusing on a PhastSystem™ separated K-casein A and B; β-casein A1, A2, A3, and B; αs1-casein Band C; β-lactoglobulin A and B; αs2-casein A; and α-lactalbumin B. Minimal preparation and a short separation time enabled many whole milk and whole casein samples to be phenotyped daily.
Stepwise regression equations derived to predict samples as homozygous or heterozygous for K-casein A and B and β-casein A1, A2, and B had coefficient of determination values of .18, .58, .82, and .72 for K-casein A and B, β-casein A1, β-casein A2, and β-casein B. Although amino acid analysis can identify whether β-casein A1, A2, or B variants are present, it cannot identify whether K-casein A and B variants are present.
Percentages of K-casein, β-casein, αs1-casein, and αs2-casein obtained with isoelectric focusing, cation-exchange fast protein liquid chromatography, and anion-exchange fast protein liquid chromatography compare well with published results. Isoelectric focusing and anion-exchange fast protein liquid chromatography methods separated K-casein into its A and B variants. Isoelectric focusing and cation-exchange fast protein liquid chromatography separated β-casein into its A1, A2, and B variants. Individual cows homozygous for K-casein A or B expressed the same amount of K-casein. When results from individual cows heterozygous for K-casein are combined, the two alleles are expressed equally; on an individual cow basis, however, some cows expressed more K-casein B than K-casein A. Individual cows homozygous for β-casein A1, A2 or B expressed the same amount of β-casein. When the results for individual cows heterozygous for β-casein are combined, the two β-casein alleles are expressed equally. In milk from individual cows typed β-casein A2B, slightly more B than A2 was expressed with cation-exchange fast protein liquid chromatography.
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Cong, Xiaojing. "Molecular Simulation Studies on the Prion Protein Variants: Insights into the Intriguing Effects of Mutations." Doctoral thesis, SISSA, 2013. http://hdl.handle.net/20.500.11767/4810.
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Prion diseases, or transmissible spongiform encephalopathies (TSE), are a group of rare fatal neurodegenerative maladies that affect humans and animals. The fundamental breakthrough in TSE research was the discovery of the "prion"⎯proteinaceous infectious particle⎯ and the verification of the “protein-only” hypothesis, which states that prions could self-propagate by converting the cellular prion protein (PrPC) into the scrapie form, PrPSc (or prions), and lead to neurodegeneration without using any nucleic acids. The concept of prions may unify neurodegenerative diseases under a common pathogenic mechanism. Indeed, growing evidence shows that TSE may share similar pathogenesis with common neurodegenerative syndromes such as Alzheimer’s disease and Parkinson’s disease, for which there are currently no cure. Today, PrP is one of the most studied models for protein misfolding mechanism and TSE serve as an excellent model for studying many other neurodegenerative diseases. Understanding the molecular mechanism of the PrP misfolding process may profoundly influence the development of diagnostics and effective therapies for neurodegenerative diseases in general.
Investigating human (Hu) PrP TSE-linked mutations (more than 50 currently identified mutations, linked to ~15% of the cases) may be very instrumental in this respect, as it can provide hints on the molecular basis of the PrPC→PrPSc conversion. These mutations cause spontaneous TSE, which are likely due to modifications in the native structure of PrPC. They are located all over the structure. Polymorphisms (i.e. non-pathogenic, naturally occurring mutations) in the PrP gene have been found to influence the etiology and neuropathology of the disease in both humans and sheep. In transgenic (Tg) mice, artificial mutations can determine the susceptibility to the infection of different prion strains. Intriguingly, mouse (Mo) PrP containing artificial mutations (denoted MoPrP chimera, hereinafter) have very different effects in vitro: some MoPrP chimera were found to resist PrPSc infection, whereas some others did not; some of the resistant MoPrP chimeras even exhibited a protective effect (known as the dominant-negative effect) over the co-expressed endogenous wild-type (WT) MoPrPC. Most mutations are located in the folded globular domain (GD) while fewer are located in the intrinsically disordered N-terminal domain (N-term). The N-term of PrPC has been suggested to serve multiple functions in vivo, which likely relies on the structural flexibility of this domain. Therefore, characterizing the structural features of the N-term is central for investigating not only the mutations in this domain, but also the physiological role of the N-term.
Based on previous studies in our lab, in this thesis we first applied molecular dynamics simulations to studying the impact of all the known Hu TSE-linked mutations in HuPrPC GD. We next applied the same approach to study the GD structure of MoPrP chimeras which contain one or two residues from Hu or sheep PrP sequence. By studying these PrP variants, we aim to identify the structural determinants of the mutants that may play a role in the PrPC→PrPSc conversion. Our calculations discovered that these mutants exhibit different structural features from those of the WT PrP GD mainly in two common regions that are likely the “hot spots” in the protein misfolding process. These features can be classified into different types that are correlated to the types of mutants (i.e. pathogenic, resistant or dominant-negative), thus hinting to the molecular mechanisms of PrPSc formation and propagation. We have then predicted the structure of the entire PrP N-term and the impact of the Hu TSE-linked mutations in this domain using a novel Monte Carlo-based simulation approach, PROFASI. PROFASI has already shown to provide structural predictions in a disordered protein such as α-synuclein. Our results are consistent with available experimental data and therefore firmly allow us to provide the first overview on the structural determinants of all Hu TSE-linked mutations in PrP.
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Li, Jiaxie. "Effects of genetic variants of k-casein and ß-lactoglobulin on heat denaturation of milk proteins and formation of protein complex." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/mq29741.pdf.
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Li, Jiaxie. "Effects of genetic variants of k-Casein and b-lactoglobulin on heat denaturation of milk proteins and formation of protein complex." Thesis, McGill University, 1997. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=27367.
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This study was based on the 462 milk samples collected from approximately 2000 cows registered in Dairy Herd Analysis Service (DHAS). Milk samples from fresh milks were phenotyped by gel electrophoresis. Milk samples were selected according to the nine possibilities of phenotype combination of $ kappa$-casein AA, AB, BB and $ beta$-lactoglobulin AA, AB and BB. Selected milk samples from fresh milks were heated at 25$ sp circ$C, 60$ sp circ$C, 70$ sp circ$C, 80$ sp circ$C and 90$ sp circ$C, respectively. Whole casein and whey protein were separated by adjusting the pH to 4.6. Quantitative determination of milk protein were performed by reverse-phase HPLC. Whole casein was separated to $ kappa$-casein ($ kappa$-Cn), $ beta$-casein ($ beta$-Cn) and $ rm alpha sb{s}$-casein ($ rm alpha sb{s}$-Cn). Whey protein was separated as immunoglobulin (Ig), bovine serum albumin (BSA), $ alpha$-lactalbumin ($ alpha$-La) and $ beta$-lactoglobulin ($ beta$-Lg). Individual milk protein fraction was quantitatively determined by relative peak area and their ratios to whey protein or casein. The denaturation of individual milk protein at different heating temperature was investigated. (Abstract shortened by UMI.)
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Gronow, Joana Verfasser], Frank [Akademischer Betreuer] [Sönnichsen, and Ulrich [Gutachter] Lüning. "Structural Stabilization of α-Helical Antifreeze Protein Variants Using the Trp-cage Protein / Joana Gronow ; Gutachter: Ulrich Lüning ; Betreuer: Frank D. Sönnichsen." Kiel : Universitätsbibliothek Kiel, 2020. http://nbn-resolving.de/urn:nbn:de:gbv:8-mods-2020-00047-1.
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Gronow, Joana [Verfasser], Frank D. [Akademischer Betreuer] Sönnichsen, and Ulrich [Gutachter] Lüning. "Structural Stabilization of α-Helical Antifreeze Protein Variants Using the Trp-cage Protein / Joana Gronow ; Gutachter: Ulrich Lüning ; Betreuer: Frank D. Sönnichsen." Kiel : Universitätsbibliothek Kiel, 2020. http://d-nb.info/1206179678/34.
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Azoulay, Eric. "Induction of apoptosis or cell cycle arrest by two human wildtype variants of the p53 protein." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0031/MQ64313.pdf.
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Baugh, Evan H. "Predicting the Effects of Protein Variants using Structural Modeling, Large-Scale Data Integration, and Machine Learning." Thesis, New York University, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10247644.
Full textAbstract:
High-throughput sequencing technologies and new computational techniques for analyzing population genetics data are rapidly improving our understanding of disease susceptibility in humans and adaptation in a wide variety of organisms. These studies often discover nonsynonymous variation with large effects as even a single amino acid change can disrupt the folding, catalytic activity, and physical interactions of proteins. Current estimates predict that every human genome contains 10,000-11,000 nonsynonymous variations and, while we cannot currently characterize all this diversity experimentally, many variants that alter protein function can be identified computationally from destabilization of structural models or amino acid conservation. Methods for annotating variant effects in genome-wide association studies and exome sequencing studies use conservation and other sequence-based features to identify damaging variants but cannot predict the effect these variants have on protein function. Recent studies of de novo variants have demonstrated the power of these methods but also the need for additional information, such as physical models from the Protein Data Bank, to identify causal variants in disease association studies.
I present VIPUR, a computational framework that integrates sequence analysis and structural modeling using the Rosetta protein modeling suite to identify and interpret deleterious protein variants. To train VIPUR, I collected 9,477 protein variants with known effects on protein function from multiple organisms and curated structural models for each variant from crystal structures and homology models. VIPUR can be applied to variants in any organism’s proteome with improved generalized accuracy (AUROC .83) and interpretability (AUPR .87) compared to other methods. I show that VIPUR’s predictions of deleteriousness match the biological phenotypes for pathogenicity in ClinVar despite being trained on a different label. I use VIPUR to interpret mutations associated with inflammation and diabetes, demonstrating the structural diversity of disrupted functional sites and improved interpretation functional effects.
Generalizable tools for interpreting genetic variants are especially needed with individualized exome sequencing, where clear indications of confident predictions are necessary to identify causal variation. I demonstrate VIPUR’s ability to select candidate variants associated with human diseases by predicting the effects of de novo variants associated with Autism Spectrum Disorders (ASD) in the Simons Simplex Collection. Compared to existing methods, VIPUR deleterious predictions have the greatest enrichment for mutations found in children with ASD. VIPUR’s predictions of deleterious effects are easily combined with other protein functional data to produce a small set of candidate genes and variants with specific mechanistic predictions.
Although designed to aid in the discovery of causal variants, VIPUR can also simulate mutations to better understand specific protein functions. The distribution of VIPUR scores across all positions in a protein can be used to highlight conserved residues and provides an overall measure of protein conservation. When applied to levoglucosan kinase, a bacterial enzyme of interest for biofuel processing, VIPUR neutral predictions have a five fold enrichment for beneficial growth mutations. While VIPUR is not designed to detect gain-of-function mutations, this enrichment suggests VIPUR scores can identify potentially beneficial mutations by removing clearly deleterious ones. When applied to TP53, a human protein that is mutated in nearly half of all cancers, VIPUR score trends highlight the most common mutations in the COSMIC database, suggesting other variants that may have similar effects on tumor growth. VIPUR and the large-scale data analysis empowering it will aid in the interpretation of protein variation by providing a detailed feature space to characterize protein functional effects and confident predictions of deleterious variation in Genome-Wide Association Studies, exome sequencing initiatives, and protein engineering.
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Klapper, Maja [Verfasser]. "Promoter variants and transcriptional regulation of the intestinal fatty acid binding protein gene (FABP2) / Maja Klapper." Kiel : Universitätsbibliothek Kiel, 2008. http://d-nb.info/1019630728/34.
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Gress, Alexander [Verfasser]. "Integration of protein three-dimensional structure into the workflow of interpretation of genetic variants / Alexander Gress." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2020. http://d-nb.info/1218075473/34.
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Apaja, P. (Pirjo). "Luteinizing hormone receptor:expression and post-translational regulation of the rat receptor and its ectodomain splice variant." Doctoral thesis, University of Oulu, 2005. http://urn.fi/urn:isbn:9514279298.
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Abstract
The luteinizing hormone receptor (LHR) is a G protein-coupled receptor (GPCR) that has a large N-terminal ligand binding ectodomain. The LHR ectodomain splice variant, expressed concomitantly with the full-length LHR in tissues, has an unknown biological function. GPCRs are a major pharmacological target, however, very little is known about the intracellular regulation of these receptors. In the present work, expression and maturation of the rat LHR and its variant were elucidated using both tissues and heterologous expression systems. A special effort was made to identify the role of developmental stage and tissue type on the LHR maturation and to find out about the molecular role of the ectodomain splice variant.
We found two sites of localization for the receptor, namely the sensory system and urogenital tissues. This was demonstrated at mRNA and protein level and by rat LHR promoter-driven β-galactosidase (β-Gal) expression in the mice. In neurons, the β-Gal co-localized with the cytochrome P450 side chain cleavage enzyme, which may indicate a novel role in the neurosteroid synthesis.
The neuronal LHR was expressed in the mature and immature protein forms in both developing and adult tissues, being able to bind hormone with similar high-affinity as gonadal receptors. In contrast, only immature receptors were detected in the fetal rat urogenital structures. A significant novel finding was substantial upregulation of the LHR in pregnant female rat adrenal glands and kidneys at a time that coincides with the differentiation of the fetal urogenital tissues.
The mice overexpressing the ectodomain splice variant showed interference in pituitary-gonadal functions and morphological changes in the urogenital tissues. The studies showed that the variant was an endoplasmic reticulum (ER)-retained soluble protein. It accumulated in juxtanuclear regions of the ER together with ER folding chaperones and was a substrate for ER associated degradation (ERAD). The co-expression of the variant with the full-length receptor decreased the amount of receptors and misrouted them to the juxtanuclear ER subcompartment.
Taken together, we suggest that the maturation of the LHR protein is developmentally and physiologically regulated at the post-translational level in tissues. The LHR ectodomain splice variant possibly modulates post-translationally the number of full-length receptors through physiological signals. Our observation of the chaperone and protein accumulation into a specific ER subcompartment may represent a protein quality control holding compartment for inefficiently/misfolded ERAD substrates.
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Chu, Ge. "PCSK9 and Its Variants: An Unbiased Global Proteomic Study to Identify Interactors and Effects on Protein Trafficking." Thesis, Université d'Ottawa / University of Ottawa, 2015. http://hdl.handle.net/10393/32988.
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Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted glycoprotein that promotes degradation of low-density lipoprotein receptors. Gain- and loss-of-function variants of PCSK9 cause hypercholesterolemia and hypocholesterolemia, respectively. Although it has been a decade since the discovery of PCSK9, its effect in terms of global protein changes and interactions still require further understanding. This study provided a global outlook at the protein changes caused by PCSK9 and its variants in human hepatic HUH7 cell line. First, a proteomics-based method for protein subcellular distribution analysis has been developed. Second, through secretome analyses, six apolipoproteins and six proteins involved in the coagulation pathway were found with >2-fold changes between wild type PCSK9 and its variants. Third, through secreted interactome analyses, a list of 159 PCSK9 interactor candidates was identified. Two interacting proteins, FASN and PSMD2, were validated and demonstrated with dynamic interacting patterns between PCSK9 and its variants.
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Deming, Brenda Boon. "Evaluating the role of lymphocyte radiosensitivity and variants in double-strand break repair genes, checkpoint kinase 2 (CHEK2) and nibrin (NBN), in the predisposition to prostate cancer : a dissertation /." San Antonio : UTHSC, 2007. http://proquest.umi.com/pqdweb?did=1425298611&sid=1&Fmt=2&clientId=70986&RQT=309&VName=PQD.
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Warren, Curtis R. "Linker region of the BRCA2 protein increases chemoresistance to cisplatin: Screen for the characterization of cancer-associated variants." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 84 p, 2009. http://proquest.umi.com/pqdweb?did=1885607671&sid=3&Fmt=2&clientId=8331&RQT=309&VName=PQD.
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Coghill, Lorraine Sheila. "Regulation of large conductance calcium- and voltage-activated potassium (BK) channel splice variants by protein kinase A." Thesis, University of Edinburgh, 2003. http://hdl.handle.net/1842/23309.
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