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1
Cloonan, Nicole, and N/A. "Sin1 and Sin1 Isoforms: An Investigation into the Biological Significance of a Novel Human Protein Family." Griffith University. School of Biomolecular and Biomedical Science, 2006. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20071102.150237.
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Stress activated protein kinase (SAPK) interacting protein 1 (Sin1) is a member of a recently characterized gene family, conserved from yeast to humans. The gene copy number is strictly conserved (one Sin1 gene per genome), and the protein may be expressed ubiquitously in mammalian tissues. The Sin1 family has been implicated in several different signal transduction pathways. Originally identified as a partial cDNA and candidate Ras inhibitor, recent functional studies have revealed interactions with an interferon (IFN) receptor subunit (IFNAR2), and the SAPK JNK. Interactions have also been described between the yeast orthologues and the phosphatidylinositol kinase
TOR2. Collectively, these data suggest that Sin1 has an important cellular role, and this study has investigated possible functions for this protein. As human Sin1 proteins have no paralogues within the genome, secondary structure homology was used to identify major domains within the protein. Four major domains within human Sin1 were deduced: an N-terminal domain containing a functional nuclear localization signal, a functional nuclear export signal, and a coiledcoil region; the conserved region in the middle that is likely to be a ubiquitin-like β-grasp protein binding domain; a Ras binding domain; and a pleckstrin homology-like domain that targets Sin1 to the plasma membrane and lipid rafts in vivo. Full and partial length EGFP constructs were used to examine the localization of human Sin1, and several isoforms derived from alternative splicing. All isoforms localized to the nucleus and nucleolus. Beyond this, Sin1α and Sin1ϒ had cytoplasmic staining, while Sin1 and Sin1β were also found at the plasma membrane and lipid rafts. Both the N-terminal domain and the conserved region in the middle were found to contribute to nuclear localization. Comparative genomic analysis between human, mouse, rat, dog, and chicken Sin1 genes revealed a number of conserved intronic regions, and the putative functions of these were predicted. Additionally, a putative promoter module within a CpG island and encompassing the transcription start site was predicted in all species. The human CpG island was found to have promoter activity in HEK293 cells. Using bioinformatics, genes that may be co-regulated with Sin1 were identified. These genes contained the Sin1 promoter module, and were found to co-express in large scale gene expression studies. Most of these genes were directly involved in the cellular response to pathogen infection, suggesting a conserved role for Sin1 in this pathway. Key biochemical functions of the Sin1 proteins were also identified, including the ability of Sin1 proteins to form dimers, and the ability of over-expressed Sin1 to induce apoptosis (mediated through the conserved region in the middle). Additionally, endogenous Sin1 protein levels were found to change following serum deprivation and hypoosmotic stress. Together, these studies have provided significant insight into the cellular role of Sin1, suggesting a role in inducing apoptosis as part of the IFN response to viral infection. The biological significance of the Sin1 proteins is discussed in the context of their predicted functions and the evolution of the protein family.
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Cloonan, Nicole. "Sin1 and Sin1 Isoforms: An Investigation into the Biological Significance of a Novel Human Protein Family." Thesis, Griffith University, 2006. http://hdl.handle.net/10072/367210.
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Stress activated protein kinase (SAPK) interacting protein 1 (Sin1) is a member of a recently characterized gene family, conserved from yeast to humans. The gene copy number is strictly conserved (one Sin1 gene per genome), and the protein may be expressed ubiquitously in mammalian tissues. The Sin1 family has been implicated in several different signal transduction pathways. Originally identified as a partial cDNA and candidate Ras inhibitor, recent functional studies have revealed interactions with an interferon (IFN) receptor subunit (IFNAR2), and the SAPK JNK. Interactions have also been described between the yeast orthologues and the phosphatidylinositol kinase TOR2. Collectively, these data suggest that Sin1 has an important cellular role, and this study has investigated possible functions for this protein. As human Sin1 proteins have no paralogues within the genome, secondary structure homology was used to identify major domains within the protein. Four major domains within human Sin1 were deduced: an N-terminal domain containing a functional nuclear localization signal, a functional nuclear export signal, and a coiledcoil region; the conserved region in the middle that is likely to be a ubiquitin-like β-grasp protein binding domain; a Ras binding domain; and a pleckstrin homology-like domain that targets Sin1 to the plasma membrane and lipid rafts in vivo. Full and partial length EGFP constructs were used to examine the localization of human Sin1, and several isoforms derived from alternative splicing. All isoforms localized to the nucleus and nucleolus. Beyond this, Sin1α and Sin1ϒ had cytoplasmic staining, while Sin1 and Sin1β were also found at the plasma membrane and lipid rafts. Both the N-terminal domain and the conserved region in the middle were found to contribute to nuclear localization. Comparative genomic analysis between human, mouse, rat, dog, and chicken Sin1 genes revealed a number of conserved intronic regions, and the putative functions of these were predicted. Additionally, a putative promoter module within a CpG island and encompassing the transcription start site was predicted in all species. The human CpG island was found to have promoter activity in HEK293 cells. Using bioinformatics, genes that may be co-regulated with Sin1 were identified. These genes contained the Sin1 promoter module, and were found to co-express in large scale gene expression studies. Most of these genes were directly involved in the cellular response to pathogen infection, suggesting a conserved role for Sin1 in this pathway. Key biochemical functions of the Sin1 proteins were also identified, including the ability of Sin1 proteins to form dimers, and the ability of over-expressed Sin1 to induce apoptosis (mediated through the conserved region in the middle). Additionally, endogenous Sin1 protein levels were found to change following serum deprivation and hypoosmotic stress. Together, these studies have provided significant insight into the cellular role of Sin1, suggesting a role in inducing apoptosis as part of the IFN response to viral infection. The biological significance of the Sin1 proteins is discussed in the context of their predicted functions and the evolution of the protein family.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
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Nilsson, Jonas. "The LRIG-family : identification of novel regulators of ErbB signaling with clinical implications in astrocytoma /." Doctoral thesis, Umeå : Department of Radiation Sciences, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-783.
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Bartee, Eric Carter. "Discovery and characterization of a novel family of human ubiquitin ligases termed Membrane Associated RING-CH (MARCH) proteins." Oregon Health & Science University, 2007. http://content.ohsu.edu/u?/etd,629.
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Ph.D.Molecular Microbiology and Immunology
Both poxviruses and γ2-herpesviruses share the K3-family of viral immune evasion proteins. These proteins are characterized by an amino-terminal RING-CH domain followed by two transmembrane domains. We analyzed several human homologues of the K3-family termed membrane-associated RING-CH (MARCH) proteins. All MARCH proteins localized to subcellular membranes while several reduced surface levels of known K3-family substrates. Thus, MARCH proteins appear to be structurally and functionally homologous to viral K3 proteins. One of the major challenges in determining the function of this family is the identification of their physiological substrates. To overcome this we created a quantitative proteomics approach which can be used to identify novel substrates for both the K3- and MARCH-families. Using stable isotope labeling by amino acids in cell culture, we compared the proteome of plasma membrane, golgi, and endoplasmic reticulum membranes in the presence and absence of K5 and MARCH-VIII. Quantitative mass spectrometric protein identification from these fractions revealed that CD316 (bone marrow stromal antigen 2), CD166 (activated leukocyte cell adhesion molecule) and syntaxin-4 were consistently underrepresented in the plasma membrane of K5 expressing cells, while CD44, CD81 (TAPA-1) and B-cell receptor-associated protein 31kDa (Bap31) were consistently underrepresented in the plasma membrane of MARCH-VIII expressing cells. Furthermore, downregulation of each of these proteins was independently confirmed. Our results both identify and characterize a novel family of human ubiquitin ligase enzymes and elucidate a novel technique which can analyze this family and be easily adapted to the analysis of other cellular enzymes viral immune modulators.
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Iwai, Akio. "Siah-1L, a novel transcript variant belonging to the human Siah family of proteins, regulates β-catenin activity in a p53-dependent manner." Kyoto University, 2005. http://hdl.handle.net/2433/144711.
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Ruane, Peter Thomas. "Functional characterisation of human EB protein family member EB2." Thesis, University of Leeds, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.550859.
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Dynamic protein filaments in eukaryotic cells make up cytoskeletal arrays that perform essential functions. Microtubules form part of the cytoskeleton, and impart structure, integrity and organisation to cells. End binding (EB) proteins are an evolutionarily conserved family which associate with the dynamic ends of microtubules, where they act to control microtubule growth and recruit other proteins that localise to this site (+ TIPs). EB2 is one of three vertebrate EB proteins but its role is unclear because it exhibits weak EB protein activity. This thesis describes molecular and cell . biological experiments designed to functionally characterise human EB2. Fourteen human cell lines were all shown to express EB2 at lower levels than EB 1, while isolation of EB2 in HeLa cells by siRNA-mediated knock down of EB 1 and EB3 confirmed reports that EB2 is outcompeted at microtubule ends by EB 1 and EB3. Analysis of individual microtubules corroborated proposals that EB2 has lower affinity for microtubule tips than EB 1, and also suggested that EB proteins interact more strongly with the proximal region of microtubule tips than the distal region. Furthermore, the + TIP CLIP-170 localised to this distal region independently of EB proteins, implicating an activatory rather than direct-coupling role for EB proteins in the recruitment of CLIP-170. Additionally, the functional effects of structural divergences in EB2 were examined by mutation. An N-terminal extension, EB2 Nose, was shown to attenuate + TIP binding through a functional interaction with 322pQ323 in the C- terminal tail of EB2. A putative SxIP motif, 25TIIp28, was identified within EB2 Nose which contributed to this attenuation. It is proposed from these findings that EB2 is autoinhibited for + TIP binding by intramolecular interactions, between EB2 Nose and 322pQ323, and between 25Tllp28 and the C-terminal EB domain. These data portray EB2 as an unproductive EB protein, and a '+ TIP spacer' function is postulated.
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Temprano, López Ana. "The lipin protein family in human adipocytes: lipid metabolism and obesity." Doctoral thesis, Universitat Rovira i Virgili, 2016. http://hdl.handle.net/10803/398025.
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Les lipins són una família conservada evolutivament de fosfatases de fosfatidat (PAP1) dependents de Mg2+, que generen diacilglicerol per a la síntesi de fosfolípids i triacilglicerol. En mamífers, la família consta de lipina-1, lipina-2 i lipina-3. Mentre en ratolins la mutació del gen Lpin1 causa lipodistròfia, les mutacions deletèries en el gen LPIN1 en humans no afecten la distribució del greix. No obstant, persones amb diabetis tipus 2 mostren nivells reduïts de l'expressió de LPIN1 i de l'activitat PAP1. Aquesta tesi estudia el paper de les lipins en el teixit adipós humà, la adipogènesi i la lipòlisi. Descobrim que la expressió de gens i proteïnes lipin és alterada en el teixit adipós de les persones amb diabetis tipus 2. Silenciant cada membre de la família lipin en la línia cel•lular humana de preadipòcits del síndrome Simpson-Golabi-Behmel (SGBS), mostrem que mentre que els tres membres tenen un paper en el primers estadis de l’adipogènesi, els preadipòcits silenciats de lipin es diferencien i acumulen lípids neutres, la qual cosa condueix a la hipòtesi de l'existència de vies alternatives per a la síntesi de triacilglicerol en adipòcits humans quan es reprimeix l'expressió de les lipin. Les lipin participen també en el reciclatge d'àcids grassos alliberats mitjançant la via lipolítica. Després de la inducció de la lipòlisi, les lipines són defosforilades i es desplacen a la membrana del reticle endoplasmàtic, on exerceixen la seva funció enzimàtica. Aquesta activació és induïda pels àcids grassos alliberats i s'inverteix amb la presència d’albúmina o triacsin C. La inducció d’adipòcits silenciats de cada lipina demostra el seu paper en el metabolisme dels lípids neutres. En resum, les lipin semblen no tenir un paper imprescindible en la adipogènesi humana però sí poden comprometre el reciclatge d'àcids grassos, important per a la homeòstasis lipídica.Las lipinas son una familia de fosfatasas de fosfatidato (PAP1) dependientes de Mg2+ evolutivamente conservadas, que generan diacilglicerol para la síntesis de fosfolípidos y triacilglicerol. En mamíferos, la familia consiste en lipina-1, lipina-2, y lipina-3. Mientras en ratones la mutación del gen Lpin1 causa lipodistrofia, las mutaciones deletéreas en el gen LPIN1 en humanos no afectan a la distribución de grasa. Sin embargo, los individuos con diabetes tipo 2 manifiestan niveles reducidos de expresión de LPIN1 y de actividad PAP1. En esta tesis doctoral se estudia la función de las lipinas en el tejido adiposo humano, la adipogénesis y la lipólisis. Descubrimos que la expresión génica y proteica de las lipinas está alterada en el tejido adiposo de individuos con diabetes tipo 2. La depleción de cada miembro de las lipinas en la línea celular humana de preadipocitos del síndrome Simpson–Golabi–Behmel (SGBS), mostró que, a pesar de que los tres miembros tienen un papel en la adipogénesis temprana, los adipocitos deplecionados de lipinas se diferencian y acumulan lípidos neutros, llevándonos a la hipótesis de la existencia de vías alternativas para la síntesis de triacilglicerol en adipocitos humanos cuando la expresión de las lipinas es reprimida. Las lipinas también intervienen en el reciclaje de los ácidos grasos liberados por la vía lipolítica. Tras la inducción de la lipólisis, las lipinas son defosforiladas y se desplazan a la membrana del retículo endoplásmico, donde ejercen su función. Esta activación es inducida por los ácidos grasos liberados, y revertida con albúmina o triacsin C. La depleción de cada lipina en adipocitos SGBS y posterior inducción de la lipólisis, demuestra su papel en el metabolismo de lípidos neutros. En resumen, las lipinas parecen no tener un papel indispensable en la adipogénesis humana pero sí comprometer el reciclaje de ácidos grasos, importante para la homeostasis lipídica.
Lipins are evolutionarily conserved Mg2+-dependent phosphatidate phosphatases (PAP1) that generate diacylglycerol for phospholipid and triacylglycerol synthesis. In mammals the Lipin family consists of lipin-1, lipin-2 and lipin-3. Whereas mutations in the Lpin1 gene cause lipodystrophy in mouse models, LPIN1 deleterious mutations in humans do not affect fat distribution. However, reduced LPIN1 expression and PAP1 activity have been described in participants with type 2 diabetes. In this doctoral thesis we investigate the roles of all lipin family members in human adipose tissue, adipogenesis and lipolysis. We found that adipose tissue gene and protein expression of the lipin family is altered in type 2 diabetes. Depletion of every lipin family member in a human Simpson–Golabi–Behmel syndrome (SGBS) pre-adipocyte cell line showed that even though all members alter early stages of adipogenesis, lipin-silenced cells differentiate and accumulate neutral lipids, pointing to the hypothesis of alternative pathways for triacylglycerol synthesis under repression of lipin expression. Lipins also have a role in the recycling of the fatty acids released by the lipolytic pathway. They become dephosphorylated upon lipolytic induction, and translocate to their active site, the endoplasmic reticulum membrane. This activation is induced by fatty acids and reversed with albumin or triacsin C. Depletion of every lipin member and subsequently stimulation of lipolysis in SGBS adipocytes revealed a role for lipins in neutral lipid metabolism. Overall, our data support that lipins may not have an indispensable role in adipogenesis, but their depletion compromise fatty acid recycling and lipid homeostasis.
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Chan, Che-man, and 陳志敏. "Functional study of spike protein of a novel human coronavirus HKU1." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hub.hku.hk/bib/B41896932.
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Chan, Che-man. "Functional study of spike protein of a novel human coronavirus HKU1." Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/B41896932.
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Rhyner, Johannes A. "The human calmodulin gene family and characterization of the calmodulin-like protein /." [S.l.] : [s.n.], 1994. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=10508.
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Rogi, Tomohiro. "Physiological function of the novel human aminopeptidases belonging to oxytocinase sub-family." Kyoto University, 2002. http://hdl.handle.net/2433/149525.
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Bedrossian, Anaid. "Structure, Expression and Function of the novel KIND Domain Family Protein very-KIND." Doctoral thesis, kostenfrei, 2008. http://www.opus-bayern.de/uni-wuerzburg/volltexte/2008/2846/.
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Palmer, Ruth Helen. "Cloning and characterisation of the PRK family : a novel family of protein kinases related to the PKC superfamily." Thesis, University College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321771.
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Samuels, Yardena Rachel. "ASPP : a novel family of proteins that specifically regulates the apoptotic function of p53." Thesis, Imperial College London, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.289842.
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Crossland, Katherine Louise. "Characterisation of a novel transmembrane protein in primary human CD4+ T-cells." Thesis, University of Newcastle upon Tyne, 2014. http://hdl.handle.net/10443/2573.
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There are two main mechanisms of tolerance, one in the thymus and one in the periphery. Anergy, a peripheral mechanism, is a state of hypo-responsiveness where T-cells fail to respond to antigenic stimulus. A breakdown in immunological self-tolerance leads to autoimmunity and so provides an exciting research area for therapeutic intervention in autoimmune disease. Differential display studies comparing anergic and activated CD4+ T-cells identified claudin domain containing protein 1 (CLDND1) to be differentially expressed between these two states. In addition, preliminary experiments performed in our lab identified CLDND1 as a potential negative regulator of CD4+ T-cell activation. The aim of this study was to identify the role of CLDND1 in CD4+ T-cells. Antibodies against CLDND1 were raised and validated before use to determine CLDND1 expression in immune cell subsets and during T-cell activation. The function of CLDND1 in T-cells was investigated using gene silencing or over-expression techniques. CLDND1 expression was also sought in the autoimmune disease, rheumatoid arthritis (RA), to identify whether CLDND1 may be involved in disease pathogenesis. Antibodies were successfully raised against CLDND1 and CLDND1 was found to be transiently up-regulated during CD4+ T-cell activation. CLDND1 gene silencing attempts, while successful at the RNA level, did not translate to a reduction in CLDND1 protein, suggesting CLDND1 may be regulated independently of gene transcription. Over-expression studies were consistent with CLDND1 being a negative regulator of T-cell proliferation or an inducer of cell death, depending on the activating stimulus used. CLDND1 expression was found to correlate with rheumatoid factor (RF) status in early RA patients and may suggest a role for CLDND1 in the disease setting. Some findings identify similarities between CLDND1 and other proteins, providing links for functional pathways and a plethora of further avenues of research.
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Le, Van Son. "The BURP domain protein family of Arabidopsis a novel component related to seed development /." [S.l.] : [s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=979554594.
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Wong, L. H. Y. "Analysis of the novel Lipid transfer protein Anchored at Membrane contact sites (LAM) family." Thesis, University College London (University of London), 2017. http://discovery.ucl.ac.uk/1560219/.
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Membrane contact sites are dynamic structures where two organelles come into close proximity to regulate and facilitate the flow of material and information between them. One type of inter-organelle communication is lipid exchange, which is essential for membrane maintenance and in response to environmental and cellular stimuli. We recently discovered a new family of Lipid transfer proteins Anchored at Membrane contact sites (LAMs) that is present in all eukaryotes. LAM proteins are integral Endoplasmic Reticulum (ER) proteins containing at least one domain that is structurally similar to the StARkin domain superfamily, a specialised fold that can bind amphipathic ligands such as lipids. The budding yeast, Saccharomyces cerevisiae, has six such proteins: Lam1p-6p. Lam1p-4p are located at contacts between the ER and the plasma membrane (PM), and Lam1p-3p are implicated in retrograde sterol traffic between the ER and PM. The PM contains a high concentration of sterol where it increases rigidity by altering the packing characteristics of the phospholipids in the bilayer. Sterol is also important in the ER, where its levels are low but it is both synthesised and sensed. However, the mechanism by which sterol traffics between the ER and the PM is unknown. This investigation characterises the phenotype of yeast delete LAM strains on Amphotericin B, a sterol sequestering antifungal agent and shows that the conserved StARkin domain of LAM proteins is responsible for resistance against Amphotericin B. Aspergillus fumigatus, a filamentous fungus, has two LAM proteins and the removal of AfLamA causes a severe growth phenotype. Also, in vitro studies indicate that LAM StARkin domains have a clear sterol transfer activity and a mutation that can diminish the function in vivo and in vitro has been identified. These findings present a new candidate protein family for intracellular sterol trafficking.
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Fisk, Dianna G. "CRP1 : founding member of a novel protein family that functions in organellar gene expression /." view abstract of download file of text, 2000. http://wwwlib.umi.com/cr/uoregon/fullcit?p9987422.
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Ali, Stuart Alvaro. "Transferrin trojan horses : a novel approach for drug delivery?" Thesis, Brunel University, 1999. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.285047.
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Sawzdargo, Marek. "Discovery of novel G protein-coupled receptor genes including human GALR3 receptor gene." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape9/PQDD_0003/MQ46146.pdf.
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Panetta, Rosemarie. "Human somatostatin receptor 5(hSSTR5) : a novel pituitary selective G protein-coupled receptor." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=40419.
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Using a combination of polymerase chain reaction (PCR) and genomic library screening, a human (h) gene for a subtype of the somatostatin (SST) receptor (SSTR), termed hSSTR5, was cloned. Human SSTR5 consists of a 363-residue polypeptide exhibiting a putative seven-transmembrane domain topology typical of G protein-coupled receptors. The receptor displays 45% to 52% sequence identity to the other four cloned hSSTR subtype genes (SSTRl-SSTR4). Pharmacological analysis of hSSTR5 revealed that this receptor bound SST-28 with a 12.6-fold greater affinity compared with SST-14, indicating that hSSTR5, unlike the other four SSTR subtypes, is SST-28 selective. hSSTR5 was negatively coupled to adenylyl cyclase via a pertussis toxin-sensitive G protein. Northern blot analysis of SSTR5 mRNA revealed a 2.4kb transcript in normal rat pituitary and GH$ sb3$ rat pituitary tumour cells and a 4.0 kb transcript in normal human pituitary. Reverse transcriptase PCR revealed expression of the hSSTR5 gene in fetal human pituitary and hypothalamus but not in human adult cerebral cortex. Expression of SSTR5 mRNA was compared with that of SSTR 1-4 mRNA in normal rat and human pituitary, purified rat somatotrophs, rodent pituitary cell lines (GH$ sb3$, GH$ sb4$C$ sb1$, AtT-20), and human secretory and nonsecretory pituitary tumours. All of these tissues expressed several SSTR gene subtypes, SSTR2 being the predominant form. Since tumour cells are monoclonal in origin, and somatotrophs are an individual subpopulation of pituitary cell, these studies have additionally demonstrated that multiple SSTR genes are expressed in individual cells. SST-28 pre-treatment of CHO-K1 cells stably expressing hSSTR5 markedly attenuated the potency of SST-28 to inhibit cAMP formation, indicating receptor desensitization. Human SSTR5 was also internalized in a time- and temperature-dependent manner upon agonist exposure. The possible role of putative residues and structural domains in the carboxyl-terminal ta
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Quinn, Caroline Frances. "Human respiratory syncytial virus F protein-induced immunosuppression : structures, mechanisms and novel vaccines." Thesis, Queen's University Belfast, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.602933.
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Respiratory syncytial virus (RSV) is a major cause of morbidity and mortality in humans. It repeatedly infects throughout life, suggesting induction of poor memory immune responses. The RSV F protein is a highly conserved antigen that was studied extensively as a vaccine both in animal models and clinical trials but has so far failed in humans. Interestingly. evidence suggested that RSV F blocks proliferation of PHA-stimulated human PBMCs in a contact-dependent and species-specific manner. Specifically, HRSV F preferentially inhibited human PBLs, while bovine RSV F preferentially inhibited bovine PBLs. We aligned multiple strains of human and bovine RSV to identify 8 species-specific residues. We hypothesized that replacing the 8 HRSV F residues with their bovine counterparts would not alter the antigenic or immunogenic structure of HRSV F, while removing the capacity of HRSV F to block human PBL proliferation. Using site-directed mutagenesis, we generated a recombinant HRSV F protein (rHRSV Fmut8) comprising the 8 bovine residues. rHRSV Fmut8 was shown to retain reactivity when tested with a panel of RSV F-specific MAbs, confirming that the antigenic structure was preserved. We subsequently generated and characterised a recombinant Sendai virus efficiently expressing our mutated HRSV F (rSeV/RSV Fmut8), found to be functional and antigenically intact. S.eV (mwine PIVI) was chosen as a vector, as it is immunogenic but non-pathogenic in humans, has vaccine potential against human parainfluenza virus type I (HPrv 1) and can be easily manipulated by reverse genetics. Following intra-nasal administration to BALB/c mice, rSeV/RSV Fmut8 induced protective immunity against RSV challenge. Human PBMC proliferation block experiments revealed that HRSV efficiently blocks inhibition, in which RSV F plays a role. If confirmed, abrogation of the human PBL proliferation block combined with its protective efficacy in vivo, suggests that rSeV/RSV Fmut8 will constitute a very interesting and novel RSV vaccine candidate.
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Dietrich, Joanna Louise. "Phosphorylation of human platelet cytosolic phospholipase A₂ by the Src-family protein tyrosine kinases." Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621659.
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Al, Gharibi Khalaf. "MMP family protein expression as prognostic biomarkers in human soft tissue sarcoma of extremities." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/thesis-title-mmp-family-protein-expression-as-prognostic-biomarkers-in-human-soft-tissue-sarcoma-of-extremities(c99f2355-5c2c-4a0c-bf40-3acace12c94a).html.
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Soft tissue sarcomas (STS) are rare human malignant neoplasms, arising mostly from stem cells within non-skeletal connective tissues. They account for approximately 1% of all human malignancies. Matrix metalloproteinases (MMPs) are enzymes involved in degradation of the extracellular matrix and their expression by cancer cells allows the cells to penetrate basement membranes and tissue matrix, thereby invading and metastasising. The most studied malignant tumours from the perspective of MMP expression and its relationship to malignant behaviour are epithelial-derived carcinomas. MMPs role in invasion and metastasis of sarcomas has been very little investigated. This is in part because of the difficulty in accumulating sufficient tumour tissue to enable statistically relevant analysis of sufficient tumours. The purpose of this thesis was to examine the expression of key MMPs - MMP-2, MMP-7, MMP-9, and MMP-14 and their inhibitors (TIMP-1 and TIMP-2) at the invasive/subcapsular edge of human malignant and benign connective tissue tumours using immunohistochemistry, a technique that allows a very high level of reaction product localisation within tumours. In three different STS types and appropriate benign equivalents, the expression of MMPs -2, -7, -9, and -14 and their inhibitors (TIMPs -1 and -2) were measured using intensity of staining and the percentage area of staining by image analysis. The results were compared between tumour types and against histological grading that is widely used as a prognostic factor. The findings from this research indicated that metalloproteinases were commonly expressed in STS and benign equivalents. There were differences in expression of some benign versus malignant neoplasms of the same group. No uniform pattern of expression of any of MMPs was observed across the tumours, but some of the data, most notably that for expression of MMP-2 and -9 indicate, a role for MMPs in malignant behaviour and some showed (e.g. MMPs -7 and -14) change in expression with the grade of malignant tumours in the same broad category. There is some evidence of an inverse relationship between MMP and appropriate TIMP expression suggesting that a failure of inhibition, as much as increased expression, is a feature of malignancy.
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Wheeler, Louise Claire. "Characterisation of RASAL, a novel Ca'2+-regulated RapGAP of the human GAP1 family." Thesis, University of Bristol, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.393010.
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Munton, Richard Paul. "Investigation of protein kinase-A dependant GABAb receptor desensitisation and characterisation of a novel family 3 G-protein coupled receptor." Thesis, University of Bristol, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.398537.
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Bardien-Kruger, Soraya. "A molecular investigation of the novel gene underlying autosomal dominant retinitis pigmentosa in a South African family." Doctoral thesis, University of Cape Town, 1999. http://hdl.handle.net/11427/26927.
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The inherited retinal degenerative disorders are a common cause of severe visual handicap in the W estem world. Retinitis pigmentosa (RP) is a group of retinopathies in which a primary feature is a progressive loss of photoreceptor and retinal pigment epithelium function. Over the last decade, investigations into the patho-physiology of RP have identified numerous disease-causing genes and loci (for a current listing refer to the web site http://www.sph.uth.tmc.edu/Retnet/). A study of a South African family with an autosomal dominant form of RP (adRP) forms the basis of this dissertation. In this family, comprising 44 individuals, the first manifestation of visual disturbance is usually evident between 20 and 30 years of age. Subsequently, another South African adRP family, consisting of 25 members, was also incorporated into this investigation. Genetic linkage analysis facilitated the mapping of the disease phenotype in the two South African adRP families to a 10 cM interval on chromosome 17q22. This novel locus, designated RP17, is the eighth identified for adRP. Haplotype construction in the two kindreds, in conjunction with multipoint analyses subsequently fine mapped RP17 to a 1 cM region between microsatellite markers D17S1604 and D17S948. Although the two families are from ethnically diverse population groups, they share the same disease-associated haplotype spanning 12 cM, which suggests that the disorder may be caused by the same pathogenic mutation in the same gene. The positional cloning approach was utilised in an endeavour to identify the RP17 gene and an attempt was made to construct a physical map of the 1 cM critical region. A contig consisting of seven yeast artificial chromosome (YAC) clones was assembled using sequence-tagged-site (STS) content mapping. In order to close a gap in the YAC contig, a bacterial artificial chromosome (BAC) library was screened and the vectorette PCR technique was used to verify overlapping sequences. This contig should provide a useful tool for the purpose of isolating genes or transcription units within the RP17 critical interval. In this regard, purified YAC DNA was isolated using pulsed-field gel electrophoresis and the cDNA selection technique was employed to generate a transcription map. This approach was applied to YAC 75Ic12 using a foetal brain cDNA library, and two rounds of selection were performed to create a sub-library for enriched cDNAs derived from this clone. Screening for the presence of contaminating sequences in the 480 transformants revealed that (i) approximately 7% of the selected clones contain COT-1 DNA and (ii) none of the clones were contaminated with yeast AB1380 DNA. Ten randomly chosen clones were sequenced and subjected to BLASTN analysis, which revealed the presence of a 23 bp contaminant, known genes as well as novel transcripts. In order to optimise efforts to isolate the adRP gene, four positional candidates residing on 17q were screened for evidence implicating them in the adRP phenotype in the two 17q22-linked families. The genes investigated were: PDEG (gamma subunit of rod phosphodiesterase), TIMP2 (tissue inhibitor of metalloproteinases-2), PKCA (protein kinase C alpha) and retinal fascin. These candidates were chosen on the basis of (i) mapping to 17q, (ii) expression in the retina and/or (iii) potential involvement in the rod phototransduction pathway. Recombination events between the adRP locus and a single strand conformation polymorphism (SSCP) in PDEG, and a restriction fragment length polymorphism (RFLP) in TIMP2 provided evidence for the exclusion of these candidate genes. A novel SSCP detected in the promoter region of retinal fascin was genotyped in the two adRP families and showed a lack of co-segregation with the disease locus. Furthermore, direct DNA sequencing of the coding regions as well as the promoter region of retinal fascin in RP affected family members did not reveal any pathogenic mutations. In addition, data is provided which suggests that PKCA does not reside on any of the YACs and BACs encompassing the RP17 critical interval. This gene is therefore unlikely to be responsible for the adRP phenotype in the two RP17-linked families. Ultimately, the work reported in this thesis may contribute to the body of knowledge on inherited retinal degenerative disorders. Moreover, this investigation should provide the basis for further study of the aetiology of RP in all families linked to the RP17 locus on chromosome 17q22. The immediate application of these molecular findings is the potential for pre-symptomatic testing of at-risk members from the two adRP kindreds.
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Ugarte, Chicote Javier. "Evolution and molecular characterization of uroplakin 3c, a novel human tetraspanin associated uroplakin protein." Doctoral thesis, Universitat Rovira i Virgili, 2016. http://hdl.handle.net/10803/387313.
Full textAbstract:
Les uroplaquines (UPKs) són proteïnes integrals de membrana que formen les plaques de l’uroteli. Tot i que aquestes plaques es troben solament a l’uroteli de mamífers, les UPKs existeixen també en vertebrats inferiors que els hi manca l’uroteli. Aquestes poden dividir-se en dos tipus, les UPK1a i UPK1b, pertanyent a la família de les tetraspanines, i les UPK2/3, composades per UPK2, UPK3a i UPK3b, les quals travessen la membrana una sola vegada i es desconeix la relació evolutiva que pugen tindre amb altres famílies de proteïnes.
Nosaltres, hem identificat 3 nous ortòlegs de les UPK2/3 (UPK3c, expressada en humans, UPK2b i UPK3d); hem identificat l’origen de les UPKs en l’ancestre comú de vertebrats i hem establert una relació evolutiva entre les UPK2/3s i el més antic dels receptors de tirosina fosfatasa (R3 PTPRs). Analitzant l’expansió dels loci genètics de l’UPK3c i les duplicacions segmentals que van tenir lloc en el goril•la, ximpanzé i humà, hem trobat que, possiblement, la transposició intracromosomal d’una de les duplicacions segmentals va ser a través d’un intermediari circular.
Mitjançant l’ús de tècniques de biologia molecular, hem trobat que l’UPK3c s’expressa en un ampli rang de teixits, principalment a la còrnia; que està N-glicosilada en còrnia humana, pterígion i a tumors de bufeta; així com, que es localitza principalment a les capes basal i intermèdia de l’uroteli de ratolí però es absent a les plaques de l’uroteli. En tumors de bufeta humà, l’expressió de l’UPK3c està augmentada en tumors de baix grau (9/11), mentre que en aquells d’alt grau (6/11) o invasius (3/9) està disminuïda.Las uroplaquinas (UPKs) son unas proteínas integrales de membrana que forman las placas del urotelio. Indistintamente de que estas placas se encuentran solamente en el urotelio de mamíferos, las UPKs también existen en vertebrados inferiores que carecen de urotelio. Las UPKs pueden ser divididas en dos tipos, por un lado UPK1a y UPK1b, quepertenecen a la familia de las tetraspaninas y por otro lado las uropalkinas UPK2/3 (UPK2, UPK3a y UPK3b), que atraviesan la membrana solo una vez y de las cuales se desconoce que estén relacionadas evolutivamente con otras familias de proteínas. Nosotros, hemos identificado 3 nuevos ortólogos de las UPK2/3 (UPK3c que se expresa en humanos, UPK2b y UPK3d); localizado el origen de las UPKs en el ancestro común de vertebrados; encontrado una relación evolutiva entre UPK2/3s y llas tirosina fosfatasas (R3 PTPRs) que son proteínas más antiguas;analizado la expansión de los loci genéticos de UPK3c y las duplicaciones segmentales que tuvieron lugar en gorila, chimpancé y humano, y encontrado que posiblemente la transposición intracromosomal de una de las duplicaciones segmentales se produjese a través de un intermediario circular. Mediante el uso de técnicas de biología molecular, hemos encontrado que UPK3c se expresada en un amplio rango de tejidos huamnos predominantemente en cornea. Además, hemos observado que UPK3c está N-glicosilada en cornea humana, pterigion y en tumores de vejiga; que UPK3c se localiza predominantemente en las capas basal e intermedia del urotelio de ratón y no se encuentra en las placas del urotelio. En muestras de tumores de vejiga humanos, pudimos observar que el numero de tumores que expresan UPK3c es mayor en tumores superficiales de bajo grado (9/11) y disminuye en los tumores de alto grado (6/11) y en los invasivos (3/9).
Uroplakins (UPKs) are integral membrane proteins that form the urothelial plaques. Despite the fact that these plaques are unique to the urinary bladder of mammals, UPKs also exist in lower vertebrates without a typical urothelium. Uroplakins can be divided into two types. UPK1a and UPK1b, which belong to the tetraspanin family and UPK2/3 uroplakins that comprises UPK2, UPK3a and UPK3b, span the membrane only once and are not known to be related to any other protein family. We have identify three novel UPK2/3 ortholog groups (UPK3c that is expressed in humans, UPK2b and UPK3d), report the origin of UPKs in the common ancestor of vertebrates and the evolutionary relationship between UPK2/3s and the more ancient tyrosine phosphatase receptors (R3 PTPRs). The joint expansion of the UPK3c genetic loci and segmental duplications in the gorilla, chimpanzee and homo and we found that intrachromosomal transposition of one of these segmental duplication could have been produced by a circular intermediate Using molecular biology techniques we found UPK3c expression widespread in human tissues, particularly strong in corneal tissue. Additionally, UPK3c is N-glycosilated in human normal corneal tissue, pterygium and bladder tumors; localizes predominately to the basal and intermediate cell layers of the mouse urothelium and is absent in the urothelial plaques. Finally in human bladder cancer the number of superficial low grade tumors expressing UPK3c assessed by immunoblot was higher than in superficial superficial high grade tumors or invasive tumors .
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Collins-De, Peyer Laurence. "Screening of a rat thymus and a human hippocampus cDNA library for a novel fyn-related oncogene." Thesis, Hong Kong : University of Hong Kong, 1999. http://sunzi.lib.hku.hk/hkuto/record.jsp?B21253870.
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Kharalkar, Shilpa S. "Identification of Novel Allosteric Regulators of Human Erythrocyte Pyruvate Kinase." VCU Scholars Compass, 2006. http://hdl.handle.net/10156/2083.
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Garami, Elizabeth. "Initial characterization of a novel human retinal gene encoding a putative Pleckstrin homology domain protein." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1995. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/MQ45438.pdf.
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Chen, Bin, and 陈斌. "Structural and functional characterization of human APPL2, a novel adaptor protein involved in insulin signaling." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B4552757X.
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Li, Hung-sing, and 李鴻陞. "Identification of polycomb group protein CBX8 as a novel tumor suppressor in human colorectal cancer." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/197534.
Full textAbstract:
Polycomb group (PcG) proteins governs the regulation of diverse cellular functions, such as cell fate decision, cell cycle progression, maintenance of embryonic stem cell pluripotency, and DNA damage repair. Although aberrant expression of PcG proteins has been frequently reported in different cancer types, CBX8 is one of the least studied PcG family members in cancer. Recently, a study showed that forced expression of CBX8 in normal human and mouse fibroblasts demonstrated that cells could bypass senescence via INK4a-ARF repression; while another report demonstrated that CBX8 was involved in MLL-AF9-linked leukemogenesis. Despite accumulating evidence on CBX8-related carcinogenic functions, the role of CBX8 in solid cancers has not been investigated thus far. This study is therefore initiated to investigate and establish the functional role of CBX8 in colorectal cancer.
In this study, expression of CBX8 in 121 pairs of human CRC samples was analyzed by immunohistochemistry; and data were correlated with different clinicopathological parameters. To evaluate the functional effects of CBX8, CBX8 overexpressed and downregulated clones were established from three CRC cell lines. The in vitro effects of CBX8 on cell proliferation, cell cycle progression and apoptosis profiles were investigated; and the effects of CBX8 on tumorigenicity in vivo were further demonstrated in mice xenograft models.
The results showed that CBX8 expression was downregulated or loss in approximately 48.8% of human colorectal tumors, and downregulated or loss of CBX8 expression were mainly observed in tumors with intermediate to later stages (stage II to IV). Moreover, expression of CBX8 showed a significant inverse correlation with colorectal tumor sizes (P < 0.0001). Ectopic expression of CBX8 in CRC cell lines resulted in inhibition of cell proliferation, clonogenic ability and anchorage-independent growth, which are hallmarks of tumorigenesis. Conversely, downregulation of CBX8 promoted proliferation and clonogenic ability. Moreover, it was found that restoring CBX8 expression could induce G0/G1 arrest of cell cycle. The tumor suppressive role of CBX8 in colorectal cells was further demonstrated in vivo through subcutaneous and orthotropic mice tumor models; followed by immuno-staining of the proliferation marker Ki-67. To unveil the possible mechanisms behind the tumor suppressing effects of CBX8, two signalling pathways commonly engaged in CRC were evaluated. At least part of the effects could be attributed to the mediation of MAPK signaling pathway; whereas the Wnt signalling was not affected by CBX8.
This study demonstrated for the first time the loss of CBX8 expression in intermediate and late stage tumors, and was the first to report the tumor suppressing ability of CBX8 in solid cancers. The effects of CBX8 in this study were different to the functional implications reported in the current literature. This functional divergence in distinct cell types suggested a dynamic role of CBX8 depending on specific cellular context.published_or_final_version
Surgery
Master
Master of Philosophy
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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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Crosby, David. "Cross-talk between tyrosine kinases and members of the protein kinase C family in human platelets." Thesis, University of Bristol, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.288410.
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Laitinen, Saara. "Family of human oxysterol binding protein homologues : ORP2 is a new regulator of cellular lipid metabolism." Helsinki : University of Helsinki, 2002. http://ethesis.helsinki.fi/julkaisut/laa/kansa/vk/laitinen/.
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Mills, Elena Claire. "Characterisation of the trypanosomatid PPEF-like phosphatases : novel members of the RDGC/PP5-related protein phosphatase family." Thesis, Imperial College London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.423156.
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Bromley, Elizabeth Verity. "Characterisation of a novel protein belonging to the WD repeat family in the protozoan parasite Trypanosoma cruzi." Thesis, London School of Hygiene and Tropical Medicine (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.289740.
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Weir, Marion. "Novel Mechanisms Governing Autoregulation of the Src Family Kinase Fyn and its Crosstalk with Protein Kinase A." ScholarWorks @ UVM, 2016. http://scholarworks.uvm.edu/graddis/592.
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ABSTRACT
Phosphorylation is a post-translational modification important for regulating protein activity and protein binding capacity. It is used in many different signaling pathways within the cell. Src Family Kinases and Protein Kinase A (PKA) are two prototyptical non-receptor tyrosine and serine/ threonine kinases, respectively, which are found in canonical signaling pathways. These two kinases are critical for signaling in essentially every cell of a multicellular organism, and are particularly important in development, cell migration and proliferation. Although both proteins have been intensely studied for many decades, an understanding of the molecular mechanisms which govern their regulation and the regulation that they effect on other proteins are still being elucidated.
Fyn, like its related Src Family Kinase members, has previously been shown to be regulated by two tyrosine phosphorylation events at residues Y420 and Y531. Y420 is located in the kinase (Src Homology 1(SH1)) domain and it is a highly-characterized intermolecular autophosphorylation site that increases the activity of the kinase. Y531 is located near the C-terminus and is phosphorylated by C-terminal Src kinase (Csk). Phosphorylation of Y531 allows it to bind to R176 in the SH2 domain in an intramolecular fashion. In this conformation Fyn has only basal activity. Since these sites are essential for regulating the activity of the kinase, we hypothesized that four novel sites of tyrosine phosphorylation in Fyn could also importantly regulate the protein. Three of the novel sites lie in the SH2 domain, and one is located in the kinase domain. Mass spectrometry, in vitro kinase assays, as well as western blot analysis aided in uncovering that these novel Fyn phosphorylation sites fine tune the activity and substrate binding of the protein.
PKA has been implicated in a multitude of signaling pathways and is particularly important in cell growth, proliferation, and migration. Fyn and PKA have classically been considered to be in separate signaling pathways. However, research over the past several decades has provided evidence that there is crosstalk that exists between the two pathways. The SFK Fyn and PKA can phosphorylate each other, thereby regulating each other's activity. Based on these data, we hypothesized the existence of downstream effectors of this relatively uncharacterized pathway. It was hypothesized that the presence of Fyn could lead to PKA activation and to differences in PKA binding partners. Through the use of co-immunoprecipitations, Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) and quantitative mass spectrometry, many proteins were found to increase their binding to PKA in the presence of Fyn. Several proteins were selected and further biochemically validated. These data suggest that the presence of Fyn could allow for PKA to more importantly interact with discrete pools of proteins within the cell to effectuate its signal transduction. Together these studies provide understanding on critical and fundamental processes by which all cells function.
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Chen, Chao-Min Amy. "Identification and characterization of I-mf, a novel myogenic repressor that interacts with MyoD family members /." Thesis, Connect to this title online; UW restricted, 1996. http://hdl.handle.net/1773/5060.
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Adair, Richard. "Investigation of protein products encoded by the human cytomegalovirus US22 family genes UL23, UL24, UL43, and US22." Thesis, University of Glasgow, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.392456.
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McKay, Paul Francis. "Cloning of the cDNA for gp200-MR6 : a novel member of the human macrophage mannose receptor family." Thesis, Imperial College London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.369208.
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Shikhagaie, Medya. "Characterization of UL1, a member of the human cytomegalovirus RL11 gene family." Doctoral thesis, Universitat Pompeu Fabra, 2011. http://hdl.handle.net/10803/53580.
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In the present study, we have approached the molecular characterization of the HCMV specific UL1. To this end a HCMV (AD169-derived HB5 background) recombinant with an HA-epitope tagged UL1 and a mutant with a full UL1 deletion in the endotheliotropic HCMV TB40/E strain were generated. Our data reveal that the UL1 is transcribed with late kinetics. pUL1 is glycosylated and localizes at the site of virus assembly and secondary envelopment in infected cells forming part of the envelope of HCMV virions. A HCMV mutant with a targeted deletion of UL1 exhibits a growth defect phenotype in retinal pigment epithelium cells but not in fibroblasts, indicating that this ORF encodes a cell-type specific tropism factor.En aquest treball hem investigat la pauta oberta de lectura de UL1 del Cytomegalovirus humà (HCMV), el gen UL1 es específic del HCMV. Hem caracteritzat la proteïna UL1 modificada amb un epítop HA en la soca HB5, derivada de AD169. L'UL1 s’expressa com una glicoproteïna que es pot detectar a les 48 i 72h post-infecció. En fibroblasts humans infectats, UL1 co-localitza al citoplasma, al lloc d’assemblatge del virió, amb proteïnes estructurals del virus. A més a més, els anàlisis de virions AD169 purificats que contenen UL1-HA mostren que UL1 és un nou constituent de l’envolta del HCMV. La delecció de UL1 en el context de la soca TB40/E del HCMV disminueix el creixement viral de manera selectiva en determinats tipus cel•lulars, suggerint que UL1 podria estar involucrat en la regulació del tropisme cel•lular del HCMV.
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Shobako, Naohisa. "Identification and characterization of a novel anti-hypertensive peptide derived from rice bran protein." Kyoto University, 2019. http://hdl.handle.net/2433/242924.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第21973号
農博第2363号
新制||農||1071(附属図書館)
学位論文||R1||N5224(農学部図書室)
京都大学大学院農学研究科食品生物科学専攻
(主査)教授 井上 和生, 教授 谷 史人, 准教授 大日向 耕作
学位規則第4条第1項該当
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Khan, Emad Ali Flood Patrick M. "Characterization of a novel protein - 3D10 - a secreted receptor form of the human osteoclast-associated receptor OSCAR." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2007. http://dc.lib.unc.edu/u?/etd,1374.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.Title from electronic title page (viewed Apr. 25, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the School of Dentistry Curriculum in Oral Biology." Discipline: Oral Biology; Department/School: Dentistry.
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Wingate, Ianthe. "Identification of potential novel roles for Hsp70/Hsp90 organising protein (Hop) using proteomic analysis in human cells." Thesis, Rhodes University, 2016. http://hdl.handle.net/10962/64758.
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Lehman, Jason Alexander. "Novel Redox and DNA-Dependent Conformational Changes in Human Ku, a DNA-Double Strand Break Repair Protein." Wright State University / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=wright1211323245.
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Bryce, Steven David. "Identification and characterisation of a novel family of human genomic sequences closely related to the Cathepsin L gene." Thesis, University of Newcastle Upon Tyne, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.309763.
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Williams, Marni. "Biochemical and structural characterization of novel drug targets regulating polyamine biosynthesis in the human malaria parasite, Plasmodium falciparum." Thesis, University of Pretoria, 2011. http://hdl.handle.net/2263/26237.
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Malaria is prevalent in over 100 countries which is populated by half of the world’s population and culminates in approximately one million deaths per annum, 85% of which occurs in sub-Saharan Africa. The combined resistance of the mosquitoes and parasites to the currently available pesticides and antimalarial chemotherapeutic agents requires the concerted effort of scientists in the malaria field to identify and develop novel mechanisms to curb this deadly disease. In this study, a thorough understanding of the role players in the polyamine pathway of the parasite was obtained, which could aid future studies in the development of novel inhibitory compounds against these validated drug targets. The uniquely bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase (AdoMetDC/ODC) of Plasmodium falciparum forms an important controlling node between the polyamine and methionine metabolic pathways. It has been speculated that the unique bifunctional association of the rate-limiting enzymes allows for the concerted regulation of the respective enzyme activities resulting in polyamine synthesis as per requirement for the rapidly proliferating parasite while the methionine levels are strictly controlled for their role in the methylation status. The results of this study showed that the enzyme activities of the bifunctional complex are indeed coordinated and subtle conformational changes induced by complex formation is suggested to result in these altered kinetics of the individual AdoMetDC and ODC domains. Studies also showed that the identification of the interaction sites between the domains, which allows for communication across the complex, may be targeted for specific interference with the enzyme activities. Furthermore, these studies showed that the current knowledge on the different subclasses of the AdoMetDC family should be re-evaluated since P. falciparum AdoMetDC shows diverse properties from orthologues and therefore points towards a novel grouping of the plasmodial protein. The extensive biochemical and biophysical studies on AdoMetDC has also provided important avenues for the crystallisation and solving of this protein’s 3D structure for subsequent structure-based identification of drug-like lead compounds against AdoMetDC activity. The application of structure-based drug design on malarial proteins was additionally investigated and consequently proved that the rational design of lead inhibitory compounds can provide important scaffold structures for the identification of the key aspects that are required for the successful inhibition of a specific drug target. Spermidine synthase, with its intricate catalytic mechanism involving two substrate binding sites for the products of the reactions catalysed by AdoMetDC/ODC, was used to computationally identify compounds that could bind within its active site. Subsequent testing of the compounds identified with a dynamic receptor-based pharmacophore model showed promising inhibitory results on both recombinant protein and in vitro parasite levels. The confirmation of the predicted interaction sites and identification of aspects to improve inhibitor interaction was subsequently investigated at atomic resolution with X-ray protein crystallography. The outcome of this doctoral study shows the benefit in applying a multidisciplinary and multinational approach for studying drug targets within the malaria parasite, which has led to a thorough understanding of the targets on both biochemical and structural levels for future drug design studies.Thesis (PhD)--University of Pretoria, 2011.
Biochemistry
unrestricted
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Wang-Heaton, Hui. "A novel role of human DNA damage checkpoint protein ATR in suppressing Ca2+ overload-induced PARP1-mediated necrosis." Digital Commons @ East Tennessee State University, 2016. https://dc.etsu.edu/etd/3171.
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Ataxia telangiectasia and Rad3-related (ATR) is well known for its regulatory role in DNA damage responses (DDR) as a checkpoint kinase that phosphorylates hundreds of protein substrates. However, its role in cellular non-DNA damage stress responses (NDDR) is unknown. Necrosis is one form of cell death and traditionally has been regarded as a passive and uncontrolled cell death. Recently, evidence has emerged to support the concept that necrosis also may occur in a programmed manner and that PARP1 can be a mediator. Active poly (ADP-ribose) polymerase 1 (PARP1) hydrolyzes nicotinamide adenine dinucleotide (NAD+) to produce poly (ADP-ribose) (PAR) polymers on target proteins or itself. As a result, hyper-activity of PARP1 may lead to necrosis by excessively depleting ATP pool which results in mitochondrial energetic collapse. On the other hand, it is known that Ca2+ overload induces necrosis, but much still remains unknown about how Ca2+ overload-induced necrosis is regulated in cells. In this study, we show that ATR, besides its hallmark regulatory role in DDR, also plays a role in NDDR by suppressing ionomycin-induced necrosis. Ionomycin as a Ca2+ ionophore can dramatically raise the intracellular level of Ca2+, leading to necrosis. We found that this Ca2+ overload-induced necrosis occurs without inducing DDR in cells. Instead, the hyper-poly(ADP-ribosyl)ation (PARylation) activity of activated PARP1 could be a reason leading to necrosis, as NAD+ supplied to media can rescue ionomycin-induced necrosis. In vitro PARylation assay also demonstrates that PARP1 hyper-activation is Ca2+ dependent. In cells, ATR-PARP1 interaction happened after ionomycin treatment. Furthermore, ionomycin treatment induces more full-length PAR polymers formed in ATR-deficient cells than in ATR-proficient cells. The interaction of kinase-dead ATR and PARP1 dramatically decreased as compared to wild-type ATR. Therefore, ATR plays a novel role in NDDR wherein it is able to suppress Ca2+ overload-induced PARP1-mediated necrosis. Ca2+ overload-induced cell death is a major cause of many human medical conditions and diseases, such as brain injury, stroke and ischemia et al. Our ongoing studies will help to define the molecular mechanisms of the anti-necrosis activities of ATR, which may support ATR as a new clinical target for therapeutic treatment of those diseases.