Дисертації: "PfpI Family Member Protein"

1

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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2

Hu, Bin. "Identification of the paralemmin protein family and initial characterization of palmdelphin, a member of the family." [S.l.] : [s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=964992736.

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3

Kan, Ming-Chung. "Analysis of CPEB Family Protein Member CPEB4 Function in Mammalian Neurons: A Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/362.

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Local protein synthesis is required for long-term memory formation in the brain. One protein family, Cytoplasmic Polyadenylation Element binding Protein (CPEB) that regulates protein synthesis is found to be important for long-term memory formation possibly through regulating local protein synthesis in neurons. The well-studied member of this family, CPEB1, mediates both translational repression and activation of its target mRNAs by regulating mRNA polyadenylation. Mouse with CPEB1 KO shows defect in memory extinction but not long-term memory formation. Three more CPEB1 homologs (CPEB2-4) are identified in mammalian system. To test if CPEB2-4 may have redundant role in replacing CPEB1 in mediating local protein synthesis, the RNA binding specificity of these homologs are studied by SELEX. The result shows CPEB2-4 bind to RNAs with consensus sequence that is distinct from CPE, the binding site of CPEB1. This distinction RNA binding specificity between CPEB1 and CPEB2-4 suggests CPEB2-4 cannot replace CPEB1 in mediating local protein synthesis. For CPEB2-4 have distinct RNA binding specificity compared to CPEB1, they are referred as CPEB-like proteins. One of CPEB-like protein, CPEB3, binds GluR2 mRNA and represses its translation. The subcellular localization of CPEB family proteins during glutamate over stimulation is also studied. The CPEB family proteins are identified as nucleus/cytoplasm shuttling proteins that depend on CRM1 for nuclear export. CPEB-like proteins share similar nuclear export ciselement that is not present in CPEB1. Over-stimulation of neuron by glutamate induces the nuclear accumulation of CPEB family proteins possibly through disrupted nuclear export. This nuclear accumulation of CPEB family protein is induced by imbalance of calcium metabolism in the neurons. Biochemical and cytological results suggest CPEB4 protein is associated with ER membrane peripherally in RNA independent manner. This research provides general description of biochemical, cytological properties of CPEB family proteins.

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4

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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5

Najmabadi, Sepideh. "Drosophila model of myosin myopathy rescued by overexpression of a TRIM-protein family member." Thesis, Högskolan i Skövde, Institutionen för hälsa och lärande, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-17919.

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Laing distal myopathy is inherited in an autosomal dominant manner usually before the age of five that initially involves the dorsiflexion in the ankles’ and in big toes to the finger extensors. Weakness of the flexor muscles in the neck is seen in most affected individuals and mild facial weakness is also often present. Hypertrophic or dilated cardiomyopathy, starting at birth to respectively second or third decade of life, is the symptom in the affected humans.This study performed on Drosophila melanogaster, has evaluated whether feeding MuRF1 enzyme (which has a similar role as ABBA enzyme) to Drosophila larvae, in different concentrations, will have a positive effect on the larvae’s muscular abilities through an analysis of their manifestation, the distance they manage to crawl and the time it takes for them to turn from a ventral up to dorsal up position.The result show no significant impact on larvae ability to turn or crawl between different groups fed with MuRF1 enzyme, nor between the two control groups, wild larvae and mutated larvae. Other studies have proven that there is a significant difference in muscular ability between wild and mutated larvae, so explanations to why this study did not manage to replicate these results were evaluated. The study found that how many days has passed since hatching has a significant impact on performance of turning and crawling for wild larvae that are not treated with enzyme.There are a number of improvement suggestions to the experimental design and the methodology to enable a proper evaluation of the research aim of this thesis. Future research on the topic should implement these and redo the experiments and measurements of this study. In addition, the quantity of larvae that reaches pupa stage should be captured to evaluate whether the MuRF1 enzyme has a positive impact on mutated larvae reaching pupation stage. The most important parts of the improvement proposals to measure the ability of larvae when they are about the same age, as this was proven with statistical significance to have an impact on crawling and turning.

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6

Lacy, Susan E. "A structural and functional analysis of cyclin interactions with the retinoblastoma protein family member P130." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0020/NQ30099.pdf.

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7

Przyborski, Jude Marek. "Analysis of protein trafficking signals of a member of the P. falciparum stevor multi-gene family." Thesis, University of Liverpool, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.404839.

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8

Dolniak, Blazej. "Functional characterisation of NIC2, a member of the MATE family from Arabidopsis thaliana (L.) Heynh." Phd thesis, [S.l.] : [s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=975992767.

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9

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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10

Blech-Hermoni, Yotam. "Roles of CUG-BP, Elav-Like Family Member 1 (CELF1), an RNA Binding Protein, During Vertebrate Heart Development." Case Western Reserve University School of Graduate Studies / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=case1417636826.

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11

Crombie, Andrea Rene. "Lysosomal integral membrane protein II, a member of the CD36 gene family : comparative analysis of structure-function relationships /." Access full-text from WCMC, 1998. http://proquest.umi.com/pqdweb?did=733079741&sid=9&Fmt=2&clientId=8424&RQT=309&VName=PQD.

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12

Einheber, Steven. "Isolation and characterization of acDNA clone encoding avian skeletal muscle C-protein : an intracellular member of the immunoglobulin superfamily /." Access full-text from WCMC, 1989. http://proquest.umi.com/pqdweb?did=744115441&sid=1&Fmt=2&clientId=8424&RQT=309&VName=PQD.

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13

DALL'ARMI, CLAUDIA. "The role of Aip4/Itch, a member of Nedd4 family E3 ligases, in the ubiquitination of proteins involved in RTKs endocytosis." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2006. http://hdl.handle.net/2108/240.

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ABSTRACT E’ stata utilizzata una nuova tecnica di approccio genomico (WISE: Whole Interactome Scanning Experiment) per cercare, nell’intero proteoma umano, proteine contenenti peptidi in grado di interagire con i domini SH3 dell’anfifisina I e dell’endofilina, due proteine coinvolte nel traffico vescicolare. Dall’analisi dei risultati sono emersi degli interattori noti quali, la dinamina e la sinaptojanina e dei nuovi possibili interattori (Langraf et al.; 2004). Tra questi ultimi è stata focalizzata l’attenzione sulla proteina Aip4/Itch, una E3 ligasi appartenente alla famiglia di Nedd4, in quanto negli ultimi anni è stato dimostrato che l’ubiquitinazione ha un ruolo sempre più importante per la regolazione del traffico vescicolare. E’ stata confermato l’interazione interazione, in vitro, tra Itch e anfifisina, dimostrando che l’amfifisina I co-immunoprecipita con Itch, in vivo; tuutavia, nonostante il forte legame l’anfifisina non viene ubiquitinata da Itch. Questo fenomeno ha fatto supporre che il legame anfifisina-Itch possa promuovere la localizzazione e l’attività enzimatica di Itch a livello delle vescicole endocitiche e dei compartimenti endosomali. Alla luce di questa ipotesi sono state testate sistematicamente diverse proteine coinvolte nell’endocitosi dei recettori tirosin chinasici per verificare quali potevano essere bersaglio dell’attività ubiquitinante di Itch, sia in vivo che in vitro. Alla fine di questo lavoro di tesi è stato dimostrato che Itch è implicata nel legame e nell’ubiquitinazione di tre proteine: Hrs, STAM2 e Eps15. Queste tre proteine sono le costituenti di un etero-complesso multivalente essenziale per lo smistamento di proteine ubiquitinate nei diversi compartimenti vescicolari, in particolare sono implicate nel traffico vescicolare tra gli endosomi tardivi e i lisosomi.
ABSTRACT We have used a recently developed genomic approach (WISE: Langraf et al.; 2004), to search the whole proteome for proteins containing peptides that could bind to the SH3 domains of amphiphysin I and endophilin, two proteins implicated in vesicle trafficking. Among the inferred candidate ligands we focused on the protein Itch, a HECT domain ubiquitin ligase that was recently shown to participate in ubiquitination processes affecting the internalization of the EGF receptor. Itch can be co-immunoprecipitated with Amphiphysin I, in vivo, suggesting that this interaction could promote the enzymatic activity of Itch in endocytic vesicles and endosome compartments. We are systematically testing a number of proteins involved in receptor endocytosis to see whether they are targets of the ubiquitination activity of Itch, in vivo or in vitro. We demonstrated that Itch is implicated in the binding and ubiquitination of three proteins HRS, STAM2, Eps15 that form a multivalent complex essential in sorting ubiquitinated

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14

Nodzon, Lisa A. "A member of the Arabidopsis thaliana XBAT family of ubiquitin protein ligases, XBAT32, is a positive regulator of lateral root development." [Gainesville, Fla.] : University of Florida, 2005. http://purl.fcla.edu/fcla/etd/UFE0013082.

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15

Low, Christopher Gah-Mun. "The role of BIRC6, a member of the inhibitor of apoptosis protein (IAP) family, in the survival of human prostate cancer cells." Thesis, University of British Columbia, 2010. http://hdl.handle.net/2429/29564.

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Prostate cancer is the most commonly diagnosed cancer and third leading cause of cancer deaths in Canadian men. Prostate cancers typically begin as androgen-dependent tumours susceptible to growth arrest/apoptosis induced by ablation of androgens. Although initially effective, androgen ablation frequently leads to the development of castration-resistant (androgen-independent) prostate cancer, which is generally also resistant to other available treatments. Development of castration-resistant prostate cancer is characteristically associated with marked increases in resistance to apoptosis. BIRC6 is a member of the Inhibitors of Apoptosis Protein (IAP) family which protects a variety of cancer cell lines from apoptosis. In the present study, we have investigated whether BIRC6 plays a role in prostate cancer and could potentially be useful as a novel therapeutic target. Analysis of a variety of human prostate cancer cell lines and clinical specimens for BIRC6 protein expression, using Western blot and immunohistochemical analyses, respectively, showed that BIRC6 protein is markedly expressed by the prostate cancer cell lines and by clinical cancer specimens, as distinct from benign prostate cells/tissue. In addition, analysis of the clinical specimens showed that elevated BIRC6 protein expression was found to be particularly associated with cancers of Gleason score 6-8 and with the development of castration-resistant disease. Specific, siRNA-induced reduction of BIRC6 expression in LNCaP cells led to a marked reduction in cell proliferation, associated with an increase in apoptosis markers and a decrease in autophagosome markers, indicating that BIRC6 plays a major protective role in the proliferation of LNCaP cells by inhibiting apoptosis and perhaps by enhancing autophagy. Taken together, the data suggest an important role for BIRC6 in prostate cancer growth and progression, particularly, in the development of treatment resistance. In conclusion, this study indicates - for the first time - that the BIRC6 gene and its product are potentially valuable targets for therapy of human prostate cancers. BIRC6-targeting drugs may be especially useful for sensitization of cancer cells in combination therapy.

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16

Curtis, David Floyd. "A Member of the Novel FIKK Family of Plasmodium falciparum Putative Protein Kinases Exhibits Diacylglycerol Kinase Activity and Is Exported to the Host Erythrocyte." Master's thesis, University of Central Florida, 2007. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/2363.

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Plasmodium falciparum is one of four species known to cause malaria in humans and is the species that is associated with the most virulent form of the disease. Malaria causes nearly two million deaths each year, many of these occurring among children in under-developed countries of the world. One reason for this is the prevalence of drug resistant strains of malaria that mitigate the efficacy of existing drugs. Hence, the identification of a new generation of pharmacological agents for malaria is extremely urgent. The recent identification of a group of novel protein kinases within the Plasmodium falciparum genome has provided researchers with a basis for what many hope to be new potential drug targets for malaria. Identified within the Plasmodium genome and a few select apicomplexans, these novel proteins have been predicted to be protein kinases based solely on certain sequence features shared with other eukaryotic protein kinases (ePKs). However, to date, no significant studies to determine the function of these novel kinases have been performed. Termed FIKKs, these proteins all possess a non-conserved N-terminal sequence that contains a Plasmodium export element (Pexel) which may target the proteins for export from the parasite and a conserved C-terminal catalytic domain containing a FIKK sequence common to all twenty members of this family. We analyzed the localization of one of the FIKK proteins, FIKK11, encoded by the PF11_0510 locus, during intraerythrocyte differentiation of P. falciparum by Western blot analysis and indirect immunofluorescence assay. Western blot analysis demonstrated that FIKK 11 is expressed within the parasite at all stages of its erythrocytic life cycle with its highest expression occurring during the schizont stage. Immunofluorescence assays showed that this protein is exported from the Plasmodium parasite into the host erythrocyte cytosol which is consistent with studies on other Plasmodium proteins that also have the Pexel motif. To determine the enzymatic activity of FIKK11, we overexpressed the recombinant protein in E. coli and then purified it. However, no protein kinase activity was detected using several commonly used protein kinase substrates including histone H1, myelin basic protein, or dephosphorylated casein. We also did not detect any kinase activity of the native enzyme using pull-down assays of the Plasmodium falciparum cell extract against those same substrates. In addition, kinase substrate peptide array analysis of FIKK11 showed no evidence of protein kinase activity either for FIKK11. Interestingly, however, we were able to detect some kinase activity using the recombinant protein alone with no substrate. The lack of the glycine triad within subdomain I of these FIKK kinases as compared with most traditional eukaryotic protein kinases may explain why we were unable to find any interactions between FIKK11 and other commonly protein kinase substrates. Of interest was the observation that the protein reproducibly exhibited what appeared to be an autophosphorylation activity when using the standard protein kinase assay. Further analyses, however, showed that FIKK11 actually possesses diacylglycerol kinase activity utilizing 1-Stearoyl-2-arachidonoyl-sn-glycerol as a substrate. This is the first evidence of diacylglycerol kinase activity in Plasmodium falciparum. Because FIKK11 is exported into the host cell and is localized on the erythrocyte membrane, its enzymatic activity may potentially have relevance in the pathophysiology of the disease.
M.S.
Department of Molecular Biology and Microbiology
Burnett College of Biomedical Sciences
Molecular and Microbiology MS

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17

Vavassori, Stefano. "Structural and functional characterization of human ERp44 : a closer look at a member of PDI family regulating protein quality control in the early secretory pathway." Thesis, Open University, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.522107.

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18

Desuzinges-Mandon, Elodie. "Rôle du domaine extracellulaire d’ABCG2 dans l’homéostasie des porphyrines." Thesis, Lyon 1, 2010. http://www.theses.fr/2010LYO10236/document.

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ABCG2 est un transporteur de la famille ABC impliqué dans le phénotype de résistance aux drogues développé par certaines cellules, par exemple les cellules cancéreuses. Ce transporteur a aussi un rôle physiologique de détoxication de composés endogènes, notamment les porphyrines, molécules indispensables mais qui présentent une toxicité potentielle. Cette toxicité nécessite une prise en charge particulière, évitant à ces composés d’être libres en solution. Dans ce contexte, nous avons fait l’hypothèse qu’ABCG2 pourrait participer à cette détoxication en limitant l’accumulation des porphyrines dans les cellules en les présentant à un partenaire extracellulaire. Nous montrons qu’ABCG2 transporte de l’hème ainsi que certains de ses dérivés et précurseurs et que ces porphyrines, contrairement aux autres substrats d’ABCG2, se fixent sur un domaine extracellulaire spécifique d’ABCG2, ECL3, composé d’environ 70 acides aminés. L’affinité d’ECL3 pour les porphyrines est de 0,5 à 3,5 μM, suffisamment affine pour permettre leur fixation après transport.Nous montrons aussi que l’albumine sérique humaine, impliquée dans la détoxication de l’hème, récupère les porphyrines fixées sur ECL3 par une interaction directe avec ABCG2. L’ensemble de ce travail a donc permis d’une part de mieux comprendre le rôle d’ABCG2 dans la régulation de l’homéostasie des porphyrines, notamment l’hème, et d’autre part, de façon originale, d’identifier le mécanisme moléculaire par lequel cette détoxication s’effectue
ABCG2 belongs to the ABC-transporter family, involved in drug resistance developed by cells, notably cancer cells. This transporter has also a physiological role of endobiotic detoxification, in particular porphyrins that are essential but potentially toxic molecules. This toxicity implies a specific handle, to avoid them to remain free in solution. In that context, we hypothesized that ABCG2 participate to this detoxification, limiting the intracellular porphyrin accumulation by presenting them to an extracellular partner. We show that ABCG2 transports heme and some of its derivatives and precursors. Interestingly, these porphyrins, unlike other ABCG2 (non-porphyric) substrates, can bind to an extracellular domain, specific of ABCG2, ECL3, 70 residues-long. ECL3 displays affinities for porphyrins in the range of 0.5 to 3.5 μM, high enough to allow their binding after transport. We also show that human serum albumin, implicated in heme detoxification, releases porphyrins bound to ECL3 by a direct interaction with ABCG2. This work established a better comprehension of ABCG2 role in porphyrin and in particular heme homeostasis regulation. In addition, our results contribute to elucidate part of the molecular mechanism by which such regulation is carried out

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19

Graf, Justin T. "Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variation." Thesis, Queensland University of Technology, 2008. https://eprints.qut.edu.au/25913/1/Justin_Graf_Thesis.pdf.

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This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation. In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation. The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity. Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes. This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.

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20

Graf, Justin T. "Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variation." Queensland University of Technology, 2008. http://eprints.qut.edu.au/25913/.

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This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation. In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation. The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity. Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes. This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.

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21

Correia, Patrícia Maria Dias. "Identification and characterization of potential therapeutic targets for spinal cord repair." Master's thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/22055.

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Mestrado em Biomedicina Molecular
Traumatic spinal cord injury (SCI) is a devastating event that leads to loss of neurological functions below the vertebral level of the lesion. As adult neurons from central nervous system (CNS) fail to regenerate when injured, the consequences of SCI are partially or totally irreversible. The lack of regeneration ability of CNS neurons has been studied for years but still no effective treatment was found for this pathology; only steroids are validated and recognized as a pharmacologic treatment attempt, but just limit the lesion extent. This work focused on finding putative candidate genes involved in regeneration that could be targeted for therapy. A bioinformatics analysis based on studies with rodent SCI models, where a regenerative treatment attempt was applied and functional recovery was observed, was performed and some common regulated genes were found in the analysed studies. KIF4A and MPP3 genes were highlighted for further experimental studies in a regenerative model: a rodent model of peripheral nervous system (PNS) injury, with crush or transection of the sciatic nerve. Our results demonstrated that KIF4A and MPP3 are expressed and regulated in the lesioned sciatic nerve and in the corresponding dorsal root ganglia (DRG). Moreover, these genes also showed protein distribution in spinal cord tissue sections, in sciatic nerve and in DRG cuts, revealing that they are neuronal specific. These results represent important remarks to instigate further studies regarding the role of these genes in regenerative processes of lesioned neuronal tissues and the possibility of becoming important therapeutic targets in spinal cord injuries or related pathologies affecting the spinal cord integrity
A lesão traumática da medula espinal é um evento devastador que leva à perda de funções neurológicas abaixo do nível vertebral da lesão. Devido à falta de capacidade regenerativa dos neurónios adultos do sistema nervoso central, quando lesionados, as consequências das lesões são parcial ou totalmente irreversíveis. A falta de capacidade de regeneração dos neurónios do SNC tem sido estudada há anos, mas ainda não foi encontrado um tratamento efetivo para esta patologia; apenas os esteroides são validados e reconhecidos como um tratamento farmacológico, mas só limitam a extensão da lesão. Este trabalho centrou-se na procura de genes hipoteticamente envolvidos em regeneração do sistema nervoso, que possam ser candidatos a alvos de terapia para lesões na medula. Foi realizada uma análise bioinformática baseada em estudos com modelos de roedores com lesão da medula espinal, onde uma tentativa de tratamento regenerativo foi aplicada e observou-se recuperação funcional, e foram levantados os genes regulados comuns aos três estudos. Os genes KIF4A e MPP3 foram destacados para estudos experimentais adicionais num modelo regenerativo: um modelo de roedor, de lesão do sistema nervoso periférico, com esmagamento ou corte do nervo ciático. Os resultados demonstraram que os genes KIF4A e MPP3 são expressos e regulados no nervo ciático lesionado e nos gânglios da raiz dorsal correspondentes. Além disso, estes genes também mostraram distribuição proteica em secções de tecido de medula espinhal, de nervo ciático e em cortes de DRG, desvendando que possam ser específicos de tecido neuronal. Estes resultados representam observações importantes para instigar estudos adicionais sobre o papel destes genes nos processos regenerativos de tecidos neuronais lesionados e a possibilidade de se tornarem alvos terapêuticos importantes para lesões ou patologias relacionadas que afetem a integridade da medula espinal.

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22

Marafona, Ana Marlene Neto. "The novel LAP1: TRF2 complex is associated to DNA damage." Master's thesis, Universidade de Aveiro, 2016. http://hdl.handle.net/10773/22000.

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Mestrado em Biomedicina Molecular
Lamin associated protein 1 (LAP1) is a type II integral membrane protein located at the inner nuclear membrane (INM). The role of LAP1 remains poorly understand, however, this protein has been associated with several cellular functions due to its interactions with lamins, phosphatase protein 1 (PP1), emerin and torsinA. Moreover, novel putative LAP1 interactors are emerging. A recent study from our group allowed the identification of several novel putative LAP1 interactors involved in telomere signaling and DNA damage responses, namely Ataxia-telangiectasia mutated (ATM), Telomeric repeat binding factor 2 (TRF2), Repressor Activator Protein 1 (RAP1), RAP1 interacting factor 1 homologue (RIF1), Mitotic arrest deficient-like1 (MAD2L1) and Mitotic arrest deficient-like1 binding protein (MAD2L1BP). Protein-protein interactions are crucial in the study of signaling pathways. In this study, TRF2 was identified as a novel LAP1 binding protein using both co-immunoprecipitations and mass spectrometry based methodologies. To determine the functional relevance of the novel complex LAP1:TRF2, HeLa cells were subjected to DNA damage using hydrogen peroxide (H2O2), namely double-stranded breaks (DSBs). In response to DSBs, the expression levels of LAP1 and TRF2 were significantly reduced. The phosphorylation of Histone 2A family member (γ-H2AX) that is considered the hallmark of DSBs was also evaluated. Upon DNA damage, LAP1 not only co-localizes with γ-H2AX in some specific points near nuclear envelope (NE) and nucleus, but also with TRF2 in the nuclear periphery. Moreover, LAP1 and TRF2 have been reported to be crucial for cell cycle progression. Therefore, we decided to pursued this issue. When the NE is reassembled, the complex is located mainly in specific regions of the NE, evidencing that TRF2 allows the attachment of chromosomes to NE membrane in somatic cells. In conclusion, our results are of paramount importance since novel functional insights regarding the novel LAP1:TRF2 complex were achieved particularly related with DNA damage response and cell cycle progression.
Proteína 1 associada com a lâmina (LAP1) é uma proteína integral da membrana do tipo II localizada na membrana nuclear interna (INM). O papel da LAP1 não é inteiramente sabido, no entanto esta proteína tem sido associada a algumas funções celulares devido às suas interações com as lâminas, proteína fosfatase 1 (PP1), emerina e torsinA. Além disso, novos putativos interactores da LAP1 estão a surgir. Um recente estudo do nosso grupo permitiu a identificação de vários novos putativos interactores da LAP1 envolvidos na sinalização dos telómeros e em respostas a danos no DNA, nomeadamente a mutação da ataxia telangiectasia (ATM), fator 2 de ligação às repetições teloméricas (TRF2), proteína 1 ativadora repressora (RAP1), fator homólogo 1 de interação com a RAP1 (RIF1), proteína 1 do checkpoint do fuso mitótico (MAD2L1) e a proteína de ligação à proteína 1 do checkpoint do fuso mitótico (MAD2L1BP). As interações proteína-proteína são cruciais no estudo das vias de sinalização. Neste estudo, a TRF2 foi identificada como uma nova proteína interatora da LAP1 utilizando tanto co-immunoprecipitação como metodologias baseadas em espectrometria de massa. Para determinar a relevância funcional do novo complexo LAP1:TRF2, células HeLa foram submetidas a danos no DNA através do peróxido de hidrogénio (H202), nomeadamente a quebras de DNA de cadeia dupla (DSBs). Em resposta a DSBs, os níveis de expressão da LAP1 e da TRF2 estavam significativamente reduzidos. A fosforilação do membro da família da histona 2A (γ-H2AX) que é considerado um biomarcador de DSBs foi também avaliada. Em resposta a danos no DNA, a LAP1 não só co-localiza com a γ-H2AX em alguns pontos específicos perto do invólucro nuclear (EN) e núcleo, mas também com TRF2 na periferia nuclear. Além disso, a LAP1 e a TRF2 têm sido reportadas como proteínas cruciais na progressão do ciclo celular. Por isso, decidimos prosseguir com esta questão. Quando o EN é remontado, o complexo está localizado principalmente em regiões especificas do EN, evidenciado que a TRF2 permite a ligação dos cromossomas à membrana do NE em células somáticas. Como conclusão, os nossos resultados são de uma importância suprema, uma vez que novas descobertas funcionais relativas ao novo complexo LAP1:TRF2 foram alcançadas, particularmente relacionadas com respostas a danos no DNA e progressão do ciclo celular.

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23

Lusito, E. "A Network-based Approach to Breast Cancer Systems Medicine." Doctoral thesis, Università degli Studi di Milano, 2015. http://hdl.handle.net/2434/265572.

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Breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer death in women. Although recent improvements in the prevention, early detection, and treatment of breast cancer have led to a significant decrease in the mortality rate, the identification of an optimal therapeutic strategy for each patient remains a difficult task because of the heterogeneous nature of the disease. Clinical heterogeneity of breast cancer is in part explained by the vast genetic and molecular heterogeneity of this disease, which is now emerging from large-scale screening studies using “-omics” technologies (e.g. microarray gene expression profiling, next-generation sequencing). This genetic and molecular heterogeneity likely contributes significantly to therapy response and clinical outcome. The recent advances in our understanding of the molecular nature of breast cancer due, in particular, to the explosion of high-throughput technologies, is driving a shift away from the “one-dose-fits-all” paradigm in healthcare, to the new “Personalized Cancer Care” paradigm. The aim of “Personalized Cancer Care” is to select the optimal course of clinical intervention for individual patients, maximizing the likelihood of effective treatment and reducing the probability of adverse drug reactions, according to the molecular features of the patient. In light to this medical scenario, the aim of this project is to identify novel molecular mechanisms that are altered in breast cancer through the development of a computational pipeline, in order to propose putative biomarkers and druggable target genes for the personalized management of patients. Through the application of a Systems Biology approach to reverse engineer Gene Regulatory Networks (GRNs) from gene expression data, we built GRNs around “hub” genes transcriptionally correlating with clinical-pathological features associated with breast tumor expression profiles. The relevance of the GRNs as putative cancer-related mechanisms was reinforced by the occurrence of mutational events related to breast cancer in the “hub” genes, as well as in the neighbor genes. Moreover, for some networks, we observed mutually exclusive mutational patterns in the neighbors genes, thus supporting their predicted role as oncogenic mechanisms. Strikingly, a substantial fraction of GRNs were overexpressed in Triple Negative Breast Cancer patients who acquired resistance to therapy, suggesting the involvement of these networks in mechanisms of chemoresistance. In conclusion, our approach allowed us to identify cancer molecular mechanisms frequently altered in breast cancer and in chemorefractory tumors, which may suggest novel cancer biomarkers and potential drug targets for the development of more effective therapeutic strategies in metastatic breast cancer patients.

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24

Bankapalli, Kondalarao. "Understanding the Role of ThiJ/DJ-1/PfpI Family Member Proteins in Regulating Redox Homeostasis, Mitochondrial Health and Lifespan in Saccharomyces cerevisiae." Thesis, 2017. http://etd.iisc.ac.in/handle/2005/4208.

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In a healthy cell, the ROS levels are stringently regulated by the action of various enzymatic or non-enzymatic antioxidant systems. Imbalance in the ROS homeostasis generates oxidative stress resulting in damage to cellular macromolecules. Besides, pro-oxidants, glyoxals which are normally generated as an intermediate compound in the glycolytic pathway and other metabolic activity are known to cause oxidative stress in the cell. Elevated oxidative stress is one of the prominent cellular aetiologies associated with premature aging, cardiovascular and retinal disorders, atherosclerosis, and several neurological disorders. Parkinson disease (PD) is one of well-known neurodegenerative diseases whose pathogenicity is correlated to mitochondrial dysfunction due to elevated oxidative stress in the neuronal cells. Several proteins which are associated with development of familial form of PD, DJ-1, a member of ThiJ/DJ-1/PfpI super family, is known to act as an oxidative sensor in humans. Interestingly, heat shock protein (Hsp)31 from S. cerevisiae which belongs to DJ-1 family was shown to provide a similar oxidative stress resistance in yeast. However, the mechanistic aspects how these family members functions as an oxidative stress sensor are not clearly defined. The main focus of my investigation is to understand the involvement of these DJ-1 proteins in regulation of redox homeostasis and mitochondrial health, which are major hallmarks in the pathogenesis of PD. My major findings demonstrate the importance of Hsp31 family proteins in protecting cells against oxidative stress, which is induced by methylglyoxal (MG). The deletion of Hsp31 leads to a compromised growth phenotype in yeast upon MG induced stress. Moreover, Hsp31 exhibited robust GSH-independent glyoxalase activity both in vivo and in vitro. Besides, the glyoxalase activity is critical for glyoxal detoxification as well as suppression of ROS levels in cells. On the other hand, in agreement with the observed growth phenotypes, Hsp34 protein possesses a very mild glyoxalase activity as compared to Hsp31. Furthermore, active site mutational analysis reveals that methylglyoxalase activity of Hsp31 protein is critical for providing protection against oxidative stress in yeast. Importantly, endogenous expression of human DJ-1 could complement the growth of yeast under oxidative and glyoxal stress conditions signifying its functional conservation across species. Mechanistically, my findings highlight that Hsp31 regulates cellular GSH and NADPH homeostasis thereby protecting cells against oxidative stress. In addition, cellular localization experiment reveals that though Hsp31 is a cytosolic protein, it predominantly localizes into mitochondria under oxidative stress conditions and protects the organelle from severe oxidative damages. Lastly, my findings uncover the role of Hsp31 paralogs in the maintenance of mitochondrial health integrity and other stress related pathways. To test their role in the mitochondrial health, I have analysed several parameters such as mass, dynamics and functionality. Interestingly, though the single deletions of these paralogs do not have significant effects over the mitochondrial phenotypes, the deletion of DJ-1 homologs in combination of hsp31 and hsp34 in yeast led to enhanced total as well as functional mitochondrial mass in cells. To address how mitochondrial mass enhancement occurs in the cells, the organelle turnover (mitophagy) was assessed. The microscopic and western analysis indicates, there was no alteration in mitophagy among the ∆hsp31∆hsp34 compared to WT. On the contrary, an enhancement in the basal levels of ROS stimulated increased biogenesis of mitochondria in ∆hsp31∆hsp34 cells was observed. Strikingly, ∆hsp31∆hsp34 cells also exhibit upregulation of mitochondrial fusion proteins resulting hyperfusion of mitochondria. Additionally, our results demonstrates that ∆hsp31∆hsp34 cells exhibited a long-term G2/M cell cycle arrest, which was rescued upon overexpression of mitochondrial fission protein, Dnm1. Lastly, absence of these paralogs in yeast, resulted in induction of apoptotic-like features in the cells and decreased lifespan in Saccharomyces cerevisiae. Altogether, my studies highlight the importance of DJ-1 class of proteins in maintaining the cellular redox status, mitochondrial integrity and cellular health in yeast. In conclusion, overall my studies highlight that Hsp31 is a robust methylglyoxalase and regulates cellular NADPH and GSH pool thereby helps in the maintenance of redox homeostasis. Hsp31 predominantly translocate into mitochondria upon oxidative stress to protect the organelle from oxidative damages. Furthermore, my findings provide the first evidence over the involvement of DJ-1 family proteins in the regulation of mitochondrial health and dynamics, cell-cycle arres and reduced lifespan in yeast.

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25

Youssef, M. M., M. A. Al-Omair, and Stephen M. Picksley. "Genetic characterisation of Escherichia coli RecN protein as a member of SMC family of proteins." 2011. http://hdl.handle.net/10454/10558.

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Yes
The proteins of SMC family are characterised by having Walker A and B sites. The Escherichia coli RecN protein is a prokaryotic member of SMC family that involved in the induced excision of Tn10 and the repair of the DNA double strand breaks. In this work, the Walker A nucleotide binding site of the E. coli RecN protein was mutated by changing the highly conserved lysine residue 35 to the aspartic acid (D), designated as recN(K35D). Reverse genetics was utilized to delete the entire recN gene (Delta recN108) or introduce the recN(K35D) gene into the E. coli chromosomal DNA. The recN(K35D) cells showed decreasing in the frequency of excision of Tn10 from gal76

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26

Kavanagh, Steven James. "Characterisation of PSC1 as an acidic rich RS domain protein (ARRS) with a conserved mammalian family member." 2006. http://hdl.handle.net/2440/59642.

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Title page, table of contents and summary only. The complete thesis in print form is available from the University of Adelaide Library.
The Acidic Rich family of RS Domain proteins (ARRS) is defined by both the presence and arrangement of conserved domains within 2 family members. Conserved regions include an RS domain, zinc finger domain, RNA binding motif and a C terminal acidic rich region. Two conserved motifs within the RRM of ARRS proteins have been defined that are not found in the RRM of other RNA binding proteins. Peri implantation stem cell 1 (pscl), the founding member of this family, was originally identified as a developmental marker differentially expressed between the Inner Cell Mass and primitive ectoderm of the mammalian embryo. Psc1 RNA is differentially up-regulated in the post gastrulation embryo and the adult, with high mRNA levels in lung, brain and kidney, and low level expression in other tissues. Comparative analysis of Psc1 to RS domain proteins known to function in mRNA processing, such as SC35 and ASF/SF2, has shown it colocalises to characteristic nuclear speckles. However, in contrast to SR proteins, Pscl localises to additional regions in the nucleus, not containing SR proteins, and to punctate regions in the cytoplasm termed cytospeckles. Further, in the absence of transcription, Psc 1 localises to regions in the nucleus which exclude nuclear speckles. Finally, unlike SC35 and ASF/SF2, which move rapidly in and out of nuclear speckles, FRAP assays show Psc1 is tethered within the nucleus. Analysis of Psc1 domain contribution to subcellular localisation and mobility shows the RRM to be both necessary and sufficient for Psc 1 cytospeckle localisation and is responsible for the nuclear tethering of Pscl. The RS domain of Pscl acts as a nuclear localisation signal and contributes to nuclear speckle localisation. The C terminal of Psc I localises with microtubules and is proposed to mediate Psc 1 cytoskeletal association. The expression of the Drosophila acidic rich RS domain protein (NP609976) is developmentally regulated in the same manner as Pscl, it has a nuclear localisation profile identical to Psc 1 and also localises to speckles in the cytoplasm, all of which support a conserved evolutionary role for Pscl and the ARRS protein family in mRNA processing and trafficking both in the nucleus and in the cytoplasm.
http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1236604
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2006

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27

Hu, Bin [Verfasser]. "Identification of the paralemmin protein family and initial characterization of palmdelphin, a member of the family / vorgelegt von Bin Hu." 2001. http://d-nb.info/964992736/34.

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28

朱學亮. "Cloning, folding and function analysis of human cyclin I protein: a new member of cyclin family." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/83004135365440026282.

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29

Hajek, Kathryn L. "Capsid assembly and envelope protein expression by the retrovirus-like element TED, a lepidopteran member of the Gypsy Family." 1997. http://catalog.hathitrust.org/api/volumes/oclc/39909580.html.

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Thesis (Ph. D.)--University of Wisconsin--Madison, 1997.
Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 128-143).

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30

Wu, Chieh-His, and 吳潔曦. "The Role of Ras Homolog Gene Family, Member A/ Rho-associated Protein Kinase Signaling Pathway in Pathogenesis of Spinocerebellar Ataxia Type 2." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/34116191554741126044.

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碩士
中山醫學大學
醫學檢驗暨生物技術學系碩士班
103
Spinocerebellar ataxia type 2 (SCA2) , an autosomal dominant neurodegenerative disease, is caused by the expansion of a CAG triplet repeats located in the N-terminal coding region of the ATXN2 gene. Alleles of the ATXN2 gene that carry 13-31 CAG-trinucleotide repeats are present in normal individuals. Contrariwise, alleles with a CAG triplet repeat number of 31 and up to approximately 200 are present in patients with SCA2. Although the detail mechanism of pathogenesis is yet to be defined, neurotoxin, especially reactive oxygen species (ROS) released from aggregated mutant proteins, and formed stress granule (SG) may play a role in the pathogenic process. In this study, the lymphoblastoid cell lines (LCLs) isolated from SCA2 patients were utilized to compare with the wild-type lymphoblastoid cells. We investigated the crucial relationship between the expression of p-ERK1/2 and autophagy. Western blot results found the endogenous p-ERK1/2 and autophagy marker protein, Atg8 (LC3 class ΙΙ) were higher in SCA2 patients. Moreover, misfolding mutant ataxin aggregation causes spinocerebellar ataxia type 2 downregulate heat shock protein reduces neurotoxicity by promoting polyglutamine protein degradation. Heat-shock protein disfunction, like Hsp60, Hsp40, Hsp70 and Hsp27 consistently suppresses the formation of polyQ inclusion bodies and their toxicity. These results highlight the possibility that chaperones facilitate neuroprotection through several distinct mechanisms, but because small, diffusible, potentially toxic polyQ assemblies could not be evaluated, an essential role for the refolding activity of chaperones cannot be ruled out. The essential functions of Rho family GTPases in regulating neuronal growth cone formation, neurite outgrowth, and nervousmsystem development suggest that Rho GTPases have an important and conserved function in mediating neuronal survival and death. Furthermore, RhoA/ROCK signaling pathway marker protein, ROCK and RhoA were higher in SCA2 cells. Rho can provoke apoptosis via activation of either p38- or JNK-dependent signaling pathways which elicit apoptosis through activation of pro-apoptotic members of the Bcl-2 family of proteins lead to neurodegenerative. Otherwise, based on the above observations we hypothesized that the aggregated mutant Ataxin-2 protein may generate stress and form SG, which subsequently up-regulate Atg8 expression levels and ultimately lead to cell autophagy to slow down neurodegenerative.

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31

Chen, Wei-Ren, and 陳威仁. "Identification of the Regulatory Autophosphorylation Site of Autophosphorylation-Dependent Protein Kinase(Auto-kinase), Evidence that Auto-kinase Belongs to a Member of PAK Family." Thesis, 1997. http://ndltd.ncl.edu.tw/handle/28049085640637917570.

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32

Chen, Wei-Jen, and 陳威仁. "Identification of the Regulatory Autophosphorylation Site of Autophosphorylation-Dependent Protein Kinase(Auto-kinase), Evidence that Auto-kinase Belongs to a Member of PAK Family." Thesis, 1997. http://ndltd.ncl.edu.tw/handle/14372604105595277445.

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碩士
長庚醫學暨工程學院
基礎醫學研究所
85
Autophosphorylation-dependent protein kinase(auto-kinase) was found existing in pig brain and liver in 1987 by Yang et al. with a unique property that its activity is regulated by autophosphorylation.Auto-kinase is a cyclic nucleotide- and calcium-independent protein kinase, and its molecular weight is 36kDa. Subsequent studies revealed that auto-kinase is a multisubstrate protein kinase which can act on several key regulatory proteins and enzymes with the following substrate consensus sequence motif:R-X-(X)-S*/T*-X3-S/T (where * is the kinase target site). However, the gene of auto-kinase has not yet been cloned and therefore, the founding of auto-kinase as a new protein kinase is still controversial. Furthermore, the autophosphorylation site(s) of auto-kinase, which play(s) a critical role in regulating its activity, has(have) not yet been identified. To elucidate these points, we purified auto-kinase was subjected to N-terminal and internal partial sequencing. Three segments of partial amino acid sequence of auto-kinase (VDGGAKTSDKQKKKAXMTDE, EKLRTIV and LQNPEI/KLTP/FI) were successfully obtained. These sequences were perfectly matched with those of a protein kinase named gamma-p21-Cdc42/Rac- activated kinase(PAK2), which was identified from human placenta cDNA library by Martin et al. in 1995, when mapped data bank. This, together with our finding that auto-kinase can be specifically recognized by an anti-peptide antibody raised against the 14-amino acid C-terminal peptide of human PAK2 demonstrated that auto-kinase is a pig homologue of human PAK2. A synthetic peptide EQSKRSTMVGTPYWMAPEVVTRK (STM peptide) corresponding to amino acid residue 397-419 of human PAK2, which possesses the substrate consensus sequence motif for auto- kinase, can be efficiently phosphorylated by auto-kinase at threonine residue. Moreover, the tryptic phosphopeptide map of 32P-STM peptide phosphorylated by auto-kinase is identical to that of 32P-autophosphorylated tryptic phosphopeptides by manual Edman degradation. These results demonstrated that Thr403 (EQSKRSTMVGTPYWMAPEVVTRK, * indicates the phosphorylation site, according to the sequence of human PAK2) is the only autophosphorylation site of auto-kinase.

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Дисертації з теми "PfpI Family Member Protein"

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