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Tesis sobre el tema "Biochemical studie"

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Publicado: 11 de marzo de 2023

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1

Sims, Lynn. "Biochemical Studies of ABCE1". Doctoral diss., University of Central Florida, 2012. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/5501.

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The growth and survival of all cells require functional ribosomes that are capable of protein synthesis. The disruption of the steps required for the function of ribosomes represents a potential future target for pharmacological anti-cancer therapy. ABCE1 is an essential Fe-S protein involved in ribosomal function and is vital for protein synthesis and cell survival. Thus, ABCE1 is potentially a great therapeutic target for cancer treatment. Previously, cell biological, genetic, and structural studies uncovered the general importance of ABCE1, although the exact function of the Fe-S clusters was previously unclear, only a simple structural role was suggested. Additionally, due to the essential nature of ABCE1, its function in ribosome biogenesis, ribosome recycling, and the presence of Fe-S within ABCE1, the protein has been hypothesized to be a target for oxidative degradation by ROS and critically impact cellular function. In an effort to better understand the function of ABCE1 and its associated Fe-S cofactors, the goal of this research was to achieve a better biochemical understanding of the Fe-S clusters of ABCE1. The kinetics of the ATPase activity for the Pyrococcus abyssi ABCE1 (PabABCE1) was studied using both apo- (without reconstituted Fe-S clusters) and holo- (with full complement of Fe-S clusters reconstituted post-purification) forms, and is shown to be jointly regulated by the status of Fe-S clusters and Mg2+. Typically, ATPases require Mg2+, as is true for PabABCE1, but Mg2+ also acts as a unusual negative allosteric effector that modulates ATP affinity of PabABCE1. Comparative kinetic analysis of Mg2+ inhibition shows differences in the degree of allosteric regulation between the apo- and holo-PabABCE1 where the apparent Km for ATP of apo-PabABCE1 increases >30 fold from ~30 [micro]M to over 1 mM when in the presence of physiologically relevant concentrations of Mg2+. This effect would significantly convert the ATPase activity of PabABCE1 from being independent of cellular energy charge to being dependent on energy charge with cellular [Mg2+]. The effect of ROS on the Fe-S clusters within ABCE1 from Saccharomyces cerevisiae was studied by in vivo 55Fe labeling. A dose and time dependent depletion of ABCE1 bound 55Fe after exposure to H2O2 was discovered, suggesting the progressive degradation of Fe-S clusters under oxidative stress conditions. Furthermore, our experiments show growth recovery, upon removal of the H2O2, reaching a growth rate close to that of untreated cells after ~8 hrs. Additionally, a corresponding increase (~88% recovery) in the ABCE1 bound 55Fe (Fe-S) was demonstrated. Observations presented in this work demonstrate that the majority of growth inhibition, induced by oxidative stress, can be explained by a comparable decrease in ABCE1 bound 55Fe and likely loss of ABCE1 activity that is necessary for normal ribosomal activity. The regulatory roles of the Fe-S clusters with ABCE1 provide the cell a way to modulate the activity of ABCE1 and effectively regulate translation based on both cellular energy charge and the redox state of the cell. Intricate overlapping effects by both [Mg2+] and the status of Fe-S clusters regulate ABCE1's ATPase activity and suggest a regulatory mechanism, where under oxidative stress conditions, the translational activity of ABCE1 can be inhibited by oxidative degradation of the Fe-S clusters. These findings uncover the regulatory function of the Fe-S clusters with ABCE1, providing important clues needed for the development of pharmacological agents toward ABCE1 targeted anti-cancer therapy.
Ph.D.
Doctorate
Biology
Sciences
Biomedical Sciences
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2

Dillingham, Mark Simon. "Biochemical studies on DNA helicases". Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312245.

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3

Paternostro, Giovanni. "Biochemical studies of cardiac hypertrophy". Thesis, University of Oxford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.337538.

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4

Stuart, E. "Biochemical studies on serotonin receptors". Thesis, University of Nottingham, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.372014.

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5

Zailaie, Abdo M. "Biochemical studies on human haemoglobin". Thesis, University of Salford, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.374518.

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6

Paton, F. M. "Biochemical studies of marine fungi". Thesis, University of Liverpool, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.382060.

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7

Yeates, Laura Catherine. "Biochemical studies of cytokinin sensitivity". Thesis, University of Cambridge, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627620.

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8

Zhang, Jing. "Biochemical studies on T5 exonuclease". Thesis, University of Sheffield, 2012. http://etheses.whiterose.ac.uk/2639/.

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9

Lester, Jill. "Biochemical and crystallographic studies of proteins". Thesis, Liverpool John Moores University, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.292313.

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10

Udell, M. N. "Biochemical studies on microbial secondary metabolites". Thesis, University of Nottingham, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.379013.

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11

Taylor, Robert William. "Mitochondrial oxidations : biochemical and molecular studies". Thesis, University of Newcastle Upon Tyne, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.358137.

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12

Slight, S. H. "Biochemical studies on the mammalian lens". Thesis, University of Salford, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.381703.

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13

Mills, Ann. "Biochemical studies on the neurotensin receptor". Thesis, Imperial College London, 1988. http://hdl.handle.net/10044/1/47181.

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14

Henzing, Alexander John. "Chemical & biochemical studies of Caspases". Thesis, University of Edinburgh, 2000. http://hdl.handle.net/1842/15007.

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Caspases are cysteine-dependent aspartate-directed proteases responsible for the proteolysis of a plethora of substrates during programmed cell death. These include structural proteins of the cytoplasm and nucleus, components of the DNA repair machinery, protein kinases, signalling proteins and regulatory proteins. Caspases are synthesised as relatively inactive zymogens, that become activated by scaffold-mediated transactivation or via cleavage by upstream proteases in an intracellular cascade. The resulting heterotetrameric enzymes possess a unique absolute requirement for aspartate at the substrate cleavage site, and recognise a tetrameric sequence within the substrate. In order to assess the role of caspases in apoptotic execution, I set out to evaluate the synthesis of novel caspase inhibitors, which would enable the detection of active caspases from apoptotic whole cell extracts. First a 2,4-dinitrophenyl probe was designed for the affinity tagging of caspases in two-dimensional gel electrophoresis. Second, biotinylated peptidylaldehydes were prepared which will enable the affinity purification of caspases from apoptotic cytosolic extracts under non-denaturing conditions. To enable biochemical studies of caspases, I developed a method, which permits the affinity purification of caspases. Apoptotic chicken hepatoma cell-line extracts were purified over an avidin column using a biotinylated probe. Finally, to permit the appraisal of the caspase proteomic variability between different cell types, and methods of apoptotic induction, and the identification of post-translational modifications of caspases, I developed a reproducible system for the identification of caspases by two-dimensional gel electrophoresis.
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15

Upcher, William R. "Biochemical studies of the cohesin complex". Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:e9a77a9c-40a5-466d-a894-c7fefeddef52.

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The accurate inheritance of genetic material depends upon the establishment and maintenance of sister chromatid cohesion. Replicated chromosomes are topologically encircled by the large, tripartite protein complex cohesin, allowing bi-orientation in mitosis. To entrap and reversibly dissociate from DNA, the annular complex structure must be disrupted at either the hinge domain between Smc1 and Smc3, or the interfaces created by the kleisin subunit Scc1 bridging the two ABC-like ATPase domains. The aim of this work was to characterise the cohesin complex loading and releasing mechanisms by examining the biochemical requirements for these processes. Although the identity of a chromosomal cofactor could not be assigned, the loading reaction was found to necessitate engagement of ATPase domains in an ATP-dependent manner. Notions of allosteric modulation of ATP binding and NBD engagement by acetylation were discredited. Likewise, a direct and stable physical association of hinge domains with NBDs was shown to be an unlikely conformational intermediate in a reaction thought to promote hinge opening for loading of cohesin onto DNA. The Smc3-Scc1 kleisin interface might be exploited during the opposing process of release of cohesin from DNA. Therefore, a novel protein- protein cross-linking system was adapted for use in S. cerevisiae, with a view to (1) confirming the well-founded role of hinge dissociation in topological entrapment, and (2) validating the Smc3- Scc1 interface as the recently conjectured exit gate. Despite promising preliminary kinetics in vitro, the SpyTag-SpyCatcher system was considerably less efficient in vivo. It was thus deemed unsuitable in its current format for investigating the process of interface dissociation in live cells. Finally, the large, cohesin complex-associated HEAT repeat protein Pds5 has been either speculated or shown to participate in all of the aforementioned processes, potentially modulating the hinge and Smc-kleisin interfaces. Acting as a regulatory node in cohesin function, it was pursued as an informative target for structural studies. Although no diffractive material could be obtained, Pds5 was confirmed to bind a short, N-terminal sequence of Scc1 and was in turn bound at its N- terminus by Wapl. Together, these findings contribute to defining the structures and states of the cohesin complex.
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16

Hayward, Caroline Irma. "Biochemical studies of cystic fibrosis antigen". Thesis, University of Edinburgh, 1987. http://hdl.handle.net/1842/18950.

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17

Russell, Janelle P. N. "Chemical and Biochemical Studies of Bacillithiol". Youngstown State University / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1347888245.

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18

Briggs, Louise Clare. "Biochemical studies of P97 AAA ATPase". Thesis, Imperial College London, 2007. http://hdl.handle.net/10044/1/11495.

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19

Ozdemir, Ahmet Yunus. "BIOCHEMICAL STUDIES OF DNA POLYMERASE THETA". Diss., Temple University Libraries, 2019. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/560412.

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Biomedical Sciences
Ph.D.
POLQ is a unique multifunctional replication and repair gene that encodes a multidomain protein with a N-terminal superfamily 2 helicase and a C-terminal A-family polymerase. Although the function of the polymerase domain has been investigated, little is understood regarding the helicase domain. Multiple studies have reported that polymerase θ-helicase (Polθ-helicase) is unable to unwind DNA. However, it exhibits ATPase activity that is stimulated by single-stranded DNA, which presents a biochemical conundrum. In contrast to previous reports, we demonstrate that Polθ-helicase (residues 1– 894) efficiently unwinds DNA with 3'–5' polarity, including DNA with 3' or 5' overhangs, blunt- ended DNA, and replication forks. Polθ-helicase also efficiently unwinds RNA-DNA hybrids and exhibits a preference for unwinding the lagging strand at replication forks, similar to related HELQ helicase. Finally, we find that Polθ-helicase can facilitate strand displacement synthesis by Polθ-polymerase, suggesting a plausible function for the helicase domain. Taken together, these findings indicate nucleic acid unwinding as a relevant activity for Pol theta in replication repair. DNA polymerase theta is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining of DNA double-strand breaks. How full-length human DNA polymerase theta performs microhomology-mediated end-joining and is regulated by the helicase and disordered central domain remains unknown. We find that the helicase upregulates DNA polymerase theta microhomology-mediated end-joining activity in an ATPase-independent manner. Using single-particle microscopy, we find that DNA polymerase theta forms large multimeric complexes that promote DNA accumulation and end-joining. We further find that the disordered central domain regulates DNA polymerase theta multimerization and governs its DNA substrate requirements for end-joining. In summary, these studies identify major regulatory functions for the helicase and central domains in DNA end-joining and the structural organization of DNA polymerase theta.
Temple University--Theses
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20

HAGO, ALMUGADAM Shawgi Yousif. "LEISHMANIA: METABOLOMICS, BIOCHEMICAL AND CLINICAL STUDIES". Doctoral thesis, Università degli studi di Ferrara, 2017. http://hdl.handle.net/11392/2487968.

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Visceral Leishmaniasis (VL) is a major health problem worldwide and both, drugs have toxic effects and parasites developed resistance therefore new compounds and drug targets are sought. In this thesis Leishmania (L) and leishmaniasis were probed both in biochemical and clinical studies. The 6-aminonicotinamide effect was analysed on L parasite growth and metabolism by metabolomics technology. The rational was pentose phosphate pathway (PPP), a major antioxidants provider, has been reported as a 6-AN target and L PPP enzymes have significant differences versus those of the mammals, thus PPP might represent a good target. The other biochemical study was conducted to characterize L transglutaminase (TGase), catalyzing irreversible protein crosslinking; if fundamental for L parasites, these enzymes could represent another promising drug target. In the thesis clinical part, subclinical L infections were evaluated in autoimmune rheumatic patients treated with immunosuppressive drugs at Ferrara Hospital in Italy. L. mexicana and L. infantum promastigotes were treated with 7.8 mM 6-AN for 24 hours and small metabolites were extracted and analysed by pHILIC-LC-MS. TGase was tested in vivo in canine L. infantum promastigotes as well as in vitro in parasite extracts using fluorescein-cadaverine. Transamidation in AS precipitated fractions was measured in microwell plates. Western blot of human L. infantum lysate and canine L. infantum extract and precipitated fractions as well as immunocytochemical analysis of canine L. infantum were performed using human TGase 2 polyclonal antibodies. L PCR and real-time PCR were performed on DNA extracted from PBMCs from 50 autoimmune rheumatic patients and 50 healthy subjects. Plasma cytokines were also measured. In both L. mexicana and L. infantum, 6-AN caused significant depletion of phosphoribosylpyrophosphate (PRPP) and nicotinate and as a result purine and pyrimidine nucleotides were reduced and their nucleobases accumulated. Glutathione, ribose-5-phosphate, 6-phosphogluconate levels and downstream PPP intermediates were similar to controls. In mammals 6-AN is converted to abnormal 6-ANAD/P by NAD+ glycohydrolase, however, in L its toxicity is only seen in mM range, in which 6-AN depletes the cellular PRPP content probably in the Preiss-Handler NAD+ salvage pathway, resulting in depletion of nucleotides required for nucleic acid biosynthesis. L NAD+ glycohydrolase might decompose NAD+ but not catalyze exchange reactions as found in other microrganisms, however, combined 13C-glucose labeling and flux analysis might be useful to ascertain the fate and action mechanism of 6-AN in L. TGase was detected and confirmed in vivo and/or in vitro in human and canine L. infantum promastigotes. The activity in the canine strain was found Ca2+-dependent and inhibited by 10mM GTP. Immunocytochemistry showed fluorescent protein and blots revealed a band of 74.6 KDa for the canine strain and two bands of 55.34 KDa and 65.87 KDa for the human strain, thus the TGase pAbs (orb2986) could permit purification of this L enzyme. Eighteen autoimmune rheumatic patients were positive for L DNA by PCR and/or quantitative PCR. No statistical difference observed in relation to ownership of a dog or the type of biological drug used. Pro-inflammatory IL-1, IL-6, IL-12(p70), IL-7, IL-15, IFN-γ and TNF-α; anti-inflammatory IL-4, IL-13; and regulatory IL-10 cytokines were markedly elevated in patients with additional increase those positive for L DNA. The high L parasitaemia detected suggests that biologic treatment can lead to cryptic VL or L infection in a latent phase which may progress to full VL in the setting of immunosuppression. Molecular screening for L using PBMC fractions and cytokine analysis should be performed before treating autoimmune rheumatic patients with biologic drugs.
La leishmaniosi viscerale (VL) è un problema sanitario mondiale e i farmaci possono dare tossicità nonché generare resistenza, perciò è importante la ricerca di nuovi composti e nuovi “drug target”. In questa tesi la Leishmania (L) è stata studiata sia dal punto di vista biochimico che clinico. Ho analizzato gli effetti antiproliferativi e metabolici della 6-aminonicotinamide. Essendo riportato come bersaglio principale della 6-AN la via dei pentosi fosfato (PPP), necessaria al sistema di difesa antiossidante della cellula e i cui enzimi in questi parassiti presentano differenze significative con quelli di mammifero, PPP potrebbe rappresentare un buon ”drug target”. Inoltre, poiché un secondo bersaglio potrebbe essere la transglutaminasi (TGasi) riportata essere importante per il parassita, si è cercato di caratterizzarla in L. infantum. Nello studio clinico, si è valutata la prevalenza di parassitosi da L, asintomatiche/subcliniche in pazienti dell’ospedale di Ferrara, affetti da patologie reumatiche autoimmuni, trattati con farmaci immunosoppressori. Promastigoti di L. mexicana e L. infantum sono stati trattati per 24 ore con 6-AN 7,8 mM e dopo estrazione i metaboliti più piccoli sono stati analizzati mediante pHILIC-LC-MS. La TGasi è stata saggiata in vivo in promastigoti di L. infantum e in vitro in lisato cellulare dopo incubazione con fluoresceina-cadaverina. L’attività è stata anche saggiata in micropiastra pure dopo frazionamento con AS. Anticorpi policlonali contro TGasi 2 umana sono stati usati nell’immunocitochimica e in Western Blot di lisati di L. infantum umana e canina e frazioni precipitate. Su DNA estratti da PBMC di 50 pazienti e 50 soggetti sani si sono effettuate PCR qualitativa e real-time. Inoltre si sono quantificate le citochine seriche. Sia in L. mexicana che L. infantum, 6-AN ha causato una diminuzione significativa di fosforibosil-pirofosfato (PRPP) e nicotinato e come conseguenza di nucleotidi mentre le nucleobasi sono aumentate. I livelli di glutatione, ribosio-5-fosfato, 6-fosfogluconato e intermedi PPP a valle erano simili ai controlli. Nei mammiferi la 6-AN è metabolizzata a 6-ANAD/P da una NAD+ glicoidrolasi, ma in L la tossicità solo in concentrazione mM, con deplezione di PRPP e conseguente calo di nucleotidi, indica che 6-AN potrebbe entrare nella via Preiss-Handler di sintesi del NAD+. In L la NAD+ glicoidrolasi potrebbe idrolizzare il NAD+ ma non catalizzare lo scambio tra nicotinamide e 6-AN, come riportato in altri microrganismi. Analisi ulteriori di flusso dopo marcatura con 13C-glucosio potrebbero chiarire il meccanismo d’azione del 6-AN in L. Si è confermata in vivo and in vitro, la presenza in L di una TGasi attiva, in promastigoti canini Ca2+-dipendente e inibita da GTP. Con l’immunocitochimica si è rivelata fluorescenza e nei blot bande di 74.6 KDa per il ceppo canino e di 55.34 e 65.87 KDa per quello umano, suggerendo che gli anticorpi usati potrebbero essere utili nella purificazione dell’enzima. In 18 pazienti si è riscontrata positività ma non evidenziate differenze significative tra possessori o meno di cani e in riferimento al tipo di farmaco somministrato. In tutti i pazienti in trattamento con farmaci biologici, erano significativamente elevate le citochine pro-infiammatorie IL-1, IL-6, IL-12(p70), IL-7, IL-15, IFN-γ e TNF-α, quelle anti- infiammatorie IL-4 e IL-13 e la regolatoria IL-10, ma un aumento maggiore era presente nei pazienti positivi per la presenza di DNA di L. L’alta parassitemia trovata suggerisce che il trattamento possa portare a VL criptica o infezione latente, con possibilità di progressione a VL conclamata in caso di evoluzione a uno stato di immunocompromissione. Si potrebbero quindi introdurre uno screening molecolare su frazioni PBMC e l’analisi di citochine prima di prescrivere questo tipo di farmaci a pazienti con patologie autoimmuni.
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21

Mijuskovic, Martina. "Structural and biochemical studies on human TAF2 /". Zürich : ETH, 2007. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=17313.

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22

Petrin, Dino P. "Molecular and biochemical studies of Trichomonas vaginalis". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0011/MQ28454.pdf.

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23

Wei, Lei. "Guinea pig osteoarthrosis : morphological and biochemical studies /". Stockholm, 1999. http://diss.kib.ki.se/1999/91-628-3361-8/.

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24

Wang, Lijun. "Biochemical studies of Mms6--a biomineralization protein". [Ames, Iowa : Iowa State University], 2007.

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25

Hemming, Richard John. "Radioautographical and biochemical studies on nucleoplasmic glycoproteins". Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=41298.

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EM radioautography was used to examine the tissue distribution of cells exhibiting nucleoplasmic labeling after being exposed to $ sp3$H-sugars or $ sp{35}$S-sulphate to indicate the general extent of the occurrence of nucleoplasmic glycoproteins within animal cells. The observation of some degree of such labeling in virtually all cells in tissues of three animal species suggests that nucleoplasmic glycoproteins are a common cellular feature. To better define the distribution and nature of the putative labeled nucleoplasmic glycoproteins, cultured cells were used as a model cell type for both quantitative EM radioautographic and biochemical studies. After exposure to $ sp3$H-sugars, all three lines of cultured cells examined exhibited significant nucleoplasmic reaction in which the euchromatin, heterochromatin and nucleoli were all labeled to some extent. Studies on isolated, envelope-depleted nuclei from myeloma cells confirmed that the molecules in the nucleoplasm itself were the source of the radioautographic reaction observed over nuclei. Biochemical analyses of fractions of isolated nuclei indicated that much of the label resided in nuclear matrix glycoproteins of different molecular weights. Lectin binding studies on nuclear matrix fractions revealed the presence of galactose, fucose, and/or sialic acid residues in proteins. Glycosidase experiments indicated that some but not all of these glycoproteins had N-linked sidechains.
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26

Tomlinson, Esther Jane. "Studies on the evolution of biochemical pathways". Thesis, University of Oxford, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.240380.

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27

Ryba, N. J. P. "Biochemical and biophysical studies of photoreceptor membranes". Thesis, University of Oxford, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.370299.

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28

Schwill, Richard. "Structural, biochemical and molecular studies on thraustochytrium". Thesis, University of Portsmouth, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.511400.

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29

Wang, Ruiqi Rachel. "Biochemical and Structural Studies of Membrane Proteins". Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10154.

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Membrane proteins live at the interface between a cell and its environment; hence, they play a variety of important physiological roles such as transmembrane transport, signal transduction, and cell adhesion. The importance of membrane proteins in biology and medicine requires that we understand their structure and function on the atomic level. In this thesis, I studied members of two different membrane protein families, namely the neuronal and keratinocyte TRPV ion channels that sense temperature changes and MP20, a member of the PMP22/EMP/MP20/claudin superfamily. Using a variety of biochemical, X-ray crystallographic and electrophysiological techniques, I addressed mechanistic questions pertaining to the regulation of thermosensitive TRPV channels by ATP and calmodulin in neurons and keratinocytes. For MP20, a protein specific for the lens of the mammalian eye, I used a vesicle assay in combination with electron microscopy (EM) to study its function, ruling out the possibility that MP20 is involved in the formation of membrane junctions. Furthermore, I made progress in expressing and crystallizing MP20 for X-ray diffraction studies. In a separate effort, I also worked on improving and expanding the use of monolayer purification and Affinity Grids, recently introduced techniques to prepare specimens for single-particle EM based on the recruitment of His-tagged proteins to nickel lipidcontaining lipid monolayers. I extended the use of these techniques by synthesizing a glutathione lipid that can be used to recruit GST-tagged proteins. A major hurdle in the use of monolayer purification techniques, however, is the extent of non-specific protein binding to the lipid monolayer. I found that incorporating PEG lipids in the monolayer appears to reduce the problem of non-specific protein binding. While it remains to be seen whether these techniques can be developed to a point at which it will be possible to recruit exclusively tagged proteins out of cell lysates, my goal is to continue to improve and expand the use of the monolayer purification and Affinity Grid techniques in hope to make single-particle EM more easily amenable to biochemists and cell biologists.
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30

Clingen, Peter H. "Biochemical studies of purine photodamage in DNA". Thesis, Queen's University Belfast, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.241386.

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31

Afrasiabi, Morteza. "Biochemical studies of erythropoietin and erythropoietin receptors". Thesis, Queen's University Belfast, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.334533.

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32

Smith, Janet G. "Biochemical and biophysical studies of plant chromatin". Thesis, Liverpool John Moores University, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.304950.

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33

Gibson, Kevin J. C. "Structural and biochemical studies on bacterial lipoglycans". Thesis, University of Newcastle Upon Tyne, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.397343.

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34

Nury, David. "Functional and biochemical studies of metallothionein localisation". Thesis, University of Newcastle Upon Tyne, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.402596.

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35

Smith, T. M. "Molecular and biochemical studies in Penicillium chrysogenum". Thesis, University of Westminster, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.371397.

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36

Rahman, Salman. "Biochemical and immunological studies on gap junctions". Thesis, University College London (University of London), 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.266786.

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37

Stanley, John Robert. "Biochemical and biophysical studies of thaumatin proteins". Thesis, University of Greenwich, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.305109.

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38

Yendle, Peter W. "Chemometric studies of biochemical and geochemical systems". Thesis, University of Bristol, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.232950.

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39

Santiapillai, Nirmala Fleurette. "Biochemical studies on the crustacean neuromuscular junction". Thesis, University of Cambridge, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.305881.

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40

Tse, Shun. "Biochemical and cellular studies of vertebrate globins". Thesis, University of East Anglia, 2015. https://ueaeprints.uea.ac.uk/58579/.

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41

Wirthensohn, David Christopher. "Biochemical and structural studies of amyloid proteins". Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/288829.

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Amyloidogenic neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD) are an important health issue. However, the underlying molecular mechanisms of the disease-related protein aggregates, that are present in humans, are only understood partially. I have used and developed biophysical methods to study the structural and biological properties of individual aggregates of Amyloid β peptide and α-Synuclein, proteins whose aggregation is associated with the development of Alzheimer's and Parkinson's disease respectively. I expanded the single aggregate visualisation through enhancement (SAVE) technique, which is a method based on the fluorescent dye Thioflavin T (ThT) that reversibly bind to the aggregates and whose fluorescence increases upon binding. I firstly explored the use of other dyes for these experiments and found that a ThT dimer has higher affinity to α-Synuclein aggregates in vitro. I then applied the SAVE method to the cerebral spinal fluid (CSF) of a cohort of AD patients and control CSF and observed no clear difference in aggregate number. However, these experiments provided insights into how antibodies bind the aggregates in human CSF. I could show, that despite altering the Ca2+ influx into both cells and vesicles, the antibody did not measurably affect the aggregate structure. To study the size specific effects of the Amyloid β 42 (Aβ42) peptide in more detail, I used and optimised gradient ultracentrifugation combined with single aggregate imaging to study the structural properties of the isolated aggregates. This aggregation kinetic independent method allowed me to compare the properties of fluorescently labelled and unlabelled Aβ42 and characterize the size dependent properties of aggregates in a single experiment. Since I could measure the relative concentration of different size aggregates it was also possible to compare the properties of single aggregates of different sizes. I then used biological assays to examine the ability of aggregates to permeabilise membranes resulting in the entry of calcium ions, and their ability to induce TNFα production in microglia cells. Both processes are thought to play key roles in the development of AD. I found that small soluble oligomers are most potent at inducing Ca2+ influx, whereas longer protofilaments are the most potent inducers of TNFα production. My results suggest that the mechanism by which aggregates damage cells changes as aggregation proceeds, as longer aggregates with different structures are formed. Protofilaments with a diameter of 1 nm or less have a structure that could make them particularly potent at causing the signalling of toll-like receptors, providing a molecular basis for their ability to induce TNFα production.
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42

Jaffé, Matthias Benno. "Biochemical studies of the Saccharomyces cerevisae kinetochore". Thesis, Massachusetts Institute of Technology, 1996. http://hdl.handle.net/1721.1/38399.

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43

Elliot-Smith, Andrea Elizabeth. "Biochemical studies of the Cdc42-ACK interaction". Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.615807.

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44

Xypnitou, Andromachi. "Biophysical, biochemical and inhibition studies of hexokinases". Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/31486.

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Hexokinase is the first enzyme in glycolysis, a major pathway for the generation of energy in all eukaryotes. Mammalian cells have four isoforms (I, II, III, IV) that have different tissue distribution and kinetic properties. Among all isoforms, human hexokinase II (hHKII) has been found to be implicated in many cancers with an increased expression which serves a dual role. First, it maintains the high glycolytic rate of malignant cells (Warburg effect) and second it prevents apoptosis when is bound to mitochondria. Trypanosoma brucei is a parasite that causes Human African Trypanosomiasis (HAT) and has two isoforms with extensive sequence similarity (98%), TbHKI (active form) and TbHK2 (inactive form). The bloodstream-form parasites (BSF) depend exclusively on glycolysis for their survival. The enzyme from both organisms is a validated target for drug-discovery against both cancer and HAT. The aim of the present study is the discovery of novel and specific inhibitors of the enzymes based on their structure. Structure-based drug discovery is commonly used in pharmaceutical companies to aid in the discovery of potent lead compounds. In silico studies were performed in this project using the known crystal structure of human hexokinase I and a model of TbHKI generated by the protein modelling tool Phyre2. The docking programs, AutoDock (AD) and AutoDock Vina (Vina), were chosen to perform the docking of ~3 million compounds to the target molecules and scoring functions calculated the predicted binding affinities of each compound. In total, 28 compounds were purchased to test on the target molecules. In the experimental part of the project, the two enzymes were cloned, expressed and purified. hHKII was successfully purified giving a high yield of active and pure protein. The protein was characterised using many biophysical methods to establish the oligomeric state, the homogeneity and the secondary structure. Crystallisation trials failed and for this reason, N and C domains of the hHKII were purified separately. Unfortunately, the domains also failed to crystallise thus SAXS data were collected and analysed to gain information of their shape at low resolution. A novel inhibition assay was developed and used to identify four weak inhibitors against full length hHKII. TbHKI was difficult to express in a soluble form as most of the protein was expressed in inclusion bodies. The purification resulted in a small amount of active protein that was used entirely for biochemical assays. Four compounds were purchased from the docking of the TbHKI model and one was found to inhibit the enzyme over 65% at 100 μM. Because the active site of both enzymes (hHKII, TbHKI) is well conserved the compounds from hHKII docking were also screened against the TbHKI. Four compounds were found to inhibit this enzyme while one of them was also an inhibitor for human isoform. The remaining three were specific for inhibition of TbHKI.
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45

Nguyen, Thi Hoang Duong. "Structural and biochemical studies of spliceosomal activation". Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.707998.

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46

Omori, Taketo. "Biochemical studies of novel glycerophospholipids in mammals". Kyoto University, 2009. http://hdl.handle.net/2433/126535.

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Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第14874号
農博第1786号
新制||農||975(附属図書館)
学位論文||H21||N4489(農学部図書室)
27296
UT51-2009-K670
京都大学大学院農学研究科応用生命科学専攻
(主査)准教授 栗原 達夫, 教授 喜多 恵子, 教授 植田 充美
学位規則第4条第1項該当
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47

Honda, Kosuke. "Biochemical and applied studies of microbial lactonases". Kyoto University, 2003. http://hdl.handle.net/2433/148965.

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Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第10237号
農博第1309号
新制||農||863(附属図書館)
学位論文||H15||N3758(農学部図書室)
UT51-2003-H658
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 清水 昌, 教授 加藤 暢夫, 教授 宮川 恒
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48

Hidalgo, Aklani-Rose Gamboa Delacruz. "Biochemical and applied studies of microbial oxidoreductases". Kyoto University, 2002. http://hdl.handle.net/2433/149519.

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Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第9838号
農博第1298号
新制||農||855(附属図書館)
学位論文||H14||N3725(農学部図書室)
UT51-2003-B378
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 清水 昌, 教授 加藤 暢夫, 教授 江﨑 信芳
学位規則第4条第1項該当
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49

Bennett, Brian. "Biochemical and spectroscopic studies of oxomolybdenum enzymes". Thesis, University of Sussex, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.358987.

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50

Henshilwood, James Andrew. "Studies towards the synthesis of the active constituents of feverfew". Thesis, University of Southampton, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.252668.

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