Dissertations / Theses on the topic 'Extrinsic proteins'

Over the past two decades a body of evidence concerning residual biological contamination on cleaned surgical instruments has accumulated. This is substantiated by the number of yearly surgery cancellations due to visible residue on instruments in surgical packs and incidences of iatrogenic Creutzfeldt-Jakob disease (iCJD). It is therefore imperative to develop a method of protein detection for use in clinical sterile services departments (SSDs) for monitoring of decontamination quality. This Thesis describes the development and use of an epifluorescence surface scanner (EFScan) technology in the assessment of proteinaceous residue on surgical instruments, by detecting protein pre-labelled with fluorescein isothiocyanate (FITC), and exploratory studies on the feasibility of label-free detection, using intrinsic protein fluorescence. Measurements using FITC labelling showed that residual protein on the order of micrograms can be found on new, single-use instruments (i.e. prior to use). This is comparable to the amount of residual protein found on retired, reusable instruments. To confirm the suitability of fluorescence techniques in the detection and quantification of proteinaceous residue, a blind, pilot study was carried out in conjunction with groups from Queen Mary University and the University of Southampton. Each University used a different labelling and detection method, and results showed good agreement between these methods. This showed that fluorescence is a suitable technique for the detection and quantification of proteinaceous contamination on surgical instruments. The next step in the project focussed on detection of contamination via intrinsic protein fluorescence from tryptophan residues, with a view to elimination of the labelling step. Intrinsic fluorescence of proteins in solution is widely characterised; however, fluorescence characteristics of solid or surface-bound protein have been little studied. Therefore, the characterisation of solid protein fluorescence and the emission characteristics of protein adsorbed onto stainless steel was undertaken. Analysis of the commonly used protein standard bovine serum albumin (BSA) showed that the two tryptophan residues it contains are highly susceptible to photo-oxidation in the solid state, resulting in conversion to the fluorescent photoproducts n-formylkynurenine (NFK) and kynurenine. Therefore, BSA is not suitable for use as a standard in the development of intrinsic fluorescence detection of surface-bound protein. The 72-tryptophan protein fibrinogen, as well as a series of other multi-tryptophan proteins, were assessed and it was found that photo-oxidation of the tryptophan residues did not occur on the irradiation timescale of 1 hour utilized. Therefore, it was concluded that lysozyme or gamma-globulins, a prominent group of serum proteins, would be more suitable candidates as a standard in subsequent research into the intrinsic detection and quantification of proteinaceous contamination. A third study explored the potential use of fluorescence in the early diagnosis of cataract. This involved the fluorescence characterisation of healthy porcine lenses and the use of UV irradiation of the lens to attempt to create cataract in vitro. There was found to be a large variation in fluorescence characteristics from lens to lens, suggesting that fluorophore concentrations can vary significantly. This implies that identification of a suitable standard for the early detection of cataract may be problematic. Attempts to create cataract resulted in the photo-oxidation pathway which had been observed in BSA, and although NFK and kynurenine play a role in cataractogenesis, the accumulation of these photoproducts is not analogous to a natural cataract. It was found that these products could be destroyed by irradiation of the lens at appropriate photo-bleaching wavelengths. However, this also destroyed intrinsic, protective fluorophores within the lens, suggesting that a light-based method of cataract treatment may not be achievable.

L'incidence, le coût et l'issue fatale dans un nombre encore trop élevé de cas font du cancer un problème majeur en santé publique. Malgré les progrès réalisés dans le développement de thérapies ciblées, la plupart des cancers rechutent, vraisemblablement à cause de l'échappement des cellules souches cancéreuses (CSC) qui survivent et régénèrent la tumeur. L'enjeu clinique en cancérologie aujourd'hui est d'éliminer les cellules souches cancéreuses en épargnant les cellules souches normales. Pour atteindre cet objectif, il est primordial de comprendre leurs mécanismes spécifiques de transformation. Nous évaluons dans mon équipe de recherche l'implication du microenvironnement dans la transformation et la résistance des CSC épithéliales, à travers les effets de facteurs solubles et de contacts cellulaires : l'enzyme CD10, et la voie des BMPs (Bone Morphogenetic Proteins).Notre équipe étudie le rôle du dialogue permanent entre la CS normale et son microenvironnement qui régule la prolifération, et la survie des CS. Nous utilisons la glande mammaire et la prostate comme systèmes modèles car ces deux types d'épithélium présentent des similitudes, ce qui nous permet d'aborder la question de l'apparition et la résistance des CSC dans deux modèles tumoraux correspondants. Des dérégulations de la voie des BMPs, comme de l'enzyme CD10 sont observées dans ces tumeurs. Enfin, nous cherchons à comprendre comment les dérégulations de la voie des BMPs apparaissent, en s'intéressant principalement aux facteurs pouvant modifier directement le microenvironnement, tels que les polluants présents dans l'environnement (bisphénols, benzoapyrène)
It has been shown for a number of cancers that a cell population characterized by stem cell (SC) properties and therapeutic resistance is likely responsible for relapse several years after treatment. Current therapies kill most of the tumor cells, but fail to eradicate the so-called cancer stem cells (CSC). Therefore a complete cure of the disease will require the eradication of the tumor-sustaining CSC. We propose to study these CSC in the context of breast cancer as the existence of CSC as already been highlighted in this epithelia.CD10 is a membrane enzyme able to cleave several peptide of the microenvironment (such as oxytocin, bombesin, enkephalin.. ) that can also interact with intracellular signalling pathway through its direct interaction with PTEN. Our results, and those of the literature, indicate that CD10 enzyme controls the fate of SC and is deregulated in normal breast and cancerous tissues. We showed that CD10 membrane expression allows the maintenance of immature cells partly through its enzymatic function that inhibits mammary stem cells differentiation. As CD10 has been described in breast cancer initiation, progression and resistance, we then decided to test the role of CD10 in tumor context. Our strategy consists in flow cytometry cell sorting for CD10+/CD10- cells to compare the functional properties of both sub-population. Only CD10+ cells are able to regenerate both CD10+ and CD10- subpopulations, and CD10+ cells exhibit higher expression of immature genes. Interestingly, modulating CD10 using stable expression of CD10 in our models and Sh strategies do not mimick the normal functions of CD10, indicating that CD10 could be more a marker of a certain population with immature properties prone to transformation rather than a driver. To better characterize the role of CD10 in luminal breast transformation, we developed a new human mammary model, initiated from immature cells to obtain transformed luminal epithelial cells and their resistant counterpart. We observed a higher level of CD10 expression during mammary epithelial cell transformation process. We then performed a microarray on CD10+ and CD10- subpopulations. Preliminary analysis seems to confirm that CD10 is a potential marker for a stem cell population prone to transformation rather than a direct driver of the cell transformation

Abstract Reindeer management in the Fennoscandian area is currently facing challenges such as degradation of winter pastures, which may lead in the most severely affected areas to a concurrent decline in reindeer herd productivity. The use of often expensive supplementary feeding to prevent production losses has increased the demand for studies on the physiological effects of nutritional restriction and supplementary feeding. The knowledge obtained from such studies could be used, for example, to monitor the condition of reindeer in studies assessing herd productivity levels in different pasture conditions and management systems or sustainable use of pasture resources. In this thesis, the effects of season, year, pasture area, body mass, pregnancy and other extrinsic and intrinsic factors on the variation of blood chemical constituents of reindeer were studied in free-ranging animals under natural foraging conditions. The studied blood chemical constituents covered a wide range of parameters related to protein, carbohydrate, lipid and mineral metabolism. The same blood chemical constituents were studied in captive reindeer under defined feeding conditions, allowing an analysis of the effects of dietary protein, energy and mineral intake on the selected blood constituents and their comparison to a conventional measure of the animals' condition, live body mass. According to the results, free-ranging reindeer showed great variation in the concentrations of blood chemical constituents compared to the reference values of domesticated ruminants. Intrinsic factors such as body mass, pregnancy and age had only a minor influence on the variation of the studied parameters, whereas extrinsic factors such as season, year and pasture area, which were characterized by marked changes in environmental and nutritional conditions, explained the majority of the variation. The results obtained from captive animals in defined feeding conditions and from free-ranging animals foraging on natural pastures led to the conclusion that blood total proteins, albumin, urea, creatinine, urea:creatinine ratio, magnesium, inorganic phosphate and, to a lesser extent, globulins and albumin:globulin ratio responded to the changes in feed quality and availability and were the most suitable blood constituents to be used as nutritional biomarkers for reindeer.

The photosynthetic water oxidation reaction is catalyzed by an inorganic Mn4OxCaClyHCO3-z cluster at the heart of the oxygen evolving complex (OEC) in photosystem II. In the absence of an atomic resolution crystal structure, the precise molecular organization of the OEC remains unresolved. Accordingly, the role of the protein and inorganic cofactors of PSII (Ca2+, HCO3- and Cl-) in the mechanism of O2-evolution await clarification. In this study, rapid 18O-isotope exchange measurements were applied to monitor the substrate-water binding kinetics as a function of the intermediate S-states of the catalytic site (i.e. S3, S2 and S1) in Triton X-100 solubilized membrane preparations that are enriched in photosystem II activity and are routinely used to evaluate cofactor requirements. Consistent with the previous determinations of the 18O exchange behavior in thylakoids, the initial 18O exchange measurements of native PSII membranes at m/e = 34 (which is sensitive to the 16O18O product) show that the ‘fast’ and ‘slowly’ exchanging substrate-waters are bound to the catalytic site in the S3 state, immediately prior to O2 release. Although the slowly exchanging water is bound throughout the entire S-state cycle, the kinetics of the fast exchanging water remains too fast in the S2, S1 [and S0] states to be resolved using the current instrumentation, and left open the possibility that the second substrate-water only binds to the active site after the formation of the S3 state. Presented is the first direct evidence to show that fast exchanging water is already bound to the OEC in the S2 state. Rapid 18O-isotope exchange measurements for Ex-depleted PSII (depleted of the 17- and 23-kDa extrinsic proteins) in the S2 state reveals a resolvable fast kinetic component of 34k2 = 120 ± 14 s-1. The slowing down of the fast phase kinetics is discussed in terms of increased water permeation and the effect on the local dielectric following removal of the extrinsic subunits. In addition, the first direct evidence to show the involvement of calcium in substrate-water binding is also presented. Strontium replacement of the OEC Ca2+-site reveals a factor of ~3-4 increase in the 18O exchange of the slowly exchanging water across the S3, S2 and S1 states while the kinetics of the fast exchanging water remain unchanged. Finally, a re-investigation of the proposed role for bicarbonate as an oxidizable electron donor to photosystem II was unable to discern any 18O enrichment of the photosynthetically evolved O2 in the presence of 18O-bicarbonate. A working model for O2-evolution in terms of these results is presented.

All life on earth depends mainly on the presence of oxygen. Largest producers of oxygen are green plants, cyanobacteria and algae. Oxygen is released from the oxygenevolving complex of photosystem II during photosynthesis and it is used in cellular respiration of all life complexes. The oxygen-evolving complex of photosystem II has the same function in each photosynthetic organism, but it has a different composition and organization of extrinsic proteins; only PsbO protein is ubiquitous in all known oxyphototrophs. Until now only low resolution electron microscopy structural models of plant PSII and crystal structures of cyanobacterial PSII are available. Higher plant extrinsic proteins (PsbP, PsbQ and PsbR) are structurally unrelated, non-homologues to the cyanobacterial extrinsic proteins (PsbO, PsbU and PsbV) and this is the reason why it is not possible to predict arrangement of these proteins on the lumenal site of higher plant PSII. Recently, models differ mainly in the structure of the oxygen-evolving complex, which could be resolved by determination of the exact binding sites for extrinsic proteins. An other question evolves: if the difference in the oxygen-evolving complex composition is the result of evolution or adaptation of photosynthetic organisms to their environment. Structural knowledge of extrinsic proteins that could help to resolve the location and subsequently the function of extrinsic proteins is still incomplete. From this case,structural analysis, interactions and probably arrangement of proteins PsbP and PsbQ was studied and is described in detail in this thesis.

The photosynthetic water oxidation reaction is catalyzed by an inorganic Mn4OxCaClyHCO3-z cluster at the heart of the oxygen evolving complex (OEC) in photosystem II. In the absence of an atomic resolution crystal structure, the precise molecular organization of the OEC remains unresolved. Accordingly, the role of the protein and inorganic cofactors of PSII (Ca2+, HCO3- and Cl-) in the mechanism of O2-evolution await clarification. In this study, rapid 18O-isotope exchange measurements were applied to monitor the substrate-water binding kinetics as a function of the intermediate S-states of the catalytic site (i.e. S3, S2 and S1) in Triton X-100 solubilized membrane preparations that are enriched in photosystem II activity and are routinely used to evaluate cofactor requirements. Consistent with the previous determinations of the 18O exchange behavior in thylakoids, the initial 18O exchange measurements of native PSII membranes at m/e = 34 (which is sensitive to the 16O18O product) show that the ‘fast’ and ‘slowly’ exchanging substrate-waters are bound to the catalytic site in the S3 state, immediately prior to O2 release. Although the slowly exchanging water is bound throughout the entire S-state cycle, the kinetics of the fast exchanging water remains too fast in the S2, S1 [and S0] states to be resolved using the current instrumentation, and left open the possibility that the second substrate-water only binds to the active site after the formation of the S3 state. Presented is the first direct evidence to show that fast exchanging water is already bound to the OEC in the S2 state. Rapid 18O-isotope exchange measurements for Ex-depleted PSII (depleted of the 17- and 23-kDa extrinsic proteins) in the S2 state reveals a resolvable fast kinetic component of 34k2 = 120 ± 14 s-1. The slowing down of the fast phase kinetics is discussed in terms of increased water permeation and the effect on the local dielectric following removal of the extrinsic subunits. In addition, the first direct evidence to show the involvement of calcium in substrate-water binding is also presented. Strontium replacement of the OEC Ca2+-site reveals a factor of ~3-4 increase in the 18O exchange of the slowly exchanging water across the S3, S2 and S1 states while the kinetics of the fast exchanging water remain unchanged. Finally, a re-investigation of the proposed role for bicarbonate as an oxidizable electron donor to photosystem II was unable to discern any 18O enrichment of the photosynthetically evolved O2 in the presence of 18O-bicarbonate. A working model for O2-evolution in terms of these results is presented.

碩士
國立屏東科技大學
水產養殖系
95
Blood coagulation is an essential immune mechanism for aquatic animals with an open circulatory system such as crustaceans. This research is aimed to comprehend the molecular characteristics of the clottable protein in white shrimp, Litopenaeus vannamei, through molecular cloning of its cDNA and tissues expression, profiles. The expression of CP transcripts under different temperature, salinity and ammonia and post-Vibrio alginolyticusc injection were also investgated. CP cDNA was obtained from the heart of the white shrimp by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA using oligonucleotide primers based on the CP sequence of tiger shrimp, Penaeus monodon, and freshwater crayfish, Pacifastacus leniusculus. The full-length cDNAs of CP consisted of 5495 bp open reading frame encode 1666 amino acids (aa) with predicted include a 14-aa signal peptide, molecular mass of the mature protein was 187.917 kDa with an estimated pI of 5.27. Two putative integrin binding motif (cell adhesion site), RGD (Arg-Gly-Asp) and three N-glycosylation site were conserved in CP of white shrimp. Sequence comparison showed that CP deduced aa of L. vannamei had overall similarities of 85% and 37% to that of P. monodon and P. leniusculus respectively. Shrimp CP was found to exist in haemocyte, heart, gill, muscle, hepatopancreans, lymph organ and intestine by RT-PCR and real-time PCR. The clotting time of V. alginolyticus injection shrimps are significant longer than un-injection ones at 12 and 24 hours. At 22 and 34 ℃ water temperature for 2 days, the clotting time is significant longer than 28 ℃ treatment, moreover, after 7 days, it is significant shorter at 22 ℃treatment and significant longer at 34 ℃compared to 28 ℃. The clotting time in salinity 35 and 45 ‰ treatment for 2 and 7 days is significant longer than other treatments. When white shrimps expose to 5 and 10 mg l-1 ammonia-N for 7 days, the clotting time is significant longer than 1 mg l-1 and control groups. In V. alginolyticus challenge tests, shrimp CP expression in haemocyte, hepatopancrans and muscle were no significantly, heart CP was sharply increased post 24 h injection, but gill CP and intestine CP expression were down-regulation post 6 and 3 h, respectively, and return to initial levels at 24 and 24 h, respectively. Shrimp were exposed to 22 oC and 34 oC water for 2 days, hemocyte CP of shrimp at 34 oC was higher than shrimp at 22 oC, but both of them were higher than control shrimp at 28 oC. Heart CP and hepatopancreans CP were significantly raised when shrimp to be placed in 34 oC wate for 2 days, but return to initial levels after 7 days. Gill CP expression was significantly decreased after 2 days exposure to 34 oC water, and significantly increased after 7 days. The increment of CP expression was detected in muscle and intestine of shrimp at 34 oC for 2 days, but the graduals decrease of CP expression was detected in muscle of shrimp at 34 oC for 7 days. In salinity trials, heamocyte CP expression was lower than others treatments at 45 ‰ for 2 day, but return to initial levels post 7 days exposure to 45 ‰. Heart CP was down-regulation at 5 ‰ salt water for 2 days, but return to initial levels after 7 days. Shrimp at 15 ‰ salt water for 2 day, its gill CP expression was significantly increased, and a recovery was detected at 7 days. The expression of hepatopancreans CP was increase significantly at 35 and 45‰ for 7 days, and no significant different expression of CP was measured in muscle and intestine. Heamocyte CP expression was significantly increased after exposure to 1, 5 and 10 mg l-1 ammonia-N for 7 days, but no significantly different was found in the tissues of heart, gill and intestine. Up-regulation of CP was detected in shrimp at 1, 5 and 10 mg l-1 ammonia-N for 2 days when compared with control group. Muscle CP of shrimp at 10 mg l-1 ammonia-N for 2 days was increased significantly, and similar up-regulation was also to be found in muscle CP of shrimp at 5 and 10 mg l-1 ammonia-N for 7 days.

博士
國立臺灣海洋大學
水產養殖學系
93
Abstract Shrimp disease caused serious economic losses in world shrimp culture industry. These may be relevant in the control of infectious disease in imtensive farming of economically important shrimp. However, it is important to understand the immune mechanism of shrimp for prevent disease. In this project, white shrimp Litopenaeus vannamei was selected as a model species. The study is to focus on the cell adhesion protein, peroxinectin, which regulates and amplifies the immune response of shrimp. The paper is to deals with the purification of peroxinectin and analysis of its immune function, cloning of peroxinectin cDNA and its expression sites, expression of peroxinection under different molt stages, foreign intruders and under different environmental stressors including water temperature, salinity, ammonia-N and nitrite-N. Peroxinectin-like is a glycoprotein with molecular mass of 83 kDa. The cell adhesive protein was a multifunctional protein with several biological activities including cell adhesion, opsonin, peroxidase and encapsulation. Peroxinectin-like lost its activity under pH 4-2 and heated in 80 oC for 10 min. The cell adhesive activity was calcium-dependent, and it activity increased by sodium alginate and β-1, 3-glucan. The cell adhesive activity of peroxinectin was not associated with proPO system. The full-length cDNA of peroxinectin was 2,415 bp. The open reading frame encodes 805 amino acids, which contained a 20 amino acid signal peptide. The predicted molecular mass of mature protein was 89.1 kDa with a pI of 7.5. Sequence comparison showed that peroxinection deduced amino acid of L. vannamei had an overall similarity of 92 % and 60 % to that of P. monodon and P. leniusculus, respectively. The 432 bp fragment (partial cDNA) and 1016 bp fragment (genomic DNA) were obtained using specific primers PE2F and PE2R. There were three introns in the 1016 bp (genomic DNA) fragment. RT-PCR and in situ hybridisation indicated that peroxinectin mRNA synthesized in granular cell and semi-granular cell. The sequence of peroxinectin contained a peroxidase domain (~500 amion acid) and amino acids residues necessary for catalytic activity of peroxidase were conserved in white shrimp peroxinectin. Two putative integrin binding motifs, RGD (Arg-Gly-Asp) and KGD (Lys-Gly-Asp) were observed in white shrimp peroxinectin The assay of cell adhesive activity indicated that integrin-binding site was KGD motif, but not RGD motif. Peroxinectin transcript up-regulated significantly after 6 h post Vibrio alginolyticus-injection. The cell adhesive activity and peroxinectin transcript increased significantly for the shrimp exposed to 5 and 10 mg l-1 ammonia-N and nitrite-N. The cell adhesive activity and peroxinectin transcript were decreased significantly for the shrimp under high temperature (34oC) and low salinity (10 ‰). There was no significant difference in peroxinectin transcript under different molt stages, fed with/without 1 g kg-1 sodium alginate, and injected with saline solution, carbon, zymosan and dead V. alginolyticus.

碩士
國立屏東科技大學
水產養殖系
93
LGBP cDNA was cloned from the haemocytes and hepatopancreas of white shrimp Litopenaeus vannamei by reverse transcription polymerase chain reaction (RT-PCR) using degenerate primers. Analysis of nucleotide sequence revealed that the cDNA clone has an open reading frame of 1101 bp encoding a protein of 367 amino acids including a 17 amino acid signal peptide. The calculated molecular mass of the mature proteins is 39.92 kDa with an estimated pI of 4.37. Two putative integrin binding motifs (cell adhesion site), RGD (Arg-Gly-Asp) and a potential recognition motif for β-(1 → 3) linkage of polysaccharides were observed in the LGBP. Sequence comparison showed that LGBP deduced amino acid of L. vannamei has an overall similarity of 95%, 92% and 61% to that of blue shrimp Litopenaeus stylirostris LGBP, tiger shrimp Penaeus monodon BGBP and crayfish Pacifastacus leniusculus LGBP, respectively. They are with conserved features including a potential polysaccharide-binding motif, a glucanase motif, and N-glycosylation sites. To evaluate exogenous and endogenous factors that affect the expressions of LGBP genes, quantitative real-time PCR was conducted. The results were as follows: During different molting stages, LGBP transcripts in haemocytes at D2/3 stage were significantly higher than that at C stage, while the LGBP transcripts expression in hepatopancreas at the C stage is the highest. LGBP transcripts in haemocytes were up-regulated significantly after 3 and 6 h post Vibrio alginolyticus-injections, but the LGBP transcripts in hepatopancreas showed no significant differences. After feeding the diet containing 1 g kg-1 sodium alginate for a week, the expressions of LGBP transcripts in hepatopancreas were significantly lower than that of control groups, while the LGBP transcripts expressions in haemocytes showed no significant differences. The LGBP transcripts expressions in haemocytes at 18 and 34 groups were significantly higher than 26 ℃group after 48h thermal treatment, while the LGBP transcripts profiles in hepatopancreas at 18 and 26 ℃groups were significantly higher than 34 ℃ group after 168 h thermal treatment. The LGBP transcripts expressions in hepatopancreas in salinity 25 ‰ were significantly higher than that in 30 ‰, while LGBP transcripts in haemocytes showed no significant differences. The LGBP transcripts expressions in hepatopancreas were increased significantly while shrimps immersed in 10 mg/l ammonia-N after 48 h, but the LGBP transcripts expression in haemocytes showed no significant differences. The LGBP transcripts in haemocytes were increased significantly while shrimps immersed in 10 mg /l nitrite-N after 48 h, and 5 and 10 mg/l nitrite-N after 168 h, while the LGBP transcripts expressions in hepatopancreas treated in 5 mg/l nitrite-N after 48 h were increased significantly.