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WIDELITZ, RANDALL BRUCE. "HEAT SHOCK PROTEIN SYNTHESIS AND THERMOTOLERANCE EXPRESSION IN RAT EMBRYONIC FIBROBLASTS (HYPERTHERMIA, GENE REGULATION)." Diss., The University of Arizona, 1986. http://hdl.handle.net/10150/183851.
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In response to a variety of hyperthermic treatments, rat embryonic fibroblasts synthesize heat shock proteins (hsps), including those with molecular weights of 68,000 (hsp 68), 70,000 (hsp 70) and 89,000 (hsp 89). Hyperthermic stresses, which produce the hsps, also cause expression of thermotolerance. The dependence of thermotolerance expression on hsp synthesis was investigated in this mammalian cell line under different heating conditions. Temperature shift experiments showed that hsp synthesis and thermotolerance expression were dependent not only on the absolute hyperthermic temperature, but also on the difference between the initial incubation temperature and the hyperthermic temperature. Small temperature differences which produced no cell killing did not cause detectable synthesis of hsp 68. Increasing the difference of the initial and hyperthermic temperatures reduced cell survival and increased the synthesis of hsp 68. Thermotolerance could be expressed by surviving cells following an initial heat stress even when both heat shock and general protein synthesis were inhibited. Cells exposed to cycloheximide were heated, incubated at their initial temperature for six hours and reheated in the presence of the drug. The inhibitor was then removed and the cells plated for colony formation. The hsps were expressed during this latter incubation period. The regulation of hsp 70 in rat fibroblasts was investigated next. Hsp 70 synthesis rates correlated with the amount of hsp 70 encoding mRNA. The time course of heat shock synthesis and general protein synthesis recovery were each dependent on the duration of the heat stress. Inhibiting protein synthesis with cycloheximide resulted initially in the accumulation of the RNA encoding hsp 70 but did not effect the normal turnover of this RNA species. The conclusions based on these findings are that thermal survival adaptation can be expressed in the absence of hsp 68 synthesis. Hsp 68 is expressed by cells that will ultimately die (see Chapter 2). The hsps do not appear to protect cells against subsequent heat stress. They may function in a repair capacity (see Chapter 3). Hsp 70 expression is primarily regulated by transcription in Rat-1 cells. Hsp 70 does not act to regulate its own turnover (see Chapter 4).
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Ott-Reeves, Ellen (Ellen Theresa). "In Situ Hybridization of 70 kD Heat Shock Protein mRNA in a Rat Model of Ethanol Self-Administration." Thesis, University of North Texas, 1994. https://digital.library.unt.edu/ark:/67531/metadc332564/.
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Sucrose fading was used to initiate self-administration of ethanol on an FR4 schedule in male Fischer 344 rats. Rats showed low response rates for ethanol alone. After administration of liquid diet containing ethanol, ethanol intake increased over levels prior to administration of the liquid diet. In situ hybridization compared mRNA for the inducible or constitutive 70 kD heat shock proteins in ethanol and nonethanol rats. Both inducible and constitutive mRNAs were found in nonethanol and ethanol tissues. In peripheral organs, radiolableling was higher in ethanol tissue. In brain regions, nonethanol tissues showed higher radiolabeling.
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O'Sullivan, Joseph C. "The effect of diazoxide upon heat shock protein expression and physiological response to hemorrhagic shock and cerebral stroke." Download the dissertation in PDF, 2006. http://www.lrc.usuhs.mil/dissertations/pdf/O'Sullivan2006.pdf.
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Blood, Alan Physics Faculty of Science UNSW. "Biological effects of GSM mobile phone microwave radiation: an investigation of gene expression." Awarded by:University of New South Wales. School of Physics, 2005. http://handle.unsw.edu.au/1959.4/22071.
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There is evidence that athermal radiofrequency radiation can alter Heat Shock Protein (HSP) expression or protein phosphorylation, or alter MAP kinase signalling. Effects of long-term exposure in brain tissue due to repeated HSP perturbation (eg an inhibition of apoptosis) have been hypothesised (French et al, 2001). This study aimed to investigate the RNA expression profile (12,000 genes) and HSP family protein expression levels after either acute 1-hour or chronic 4-day intermittent exposures to simulated GSM radiation in a human primary fibroblast model. The results found minimal or no effects of GSM. Flasks were exposed to 900 MHz (217 Hz modulation) at 0.18 W/kg SAR within a Transverse Electromagnetic Mode chamber (TEM cell). Cultures rested for 2 hours before exposures. Affymetrix U95A microarray analysis of a single pilot set of experiments showed that about 40 genes were reported as upregulated >=2.5 fold in each condition. There was no evidence of altered expression of any MAPK-associated genes. Target genes reported in both conditions (CBFA2T1, ZNF148, ITGA1), and genes altered in one condition (CCS, PLEC1, BIRC5), and marginally altered HSP72 were selected for PCR analysis. No other members of the HSP family were altered. In three replicate experiments assayed by real-time PCR, six genes were either unchanged or showed randomly variable expression. However HSP72 RNA showed possible consistent slight upregulation of 1.37 +/- 0.21 in the chronic condition. Western immunoblots of HSP-60, -70, -72 and -V90 proteins showed no significant changes 5 hours after exposure. In preliminary studies using a serum starvation protocol, ERK-1 phosphorylation was unaltered after 5 or 30 minutes GSM (single experiments). When flasks were transiently cooled, ERK-1 phosphorylation was increased 20 minutes later, indicating a source of artefact in some protocols. An inflammatory challenge experiment with a low-dose of the cytokine IL-1???? found that acute GSM exposure post-challenge inhibited NF????B-mediated GRO???? induction by 1.5 fold (2 experiments). Preconditioning with mild heat induces transient inhibition of both NF????B signalling and apoptosis. Other studies indicate that EMF exposures similarly evoke cytoprotection. It is suggested that GSM evoked cytoprotective signalling in this inflammatory model.
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Chao, Sheng-Hao. "Heat Shock Proteins in Ascaris suum." Thesis, University of North Texas, 1995. https://digital.library.unt.edu/ark:/67531/metadc279311/.
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Ascaris suum were exposed to a number of stressors, including heavy metals and both high (40°C) and low (18°C) temperatures. The 70kD and 90kD heat shock proteins (HSPs) in the different A. suum tissues were analyzed by Western blot and quantitated by Macintosh Image Program.
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Wagstaff, Marcus James Dermot. "The neuroprotective effect of the heat shock proteins." Thesis, University College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.267150.
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Promisel, Carol Juanita. "Heat shock proteins in Mojave Desert dragonflies." CSUSB ScholarWorks, 1994. https://scholarworks.lib.csusb.edu/etd-project/910.
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Heydari, Ahmad R. Richardson Arlan. "The effect of age and caloric-restriction on the expression of heat shock proteins in rat hepatocytes." Normal, Ill. Illinois State University, 1990. http://wwwlib.umi.com/cr/ilstu/fullcit?p9115227.
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Thesis (Ph. D.)--Illinois State University, 1990.<br>Title from title page screen, viewed November 29, 2005. Dissertation Committee: Arlan Richardson (chair), Marjorie A. Jones, Lynne A. Lucher, Anthony J. Otsuka, Brian J. Wilkinson. Includes bibliographical references (leaves 168-187) and abstract. Also available in print.
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Fredriksson, Åsa. "On the role of protein oxidation and heat shock proteins in senescence and fitness /." Göteborg : Göteborg University, 2006. http://www.loc.gov/catdir/toc/fy0708/2006421399.html.
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Delaney, John Michael. "Regulation and function of the heat shock response in Escherichia coli." Diss., The University of Arizona, 1989. http://hdl.handle.net/10150/184776.
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The heat shock response is a highly conserved genetic mechanism which is induced by a wide range of environmental stimuli. Although intensively studied in both prokaryotes and eukaryotes, no regulatory mechanism has been identified by which the environmental stimuli affect expression of the heat shock genes. In addition, although many inducers of the heat shock response are known to cause DNA damage, the role of heat shock in repair of DNA damage remains unclear. Mutants of Escherichia coli defective in the recA, uvrA, and xthA genes are more sensitive to heat than wild type. However, these mutants are able to develop thermotolerance, suggesting that thermotolerance is an inducible response capable of repairing heat-induced DNA damage independent of recA, uvrA, and xthA. Thermotolerance itself is shown to depend on the dnaK gene, directly linking the E. coli heat shock response to thermotolerance. In addition, the dnaK mutant is sensitive to heat and H₂O₂, but not to UV suggesting that the DnaK protein may function to protect cells from the specific DNA damage caused by heat and H₂O₂. An E. coli grpE mutant was found to be substantially more resistant to 50°C heat treatment than wild type. However, grpE⁻ cells have the same H₂O₂ and UV sensitivity as wild type. This implies that the conditions, for which a grpE mutation is beneficial, are unique to heat exposure and are not caused by H₂O₂ or UV exposure. Furthermore, heat shock protein synthesis occurs sooner in the grpE mutant than in wild type, indicating that the grpE gene product of E. coli may act as a negative regulator of the heat shock response. An adenyl cyclase deletion mutant of E. coli (cya) failed to exhibit a heat shock response even after 30 min. at 42°C. Furthermore, a presumptive cyclic AMP receptor protein (CRP) binding site exists within the promoter region of the E. coli htpR gene. Together, these results suggest that the cya gene may regulate the heat shock response, through cyclic AMP, by directly affecting the level of expression of the heat shock sigma factor.
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Odunuga, Odutayo Odutola. "Molecular characterization of the tetratricopeptide repeat-mediated interactions of murine stress-inducible protein 1 with major heat shock proteins." Thesis, Rhodes University, 2003. http://hdl.handle.net/10962/d1007724.
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Murine stress-inducible protein 1 (mSTI1) is a co-chaperone that is homologous with the human heat shock protein 70 (Hsp70)/heat shock protein 90 (Hsp90)-organizing protein (Hop). The two proteins are homologues of the highly conserved stress-inducible protein 1 (STI1) family of co-chaperones. The STI1 proteins interact directly and simultaneously at some stage, with Hsp70 and Hsp90 in the formation of the hetero-multi-chaperone complexes that facilitate the folding of signal transducing kinases and functional maturation of steroid hormone receptors. The interactions of mSTI1 with both Hsp70 and Hsp90 is mediated by a versatile structural protein-protein interaction motif, the tetratricopeptide repeat (TPR). The TPR motif is a degenerate 34-amino acid sequence a-helical structural motif found in a significant number of functionally unrelated proteins. This study was aimed at characterizing the structural and functional determinants in the TPR domains of mSTI1 responsible for binding to and discriminating between Hsp70 and Hsp90. Guided by data from Hop's crystal structures and amino acid sequence alignment analyses, various biochemical techniques were used to both qualitatively and quantitatively characterize the contacts necessary for the N-terminal TPR domain (TPR1) of mSTI1 to bind to the C-terminal EEVD motif of heat shock cognate protein 70 (Hsc70) and to discriminate between Hsc70 and Hsp90. Substitutions in the first TPR motif of Lys⁸ or Asn¹² did not affect binding of mSTI1 to Hsc70, while double substitution of these residues abrogated binding. A substitution in the second TPR motif of Asn⁴³ lowered but did not abrogate binding. Similarly, a deletion in the second TPR motif coupled with a substitution of Lys⁸ or Asn¹² reduced but did not abrogate binding. Steady state fluorescence and circular dichroism spectroscopies revealed that the double substitution of Lys⁸ and Asn¹² resulted in perturbations of inter-domain interactions in mSTl1. Together these results suggest that mSTI1-Hsc70 interaction requires a network of electrostatic interactions not only between charged residues in the TPR1 domain of mSTI1 and the EEVD motif of Hsc70, but also outside the TPR1 domain. It is proposed that the electrostatic interactions in the first TPR motif collectively made by Lys⁸ and Asn¹² define part of the minimum interactions required for successful mSTI1-Hsc70 interaction. In the first central TPR domain (TPR1A), single substitution of Lys³°¹ was sufficient to abrogate the mSTI1-Hsp90 interaction. Using a truncated derivative of mSTI1 incapable of binding to Hsp90, residues predicted by crystallographic data to determine Hsp70 binding specificity were substituted in the TPR1 domain. The modified protein had reduced binding to Hsc70, but showed significant binding capacity for Hsp90. In contrast, topologically equivalent substitutions on a truncated derivative of mSTI1 incapable of binding to Hsc70 did not confer Hsc70 specificity on the TPR2A domain. These data suggest that binding of Hsc70 to the TPR1 domain is more specific than binding of Hsp90 to the TPR2A domain. In addition, residues C-terminal of helix A in the second TPR motif of mSTI1 were shown to be important in determining specific binding to Hsc70. Binding assays using surface plasmon resonance spectroscopy showed that the affinities of binding of mSTI1 to Hsc70 and Hsp90 were 2 μM and 1.5 μM respectively. Preliminary in vivo studies revealed differences in the dynamics of binding of endogenous and exogenous recombinant mSTI1 with Hsc70 and Hsp90. The outcome of this study poses serious implications for the mechanisms of mSTI1 interactions with Hsc70 and Hsp90 in the cell.
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Berry, Yoke. "The effect of pulsed electromagnetic fields on protein unfolding." Access electronically, 2005. http://www.library.uow.edu.au/adt-NWU/public/adt-NWU20060713.142625/index.html.
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Patel, Yogesh Jayendra Kumar. "Analysis of mutations in superoxide dismutase-1 and the protective effect of heat shock proteins against mutant-SOD1 toxicity." Thesis, University College London (University of London), 2006. http://discovery.ucl.ac.uk/1445899/.
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Genetic studies have revealed over 100 mutations in the gene encoding superoxide dismutase type-1 (SOD1) that cause Familial Amyotrophic Lateral Sclerosis (FALS). For the purpose of this thesis an in vitro model has been developed by stably over-expressing wild type (wt), G93A or G93R mutant SOD1 in neuronally derived ND7 cell line. It was found that wt-SOD1 could provide protection against a range of cell stresses including serum removal (plus retinoic acid) IFN-gamma, staurosporine camptothecin ischemia/reoxygenation glutamate and hydrogen peroxide (H2O2). In contrast, both mutant forms of SOD1 enhanced cell death with the G93R mutation being the more severe of the two mutations tested. Hence, the disease-associated mutations convert wt-SOD1 from a protective anti-apoptotic protein to a pro-apoptotic form. Most of the above stresses are FALS relevant stresses, which primarily induce apoptotic cell death in ND7 cells, implicated in FALS pathology. The neuroprotective effect of various heat shock proteins (Hsps) in the above system was studied, utilising a Herpes Simplex Virus (HSV) - based gene delivery system. For the first time, it was demonstrated that in an in vitro model of mutant-SOD1-induced toxicity, the exogenous expression of Hsp27 and/or Hsp70 protects both G93A and G93R-SOD1-mutant expressing cells, under all the stresses tested, and the dual expression of Hsp27 and Hsp70 is more effective in protecting against mutant-SOD1 cytotoxicity than either Hsp individually as assessed by trypan blue and TUNEL analysis. In addition, G93A and G93R- SOD1 mutant expressing cells were markedly protected by caspase-8 and caspase-9 inhibition. However no additive protective effect of Hsp and caspase inhibitor was observed. To further investigate the protective effect of wt-SOD1 and damaging effect of the mutant, an HSV-based gene delivery system was utilised. Primary cultures of dorsal root ganglia (DRGs) from postnatal rats, wild-type mice, transgenic Hsp27 and transgenic Hsp70 mice were infected with SOD1 viruses for wt-SOD1 and G93R-mutant SOD1 and subjected to stresses of NGF withdrawal, IFN-gamma and staurosporine treatment. Finally, the DRG experiments were repeated with additional delivery of Hsps via HSV vectors to further investigate the protective effect of the Hsps. These experiments provided further evidence on the protective role of Hsp27 and/or Hsp70 against mutant-SOD1 induced toxicity.
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Kühnel, Annett [Verfasser], Gabriele [Akademischer Betreuer] [Gutachter] Multhoff, Agnes [Gutachter] Görlach, and Johannes [Gutachter] Buchner. "The effect of heat shock factor 1 (HSF-1) and heat shock proteins Hsp70 and Hsp27 on radiosensitivity and immunogenicity / Annett Kühnel. Betreuer: Gabriele Multhoff. Gutachter: Gabriele Multhoff ; Agnes Görlach ; Johannes Buchner." München : Universitätsbibliothek der TU München, 2016. http://d-nb.info/1111776385/34.
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Rassadi, Roozbeh. "The effect of stress on nuclear protein transport : classical nuclear protein transport versus the nuclear transport of heat shock proteins." Thesis, McGill University, 1999. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=33476.
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The stress response is conserved among eukaryotes and affects different cellular functions including protein transport. Here, we have investigated the effect of different types of stress on classical nuclear protein import as well as nuclear import of Ssa4p family of heat shock proteins in Saccharomyces cerevisiae.<br>Under normal conditions, Aequorea victoria green fluorescent protein (GFP), carrying a classical nuclear localization sequence (cNLS-GFP) is nuclear. However, cNLS-GFP equilibrates throughout the cell upon exposure to heat, ethanol, H2O2 or starvation. Redistribution of the small GTPase Gsp1p, a soluble nuclear transport factor, correlates with cNLS-GFP equilibration. This suggests that a collapse of the Gsp1p gradient underlies the inhibition of classical nuclear protein import. In contrast to cNLS-GFP, the cytoplasmic heat shock protein Ssa4p accumulates in nuclei when classical nuclear import is inhibited. The N-terminal 236 amino acid residues of Ssa4p are sufficient for nuclear localization of Ssa4p-GFP upon heat and ethanol stress. The nuclear localization of Ssa4p(1--236)-GFP requires components of Gsp1-GTPase system, but is independent of Srp1p, the cNLS receptor.<br>Ssa4p(16--642)-GFP accumulates in nuclei of starving cells, mediated by a hydrophobic stretch of amino acid residues in its N-terminal domain. This nuclear localization is reversible upon addition of fresh medium and its export is sensitive to oxidants and temperature-dependent.
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Zourlidou, Alexandra. "Analysis of mutations in alpha-synuclein and the protective effect of heat shock proteins in a model of alpha-synuclein-induced toxicity." Thesis, University College London (University of London), 2005. http://discovery.ucl.ac.uk/1446825/.
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Genetic studies have revealed three mutations (A30P, A53T and E46K) in alpha-synuclein (alpha-Syn) that cause Parkinson's disease (PD) in a small number of pedigrees with autosomal dominant inheritance. For the purpose of this thesis an in vitro model has been developed by stably over-expressing wild type (wt), A30P or A53T mutant alpha-Syn in ND7 neuronal cells. Wt alpha-Syn can enhance cell death in response to ischaemia/reoxygenation or staurosporine treatment whilst protecting against serum removal and dopamine-induced cell death in this system. In contrast, both mutant forms of alpha-Syn enhance cell death. The above stresses were used to induce primarily apoptotic cell death, implicated in PD pathology. Hence, the PD-associated mutations convert alpha-Syn from a protein which could modulate cell death differently in different circumstances to forms which are deleterious in response to various stresses. Subsequently, the neuroprotective effect of various heat shock proteins (hsps) in the above system was studied, utilising a Herpes Simplex Virus-based gene delivery system. For the first time, it was demonstrated that in an in vitro mammalian model of alpha-Syn-induced toxicity over-expression of hsp27 protects, under all the stresses tested, both wt and mutant alpha-Syn expressing cells, as assessed by multiple apoptotic/necrotic death assays. Interestingly, A30P alpha-Syn expressing cells were markedly protected by caspase-8 and caspase-9 inhibition as well as by hsp27 over-expression. No synergy between hsp27 and the caspase inhibitors was observed. In addition, hsp70 conferred protection only to wt alpha-Syn expressing cells exposed to ischaemia whereas hsp56 had no protective role in this system. Hence, hsp27 was neuroprotective by interfering with the enhanced caspase-dependent cell death resulting from mutant A30P alpha-Syn over-expression. Finally, studies of the mitochondrial status in this system were performed to further explore the site of action of hsp27. Hsp27 reduced significantly the mitochondrial membrane potential loss in stressed A30P mutant alpha-Syn cells and this correlates well with their enhanced cell survival. These findings suggest that hsp27 has a novel neuroprotective role against mutant alpha-Syn toxicity and this is achieved by interfering with the caspase cascade and mechanisms modulating the mitochondrial membrane potential.
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Granger, Monique. "Expression of genes encoding bacteriocin ST4SA as well as stress proteins by Enterococcus mundtii ST4SA exposed to gastro-intestinal conditions, as recorded by real-time polymerase chain reaction (PCR)." Thesis, Stellenbosch : Stellenbosch University, 2007. http://hdl.handle.net/10019.1/19598.
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Thesis (MSc)--University of Stellenbosch, 2007.<br>ENGLISH ABSTRACT: The tolerance of Enterococcus mundtii ST4SA to stressful gastro-intestinal conditions in
humans and animals is vital to its success as a probiotic. The need for new effective
probiotics with stronger inhibitory (bacteriocin) activity has arisen due to the increasing
number of antibiotic resistant pathogens. Enterococci are used in the fermentation of
sausages and olives, cheese making and as probiotics. Their role as opportunistic
pathogens in humans makes them a controversial probiotic (Moreno et al., 2005).
Enterococci occur naturally in the gastro-intestinal tract which renders them intrinsic acid
and bile resistance characteristics. E. mundtii ST4SA produces a 3950 Da broad-spectrum
antibacterial peptide active against Gram-positive and Gram-negative bacteria, and
viruses. The bacteria include Enterococcus faecalis, Streptococcus spp., Pseudomonas
aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae and Staphylococcus
aureus. E. mundtii ST4SA inactivates the herpes simplex viruses HSV-1 (strain F) and
HSV-2 (strain G), a measles virus (strain MV/BRAZIL/001/91, an attenuated strain of
MV), and a polio virus (PV3, strain Sabin).
This study focuses on the genetic stability of E. mundtii ST4SA genes when exposed to
stress factors in the human and animal gastrointestinal tract. Based on results obtained by
real-time PCR, the expression of genes encoding bacST4SA, RecA, GroES and 23S
rRNA by E. mundtii ST4SA were not affected when the cells were exposed to acid, bile
and pancreatic juice. This suggests that these genes of E. mundtii ST4SA will remain
stable in the intestine. This could indicate that other genes of E. mundtii ST4SA could
remain stable in the host. Further studies on the stability of genes encoding antibiotic
resistance and virulence factors should be conducted to determine their stability and
expression in the host in stress conditions. Concluded from this study, E. mundtii ST4SA
is an excellent probiotic strain.<br>AFRIKAANSE OPSOMMING: Enterococcus mundtii ST4SA se weerstandsvermoë teen stresvolle gastrointestinale
kondisies is essensieel vir die sukses van hierdie organisme as ‘n probiotikum. Die
aanvraag vir nuwe, meer effektiewe probiotika met sterker inhibitoriese (bakteriosien)
aktiwiteit is as gevolg van die toename in antibiotikum weerstandbiedende patogene.
Enterococci word algemeen gebruik as probiotika, sowel as in die fermentasie van worse,
olywe en kaas. Hulle rol as oppertunistiese patogene in mense veroorsaak kontroversie as
gevolg van hul toenemende gebruik as probiotika. Enterococci is deel van die natuurlike
mikroflora in die gastrointestinale weg van mense en diere. Dit verleen aan hierdie
spesies ‘n natuurlike weerstandsvermoë teen maagsure, galsoute en pankreatiese
afskeidings. E. mundtii ST4SA produseer ‘n 3950 Da wye spektrum anti-bakteriese
peptied, aktief teen Gram positiewe en Gram negatiewe bakterieë sowel as virusse.
Hierdie bakterieë sluit Enterococcus faecalis, Streptococcus spp., Pseudomonas
aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae en Staphylococcus
aureus in. E. mundtii ST4SA inaktiveer die herpes simpleks virus HSV-1 en HSV-2, ‘n
masels virus (MV/BRAZIL/001/91), en ‘n polio virus (PV3, stam Sabin).
Hierdie studie fokus op die genetiese stabiliteit van E. mundtii ST4SA gene, wanneer
hulle blootgestel word aan stress faktore in die mens en dier gastrointestinale weg.
“Intydse” PKR data gebasseer op die uitdrukking van die bacST4SA, RecA, GroES en
23S rRNA gene in stresvolle kondisies dui aan dat E. mundtii ST4SA nie geaffekteer
word wanneer die sel blootgestel word aan suur, gal en pankreatiese vloeistowwe nie.
Hierdie resultate dui aan dat hierdie gene van E. mundtii ST4SA stabiel sal bly in die
intestinale weg van die mens en dier. Dit kan aandui dat ander gene van E. mundtii
ST4SA soos die wat kodeer vir virulensie faktore en antibiotikum se weerstandsvermoë
stabiel mag bly in die gasheer. Verdere studies wat fokus op die stabiliteit van gene wat
kodeer vir antibiotikum weerstandbiedendheid en virulensie faktore moet uitgevoer word
om hulle stabiliteit en uitdrukking in die gasheer te bepaal. Bevindings van hierdie studie
dui aan dat E. mundtii ST4SA goeie potensiaal het as ‘n probiotikum.
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Wang, Sihong. "Kinetics study of heat shock protein 70 expression." 2003. http://wwwlib.umi.com/cr/utexas/fullcit?p3116221.
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Sedger, Lisa Michele. "The role of heat shock proteins in virus infection." Phd thesis, 1995. http://hdl.handle.net/1885/144079.
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"Heat shock protein 70 expression in silver sea bream (Sparus sarba) tissues: effects of hormones and salinity." 2001. http://library.cuhk.edu.hk/record=b5890635.
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Ng Ho Yuen Andus.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.<br>Includes bibliographical references (leaves 105-131).<br>Abstracts in English and Chinese.<br>Chapter I --- Title page --- p.i<br>Chapter II --- Thesis committee --- p.ii<br>Chapter III --- Acknowledgement --- p.iii<br>Chapter IV --- Abstract --- p.v<br>Chapter V --- Abstract (Chinese version) --- p.vii<br>Chapter V --- Table of contents --- p.ix<br>Chapter VI --- List of abbreviations --- p.xv<br>Chapter VII --- List of figures --- p.xviii<br>General introduction --- p.1<br>Chapter Chapter 1: --- Literature review --- p.5<br>Chapter 1.1. --- Heat shock proteins (HSPs) --- p.6<br>Chapter 1.1.1. --- Introduction --- p.6<br>Chapter 1.1.2. --- The various heat shock proteins --- p.8<br>Chapter 1.1.2.1. --- HSP100s --- p.8<br>Chapter 1.1.2.2. --- HSP90s --- p.9<br>Chapter 1.1.2.3. --- HSP70s --- p.12<br>Chapter 1.1.2.3.1. --- ATPase reaction cycle of HSP70 and protein folding --- p.13<br>Chapter 1.1.2.3.2. --- Protein translocation --- p.14<br>Chapter 1.1.2.3.3. --- Selective lysosomal proteolysis --- p.16<br>Chapter 1.1.2.4. --- HSP60s --- p.16<br>Chapter 1.1.2.5. --- Small HSPs --- p.17<br>Chapter 1.1.2.6. --- Ubiquitin --- p.19<br>Chapter 1.1.3. --- HSP studies in fish --- p.21<br>Chapter 1.1.3.1. --- In vivo works --- p.21<br>Chapter 1.1.3.2. --- In vitro works --- p.23<br>Chapter 1.2. --- Growth hormone / prolactin family in teleostean fishes --- p.26<br>Chapter 1.2.1. --- Introduction --- p.26<br>Chapter 1.2.2. --- Growth hormone (GH; somatotropin) --- p.29<br>Chapter 1.2.2.1. --- Structure --- p.29<br>Chapter 1.2.2.2. --- Actions --- p.29<br>Chapter 1.2.2.3. --- Insulin-like Growth Factors (IGFs; somatomedins) --- p.31<br>Chapter 1.2.3. --- Prolactin (PRL) --- p.34<br>Chapter 1.2.3.1. --- Structure --- p.34<br>Chapter 1.2.3.2. --- Actions --- p.35<br>Chapter 1.2.4. --- Somatolactin (SL) --- p.37<br>Chapter 1.2.4.1. --- Structure --- p.37<br>Chapter 1.2.4.2. --- Actions --- p.38<br>Chapter 1.2.5. --- Growth hormone receptor (GH-R) and prolactin receptor (PRL-R) --- p.39<br>Chapter 1.3. --- Cortisol in teleostean fishes --- p.41<br>Chapter 1.4. --- Salinity adaptation in teleosts --- p.44<br>Chapter Chapter 2: --- Effect of in vitro thermal shock on HSP70 expression in whole blood of Sparus sarba --- p.46<br>Chapter 2.1. --- Introduction --- p.47<br>Chapter 2.2. --- Materials and methods --- p.49<br>Chapter 2.2.1. --- Overall experimental design --- p.49<br>Chapter 2.2.2. --- Experimental fish --- p.49<br>Chapter 2.2.3. --- Blood sampling and preparation --- p.49<br>Chapter 2.2.4. --- Thermal stress regimes --- p.50<br>Chapter 2.2.5. --- Protein extraction --- p.51<br>Chapter 2.2.6. --- Protein quantification --- p.51<br>Chapter 2.2.7. --- Indirect enzyme-linked immunosorbent assay (ELISA) --- p.52<br>Chapter 2.2.8. --- Protein gel electrophoresis and immunoblotting (Western blotting) --- p.54<br>Chapter 2.2.9. --- Statistical analysis --- p.55<br>Chapter 2.3. --- Results --- p.56<br>Chapter 2.3.1. --- Validation of indirect ELISA --- p.56<br>Chapter 2.3.2. --- Effect of in vitro thermal shock on HSP70 expression in whole blood of Sparus sarba --- p.56<br>Chapter 2.4. --- Discussion --- p.60<br>Chapter 2.5. --- Conclusion --- p.64<br>Chapter Chapter 3: --- Effects of hormones on HSP70 expression in whole blood of Sparus sarba in vitro --- p.65<br>Chapter 3.1. --- Introduction --- p.66<br>Chapter 3.2. --- Materials and methods --- p.68<br>Chapter 3.2.1. --- Overall experimental design and experimental fish --- p.68<br>Chapter 3.2.2. --- Hormone treatments --- p.59<br>Chapter 3.2.3. --- "Protein extraction and quantification, indirect ELISA，gel electrophoresis, and immunoblotting (Western blotting)" --- p.70<br>Chapter 3.2.4. --- Statistical analysis --- p.70<br>Chapter 3.3. --- Results --- p.71<br>Chapter 3.3.1. --- Effect of Cortisol on HSP70 levels in whole Blood --- p.71<br>Chapter 3.3.2. --- Effect of recombinant bream growth hormone on HSP70 levels in whole blood --- p.71<br>Chapter 3.3.3. --- Effect of recombinant bream insulin-like growth factor-I on HSP70 levels in whole blood --- p.71<br>Chapter 3.3.4. --- Effect of ovine prolactin on HSP70 levels in whole blood --- p.72<br>Chapter 3.4. --- Discussion --- p.81<br>Chapter 3.4.1. --- Effect of Cortisol on HSP70 levels in whole Blood --- p.81<br>Chapter 3.4.2. --- Effect of recombinant bream growth hormone on HSP70 levels in whole blood --- p.83<br>Chapter 3.4.3. --- Effect of recombinant bream insulin-like growth factor-I on HSP70 levels in whole blood --- p.85<br>Chapter 3.4.4. --- Effect of ovine prolactin on HSP70 levels in whole blood --- p.86<br>Chapter 3.5. --- Conclusion --- p.88<br>Chapter Chapter 4: --- Effect on HSP70 expression in whole blood of Sparus sarba acclimated to various salinities --- p.89<br>Chapter 4.1. --- Introduction --- p.90<br>Chapter 4.2. --- Materials and methods --- p.92<br>Chapter 4.2.1. --- Overall experimental design and experimental fish --- p.92<br>Chapter 4.2.2. --- "Protein extraction and quantification, indirect ELISA, gel electrophoresis, and immunoblotting (Western blotting)" --- p.92<br>Chapter 4.2.3. --- Statistical analysis --- p.93<br>Chapter 4.3. --- Results --- p.94<br>Chapter 4.4. --- Discussion --- p.97<br>Chapter 4.5. --- Conclusion --- p.100<br>Chapter Chapter 5: --- General discussion and conclusion --- p.101<br>References --- p.105
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"In vitro study of hormonal regulation of heat shock protein 70 expression in sea bream." 2003. http://library.cuhk.edu.hk/record=b6073530.
Full textAbstract:
Zhou Liran.<br>"June 2003."<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2003.<br>Includes bibliographical references (p. 182-216).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Mode of access: World Wide Web.<br>Abstracts in English and Chinese.
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"Roles of prolactin in salinity adaptation, Hsp70 expression and apoptosis in sparus sarba." Thesis, 2007. http://library.cuhk.edu.hk/record=b6074513.
Full textAbstract:
Also, the branchial hsp70 levels in fish following chronic salinity acclimation and abrupt hypo-osmotic exposure to 6 ppt were assessed by Western blotting. Upon chronic salinity acclimation, the lowest branchial hsp70 level was found in fish cultured in an iso-osmotic salinity of 12 ppt and the highest was in 50 ppt and 6 ppt environments. Freshwater acclimation resulted in return to lower hsp70 level. The results indicated that iso-osmotic salinity would bring about the least stress level while 50 ppt and 6 ppt were the most stressful salinities to Sparus sarba as indicated by using hsp70 expression as a biomarker of stress. Compared to 50 ppt and 6 ppt, the stress level of fish in fresh water was lower. On the other hand, Sparus sarba exhibited a significant increase in branchial hsp70 level immediately after abrupt hypo-osmotic exposure to 6 ppt when compared with seawater fish sampled at the same time point and increased hsp70 level was sustained throughout the sampling period, indicating the exposure was stressful to the fish.<br>In the present study, pituitary and serum levels of prolactin in a marine teleost, Sparus sarba, chronically acclimated to various salinities: fresh water (0 ppt), hypo-osmotic (6 ppt), iso-osmotic (12 ppt), normal seawater (33 ppt) and hypersaline (50 ppt) or abruptly exposed to a hypo-osmotic environment of 6 ppt were quantified by the developed peptide-based indirect ELISAs. Progressive increases in pituitary and serum prolactin were found as chronic salinity acclimation progressed from seawater to fresh water. Also, prolactin secretion was immediately induced by abrupt hypo-osmotic exposure to 6 ppt and remained significantly elevated up to 5 days post-exposure to 6 ppt. The results underline the importance of prolactin in marine teleosts kept in fresh water or waters of low salinity. However, there was no significant difference in pituitary prolactin during the course of the abrupt hypo-osmotic exposure experiment. The results may indicate that prolactin might be secreted rapidly from pituitary in large quantities to cope with abrupt exposure to a low-salinity environment.<br>In the present study, the effects of pharmacological drugs on prolactin levels in pituitary and serum of Sparus sarba were investigated. An increase in prolactin synthesis and release but a decrease in branchial hsp70 expression were found after treatment with sulpiride, a DA-D2 receptor antagonist. In contrast, a reduction in prolactin levels in pituitary and serum but an elevation in hsp70 level in gill were observed following administration of bromocriptine, a DA-D2 receptor agonist. Since hsp70 expression indicates the stress levels, the results of these studies supported the notion that increased prolactin synthesis and release might be related to a reduced stress state and prolactin might have a protective effect on stress tolerance in fish.<br>Lastly, the role of prolactin in regulating apoptosis in Sparus sarba branchial cells was examined. Successful induction of apoptosis was indicated by an increase in the apoptotic parameter caspase-3 activity in primary cultures of Sparus sarba branchial cells treated with camptothecin, a specific inducer of apoptosis. In this study, prolactin was shown to be anti-apoptotic in Sparus sarba branchial cells as co-treatment with ovine prolactin (oPRL) and camptothecin has been observed to attenuate the elevated caspase-3 activity in gill cell primary cultures. Also, prolactin was found to protect the branchial cells from apoptosis by maintaining the hsp70 level in the cells treated with camptothecin.<br>The objectives of the present study were to investigate the roles of prolactin in salinity adaptation, hsp70 expression and apoptosis in silver sea bream (Spaurs sarba). Firstly, specific peptide-based indirect ELISAs were developed for pituitary and serum prolactin of Sparus sarba. These assays had been validated by parallelism between the dilution response curves using serially diluted pituitary homogenate and serum sample with the standard curves of the synthetic peptide derived from the amino acid sequence of black sea bream (Acanthopagrus schlegelii ) prolactin.<br>Ng, Ho Yuen Andus.<br>"September 2007."<br>Adviser: N. Y. S. Woo.<br>Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4567.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2007.<br>Includes bibliographical references (p. 143-189).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Abstracts in English and Chinese.<br>School code: 1307.
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Chiu, Chi-chou, and 邱啟洲. "Effects of Amino Acid Analog on Biosynthesis of Soybaen Class I Low-molecular-weight Heat Shock Proteins: Physiological Biochemical Analysis and Their Thermotolerance." Thesis, 1997. http://ndltd.ncl.edu.tw/handle/08445131916491706744.
Full textAbstract:
碩士<br>國立臺灣大學<br>植物學系<br>85<br>Class I low-molecular-weight heat shock proteins (LMW HSPs)
which are likely to promote thermotolerance in soybean seedlings
are induced by heat shock treatment as well as
azetidine-2-carboxylic acid (Aze), a proline analog. The amount
of class I LMW HSPs induced by 10 mM Aze for 24 hours was
equivalent to that induced by 40℃for 2 hours. However,
these class I LMW HSPs induced by Aze treatment had no effect on
thermotolerance of seedlings.
When soybean seedlings, treated with 10 mM Aze for 6 hours, were
then incubated with several different amino acids for 24
hours, respectively, all seedlings of each treatment accumulated
certain amount of class I LMW HSPs. However, only the seedlings
incubated with proline subsequently acquired
thermotolerance. In these seedlings, about 40% of class I
LMW HSPs were found in post-ribosomal supernatant (PRS) fraction
of total protein.On the other hand, only around 10% of class I
LMW HSPs induced by subsequent incubated with other amino
acids (e.g., Gly, Phe, Cys and Gln) were found in PRS.
Class I LMW HSP complex from the soybean seedlings treated with
Aze and subsequently with proline reduced denaturation of PRS
of soybean seedlings at high temperature. Analysis of
amino acid composition indicated that Aze was not incorporated
into class I LMW HSP (pI 6.2, MW 17.3), but was found in
protein which was not inducible during heat shock.
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Liu, Yi-Hsin, and 劉依欣. "Physiological function assay of class I small heat shock proteins, OsHSP16.9A and OsHSP18.0." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/05326560564023071824.
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碩士<br>國立中央大學<br>生命科學研究所<br>99<br>Recently global warming is threatening the crop yield. It becomes a critical issue to improve tolerance of rice to harsh environment as well as crop yield. Rice (Oryza sativa) is one of the most important crops in the world and feeds nearly 50% of the world’s population. Besides, it can be a model plant for providing insights into gene functions of monocotyledonous plants. Based on our previous data, we found that rice class I small heat shock proteins (sHSP-CIs) selectively expressed during seed maturation (OsHSP16.9A) and Cu or Cd treatment (OsHSP18.0). To further characterize physiological function of sHSP-CIs, we constructed two transgenic rice plants constitutively overexpressing OsHSP16.9A (OsHSP16.9A-OE) and OsHSP18.0 (OsHSP18.0-OE) and three transgenic rice plants repressing OsHSP16.9A (Oshsp16.9A-RNAi), OsHSP18.0 (Oshsp18.0-RNAi), and sHSPs (Osshsp-RNAi). Our results indicated that Oshsp16.9A-OE seeds and seedlings and Oshsp18.0-OE seedlings had higher thermotolerance than wild type during heat stress. Besides, Osshsp-RNAi plants were sensitive to heat stress. Interesting, gain- and loss-of-function analyses identified that sHSPs involved in rice growth and development. Osshsp-RNAi plants were shown increased in tiller number and delayed in growth. These results suggest that sHSPs can not only function as chaperone to protect denatured proteins but also involve in rice growth process. Furthermore, OsHSP18.0-OE plants showed more resistance than OsHSP16.9A-OE and wild-type plants under Cu and Cd treatment. Similarly, OsHSP18.0-OE plants were less oxidative damage. In addition, OsHSP18.0-OE plants reduced Cu-induced membrane damage. These results indicated OsHSP18.0-OE play important role in heavy metal stress.
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BOROVANSKÁ, Michaela. "Physiological and molecular adaptations during diapause development and overwintering in a heteropteran bug, Pyrrhocoris apterus." Doctoral thesis, 2009. http://www.nusl.cz/ntk/nusl-44284.
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In this thesis I present complex experimental data on the physiological and molecular adaptations during diapause development and overwintering in a linden bug, Pyrrhocoris apterus (Heteroptera, Pyrrhocoridae). I focus on adjustments of the enzymatic complement, which is involved in the biosynthesis of cryoprotectants, and heat shock proteins, which are expressed in response to temperature stress.
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Yang, Jin-Shiou, and 楊金修. "THE EFFECT OF THERAPEUTIC ULTRASOUND ON HEAT SHOCK PROTEINS INDUCTION IN SKELETAL MUSCLES." Thesis, 1997. http://ndltd.ncl.edu.tw/handle/94960481170623052287.
Full textAbstract:
碩士<br>高雄醫學院<br>醫學研究所<br>85<br>Therapeutic ultrasound (US) has been widely used in the
treatment of musculoskeletal injuries for decades. Despite
many physiological effects have been reported to verify the
value of US in clinic, very few researches confirmed the real
mechanisms. For years, it has been well-known that heat shock
proteins (HSPs) play important roles in cellular protection by
resistance to environmental stresses or en- hancing the cell
repair. The main purpose of this study is to investigate the
relationship of the synthesis of HSPs and ultrasound. In
this study, adult male Sprague Dawley rats were employed. For
every rat, right hindlimb was true-sonicated, while left one was
mock-sonicated as control. The muscle specimens were removed 15
hours after last sonication. The detection of HSP72 was
performed by polyacrylamide gel electrophoresis, Western blot,
and immunochemical stain by monoclonal anti-HSP72 antibody.
The study was designed to verify the fact that US can increase
HSP72 synthesis in the skeletal muscle, and to determine the
appro- priate dose of US on enhancement of HSP72 synthesis. The
content of this study is as follows. 1. Detect the levels of
HSP72 in gastrocnemius muscle after sonication at different
intensities of 1/2 W/ｃ㎡、2/3 W/ｃ㎡、5/6 W/ｃ㎡、 1 W/ｃ
㎡. The muscular temperature was also detected. 2. Compare the
levels of HSP72 in gastrocnemius muscle after sonication at
the appropriate intensity obtained from the above result for 5
min, 10 min, 15 min, 20 min. 3. According to the established
rat model in studying heat shock response, we compared the
level of HSP72 in gastrocnemius muscle after sonication at
the appropriate intensity obtained from the above result
with that after whole-bodily heated by means of electric heating
pad. 4. In the mode-dependent study, we compared the level of
HSP72 in gastrocnemius muslce after sonicatio nwith the
pulse or continuous mode. 5. To observe the accumulative
effect, rats were assigned into two groups to receive 3
times and 5 times sonications respectively. Then the levels
of HSP72 were detected. 6. The gastrocnemius muscles sonicated
with the above appropriate dose were removed after one day
and seven days respectively for mor- phological changes.
The results showed that 1) 2/3 W/ｃ㎡、 5/6 W/ｃ㎡，and 1 W/ｃ㎡
induce more HSP72 synthesis, but the effect of 1/2 W/ｃ㎡ in
HSP72 induction was unapparent. The difference of the muscular
temperature changes between the groups receiving 1/2 W/ｃ㎡
and 2/3 W/ｃ㎡ treatment respectively was significant; 2) In
the duration-dependent study, we found that the amount of HSp72
synthesis significantly increased in the group of receiving 20
min sonication; 3) US induced more HSP72 synthesis as the
electric heating pad does; 4) The rats which were sonicated in
t continuous mode enhanced apparently HSP72 synthesis; 5)
The rats which were sonicated daily for 5 days can induce more
HSP72 synthesis. Finally, 6)Light microscopy demonstrated no
necrosis or inflammatory conditions between mock-and true-
sonicated muscles. In conclusion, application of 1 MHz
therapeutic ultrasound at the average intensity of 2/3 W/ｃ㎡
for 20 minutes in the continuous mode is an appropriate dose in
enhancing HSP72 synthesis. The mechanism of HSP72 induction
with US is due to thermal effect. The dose-dependent response
and accumulative effect were present. Apparently, HSP72 is an
index on the thermal effects of US. The rat model is a reliable
model in studying local heat shock response of mammals in
skeletal muscle.
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Jin, Zong Luo, and 靳宗洛. "Class I low molecular weight heat shock proteins in soybean:purification, characterization, physiological biochemical function analysis and immunolocalization." Thesis, 1995. http://ndltd.ncl.edu.tw/handle/55153889313229920168.
Full textAbstract:
博士<br>國立臺灣大學<br>植物學研究所<br>83<br>Examination of an ammonium sulfate-enriched fraction (70-100%
saturation) of heat-shock proteins (HSPs) by nondenaturing polyacrylamide gel electrophoresis revealed the presence of a high molecular mass complex (280kD) in soybean (Glycine max) seedlings.
This complex cross-reacted with antibodies raised against soybean class I low-molecular-mass (LMW) HSPs. Dissociation of the complex by denaturing polyacrylamide gel electrophoresis showed the complex to contain at least 15 polypeptides of the 15- to 18-kD class I LMW HSPs that could be detected by staining, radiolabeling, and western blotting. A similar LMW-HSP complex was observed in mung bean (Vigna radiata L.; 295 kD), in pea (Pisum sativum L.; 270kD), and in rice (Oryza sativa L.; 310 kD). The complex was stable under high salt conditions (250 mM KCl), and the integrity was not affected by 1% Nonidet P-40 and 3.mu.g/mL RNase treatment. The size of the isolated HSP complex in vitro was conserved to 55.degree.C; however, starting at 37.5.degree.C, it changed to higher molecular forms in the presence of soluble proteins. The
isolated HSP complex was able to protect up to 75% of the soluble proteins from heat denaturation in vitro.
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Liu, Kuo-Yang, and 劉國揚. "The Effect of High Intensity Interval Training on Heat Shock Proteins Expression in Tendon." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/25372444481336872763.
Full textAbstract:
碩士<br>高雄醫學大學<br>運動醫學系碩士在職專班<br>104<br>The effect of High-Intensity Interval Training (HIIT) on the Achilles tendon is not completely understood. Heat shock protein (HSP) is produced by acute exercise-induced radical oxygen species (ROS) in humans, upregulated in heat shock response, an indicator of cellular stress, acting as chaperone protein and stabilizing new protein or help-ing to refold impaired protein. We hypothesized that supplementation of Vitamin C in HIIT may modulate the gait and expression of HSPs & IGF-1 (insulin-like growth factor 1) in Achilles tendon. Twenty-eight male Sprague-Dawley male rats were randomly divided into the fol-lowing four groups including control (C, n=7), HIIT (H, n=7), control Vitamin C (CV, n=7), HIIT Vitamin C (HV, n=7). The amount of Vitamin C was 500mg/kg/day for 3 weeks in the CV and HV groups. The H & HV groups received HIIT by treadmill for 2 weeks. Statistical significance was set at p<0.05. Hindlimb footprints of the H group re-vealed on-toe pattern. The parameters of Cat-Walk test, including swing duration, step cycle, duty factor and step length, were signifi-cantly different in groups (p<0.05). Comparing HV to H, only swing duration showed significance (p<0.05). HSP27, HSP70 and IGF-1 of the Achilles tendon expressed elevation in the H group (p<0.05), but remained near to basal level in the HV group (p<0.05). Immunofluo-rescent analysis of HSP70 in the Achilles tendon showed positive cy-toplasmic localization in many cells of the H group, but only in a few cells of the HV group. H group showed decreased duty factor and on-toe gait of locomotion indicating the alteration of gait compared to control. H group showed dramatically increased HSP70 and HSP27 in Achilles tendon indicating the possibility of cellular stress after HIIT. After Vitamin C consumption, the supposed dramatic increasing ex-pression of HSPs in H group has been diminished indicating the im-portant role of ROS in HSP induction during HIIT. The increased IGF-1 secretion in H group contributed to protein synthesis in Achilles tendon indicating adaptation of tendon after HIIT. After vitamin C consumption, the supposed dramatic increasing secretion of IGF-1 in H group has been diminished indicating the important role of ROS in IGF-1 secretion during HIIT. HIIT causes adaptation in the Achilles tendon. Whether HIIT causes Achilles tendon impairment or functional improvement is uncertain according to the alteration of gait. Imbalance between production and clearance of ROS can be withstood by Vita-min C supplementation.
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Kuo, Mei-Fang, and 郭美芳. "Effect of preconditioning heat shock treatment in mitochondrial proteins of liver in septic rats." Thesis, 2003. http://ndltd.ncl.edu.tw/handle/41715205297777322707.
Full textAbstract:
碩士<br>高雄醫學大學<br>醫學研究所<br>91<br>Sepsis is regarded as a major initiator of multiple organ dysfunction syndrome that remains a leading cause of mortality in medical critical care unit. Mitochondrial dysfunction plays an important role in the cascade. Our previous studies have shown that the ATP synthesis and the mitochondrial respiratory enzyme activities were all down-regulated in the liver of experimental septic rats. In addition, heat shock response was demonstrated to the protective effect against sepsis in rats while the mechanism is still a mystery. Accordingly, the present study was designed to investigate the proteomic alternation of hepatic mitochondria during sepsis, and explore the molecular protective mechanism of preconditioning heat shock treatment.
Spraque-Dawley rats were used as the experimental animal. They were divided into two groups: non-heated and heated. Rats of heated group were whole-bodily heated to 41.5±0.5 °C for 15 min to induce the over-expression of heat shock proteins (Hsps24) hr before sepsis induction. Sepsis was induced by cecal ligation and puncture (CLP). The rats were sacrificed 9h and 18h after CLP that was defined as early and late phase of sepsis, respectively. The mitochondrial proteins of the liver were extracted and separated by two-dimensional electrophoresis with broad immobilized pH gradient strip (PH 3-10) and SDS-PAGE. The protein spots were visualized with silver stain and analyzed by Bio-2D software.
Results of Western blot and immunochemical analysis showed that Hsp72 was over-expressed maximally at 24 and sustained till 42 hours after heat shock treatment. Hsp72 was not induced in early and late sepsis, while heated septic rats all showed Hsp72 over-expression. Results of two-dimensional electrophoresis analysis showed that around 120 spots could be separated and visualized distinctly. Among them, 3 spots with same molecular weight (56.4KD) were significantly altered in septic specimens, named MP1 (PI 7.5), MP2 (PI 8.0) and MP3 (PI 8.5). MP1 and MP2 were down-regulated in sepsis while MP3 was up-regulated coincidently. Interestingly, heat shock treatment could reverse the phenomenon. Analyzed by LC/MS/MS, the 3 spots were revealed to be an identical enzyme: aldehyde dehydrogenase 2 (EC 1.2.1.3). However, RT-PCR assay of the enzyme revealed no significance in all specimens. The enzyme activity assay showed that aldehyde dehydrogenase 2 activity was down-regulated in non-heated late septic rats and reversed in heated late septic rats. But the activities between heated and non-heated early septic rats were not different significantly.
In conclusion, we suggest that post-translation modification of aldehyde dehydrogenase 2 may play a functional role in the pathogenesis of sepsis and provide a novel protective mechanism of heat shock treatment.
30
Roberts, Deirdre. "Heat-shock protein expression in Mytilus californianus : seasonal and tidal height comparisons." Thesis, 1995. http://hdl.handle.net/1957/35320.
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Voyer, Janine. "Effect of heat shock factor inhibitor, KNK437, on stress-induced hsp30 gene expression in Xenopus laevis A6 cells." Thesis, 2008. http://hdl.handle.net/10012/3630.
Full textAbstract:
Prokaryotic and eukaryotic organisms respond to various stresses with the production of heat shock proteins (HSPs). HSPs are molecular chaperones that bind to unfolded proteins and inhibit their aggregation as well as maintaining their solubility until they can be refolded to their original conformation. Stress-inducible hsp gene transcription is mediated by the heat shock element (HSE), which interacts with heat shock transcription factor (HSF). In this study, we examined the effect of KNK437 (N-formyl-3,4-methylenedioxy-benzylidene-g-butyrolactam), a benzylidene lactam compound, on heat shock, sodium arsenite, cadmium chloride and herbimycin A-induced hsp gene expression in Xenopus laevis A6 kidney epithelial cells. In studies limited to mammalian cultured cells, KNK437 has been shown to inhibit HSE-HSF1 binding activity and stress-induced hsp gene expression. In the present study, western and northern blot analysis revealed that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP30 protein and hsp30 mRNA, respectively. Western blot analysis also determined that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP70 protein. Pre-treatment of A6 cells with 100 µM KNK437 inhibited stress-induced hsp30 mRNA as well as HSP30 and HSP70 protein accumulation. Immunocytochemistry and confocal microscopy were used to confirm the results gained from western blot analysis as well as determine the localization of HSP30 accumulation in A6 cells. A 2 h heat shock at 33°C resulted in the accumulation of HSP30 in the mostly in the cytoplasm with a small amount in the nucleus. Heat shock at 35°C resulted in substantial HSP30 accumulation in the nucleus. This is in contrast with A6 cells treated for 14 h with 10 µM sodium arsenite, 100 µM cadmium chloride and 1 µg/mL herbimycin A, where HSP30 accumulation was found only in the cytoplasm and not in the nucleus. A 6 h pre-treatment with 100 µM KNK437 completely inhibited the accumulation of HSP30 in A6 cells heat shocked at 33 or 35°C as well as cells treated with 1 µg/mL herbimycin A. The same pre-treatment with KNK437 resulted in a 97-100% decrease in HSP30 accumulation in A6 cells treated with 10 µM sodium arsenite or 100 µM cadmium chloride. These results show that KNK437 is effective at inhibiting both heat shock and chemical induced hsp gene expression in amphibian cells.
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Khamis, Imran. "Effect of combined sodium arsenite and cadmium chloride treatment on heat shock protein gene expression in Xenopus laevis A6 kidney epithelial cells." Thesis, 2013. http://hdl.handle.net/10012/7916.
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Sodium arsenite and cadmium chloride are two widespread environmental toxicants which have deleterious effects on living organisms. At the cellular level, sodium arsenite and cadmium chloride cause oxidative stress, dysregulation of gene expression, apoptosis, and the unfolding of protein. Furthermore, both chemical stressors individually have the ability to induce heat shock protein (HSP) accumulation. HSPs are molecular chaperones that aid in protein folding, translocation and in preventing stress-induced protein aggregation. Previously, our laboratory demonstrated that treatment of A6 kidney epithelial cells of the frog Xenopus laevis, with either cadmium chloride or sodium arsenite plus a concurrent mild heat shock resulted in an enhanced accumulation of HSPs that was greater than found with the sum of the individual stressors. To the best of our knowledge, no information is available to date on the effect that these two chemical stressors have in combination on HSP accumulation in aquatic organisms. The present study examined the effect of simultaneous sodium arsenite and cadmium chloride treatment on the pattern of HSP30 and HSP70 accumulation in Xenopus A6 cells. Immunoblot analysis revealed that the relative levels of HSP30 and HSP70 accumulation in A6 cells treated concurrently with sodium arsenite and cadmium chloride for 12 h were significantly higher than the sum of HSP30 or HSP70 accumulation from cells subjected to the treatments individually. For instance, the combined 10 µM sodium arsenite plus 100 µM cadmium chloride treatment resulted in a 3.5 fold increase in HSP30 accumulation and a 2.5 fold increase in HSP70 accumulation compared to the sum of the stressors individually. This finding suggested a synergistic action between the two stressors. Pretreatment of cells with KNK437, an HSF1 inhibitor, inhibited the combined sodium arsenite- and cadmium chloride-induced accumulation of HSP30 and HSP70 suggesting that this accumulation of HSPs may be regulated, at least in part, at the level of transcription. Immunocytochemical analysis employing the use of laser scanning confocal microscopy (LSCM) revealed that simultaneous treatment of cells with the two stressors induced HSP30 accumulation primarily in the cytoplasm in a punctate pattern with some dysregulation of F-actin structure. Increased ubiquitinated protein accumulation was observed with combined 10 µM sodium arsenite and 10, 50 or 100 µM cadmium chloride treatment compared to individual stressors suggesting an impairment of the ubiquitin-proteasome degradation system. Finally, while incubation of A6 cells with 1 µM sodium arsenite plus 10 µM cadmium chloride did not induce a detectable accumulation of HSPs, the addition of a 30 °C mild heat shock resulted in a strong accumulation of HSP30 and HSP70. This study has demonstrated that concurrent sodium arsenite and cadmium chloride treatment can enhance HSP accumulation. Since HSP accumulation is triggered by proteotoxic stress, these findings are relevant given the fact that aquatic amphibians in their natural habitat may be exposed to multiple chemical stressors simultaneously.
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Santos, Diogo Pires dos. "The potential therapeutic effect of heat shock protein 90 inhibition in chronic myeloid leukemia." Master's thesis, 2018. http://hdl.handle.net/10316/82142.
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Trabalho Final do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina<br>A Proteína de Choque Térmico 90 (HSP90) facilita a maturação, estabilidade, atividade e enrolamento intracelular de mais de 200 proteínas, designadas “proteínas clientes”. Nas células cancerígenas, a HSP90 ajuda a superar múltiplos stresses ambientais, incluindo instabilidade genómica/aneuploidia, stress proteotóxico, necessidades nutricionais aumentadas, níveis de oxigénio reduzidos, e ajudam na evasão ao sistema imune. Uma das proteínas clientes da HSP90 é a proteína BCR-ABL, uma oncoproteína responsável pela Leucemia Mieloide Crónica (LMC). A Alvespimicina (17-DMAG) é um inibidor da HSP90 que tem melhores propriedades farmocinéticas e menos efeitos secundários comparando com outras ansamicinas benzoquinonas. Este trabalho tem por objetivo o estudo dos efeitos do 17-DMAG em linhas celulares de LMC (sensível e resistentes ao Imatinib) e explorar o papel da família das HSP na sensibilidade ao Imatinib. Neste contexto, utilizámos três linhas celulares de LMC: a linha K562, sensível ao Imatinib, e as linhas K562-RC e K562-RD, resistentes ao Imatinib. As células foram incubadas na ausência e na presença de concentrações crescentes de 17-DMAG (de 1 a 1000 nM) em administração única. As curvas dose-resposta foram determinadas pelo teste da resazurina. A morte celular foi determinada por microscopia (coloração May-Grunwald Giemsa) e por citometria de fluxo (CF), usando dupla fixação de Anexina V e Iodeto de Propídeo (IP). A sonda Apostat foi usada para avaliar os níveis de expressão das caspases e a sonda JC-1 para determinar o potencial de membrana mitocondrial por CF. O ciclo celular foi avaliado por CF, usando o teste PI/RNase. Os níveis de expressão proteica da família das HSP foram analisados por western blot. Os nossos resultados mostraram que o 17-DMAG induz uma redução da atividade metabólica das linhas celulares, dependente do tempo, da dose e da linha celular, com um IC50 de 50 nM para as células K562 e inferior a 50 nM para as células K562-RC e K562-RD, após 48 horas de tratamento. Este composto induz morte celular predominantemente por apoptose, confirmado pela análise morfológica, CF, e pelo aumento da razão JC-1 Monómeros/Agregados. A análise do ciclo celular mostrou que o 17-DMAG induz bloqueio do ciclo celular em G0/G1 nas células K562 e K562-RC e em G2/M nas células K562-RD. A análise proteica das HSP mostrou que as células K562 têm ligeiramente mais expressão de HSP90 que K562-RC e K562-RD e o tratamento de 17-DMAG induziu um aumento significativo da expressão de HSP70. Em conclusão, os nossos resultados sugerem que a inibição de HSP90 pelo 17-DMAG poderá constituir uma potencial nova abordagem terapêutica da LMC, mesmo em casos de resistência ao Imatinib.<br>Heat Shock Protein 90 (HSP90) facilitates the maturation, stability, activity and intracellular folding of more than 200 proteins, called 'client proteins'. In cancer cells, HSP90 helps to overcome multiple environmental stresses, including genomic instability/aneuploidy, proteotoxic stress, increased nutrients demand, reduced oxygen levels, and to prevent destruction by the immune system. One of these client proteins of HSP90 is BCR-ABL, the oncoprotein responsible for Chronic Myeloid Leukemia (CML). Alvespimycin (17-DMAG) is a HSP90 inhibitor that has better pharmacokinetic properties and fewer side-effects compared to others benzoquinone ansamycins. This work aims to study the potential therapeutic effect of 17-DMAG in CML cell lines (sensitive and resistant to imatinib) and to explore the role of HSP family in the sensitivity to Imatinib. In this context, we used three CML cell lines: the K562 cells, sensitive to Imatinib, and the K562-RC and K562-RD cells, resistant to Imatinib. Cells were incubated in the absence and presence of increasing concentrations of 17-DMAG (from 1 to 1000 nM) in single dose. The dose-response curves were determined by the resazurin assay. Cell death was determined through microscopy (May-Grunwald Giemsa staining) and flow cytometry (FC), using Annexin V and Propidium Iodide (PI) double staining. The Apostat Probe was used to evaluate caspase expression levels and the JC-1 probe to determine the mitochondrial membrane potential, through FC. Cell cycle was evaluated with FC, using PI/RNase assay. The protein expression levels of HSP family were assessed with western blot. Our results showed that 17-DMAG induces a reduction in cell lines metabolic activity in a time, dose and cell type dependent manner, with an IC50 of 50 nM for K562 and less than 50 nM for the K562-RC and K562-RD cell lines, after 48 hours of treatment. This compound induces cell death predominantly by apoptosis, confirmed through morphological analysis, FC and by the increase in JC-1 Monomers/Aggregates ratio. Apart from the cytotoxic effect, the cell cycle analysis showed that 17-DMAG induces cell cycle arrest in G0/G1 for K562 and K562-RC, and in G2/M for K562-RD. The HSP protein analysis showed that K562 have slightly more expression of HSP90 than K562-RC and K562-RD cells and the treatment with 17-DMAG induced a significant increase in HSP70 expression. In conclusion, our results suggest that inhibition of HSP90 by 17-DMAG could be used as a new potential approach in the treatment of CML, even in cases of Imatinib resistance.
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Snyman, Marisha. "Salicylic acid-mediated potentiation of Hsp70 in tomato seedlings is modulated by heat shock factors." Thesis, 2012. http://hdl.handle.net/10210/6172.
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Ph.D.<br>In plants, salicylic acid (SA) is a signaling molecule that regulates disease resistance responses such as systemic acquired resistance (SAR) and the hypersensitive response (HR), and has been implicated in both basal and acquired thermotolerance. It has also been shown that SA enhances heat-induced Hsp/Hsc70 accumulation in plants. In this study, temperature studies revealed that heat shock (HS) at 40 °C for 30 min significantly induced Hsp/Hsc70 accumulation in 3-week old tomato (UC82B) seedlings. Time- and dose-responsive studies showed that 0.1 mM SA for 17 hrs was unable to induce Hsp/Hsc70 but in combination with HS significantly (P > 0.001) potentiated this response. To investigate the mechanism of SA-mediated, heat-induced Hsp/Hsc70 potentiation, tomato seedlings were treated with either SA alone, HS or both, before analyses of hsp70 mRNA, Hsf DNA-binding and gene expression of hsp70, hsfAl, hsfA2 and hsfEll. SA alone established Hsf DNA-binding, but was not accompanied by increased Hsp70 accumulation or expression of hsp70 mRNA. SA had no significant effect on hsfA2 and hsf81 gene expression, but increased the basal levels of hsfAl. In heat-shocked plants, Hsf DNA-binding was enhanced, and increased hsfAl, hsfA2 and hsfB1 expression preceded accumulation of Hsp70. The combined treatment of SA and HS resulted in potentiated Hsf DNA-binding, enhanced expression of hsp70, hsfAl, hsfA2 and hsfB1, leading to potentiated levels of Hsp/Hsc70. Since increased hsp70 and hsf gene expression coincided with increased levels of Hsp70 accumulation, it is likely that the SA-mediated potentiation of Hsp70 is due to the ability of SA to regulate Hsfs during HS. This study therefore proposes a mechanism for the potentiation of Hsp70 by SA in the presence of heat, which might contribute to our understanding of the role SA plays in the heat shock response and thermotolerance.
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Alho, Donovan Brendon. "Effects of elevated Hsp70 on apoptotic events in hydrogen peroxide treated tobacco cells." Thesis, 2008. http://hdl.handle.net/10210/444.
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Programmed cell death (PCD) is a universal event experienced by all eukaryotes and is an essential process for the maintenance of regular homeostasis. The careful regulation of PCD pathways is beneficial to all organisms and a better understanding of various PCD components and their interactions may enhance overall quality of life and increase longevity of defective organisms. Since a boom of interest was shown towards the study of PCD approximately thirty years prior, a comprehensive understanding of the underlying mechanisms of apoptosis currently exists. Many of the key features involved in PCD have been shown to be conserved across the animal and plant kingdoms and although the overall process of cell death appears to occur in a similar manner in both plants and animals, subtle variations have been identified between these two kingdoms regarding various mechanisms of apoptosis. The major component of cell death in plants and animals appears to be the mitochondria where most of the PCD points of control converge. Heat shock proteins (Hsps) play a vital role in the regulation of PCD and act at an array of points along the PCD pathway. One of the crucial events of PCD is the release of cytochrome c from the mitochondria which proceeds to activate the protease cascade. Hsp70 has been shown to attenuate apoptosis by specifically preventing cytochrome c release into the cytosol of animal cells. A similar correlation between increases of Hsp70 and decreased PCD has been identified in plants, the mechanism of which remains unclear. The objective of this study was to investigate the interaction between increased Hsp70 accumulation and decreased PCD, as indicated by reduced cytochrome c translocation and DNA laddering. The major observations of this study proceeded to show that an increased expression of Hsp70, induced by a mild heat shock, was able to protect tobacco cells against H2O2-mediated cell death. This protection appeared to commence at a mitochondrial level as cytochrome c translocation was clearly inhibited and was confirmed by the absence of downstream DNA laddering. The major findings obtained from this study provided a clearer picture of the mechanisms surrounding the cytoprotective properties of thermotolerant cells, the implications of which are beneficial to post harvest industries, as the ability to postpone PCD provides an advantage enables prolonged shelf lives of various crops.<br>Dr. Marianne J. Cronjé
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Bhamjee, Rabia Ahmid. "Comparing suppression subtractive hybridization and bioinformatics approaches for analyzing functional gene expression in Arabidopsis thaliana following a heat shock treatment." Thesis, 2012. http://hdl.handle.net/10210/4692.
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M.Sc.<br>Since plants are stationary, their immune systems have adapted to their environments to enable them to overcome or respond appropriately to various environmental, physical and physiological stresses that they may encounter by developing complicated defense mechanisms. The plant defense response activates complex biochemical and structural changes in plant cells. Heat stress per se, appears to be a priority stress response in plants, and increased disease susceptibility may be a result of this response. In this study, altered gene expression levels mediated by a heat treatment in Arabidopsis thaliana seedlings were analyzed. Seedlings were exposed to a heat stress of 42C for 30 minutes, followed by a 2.5 hour recovery period at 25ºC. RNA that was isolated from the heat stress treated plants as well as control plants (untreated) was used to perform suppression subtractive hybridization (SSH) in order to obtain a forward and a reverse DNA library. The forward SSH library represented the genes that were up-regulated due to the heat shock and the reverse SSH library represented the down-regulated genes. Sequenced clones from these libraries were BLAST against the A. thaliana genome using the Genbank database and the Accession numbers retrieved were then used for Bioinformatics analysis to obtain functionality of the genes found. The bioinformatics tools used were TAIR tools, DAG graphs and FatiGO and genes were categorized into the biological processes, molecular functions and cellular components. The TAIR tools and FatiGO were then used to analyze microarray data obtained for a similar study, in order to compare the genes identified with SSH. The genes coding for photosystem IID, serine-type peptidase, phospholipase D α, a S-locus protein kinase, regulator of chromosome condensation (RCC1) and Glucose-6-phosphate translocator are prominently up-regulated whilst other genes encoding photosystem I, plastocyanin-like mavicyanin, carbohydrate trans-membrane transporter MSS1, zinc finger C3HC4 ring family protein, ubiquitin conjugating enzyme 35 (UBC35) and integral membrane family proteins are significantly down-regulated. The FatiGO results helped to assign functionality to the genes that were found. For the SSH forward library, the cellular protein metabolic pathway was the most highly expressed term (19.21%), whereas in the microarray data, the term „positive regulation of response to stimulus‟ and membrane disassembly had a 100% expression. The reverse SSH data (down-regulation) found phosphate metabolic process as the most highly expressed term with an expression of 44.36% ix and the microarray data (negative fold-change) found the term photorespiration to be the most highly expressed with 93.54% expression. These high levels of negative expression indicate the down-regulation of these processes in the cell during heat shock. From these results it can be assumed that at the onset of a heat stress, the plant‟s immediate response is to activate pathways of regulation as a response to the stimulus as a self-protection mechanism, and repress other pathways such as photorespiration in order to preserve its energy such as ATP. These findings suggest that the plant is well equipped to overcome stress in its environment by activation/repression of specific organelles and pathways in the system, in order to maintain its equilibrium. Studies such as these can prove to be helpful to solve the interesting question of how a plant overcomes various environmental stresses in order to prevent disease susceptibility.
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Kuun, Karolina. "Thermotolerance and Ralstonia solanacearum infection: implications for phenylpropanoid metabolism in Lycopersicon esculentum." Thesis, 2012. http://hdl.handle.net/10210/6771.
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M.Sc.<br>Field grown plants are constantly challenged with a variety of stressful factors, such as high temperatures, drought and pathogen infection that adversely affect crop production and quality. These stresses seldom occur as single entities in plants and in warm climates, heat stress is often a common dominator in combinatorial stress. The heat shock (HS) response in plants has priority over other stress responses, including the pathogen-induced stress response. Activation of the HS response prevents the normal plant defence strategy, leaving the plant vulnerable to pathogen attack. However, prior exposure to elevated temperatures confers protection from subsequent, otherwise lethal, temperatures (thermotolerance) and a variety of other stress conditions including heavy-metals, chilling injury and certain pathogens (cross tolerance). In general, litterature supports a central role for heat shock proteins (HSP), in particular the 70 kDa HSP (Hsp70), in thermotolerance. Incompatible host-pathogen interactions lead to the activation of an array of defence mechanisms, including the promotion of phenylpropanoid metabolism. Phenylalanine ammonia-lyase is a key regulator of this metabolic pathway, influencing the production of salicylic acid, lignin and phytoalexins among other essential defence products. In this study it was hypothesised that prior exposure to non-lethal HS confers protection from subsequent heat-related suppression of the phenylpropanoid pathway, induced as a defence mechanism during an incompatible plant-pathogen interaction. This hypothesis was verified by analysing the effect of thermotolerance on pathogen-related stimulation of PAL promoter activity, enzyme activity and lignin deposition. The tomato, Lycopersicon esculentum cultivar UC82B and Ralstonia solanacearum, the causative agent of bacterial wilt, were used as host-pathogen model. Specific objectives in the study were: (1) Development of PAL promoter-GUS reporter transformed Lycopersicon esculentum. (2) Establishment of a thermotolerance protocol that ensures optimal Hsp70 levels at subsequent HS. (3) Evaluation of the influence of prior heat treatment on phenylpropanoid metabolism after exposure to HS in combination with Ralstonia solanacearum. Results obtained support the hypothesis indicating that thermotolerance protects phenylpropanoid metabolism, in particular PAL promoter and enzyme activity, and to a certain extent lignin production, induced by avirulent Ralstonia solanacearum during a second severe HS. In contrast, HS without a prior heat treatment, suppressed phenylpropanoid metabolism. The protective potential of prior heat treatment during subsequent infection under hyperthermic conditions support the application of HSP in the development of novel plant protection strategies.
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Kresfelder, Tina. "Hsp70 induction and hsp70 gene polymorphisms as indicators of acclimatization under hyperthermic conditions." Thesis, 2008. http://hdl.handle.net/10210/384.
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Acclimatization is a process which occurs as a result of repeated mild increases in core temperature, which allows an organism to carry out increased work in the heat due to better heat dissipation (Moseley, 1997). In order to prevent the occurrence of heat illness, it is necessary for individuals who perform work in hot, humid environments to undergo acclimatization. Exposure to different types of stimuli, such as heat exercise and oxidative stress, results in the formation of proteins in the cells which are known as heat shock protein (HSP) (Powers et al, 2001). The main function of HSP is to act as molecular chaperones. Specifically, the 70 kDa HSP, known as Hsp70, play an important part in cellular protection and adaptation during and following exposure to stressful events. Two prominent members of the Hsp70 family are Hsp72, which is the inducible form of Hsp70, and Hsp73, which is the constitutively synthesized form of the protein. Acquisition of thermotolerance in vitro in Chinese hamster fibroblast cells is accompanied by increased Hsp72 levels during a heating episode, indicating that Hsp72 tends to target proteins which have been damaged by a stress situation more than Hsp73 (Li and Werb, 1982). A number of different hsp70 genes are found, which include hsp70-1, hsp70-2 (3¢utr and pst I) and hsp70-hom (Milner and Campbell, 1990). Both the hsp70-1 and hsp70-2 genes encode the primary heat-induced Hsp70 protein as an identical protein product (Hunt and Morimoto, 1985). Following heat shock, no increase in the hsp70-hom mRNA levels is observed (Milner and Campbell, 1990). The use of the Hsp70 protein and hsp70 gene polymorphisms as markers of acclimatization were investigated by subjecting twenty-two individuals to exercise in a hot, humid environment. These individuals were exposed to an initial heat stress, where they participated in a step test at an external work rate of 70 W, followed by participation in an acclimatization program which involved exercising at various combinations of 35 W and 70 W of the external work rate. After acclimatization, the individuals were exposed to a second heat stress, identical to the initial heat stress. The Hsp70 levels both before and after acclimatization were determined in response to heat stress in blood serum by means of the StressXpress ELISA Kit (Stressgen Biotechnologies) and in human monocytes by means of Western blots, using a mouse antiñHsp70 monoclonal primary antibody (Stressgen Biotechnologies) and a goat antiñmouse IgG (H+L) peroxidase conjugated secondary antibody. The hsp70 gene polymorphisms were determined by means of polymerase chain reaction (PCR), using primers specific to the hsp70-1, hsp70-2 (3¢utr), pst I and hsp70-hom genes, so that the genotype combinations for each individual were determined. Blood type was also assessed. It was found that the basal serum Hsp70 levels in individuals who exhibited the ability to acclimatize decreased in response to the acclimatization program, which allowed more Hsp70 to be induced in response to the second heat stress compared to the initial heat stress. The individuals who were unable to acclimatize showed increased basal serum Hsp70 levels in response to acclimatization, which prevented these individuals from inducing high Hsp70 levels in response to the second heat stress. The Hsp70 induced in the monocytes during this program followed the same pattern in both the individuals able to acclimatize and those who were unable to acclimatize, and can therefore not be used as a marker of acclimatization. For the female participants, the current menstrual phase of each woman had to be taken into account, as this had an affect on the core temperature and therefore influenced the division of the female participants into their respective groups. These were the group of individuals who demonstrated the ability to acclimatize or the group of individuals who were unable to acclimatize. The use of oral contraceptives also had to be taken into account, as this too had an influence on the core temperature and therefore also affected the division of the individuals into the group who demonstrated the ability to acclimatize or those who were unable to acclimatize. Cyclic changes during the menstrual cycle may have also changed the Hsp profile. Regarding the hsp70 gene polymorphisms, the A/A, P2 P2 and A1 A1 genotype combination was not present in any of the individuals who were unable to acclimatize, however six of the individuals who showed the ability to acclimatize possessed this genotype combination. The level of induced Hsp70 levels present in the serum of individuals able to acclimatize and the presence or absence of the A/A, P2 P2 and A1 A1 genotype combination therefore have the potential to be used as markers of acclimatization.<br>Dr. M. Cronje
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Snyman, Marisha. "Antioxidant-mediated effects on Hsp70/Hsc70 accumulation and related events in differentially treated tobacco cells." Thesis, 2008. http://hdl.handle.net/10210/419.
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Initially, protoplasts were isolated to detect various parameters using flow cytometric analysis. The most efficient ratio of cells to enzyme solution, for digestion of cell walls, needed to be established. To detect whether the time of incubation with the enzyme solution influenced the state or viability of the protoplasts, they were observed periodically under the light microscope during digestion at different concentrations of enzyme solution. After 2 h digestion with light swirling every 20 min, the protoplasts were still intact (Figure 1), and viable as detected with Trypan blue staining (results not shown). Increasing the digestion period led to a decrease in cell membrane integrity. The size of the protoplasts varied between 60 mm and 90 mm. Figure 1 shows the difference between cells before digestion with an enzyme solution and protoplasts after digestion. Size determination of protoplasts was important since the flow tip of the flow cytometer is limited to 100 mm and if the protoplasts exceeded this size, could lead to blockages in the flow tip of the flow cytometer, with ineffective readings and a time consuming clean-up process.<br>Dr. Marianne J. Cronje
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SONKO, PASAIKOU, and 宋鵬飛. "Status of Anisakis Infection in Scomber australasicius and Trichiurus lepturus investigated by PCR-RFLP and the Nematocidal Effect of Wasabi Paste and Ally-isothiocyanate Assessed by Heat Shock Proteins expressions." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/75055973063704368517.
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碩士<br>臺北醫學大學<br>醫學科學研究所<br>104<br>The Anisakis (Nematodes, Anisakidae) is the main causative agent of the human zoonotic disease Anisakidosis. There are nine (9) species, which are morphologically categorized into two group: Anisakis Clade I and Clade II. There are increased consumption of Sashimi and Sushi (raw fish fillet) among Taiwanese due to globalized exotic ethnic dishes and vitamins preservation. Therefore, this diets changes may increase risks of anisakiasis acquisition through consumption of raw fish fillet. Thus, it remained necessary to investigate the status of Anisakis infection to explore the potential risky fishes species.Two host fish species, Scomber australasicus and Trichiurus lepturus were investigated in the present study. Anisakis larvae collected from these fishes were morphological differentiation from other larvae using a light microscope and genetically analyzed with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using the internal transcribed spacer (ITS-1 and ITS-2) regions to identify the species. The survival rate, median lethal time (LT50) and mRNA expression of Heat Shock Protein (HSP) were determined using Kaplan Meier curves and quantitative real time PCR (qRT-PCR), respectively. A total of 1302 anisakis third stage larvae (L3) were collected from the host species S. australasicus (n=26) and T. lepturus (n=10) with a mean of 21.62 (± 27.61) and 74.00 (± 62.16) respectively. Estimated total of 40% (n= 215 and 336 respectively) larvae were randomly collected from each hosts species and analyzed with PCR-RFLP, 83.72% was identified as A. pegreffi, 6.05% as A. typica, 0.93% as A. physteris and 8.84% as hybrid genotype ( A. pegreffii + A. simplex [s.s]) in S. australasicus host and 86.31% as A. pegreffi, 2.38% as A. typica, and 11.31% for hybrid genotype ( A. pegreffii + A. simplex [s.s]) in T. lepturus host. The survival rate of A. pegreffi larva treated with wasabi paste shown a significant difference with a hazards mortality of 3.1 and 3.8 percent per minute’s treated with the 1g/ml and 2g/ml of Wasabi paste in PBS respectively. Relatively, the two groups show a significant difference (P=0.0227) and high significant difference from the control group (P<0.0001). The mean lethal time estimated for the two treatment group shown 30 and 45minutes respectively. The AITC has high toxicity to larvae, with the mean lethal time estimated to be 16, 25 and 34minutes for 10-, 20- and 30-Fold dilution respectively. Compared to the control (Oil), AITC shows significant difference 10-Fold (P=0.0001), 20-F0ld (P=0.0003) and 30-Fold (P=0.0108). Both concentration groups also shown significant difference (P<0.05). When larva treatment with WSP, the HSP90 mRNA transcription is gradually expressed and reach to the peak at 12mins post-treatments and declined (P=0.0077).The HSP70 mRNA transcribed in all the treatments time point (P<0.05) post-exposure with Wasabi Paste. The HSP90 protein does not significantly express (P=0.5633) and whereas the HSP70 protein translation gradually increased significantly in a time-dependent manner and reaches its peak at 18mins (P<0.05). When the parasite larva was treated with 10-Fold AITC, the HSP90 mRNA expression gradually decrease (P=0.01132, R2=0.7094) and significantly downregulated at 12mins compared to the control (P<0.05). The HSP70 mRNA expression basally upregulated in all the treatment time point and significantly increase in 12mins post-exposure to AITC compared to the control (P=0.0389). The HSP90 proteins significantly upregulated at 2mins (P=0.0249) and gradually downregulated (P>0.05) whereas the HSP70 proteins significantly increased in 2mins, 4mins, 6 mins and 12mins (P<0.05). Thus, wasabi and AITC are highly toxic and induced stress sensor protein to the anisakis larvae, which is evident by high expression of HSP70 mRNA at all treatments time point.