1,720,967 research outputs found
The role of molecular chaperones in Escherichia coli cells subjected to gold nanoparticles induced stress
No full textPhD (Biochemistry)Department of Biochemistry and MicrobiologyColloidal suspensions of gold particles of nanometre (nm) sizes are termed gold nanoparticles (AuNPs). Although stable, AuNPs have been reported to be toxic to E. coli cells by collapsing the bacterial cell membranes and promoting protein misfolding. An understanding of biodistribution in drug delivery and the effects AuNPs have on the function and structure of proteins such as heat shock proteins is important. Heat shock proteins facilitate protein folding and are particularly important during cellular stress. At high concentrations, AuNPs are thought to promote protein aggregation. Heat shock proteins are thought to alleviate cell stress induced by AuNPs. This study explored the role of heat shock proteins in conferring cytoprotection to E. coli against the effects of AuNPs. Citrate-AuNPs were synthesized and their integrity was validated at 520 nm by ultraviolet-visible-near infrared spectroscopy (UV-Vis-NIR). Crystallinity was confirmed by X-Ray diffraction (XRD), while dynamic light scattering (DLS) estimated the size distribution at 13 nm. Furthermore, transmission electron microscopy (TEM) and scanning electron microscope (SEM) revealed the spherical shape and crystal lattice surface morphology of citrate-AuNPs respectively. A complementation assay was conducted using cells deficient of DnaK function (E. coli ΔdnaK52). E. coli ΔdnaK52 was transformed with a recombinant dnaK before examining both DnaK deficient and transformed cells using TEM. E. coli O157:H7 was exposed to citrate-AuNPs (0 – 50 μg/ml) and allowed to grow at 37 oC before protein expression was analysed using electrophoresis followed by LC-MS analysis. This led to the identification of highly expressed proteins such as DnaK, GAPDH, ClpX, DnaJ, and GroEL. Subsequent co-affinity assay revealed possible interaction protein partners of DnaK. These were identified as ClpB, HtpG, GroEL, DnaJ, and SurA proteins. Furthermore, circular dichroism and fluorescence spectroscopy established that recombinant DnaK is stable at citrate-AuNPs concentrations less than 10 μg/ml and the protein was unstable at concentrations beyond 10 μg/ml citrate-AuNPs. In addition, the ATPase activity of recombinant DnaK increased in the presence of citrate-AuNPs at 2.5 μg/ml. The ability of DnaK to suppress aggregation of MDH in vitro was abrogated by the presence of >10 μg/ml citrate-AuNPs. The findings suggest that at low concentrations (10 μg/ml), citrate-AuNPs destabilizes protein conformation and function. Altogether the findings suggest that DnaK in cooperation with its network partners is implicated in E. coli cytoprotection against citrate-AuNPs toxicity.NR
Characterization of heat shock protein 70-z (PfHsp70-z) from plasmodium falciparium
Get PDFPhD (Biochemistry)Department of BiochemistryMalaria is a parasitic disease that accounts for more than 660 thousand deaths annually,
mainly in children. Malaria is caused by five Plasmodium species P. ovale, P. vivax, P. malariae,
P. falciparum and P. knowlesi. The most lethal cause of cerebral malaria is P. falciparum. The
parasites have been shown to up-regulate some of their heat shock proteins (Hsp) in response
to stress. Heat shock protein 70 (called DnaK in prokaryotes) is one of the most prominent
groups of chaperones whose role is central to protein homeostasis and determines the fate
of proteins. Six Hsp70 genes are represented on the genome of P. falciparum. The Hsp70
genes encode for proteins that are localised in different sub-cellular compartments. Of these
two occur in the cytosol, PfHsp70-z and PfHsp70-1; two occur in the endoplasmic reticulum,
PfHsp70-2 and PfHsp70-y; one in the mitochondria, PfHsp70-3 and one exported to the red
blood cell cytosol, PfHsp70-x. PfHsp70-1 is a well characterized canonical Hsp70 involved in
prevention of protein aggregation and facilitates protein folding. Little is known about
PfHsp70-z. PfHsp70-z was previously shown to be an essential protein implicated in the
folding of proteins possessing asparagine rich repeats. However, based on structural evidence
PfHsp70-z belongs to the Hsp110 family of proteins and is thought to serve as a nucleotide
exchange factor (NEF) of PfHsp70-1. The main aim of this study is to elucidate the functional
roles of PfHsp70-z as a chaperone and its interaction with PfHsp70-1. In the current study,
PfHsp70-z was cloned and expressed in E. coli JM109 cells. This was followed by its purification
using nickel chromatography. The expression of PfHsp70-z in parasites cultured in vitro was
investigated and its association with PfHsp70-1 was explored using a co-immuno
precipitation assay. PfHsp70-z expression in malaria parasites is up regulated by heat stress
and the protein is heat stable based on investigations conducted using Circular Dichroism.
Furthermore, the direct interaction between recombinant forms of PfHsp70-z and PfHsp70-1
were investigated using slot blot and surface plasmon resonance assays. PfHsp70-z was
observed to exhibit ATPase activity. In addition, the direct interaction between PfHsp70-z and
PfHsp70-1 is promoted by ATP. Based on limited proteolysis and tryptophan fluorescence
analyses, PfHsp70-z binds ATP to assume a unique structural conformation compared to the
conformation of the protein bound to ADP or in nucleotide-free state. PfHsp70-z was able to
suppress the heat-induced aggregation of malate dehydrogenase and luciferase in vitro.
Interestingly, while ATP appears to modulate the conformation of PfHsp70-z, the chaperone
function of PfHsp70-z was not influenced by ATP. Altogether, these findings suggest that
Characterization of Heat Shock Protein 70-z (PfHsp70-z) from Plasmodium falciparum
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PfHsp70-z serves as an effective peptide substrate holding chaperone. In addition, PfHsp70-z
may also serve as the sole nucleotide exchange factor of PfHsp70-1. The broad spectrum of
functions of this protein, could explain this PfHsp70-z is an essential protein in malaria
parasite survival. This is the first study to show that PfHsp70-z possess independent
chaperone activity and that it interacts with its cytosolic counterpart, PfHsp70-1 in a
nucleotide dependent fashion. Furthermore, the study shows that PfHsp70-z is a heat stable
molecule and that it is capable of forming high order oligomers
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
Characterisation of the cytosolic Plasmodium falciparum Hsp90 and Hsp70-z chaperones
No full textThesis (MSc)--Stellenbosch University, 2023.ENGLISH ABSTRACT: The Plasmodium falciparum parasite is the most lethal form of malaria known to be infectious to humans and is responsible for the majority of malaria related deaths. The survival of the parasites throughout their complex life cycle inside both the mosquito vector and the human host, is dependent on the action of various molecular chaperones. Heat shock proteins (Hsps) are a group of molecular chaperones that regulate several cellular processes within the parasite. Most importantly, they play an essential role in maintaining the proteome integrity within P. falciparum, by facilitating the folding of proteins into their functional structures. This is required to maintain host infectivity, mediate malaria pathogenicity and enable parasitic survival. The major cytosolic chaperones, P. falciparum Hsp90 (PfHsp90) and Hsp70-z (PfHsp70-z), are known for their crucial function in the growth, development and survival of the parasite, particularly in the intra-erythrocytic stage of their life cycle within the human host. Hsp90 also collaborates with a variety of co-chaperone and chaperone partner proteins that assist in their functional protein folding cycle. Although the functional partnerships with Hsp90 have been extensively studied in eukaryotic organisms, they are not well established in the protozoan parasites. Therefore, targeting the parasite’s robust protein quality control mechanism imperative for their survival, is challenging. The main aim of this study was to establish and characterise the chaperone complex association between the cytosolic PfHsp90 and PfHsp70-z proteins. Recombinant full-length PfHsp90 and PfHsp70-z were expressed in Escherichia coli XL1 Blue cells and purified using immobilised metal affinity chromatography and size exclusion chromatography. Bioinformatics analysis predicted a direct association between PfHsp90 and PfHsp70-z, which was also validated in vitro, for the first time, through surface plasmon resonance. In addition, their direct interaction displayed slightly greater binding kinetics in a nucleotide-free state when compared to their nucleotide-bound state in the presence of ATP or ADP. Thermal stability analysis of PfHsp90 and PfHsp70-z revealed that both proteins were resilient to heat stress above 50°C, which corresponds to their heat shock protein characteristic of withstanding extreme physiological stressors. Interestingly, it was also observed that the thermal stability of the chaperone proteins were further enhanced when complexed together. The functionality of the complex association was further elucidated by assessing their ability to suppress the aggregation of a client protein namely, malate dehydrogenase (MDH). The chaperone complex was found to efficiently suppress the aggregation of the client protein by ~80%. However, it was also noted that the independent function of PfHsp90 was more effective at suppressing the aggregation of MDH when compared to the complex. Overall, this study provided the first evidence of a direct and functional association between cytosolic PfHsp90 and PfHsp70-z. These findings suggest that the chaperone complex performs a cytoprotective role during the development and survival of the P. falciparum parasite throughout their life cycle. Therefore, further elucidating the parasite’s protein folding machinery and their functional partnerships, may provide novel targets that can be utilised to weaken their protective mechanism and help combat malaria.AFRIKAANSE OPSOMMING: Die Plasmodium falciparum parasiet is die dodelikste vorm van malaria wat bekend is en is
verantwoordelik vir die meeste malariaverwante sterftes in die wêreld. Die oorlewing van die
parasiete gedurende hul komplekse lewensiklus binne beide die muskietvektor en die menslike
gasheer, is afhanklik van die werking van verskillende molekulêre chaperones. Hitteskokproteïene
(Hsps) is 'n groep molekulêre chaperones wat verskeie sellulêre prosesse binne die parasiet
reguleer. Die belangrikste is dat hulle 'n noodsaaklike rol speel in die handhawing van die
proteoomintegriteit binne P. falciparum, deur die vou van proteïene in hul funksionele strukture te
vergemaklik. Dit is nodig om gasheerinfektiwiteit te handhaaf, malariapatogenisiteit te bemiddel en
parasitiese oorlewing moontlik te maak. Die belangrikste sitosoliese chaperones, P. falciparum
Hsp90 (PfHsp90) en Hsp70-z (PfHsp70-z), is bekend vir hul belangrike funksie in die groei,
ontwikkeling en oorlewing van die parasiet, veral in die intra-eritrositiese stadium van hul
lewensiklus binne die menslike gasheer. Hsp90 werk ook saam met 'n verskeidenheid mede-chaperone-
en chaperone-vennootproteïene wat help met hul funksionele proteïenvousiklus. Alhoewel die
funksionele vennootskappe met Hsp90 omvattend in eukariotiese organismes bestudeer is, is hulle nie
goed gevestig in die protosoë parasiete nie. Daarom is dit 'n uitdaging om die parasiet
se robuuste proteïengehaltebeheermeganisme te teiken wat noodsaaklik is vir hul oorlewing. Die
hoofdoel van hierdie studie was om die chaperone komplekse assosiasie tussen die sitosoliese
PfHsp90 en PfHsp70-z proteïene vas te stel en te karakteriseer. Rekombinante vollengte PfHsp90 en
PfHsp70-z is uitgedruk in E. coli XL1 Blou selle en gesuiwer met behulp van geïmmobiliseerde metaal
affiniteit chromatografie en grootte uitsluiting chromatografie. Bioinformatika-analise het 'n
direkte verband tussen PfHsp90 en PfHsp70-z voorspel, wat ook vir die eerste keer in vitro
gevalideer is deur oppervlakplasmonresonansie. Daarbenewens het hul direkte interaksie groter
bindende kinetika in 'n nukleotiedvrye toestand getoon in vergelyking met hul nukleotiedgebonde
toestand in die teenwoordigheid van ATP of ADP. Termiese stabiliteitsanalise van PfHsp90 en
PfHsp70-z het aan die lig gebring dat albei proteïene bestand was teen hittestres bo 50°C, wat
ooreenstem met hul hitteskokproteïen wat kenmerkend is van die weerstand teen uiterste fisiologiese
stressors. Interessant genoeg was dit ook waargeneem dat die termiese stabiliteit van die chaperone
proteïene verder verbeter is wanneer hulle saam gekompleks is. Die funksionaliteit van die
komplekse assosiasie is verder toegelig deur hul vermoë om die samevoeging van 'n kliëntproteïen te
onderdruk, naamlik malaatdehidrogenase (MDH), te beoordeel. Daar is gevind dat die chaperone-
kompleks die samevoeging van die kliëntproteïen doeltreffend onderdruk met ~80%. Daar is egter ook
opgemerk dat die onafhanklike funksie van PfHsp90 meer effektief was om die
samevoeging van MDH te onderdruk in vergelyking met die kompleks. Oor die algemeen het hierdie
studie die eerste bewys gelewer van 'n direkte en funksionele verband tussen sitosoliese PfHsp90 en
PfHsp70-z. Hierdie bevindings daarop dat die chaperone-kompleks 'n sitoprotektiewe rol speel tydens
die ontwikkeling en oorlewing van die P. falciparum parasiet gedurende hul lewensiklus. Daarom kan
die verdere toeligting van die parasiet se proteïenvoumasjinerie en hul funksionele vennootskappe
nuwe teikens bied wat gebruik kan
word om hul beskermingsmeganisme te verswak en malaria te help bekamp.Master
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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