1,721,025 research outputs found
Characterization of new putative adhesion factors in Shiga toxin-producing Escherichia coli
No full textWorldwide nearly 2.8 million people are infected by STEC with acute illness, among them approximately 0.14% lead to HUS and 0.008% lead to death. Shiga toxins are essential virulence factors of STEC, but also other factors have been reported to be involved in virulence of such bacteria. Adherence to the target intestinal epithelial cell is an important initial step to establish STEC infection, with intimin is the major adhesin. Besides intimin, STEC express fimbrial proteins such as long polar fimbriae (LPF) which also have a role as adhesion factors. For some fimbrial and putative fimbrial proteins the function in virulence is still not clear. The aim of this study was to clone the putative fimbrial operon lpfB1 into a wild type STEC strain and analyse expression of fimbrial operon genes in this strain under different growth conditions.
First PCR primers for the lpfB1 operon and one of the genes within the operon were designed, based on the nucleotide sequence of the operon from a genome sequenced strain. After optimization of the PCR, the operon with up- and downstream adjacent sequence was amplified. The identity of the PCR product was confirmed by gel electrophoresis and Sanger sequencing. The PCR product was then inserted in a cloning vector pCR-4 TOPO, which was first transformed in TOP10 competent cells. This resulted in TOP10 cells with lpfB1 insert-vector. Plasmid was extracted from the transformed cells and plasmid with the lpfB1 insert (named pDA00) and plasmid without insert (named pDA00) were amplified with PCR and performed electrophoresis to confirm the presence of lpfB1 operon. These plasmids (pDA00 and pDA09) were further cloned into a clinical wild type strain STEC O103:H2, which had lost its stx genes. Transformed cells were again tested with PCR and electrophoresis to confirm the lpfB1 operon. Transformed bacterial cells were then cultured in LB broth and SILACE broth. From these cultures RNA was extracted, and reverse transcribed to cDNA using random primers. The amplification efficiency of the real-time PCR for the lpfB1C gene, and the housekeeping gene rpoA was tested, and found to be 99% and 105% respectively. Then cDNA was amplified by real time PCR of the lpfB1C gene, using rpoA as reference, to study the expression of the operon under different growth conditions. The reference gene rpoA was expressed when grown both in LB and SILACE broth, while the lpfB1C gene was not found to be expressed in any of the two growth conditions tested. There could be several reasons why the lpfB1C gene was not expressed in this study, but due to time constrains there was not sufficient time to investigate why the lpfB1C gene was not expressed.
In conclusion, PCR amplification and cloning of the lpfB1 operon were successful, but the operon was not expressed under the two growth conditions tested. Nevertheless, this study has expanded further possibilities to analyze the expression of long polar fimbriae in non-O157 STEC
Characterization of new putative adhesion factors in Shiga toxin-producing Escherichia coli
No full textWorldwide nearly 2.8 million people are infected by STEC with acute illness, among them approximately 0.14% lead to HUS and 0.008% lead to death. Shiga toxins are essential virulence factors of STEC, but also other factors have been reported to be involved in virulence of such bacteria. Adherence to the target intestinal epithelial cell is an important initial step to establish STEC infection, with intimin is the major adhesin. Besides intimin, STEC express fimbrial proteins such as long polar fimbriae (LPF) which also have a role as adhesion factors. For some fimbrial and putative fimbrial proteins the function in virulence is still not clear. The aim of this study was to clone the putative fimbrial operon lpfB1 into a wild type STEC strain and analyse expression of fimbrial operon genes in this strain under different growth conditions.
First PCR primers for the lpfB1 operon and one of the genes within the operon were designed, based on the nucleotide sequence of the operon from a genome sequenced strain. After optimization of the PCR, the operon with up- and downstream adjacent sequence was amplified. The identity of the PCR product was confirmed by gel electrophoresis and Sanger sequencing. The PCR product was then inserted in a cloning vector pCR-4 TOPO, which was first transformed in TOP10 competent cells. This resulted in TOP10 cells with lpfB1 insert-vector. Plasmid was extracted from the transformed cells and plasmid with the lpfB1 insert (named pDA00) and plasmid without insert (named pDA00) were amplified with PCR and performed electrophoresis to confirm the presence of lpfB1 operon. These plasmids (pDA00 and pDA09) were further cloned into a clinical wild type strain STEC O103:H2, which had lost its stx genes. Transformed cells were again tested with PCR and electrophoresis to confirm the lpfB1 operon. Transformed bacterial cells were then cultured in LB broth and SILACE broth. From these cultures RNA was extracted, and reverse transcribed to cDNA using random primers. The amplification efficiency of the real-time PCR for the lpfB1C gene, and the housekeeping gene rpoA was tested, and found to be 99% and 105% respectively. Then cDNA was amplified by real time PCR of the lpfB1C gene, using rpoA as reference, to study the expression of the operon under different growth conditions. The reference gene rpoA was expressed when grown both in LB and SILACE broth, while the lpfB1C gene was not found to be expressed in any of the two growth conditions tested. There could be several reasons why the lpfB1C gene was not expressed in this study, but due to time constrains there was not sufficient time to investigate why the lpfB1C gene was not expressed.
In conclusion, PCR amplification and cloning of the lpfB1 operon were successful, but the operon was not expressed under the two growth conditions tested. Nevertheless, this study has expanded further possibilities to analyze the expression of long polar fimbriae in non-O157 STEC
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
Phaeodactylum tricornutum tolerance to cadmium and copper
Get PDFIndustrialiseringen har ført til økt utslipp av tungmetaller i både akvatiske og terrestriske miljøer,
og dette kan ha stor påvirkning på levende organismer, inkludert marine mikroalger. Til tross for
mikrogalgers fundamentale roller i marine økosystemer, er kunnskapen vår om deres sensitivitet
og toleranse for miljøfaktorer og forurensinger, inkludert tungmetaller, begrenset.
Målet for denne masteroppgaven var å undersøke effektene av kadmium og kobber eksponering i
den marine kiselalgen Phaeodactylum tricornutum. Dette er verdifullt for å kunne forstå hvordan
økene nivåer av tungmetaller i miljøet vil påvirke mikroalgesamfunn og for å kunne evaluere
potensialet for å kunne bruke P. tricornutum innen bioremedieringsteknologi. Ingen signifikante
fysiologiske påvirkninger ble observert for eksponeringsnivåer på 0.1 og 0.5 mg/L Cd2+, mens
behandling med 2.5 og 12.5 mg/L Cd2+ resulterte i redusert vekst, redusert fotosyntetisk aktivitet,
økt celledød, reduserte nivåer av nøytrale lipider, og endringer i elementinnhold. Kadmiumopptak
i cellene ble vist å øke lineært med økende nivåer av kadmium i vekstmediet.
Genekspresjonsanalyse viste oppregulering av flere gener som er antatt å være relatert til kadmium
transport og detoksifisering i P. tricornutum. Cellene viste lavere toleranse for kobbereksponering,
sammenlignet med kadmium, med signifikant reduksjon av vekst og fotosyntetisk aktivitet som
respons på kobbernivåer under 1 mg/L. Sammen indikerer disse funnene at P. tricornutum kan
tolerere høye nivåer av kadmium eksponering og viser potensiale for bruk innen
bioremedieringsteknologi.The industrialization has led to an increase in heavy metal pollution both in aquatic and terrestrial
environments and this can have major impacts on living organisms, including marine microalgae.
Despite the fundamental role of microalgae in marine ecosystems, our knowledge of their
sensitivity and tolerance to environmental factors and pollutants, including heavy metals, is limited.
The goal of this thesis was to investigate the effects of cadmium and copper exposure in the marine
diatom Phaeodactylum tricornutum. This is important to understand how rising levels of heavy
metals in the environment will influence microalgal communities and for evaluating the potential
of using P. tricornutum in bioremediation technologies. No significant physiological responses
were observed at exposure levels of 0.1 and 0.5 mg/L Cd2+, while treatment with 2.5 and 12.5 mg/L
Cd2+ resulted in reduced growth, reduced photosynthetic activity, increased cell death, reduced
neutral lipid content, and alterations in cellular content of elements. The cadmium uptake by the
cells was demonstrated to increase linearly with increasing levels of cadmium in the growth
medium. Gene expression profiling showed upregulation of several genes believed to be associated
with transport and detoxification of cadmium in P. tricornutum. The P. tricornutum cells showed
lower tolerance to copper exposure compared to cadmium, with significant reduction in growth
and photosynthetic activity in response to copper levels below 1 mg/L. Altogether, the results
indicate that P. tricornutum can tolerate high levels of cadmium exposure and show potential for
applications within the bioremediation technology
Testing the sensitivity of Phaeodactylum tricornutum to environmental conditions and pollutants
Get PDFKiselalger har betydelig økologisk betydning som primærprodusenter da de er ansvarlige for mer enn 20% av global karbonfiksering og er viktige komponenter i den marine næringskjeden. Deres unike egenskaper gjør dem til attraktive kandidater for ulike bioteknologiske formål. Derfor er det essensielt å forstå hvordan kiselalger reagerer på miljøforandringer og å finne optimale vekstforhold som kan gi bedre utbytte i industrien. Denne avhandlingen undersøker responsen til kiselalgen Phaeodactylum tricornutum på økte konsentrasjoner av NaCl og MgCl2, og på per- og polyfluoralkylstoffer (PFASer).
Saliniteten i sjøvann varierer betydelig i kystnære strøk, og kiselalger må tilpasse seg disse variasjonene. I den første delen av avhandlingen undersøkes virkningene av forhøyede nivåer av NaCl og MgCl2 på vekst, celledød, fotosyntetisk effektivitet og kapasitet, samt genuttrykk. Veksten avtok med økende saltkonsentrasjoner, med signifikant vekstreduksjon mellom 0,428 og 0,856 M for eksponeringer i 96 timer og én måned. Celledød inntraff for konsentrasjoner over 1,712 M innen 24 timer, og ble påvist ved hjelp av flowcytometri og propidium-jodidfarging. Ingen forskjeller i vekst ble observert mellom korttids- og langtidseksponering, noe som indikerer fravær av veksttilpasning etter langtidseksponering. Puls-amplitude-modulasjons-fluorometri viste økt fotosyntetisk effektivitet og kapasitet for begge salter. Målinger av osmolalitet indikerte flere partikler og høyere salinitet av NaCl-løsninger sammenlignet med MgCl2. Genuttrykk ble analysert ved revers transkripsjon-kvantitativ polymerasekjedereaksjon (RT-qPCR) og RNA-sekvensering. Funnene viste næringsmangel i den ueksponerte kontrollen og antydet at endringer i ionetransport ble indusert for å motvirke salinitetstress. Samlet sett viser resultatene at NaCl er mer potent enn MgCl2 ved samme kloridkonsentrasjon, noe som antyder at Na+ kan ha en større rolle i toksisiteten enn Cl-.
De siste årene har effekten av PFASer på mennesker, samt regulering av stoffene, fått mye oppmerksomhet. Til tross for at sjøvann antas å være det endelige oppsamlingsstedet for PFASer, har få studier undersøkt virkningene på primærprodusenter nederst i den marine næringskjeden. Den andre delen av denne avhandlingen undersøker effekten av de to vanligste påviste PFASene på vekst av P. tricornutum. Relativ fluorescens ble målt med plateleser og ble brukt som en indikator på vekst. EC50 for 96 timers inhibering av relativ fluorescens ble funnet ved ca. 160 mg/l for perfluoroktansyre (PFOA) og 80 mg/l perfluoroktansulfonat (PFOS). Dette antyder biomagnifisering i den marine næringskjeden, som til slutt kan påvirke mennesker. Videre forskning er nødvendig for å forstå de biologiske mekanismene bak denne toleransen og den toksiske virkemåten til PFASer i kiselalger og i P. tricornutum.Diatoms hold significant ecological importance as primary producers responsible for more than 20% of global carbon fixation and they are essential components of the marine food chain. Their unique traits make them potential candidates for various biotechnological applications. Understanding how diatoms respond to changing environments and identifying optimal conditions for growth and productivity is of great value. This thesis examines the responses of the diatom Phaeodactylum tricornutum to increasing concentrations of NaCl and MgCl2, and to per- and polyfluoroalkyl substances (PFASs).
Seawater salinity fluctuates substantially in the habitats of P. tricornutum, and the diatom must adapt to tolerate these changes. The first part of the thesis investigates the effects of elevated levels of NaCl and MgCl2 on growth, cell death, photosynthetic efficiency and capacity, and gene expression. Growth declined with increasing salt concentrations, with significant decreases between 0.428 and 0.856 M for 96 hour and long-term exposure. The 24 hour cell death was found for concentrations above 1.712 M using flow cytometry and propidium iodide staining. Short-term and long-term effects on growth were compared, indicating no adaption. Pulse-amplitude-modulation (PAM) fluorometry revealed increased photosynthetic efficiency and capacity for both salts. Measurements of osmolalities indicated more particles and higher salinity of the NaCl solutions compared to MgCl2. Gene expression was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RNA sequencing, revealing nutrient depletion in the non-exposed control and changes in ion flux as a mechanism to counteract salinity stress. Overall results showed that NaCl is more potent than MgCl2 at the same chloride concentration, suggesting that Na+ may contribute more to toxicity than Cl-.
In recent years, the adverse effects of PFASs on humans and the regulation of the contaminants have received much attention. Despite seawater being hypothesized as the final sink for PFASs, few studies have examined the impact on primary producers at the base of the marine food chain. The second part of this thesis investigates the effects of the two most commonly detected PFASs on growth of P. tricornutum. Relative fluorescence was measured using a plate reader and used as an indicator for growth. The 96 hour EC50 for inhibition of relative fluorescence was found at approximately 160 mg/L for perfluorooctanoic acid (PFOA) and 80 mg/L for perfluorooctane sulfonate (PFOS). Tolerance to these high concentrations suggests magnification up the marine food chain, ultimately affecting humans. Further research is necessary to understand the biological mechanisms behind this tolerance and the toxic mode of action of PFASs in diatoms
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
- …
