The role of ClpP, RpoS and CsrA in growth and filament formation of Salmonella enterica serovar Typhimurium at low temperature

BACKGROUND: Salmonellae are food-borne pathogens of great health and economic importance. To pose a threat to humans, Salmonellae normally have to cope with a series of stressful conditions in the food chain, including low temperature. In the current study, we evaluated the importance of the Clp proteolytic complex and the carbon starvation protein, CsrA, for the ability of Salmonella Typhimurium to grow at low temperature. RESULTS: A clpP mutant was severely affected in growth and formed pin point colonies at 10°C. Contrary to this, rpoS and clpP/rpoS mutants were only slightly affected. The clpP mutant formed cold resistant suppressor mutants at a frequency of 2.5 × 10(−3) and these were found not to express RpoS. Together these results indicated that the impaired growth of the clpP mutant was caused by high level of RpoS. Evaluation by microscopy of the clpP mutant revealed that it formed filamentous cells when grown at 10°C, and this phenotype too, disappered when rpoS was mutated in parallel indicating a RpoS-dependency. A csrA (sup) mutant was also growth attenuated a low temperature. An rpoS/csrA (sup) double mutant was also growth attenuated, indicating that the phenotype of the csrA mutant was independent from RpoS. CONCLUSIONS: The cold sensitivity of clpP mutant was associated with increased levels of RpoS and probably caused by toxic levels of RpoS. Although a csrA mutant also accumulated high level of RpoS, growth impairment caused by lack of csrA was not related to RpoS levels in a similar way

Investigation of the gut-associated lymphoid tissue in sheep with emphasis on natural killer cells

The gut-associated lymphoid tissue (GALT) of sheep has been investigated for decades, and several immune cells receding within these structures have been described. The GALT of sheep has specialized compartments described to contain myeloid and lymphoid cells. The newly developed antibody against ovine NCR1 has been shown to label lymphocytes with conventional attributes of NK cells. However, the precise localization, phenotype and activation of NCR1+ cells in the small and large intestinal lymphoid compartments remain unknown. Investigations presented in this thesis were done using different types of immuno-labelling on tissue sections and in flow cytometry. The NCR1+ cells were detected in the gut of foetuses from day 70 in the gestation, and these cells resided in compartments where T cells usually are seen; the inter-follicular area, dome and lamina propria. The NCR1+ cells of the ovine foetal gut showed an increasing fraction of c-kit expression in the last period before birth. The localization of NCR1+ cells described in the three papers is stable in foetus, normal unchallenged lambs and during cryptosporidiosis. The studies have revealed two separate phenotypic subtypes, both exhibiting characteristics of conventional NK cells. However, based on the new classification of innate lymphoid cells described in mouse and human, some of the NCR1+ cells could be grouped as a subtype featuring regulatory and less cytotoxic functions, designated ILC22. From the youngest group of foetuses investigated we found NCR1+ cells to proliferate, whereas this fraction of cells decreased to a minimum and were almost absent in the juvenile lambs. During an experimental infection with Cryptosporidium parvum we found the NCR1+ cells to be faithful to their compartmentalization and showed an elevation of activation status. The results presented in this thesis may be useful for comparative considerations and for general understanding of NCR1+ cells in the sheep. It may be a basis for further investigations of the GALT and immune responses in the gut of sheep.Tarmassosiert lymfatisk vev har vært undersøkt i flere tiår, og dette lymfatiske vevet har spesialiserte områder, inneholdende myeloide og lymfoide celler. Det nylige utviklede antistoffet mot sau NCR1 er blitt vist å merke lymfocytter med klassiske karakteristika kjent for dreper(NK) celler. Det er imidlertid ennå ikke blitt beskrevet lokalisasjon, fenotype eller aktiveringsstatus hos NCR1+ celler i tynn- og tykktarmens lymfoide områder hos drøvtyggere. Undersøkelser som er presentert i denne avhandlingen ble gjort ved hjelp av ulike typer immunmerking på snitt av vev og i væskestrømcytometri. NCR1+ celler ble funnet i tarm hos fostre fra 70 dager i drektigheten og ble funnet i områder hvor T celler vanligvis er lokalisert; interfollikulær områder, dome og lamina propria. NCR1+ celler som ble funnet i sauefosterets tarm viste å ha økende uttrykk av c-kit i den siste perioden før fødsel. Studiene har avslørt to separate fenotypiske subtyper, der begge innehar typiske karakteristiske trekk av klassiske NK celler. Det er imidlertid kommet en ny klassifisering av medfødte lymfoide celler, som kan føre til at noen av de NCR1+ cellene kan bli klassifisert som en subtype vist å være mer regulatorisk og mindre cytotoksisk, kalt ILC22. Vi fant prolifererende NCR1+ celler i de yngste fostrene, der andelen av disse sank til et minimum mot fødsel og var nesten fraværende i unge lam. Lokaliseringen av NCR1+ celler beskrevet i de tre artiklene er stabil i fostre, normale friske lam og i cryptosporidieinfiserte lam. I løpet av en eksperimentell infeksjon med Cryptosporidium parvum fant vi at de NCR1+ cellene viste uendret lokalisasjon i tarmen og en økning av aktiveringsstatus. Resultatene som er presentert i denne avhandlingen kan være nyttige for komparative betraktninger og for å få en generell forståelse av NCR1+ celler i sau. Det kan være et grunnlag for videre undersøkelser hos tarmassosiert vev og immunrespons hos sau

Modeling Equity, Satisfaction and Loyalty

The link between customer satisfaction and loyalty has had a tremendous impact on managerial decision-making and academic works over the years. Despite the lack of empirical research supporting this link, its existence seems to be assumed, independent of place and time. Across customer loyalty studies, however, the achieved explained variance in the customer loyalty construct is typically rather low, indicating other possible explanations than customer satisfaction to account for customers’ loyalty to service providers. At the same time, we observe that customers’ demands are changing at an increasingly high pace; customers now expect to interact with service providers in a multitude of ways simultaneously, posing new challenges to service managers striving to establish and maintain a loyal customer base. Triggered by marketing’s shift in focus from a transaction to a relationship orientation, this development has further escalated due to the rapid infusion of new technology into service industries, demonstrating as such the need to explore alternative determinants of customer loyalty in various situations. In the literature, alternative determinants or intervening variables have been classified as intra-psychological, contextual or situational factors; in this dissertation, we investigate such alternative determinants and intervening variables in order to explain customer loyalty in various situations. The overall research objective of the dissertation has been, then, to gain insight into the consequences for customer satisfaction and loyalty modeling of these rapidly changing customer demands, to develop models accordingly, and to test these models empirically. Our approach resulted in five studies conducted in different service industries, with data collected through the Norwegian Customer Satisfaction Barometer survey and related research projects

Non-essential genes form the hubs of genome scale protein function and environmental gene expression networks in Salmonella enterica serovar Typhimurium

Background Salmonella Typhimurium is an important pathogen of human and animals. It shows a broad growth range and survives in harsh conditions. The aim of this study was to analyze transcriptional responses to a number of growth and stress conditions as well as the relationship of metabolic pathways and/or cell functions at the genome-scale-level by network analysis, and further to explore whether highly connected genes (hubs) in these networks were essential for growth, stress adaptation and virulence. Results De novo generated as well as published transcriptional data for 425 selected genes under a number of growth and stress conditions were used to construct a bipartite network connecting culture conditions and significantly regulated genes (transcriptional network). Also, a genome scale network was constructed for strain LT2. The latter connected genes with metabolic pathways and cellular functions. Both networks were shown to belong to the family of scale-free networks characterized by the presence of highly connected nodes or hubs which are genes whose transcription is regulated when responding to many of the assayed culture conditions or genes encoding products involved in a high number of metabolic pathways and cell functions. The five genes with most connections in the transcriptional network (wraB, ygaU, uspA, cbpA and osmC) and in the genome scale network (ychN, siiF (STM4262), yajD, ybeB and dcoC) were selected for mutations, however mutagenesis of ygaU and ybeB proved unsuccessful. No difference between mutants and the wild type strain was observed during growth at unfavorable temperatures, pH values, NaCl concentrations and in the presence of H2O2. Eight mutants were evaluated for virulence in C57/BL6 mice and none differed from the wild type strain. Notably, however, deviations of phenotypes with respect to the wild type were observed when combinations of these genes were deleted. Conclusion Network analysis revealed the presence of hubs in both transcriptional and functional networks of S. Typhimurium. Hubs theoretically confer higher resistance to random mutation but a greater susceptibility to directed attacks, however, we found that genes that formed hubs were dispensable for growth, stress adaptation and virulence, suggesting that evolution favors non-essential genes as main connectors in cellular networks

Teknologi eller personlig service: Hvordan påvirkes kundenes lojalitet?

Artikkelen har tidligere vært publisert i Magma, 10/2009.Det er veldokumentert at bruk av teknologi kan redusere kostnader, men få studier har sett nærmere på teknologiens innvirkning på forbrukernes atferd. I denne studien anvender vi en anerkjent modell fra tjenestemarkedsføringen i en teknologikontekst. Vi belyser hvordan etablerte relasjoner mellom variablene kundetilfredshet, emosjonell (affektiv) og rasjonell (kalkulativ) tilknytning samt lojalitet blir påvirket av at tjenesten leveres gjennom bruk av teknologi versus gjennom personlig service. Vi presenterer to alternative perspektiv på teknologiens rolle. Det første perspektivet er basert på antagelsen om at teknologi forandrer alt, og at relasjonene mellom variablene som leder til lojalitet, blir fullstendig forandret. Det andre perspektivet innebærer at teknologi ikke er annet enn hvilken som helst kontekst, og at den rollen teknologi spiller for forbrukernes atferd, kun er indirekte gjennom allerede eksisterende drivere av lojalitet. En tverrsnittsstudie gjennomført i banknæringen viser at teknologi ikke forandrer alt. Den klassiske oppfatningen av hvordan kundene evaluerer tjenester samt årsakene til lojalitet, finnes igjen i teknologikonteksten. Interessant nok ser det ut til at teknologi ikke har en direkte effekt på lojalitet, men at teknologiens rolle er indirekte og mediert gjennom kundens tilknytning til serviceleverandøren. Imidlertid er det den affektive tilknytningen som synes viktig når kundens lojalitet til serviceleverandøren formes

Transcriptional changes induced by bevacizumab combination therapy in responding and non-responding recurrent glioblastoma patients

Background: Bevacizumab combined with chemotherapy produces clinical durable response in 25-30% of recurrent glioblastoma patients. This group of patients has shown improved survival and quality of life. The aim of this study was to investigate changes in gene expression associated with response and resistance to bevacizumab combination therapy.Methods: Recurrent glioblastoma patients who had biomarker-accessible tumor tissue surgically removed both before bevacizumab treatment and at time of progression were included. Patients were grouped into responders (n = 7) and non-responders (n = 14). Gene expression profiling of formalin-fixed paraffin-embedded tumor tissue was performed using RNA-sequencing.Results: By comparing pretreatment samples of responders with those of non-responders no significant difference was observed. In a paired comparison analysis of pre- and posttreatment samples of non-responders 1 gene was significantly differentially expressed. In responders, this approach revealed 256 significantly differentially expressed genes (72 down-and 184 up-regulated genes at the time of progression). Genes differentially expressed in responders revealed a shift towards a more proneural and less mesenchymal phenotype at the time of progression.Conclusions: Bevacizumab combination treatment demonstrated a significant impact on the transcriptional changes in responders; but only minimal changes in non-responders. This suggests that non-responding glioblastomas progress chaotically without following distinct gene expression changes while responding tumors adaptively respond or progress by means of the same transcriptional changes. In conclusion, we hypothesize that the identified gene expression changes of responding tumors are associated to bevacizumab response or resistance mechanisms

Identification of Metabolic Pathways Essential for Fitness of <i>Salmonella</i> Typhimurium <i>In Vivo</i>

Bacterial infections remain a threat to human and animal health worldwide, and there is an urgent need to find novel targets for intervention. In the current study we used a computer model of the metabolic network of Salmonella enterica serovar Typhimurium and identified pairs of reactions (cut sets) predicted to be required for growth in vivo. We termed such cut sets synthetic auxotrophic pairs. We tested whether these would reveal possible combined targets for new antibiotics by analyzing the performance of selected single and double mutants in systemic mouse infections. One hundred and two cut sets were identified. Sixty-three of these included only pathways encoded by fully annotated genes, and from this sub-set we selected five cut sets involved in amino acid or polyamine biosynthesis. One cut set (asnA/asnB) demonstrated redundancy in vitro and in vivo and showed that asparagine is essential for S. Typhimurium during infection. trpB/trpA as well as single mutants were attenuated for growth in vitro, while only the double mutant was a cut set in vivo, underlining previous observations that tryptophan is essential for successful outcome of infection. speB/speF,speC was not affected in vitro but was attenuated during infection showing that polyamines are essential for virulence apparently in a growth independent manner. The serA/glyA cut-set was found to be growth attenuated as predicted by the model. However, not only the double mutant, but also the glyA mutant, were found to be attenuated for virulence. This adds glycine production or conversion of glycine to THF to the list of essential reactions during infection. One pair (thrC/kbl) showed true redundancy in vitro but not in vivo demonstrating that threonine is available to the bacterium during infection. These data add to the existing knowledge of available nutrients in the intra-host environment, and have identified possible new targets for antibiotics