Outer Membrane Protease/Adhesin PgtE of Salmonella enterica: Role in Salmonella-Host Interaction

Salmonella enterica serovar Typhimurium is a common cause of gastroenteritis in humans and, occasionally, also causes systemic infection. During systemic infection an important characteristic of Salmonella is its ability to survive and replicate within macrophages. The outer membrane protease PgtE of S. enterica is a member of the omptin family of outer membrane aspartate proteases, which are beta-barrel proteins with five surface-exposed loops. The main goals of this study were to characterize biological substrates and pathogenesis-associated functions of PgtE and to determine the conditions where PgtE is fully active. In this study we found that PgtE requires rough lipopolysaccharide (LPS) to be functional but is sterically inhibited by the long O-antigen side chain in smooth LPS. Salmonella isolates normally are smooth with a long oligosaccharide O-antigen, and PgtE remains functionally cryptic in wild-type Salmonella cultivated in vitro. Interestingly, our results showed that due to increased expression of PgtE and to reduced length of the LPS O-antigen chains, the wild-type Salmonella expresses highly functional PgtE when isolated from mouse macrophage-like J774A.1 cells. Salmonella is thought to be continuously released from macrophages to infect new ones, and our results suggest that PgtE is functional during these transient extracellular growth phases. Six novel host protein substrates were identified for PgtE in this work. PgtE was previously known to activate human plasminogen (Plg) to plasmin, a broad-spectrum serine protease, and in this study PgtE was shown to interfere with the Plg system by inactivating the main inhibitor of plasmin, alpha2-antiplasmin. PgtE also interferes with another important proteolytic system of mammals by activating pro-matrix metalloproteinase-9 to an active gelatinase. PgtE also directly degrades gelatin, a component of extracellular matrices. PgtE also increases bacterial resistance against complement-mediated killing in human serum and enhances survival of Salmonella within murine macrophages as well as in the liver and spleen of intraperitoneally infected mice. Taken together, the results in this study suggest that PgtE is a virulence factor of Salmonella that has adapted to interfere with host proteolytic systems and to modify extracellular matrix; these features likely assist the migration of Salmonella during systemic salmonellosis.

Characterization of inhibition of platelet function by paracetamol and its interaction with diclofenac and parecoxib

Aim: To characterize the inhibition of platelet function by paracetamol in vivo and in vitro, and to evaluate the possible interaction of paracetamol and diclofenac or valdecoxib in vivo. To assess the analgesic effect of the drugs in an experimental pain model. Methods: Healthy volunteers received increasing doses of intravenous paracetamol (15, 22.5 and 30 mg/kg), or the combination of paracetamol 1 g and diclofenac 1.1 mg/kg or valdecoxib 40 mg (as the pro-drug parecoxib). Inhibition of platelet function was assessed with photometric aggregometry, the platelet function analyzer (PFA-100), and release of thromboxane B2. Analgesia was assessed with the cold pressor test. The inhibition coefficient of platelet aggregation by paracetamol was determined as well as the nature of interaction between paracetamol and diclofenac by an isobolographic analysis in vitro. Results: Paracetamol inhibited platelet aggregation and TxB2-release dose-dependently in volunteers and concentration-dependently in vitro. The inhibition coefficient was 15.2 mg/L (95% CI 11.8 - 18.6). Paracetamol augmented the platelet inhibition by diclofenac in vivo, and the isobole showed that this interaction is synergistic. Paracetamol showed no interaction with valdecoxib. PFA-100 appeared insensitive in detecting platelet dysfunction by paracetamol, and the cold-pressor test showed no analgesia. Conclusions: Paracetamol inhibits platelet function in vivo and shows synergism when combined with diclofenac. This effect may increase the risk of bleeding in surgical patients with an impaired haemostatic system. The combination of paracetamol and valdecoxib may be useful in patients with low risk for thromboembolism. The PFA-100 seems unsuitable for detection of platelet dysfunction and the cold-pressor test seems unsuitable for detection of analgesia by paracetamol.

Interaction of alcohol and smoking in the pathogenesis of upper digestive tract cancers - possible chemoprevention with cysteine

Background: Alcohol consumption and smoking are the main causes of upper digestive tract cancers. These risk factors account for over 75% of all cases in developed countries. Epidemiological studies have shown that alcohol and tobacco interact in a multiplicative way to the cancer risk, but the pathogenetic mechanism behind this is poorly understood. Strong experimental and human genetic linkage data suggest that acetaldehyde is one of the major factors behind the carcinogenic effect. In the digestive tract, acetaldehyde is mainly formed by microbial metabolism of ethanol. Acetaldehyde is also a major constituent of tobacco smoke. Thus, acetaldehyde from both of these sources may have an interacting carcinogenic effect in the human upper digestive tract. Aims: The first aim of this thesis was to investigate acetaldehyde production and exposure in the human mouth resulting from alcohol ingestion and tobacco smoking in vivo. Secondly, specific L-cysteine products were prepared to examine their efficacy in the binding of salivary acetaldehyde in order to reduce the exposure of the upper digestive tract to acetaldehyde. Methods: Acetaldehyde levels in saliva were measured from human volunteers during alcohol metabolism, during tobacco smoking and during the combined use of alcohol and tobacco. The ability of L-cysteine to eliminate acetaldehyde during alcohol metabolism and tobacco smoking was also investigated with specifically developed tablets. Also the acetaldehyde production of Escherichia coli - an important member of the human microbiota - was measured in different conditions prevailing in the digestive tract. Results and conclusions: These studies established that smokers have significantly increased acetaldehyde exposure during ethanol consumption even when not actively smoking. Acetaldehyde exposure was dramatically further increased during active tobacco smoking. Thus, the elevated aerodigestive tract cancer risk observed in smokers and drinkers may be the result of the increased acetaldehyde exposure. Acetaldehyde produced in the oral cavity during ethanol challenge was significantly decreased by a buccal L-cysteine -releasing tablet. Also smoking-derived acetaldehyde could be totally removed by using a tablet containing L-cysteine. In conclusion, this thesis confirms the essential role of acetaldehyde in the pathogenesis of alcohol- and smoking-induced cancers. This thesis presents a novel experimental approach to decrease the local acetaldehyde exposure of the upper digestive tract with L-cysteine, with the eventual goal of reducting the prevalence of upper digestive tract cancers.

Ionosphere-atmosphere interaction during solar proton events

Among the most striking natural phenomena affecting ozone are solar proton events (SPE), during which high-energy protons precipitate into the middle atmosphere in the polar regions. Ionisation caused by the protons results in changes in the lower ionosphere, and in production of neutral odd nitrogen and odd hydrogen species which then destroy ozone in well-known catalytic chemical reaction chains. Large SPEs are able to decrease the ozone concentration of upper stratosphere and mesosphere, but are not expected to significantly affect the ozone layer at 15--30~km altitude. In this work we have used the Sodankylä Ion and Neutral Chemistry Model (SIC) in studies of the short-term effects caused by SPEs. The model results were found to be in a good agreement with ionospheric observations from incoherent scatter radars, riometers, and VLF radio receivers as well as with measurements from the GOMOS/Envisat satellite instrument. For the first time, GOMOS was able to observe the SPE effects on odd nitrogen and ozone in the winter polar region. Ozone observations from GOMOS were validated against those from MIPAS/Envisat instrument, and a good agreement was found throughout the middle atmosphere. For the case of the SPE of October/November 2003, long-term ozone depletion was observed in the upper stratosphere. The depletion was further enhanced by the descent of odd nitrogen from the mesosphere inside the polar vortex, until the recovery occurred in late December. During the event, substantial diurnal variation of ozone depletion was seen in the mesosphere, caused mainly by the the strong diurnal cycle of the odd hydrogen species. In the lower ionosphere, SPEs increase the electron density which is very low in normal conditions. Therefore, SPEs make radar observations easier. In the case of the SPE of October, 1989, we studied the sunset transition of negative charge from electrons to ions, a long-standing problem. The observed phenomenon, which is controlled by the amount of solar radiation, was successfully explained by considering twilight changes in both the rate of photodetachment of negative ions and concentrations of minor neutral species. Changes in the magnetic field of the Earth control the extent of SPE-affected area. For the SPE of November 2001, the results indicated that for low and middle levels of geomagnetic disturbance the estimated cosmic radio noise absorption levels based on a magnetic field model are in a good agreement with ionospheric observations. For high levels of disturbance, the model overestimates the stretching of the geomagnetic field and the geographical extent of SPE-affected area. This work shows the importance of ionosphere-atmosphere interaction for SPE studies. By using both ionospheric and atmospheric observations, we have been able to cover for the most part the whole chain of SPE-triggered processes, from proton-induced ionisation to depletion of ozone.

Hybrid Modelling of Titan's Interaction with the Magnetosphere of Saturn

In this dissertation we study the interaction between Saturn's moon Titan and the magnetospheric plasma and magnetic field. The method of research is a three-dimensional computer simulation model, that is used to simulate this interaction. The simulation model used is a hybrid model. Hybrid models enable individual tracking or tracing of ions and also take into account the particle motion in the propagation of the electromagnetic fields. The hybrid model has been developed at the Finnish Meteorological Institute. This thesis gives a general description of the effects that the solar wind has on Earth and other planets of our solar system. Planetary satellites can also have similar interactions with the solar wind but also with the plasma flows of planetary magnetospheres. Titan is clearly the largest among the satellites of Saturn and also the only known satellite with a dense atmosphere. It is the atmosphere that makes Titan's plasma interaction with the magnetosphere of Saturn so unique. Nevertheless, comparisons with the plasma interactions of other solar system bodies are valuable. Detecting charged plasma particles requires in situ measurements obtainable through scientific spacecraft. The Cassini mission has been one of the most remarkable international efforts in space science. Since 2004 the measurements and images obtained from instruments onboard the Cassini spacecraft have increased the scientific knowledge of Saturn as well as its satellites and magnetosphere in a way no one was probably able to predict. The current level of science on Titan is practically unthinkable without the Cassini mission. Many of the observations by Cassini instrument teams have influenced this research both the direct measurements of Titan as well as observations of its plasma environment. The theoretical principles of the hybrid modelling approach are presented in connection to the broader context of plasma simulations. The developed hybrid model is described in detail: e.g. the way the equations of the hybrid model are solved is shown explicitly. Several simulation techniques, such as the grid structure and various boundary conditions, are discussed in detail as well. The testing and monitoring of simulation runs is presented as an essential routine when running sophisticated and complex models. Several significant improvements of the model, that are in preparation, are also discussed. A main part of this dissertation are four scientific articles based on the results of the Titan model. The Titan model developed during the course of the Ph.D. research has been shown to be an important tool to understand Titan's plasma interaction. One reason for this is that the structures of the magnetic field around Titan are very much three-dimensional. The simulation results give a general picture of the magnetic fields in the vicinity of Titan. The magnetic fine structure of Titan's wake as seen in the simulations seems connected to Alfvén waves an important wave mode in space plasmas. The particle escape from Titan is also a major part of these studies. Our simulations show a bending or turning of Titan's ionotail that we have shown to be a direct result of the basic principles in plasma physics. Furthermore, the ion flux from the magnetosphere of Saturn into Titan's upper atmosphere has been studied. The modelled ion flux has asymmetries that would likely have a large impact in the heating in different parts of Titan's upper atmosphere.

Emotional experience in psychotherapeutic interaction : Conversation analytical study on cognitive psychotherapy

The dissertation examines how emotional experiences are oriented to in the details of psychotherapeutic interaction. The data (57 audio recorded sessions) come from one therapist-patient dyad in cognitive psychotherapy. Conversation analysis is used as method. The dissertation consists of 4 original articles and a summary. The analyses explicate the therapist s practices of responding to the patient s affective expressions. Different types of affiliating responses are identified. It is shown that the affiliating responses are combined with, or build grounds for, more interpretive and challenging actions. The study also includes a case study of a session with strong misalignment between the therapist s and patient s orientations, showing how this misalignment is managed by the therapist. Moreover, through a longitudinal analysis of the transformation of a sequence type, the study suggests that therapeutic change processes can be located to sequential relations of actions. The practices found in this study are compared to earlier research on everyday talk and on medical encounters. It is suggested that in psychotherapeutic interaction, the generic norms of interaction considering affiliation and epistemic access, are modified for the purposes of therapeutic work. The study also shows that the practices of responding to emotional experience in psychotherapy can deviate from the everyday practices of affiliation. The results of the study are also discussed in terms of concepts arising from clinical theory. These include empathy, validation of emotion, therapeutic alliance, interpretation, challenging beliefs, and therapeutic change. The therapist s approach described in this study involves practical integration of different clinical theories. In general terms, the study suggests that in the details of interaction, psychotherapy recurrently performs a dual task of empathy and challenging in relation to the patient s ways of describing their experiences. Methodologically, the study discusses the problem of identifying actions in conversation analysis of psychotherapy and emotional interaction, and the possibility to apply conversation analysis in the study of therapeutic change.

Moral Professionalism in Interaction : Educators' Relational Moral Voices in Urban Schools

The aim of this study was to investigate educators relational moral voices in urban schools and to listen to what they told about moral professionalism and moral practices in challenging urban schools. Their relational moral voices were investigated through the following three questions: 1. What are the educators moral voices in relation to themselves and other people? 2. What are the educators moral voices in relation to their work and society? 3. What kind of interaction process lies between the educators moral voices and the urban school context? The research data of this study were gathered in four urban schools in Jyväskylä and Helsinki. The research schools were chosen for this study according to the criteria of the international Socrates Comenius project called Leading Schools Successfully in Challenging Urban Context: Strategies for Improvement. This study formed part of this project, which investigated successful urban schools as challenging learning environments in nine European countries and explored the principals success in leadership in particular. The data, which included 37 narratively constructed interviews with four principals and key informants selected by the principals, were gathered in interviews conducted in 2006. In other words, the data comprised three interviews with each of four principals, and interviews with two teachers, two parents, and two pupils from each school. In addition, the school deacon from one school was also interviewed. Furthermore, part of the data from one of the research schools included a medium report of the school deacon s work. This study combined the case study method, the narrative approach and the critical incident technique as the methodological framework. In addition, all of these methods served as practical tools for both analyzing and reporting the data. The educators' narrations and the results of the study appear in the original articles (Hanhimäki & Tirri 2008; Hanhimäki 2008b; Hanhimäki & Tirri 2009; Hanhimäki 2008a). The educators moral voices in relation to themselves and other people emerged through the main themes of moral leadership, the development and evaluation process, moral sensitivity, gender, values, and student well-being. The educators moral voices in relation to their work and society emerged through the main themes of multiprofessional cooperation, families and parental involvement, and moral school culture. The idea of moral interaction connected moral professionalism and the methodological combination of this study, which together emphasized social interaction and the creation of understanding and meaning in this interaction. The main point of this study was to state that the educators moral voices emerged in the interaction between the educators themselves and the urban school context. In this interaction, the educators moral professionalism was constructed and shaped in relation to themselves, other people, their work and society. The loudest relational moral voices heard through the main themes were those of caring, cooperation, respect, commitment, and professionalism. When the results were compared to the codes of ethics which guided these educators moral professional work, the ethical principles and values of the codes were clearly visible in their moral practices. The loudest message from the educators narration could be summarized in the words caring, respect and cooperation: at its best, there is just a human being and a human being with caring, respect and cooperation between them. The results of this study emphasize the need for practical approaches such as case studies and the narrative approach in teacher education to encourage educators to become moral professionals capable of meeting the needs of people of varied backgrounds. In addition, opportunities for moral, religious and spiritual education should be noticed and utilized in the plural interaction of urban schools when nurturing pupils and creating a moral school culture. Furthermore, multiprofessional cooperation and parents as the school s primary cooperation partner are needed to carry out the shared duty of moral education in urban schools. Keywords: moral professionalism, educator, relational moral voice, interaction, urban school

Therapeutic change in interaction

A process of change within a single case of cognitive-constructivist therapy is analyzed by means of conversation analysis (CA). The focus is on a process of change in the sequences of interaction, which consist of the therapist’s conclusion and the patient’s response to it. In the conclusions, the therapist investigates and challenges the patient’s tendency to transform her feelings of disappointment and anger into self-blame. Over the course of the therapy, the patient’s responses to these conclusions are recast: from the patient first rejecting the conclusion, to then being ambivalent, and finally to agreeing with the therapist. On the basis of this case study, we suggest that an analysis that focuses on sequences of talk that are interactionally similar offers a sensitive method to investigate the manifestation of therapeutic change. It is suggested that this line of research can complement assimilation analysis and other methods of analyzing changes in a client’s talk.