Molecular basis and regulation of insect pathogenicity in plant-beneficial pseudomonads

Les bactéries du genre Pseudomonas ont la capacité étonnante de s'adapter à différents habitats et d'y survivre, ce qui leur a permis de conquérir un large éventail de niches écologiques et d'interagir avec différents organismes hôte. Les espèces du groupe Pseudomonas fluorescens peuvent être facilement isolées de la rhizosphère et sont communément connues comme des Pseudomonas bénéfiques pour les plantes. Elles sont capables d'induire la résistance systémique des plantes, d'induire leur croissance et de contrer des phytopathogènes du sol. Un sous-groupe de ces Pseudomonas a de plus développé la capacité d'infecter et de tuer certaines espèces d'insectes. Approfondir les connaissances sur l'interaction de ces bactéries avec les insectes pourraient conduire au développement de nouveaux biopesticides pour la protection des cultures. Le but de cette thèse est donc de mieux comprendre la base moléculaire, l'évolution et la régulation de la pathogénicité des Pseudomonas plante-bénéfiques envers les insectes. Plus spécifiquement, ce travail a été orienté sur l'étude de la production de la toxine insecticide appelée Fit et sur l'indentification d'autres facteurs de virulence participant à la toxicité de la bactérie envers les insectes. Dans la première partie de ce travail, la régulation de la production de la toxine Fit a été évaluée par microscopie à épifluorescence en utilisant des souches rapportrices de Pseudomonas protegens CHA0 qui expriment la toxine insecticide fusionnée à une protéine fluorescente rouge, au site natif du gène de la toxine. Celle-ci a été détectée uniquement dans l'hémolymphe des insectes et pas sur les racines des plantes, ni dans les milieux de laboratoire standards, indiquant une production dépendante de l'hôte. L'activation de la production de la toxine est contrôlée par trois protéines régulatrices dont l'histidine kinase FitF, essentielle pour un contrôle précis de l'expression et possédant un domaine "senseur" similaire à celui de la kinase DctB qui régule l'absorption de carbone chez les Protéobactéries. Il est donc probable que, durant l'évolution de FitF, un réarrangement de ce domaine "senseur" largement répandu ait contribué à une production hôte-spécifique de la toxine. Les résultats de cette étude suggèrent aussi que l'expression de la toxine Fit est plutôt réprimée en présence de composés dérivés des plantes qu'induite par la perception d'un signal d'insecte spécifique. Dans la deuxième partie de ce travail, des souches mutantes ciblant des facteurs de virulence importants identifiés dans des pathogènes connus ont été générées, dans le but d'identifier ceux avec une virulence envers les insectes atténuée. Les résultats ont suggéré que l'antigène O du lipopolysaccharide (LPS) et le système régulateur à deux composantes PhoP/PhoQ contribuent significativement à la virulence de P. protegens CHA0. La base génétique de la biosynthèse de l'antigène O dans les Pseudomonas plante-bénéfiques et avec une activité insecticide a été élucidée et a révélé des différences considérables entre les lignées suite à des pertes de gènes ou des acquisitions de gènes par transfert horizontal durant l'évolution de certaines souches. Les chaînes latérales du LPS ont été montrées comme vitales pour une infection des insectes réussie par la souche CHA0, après ingestion ou injection. Les Pseudomonas plante-bénéfiques, avec une activité insecticide sont naturellement résistants à la polymyxine B, un peptide antimicrobien modèle. La protection contre ce composé antimicrobien particulier dépend de la présence de l'antigène O et de la modification du lipide A, une partie du LPS, avec du 4-aminoarabinose. Comme les peptides antimicrobiens cationiques jouent un rôle important dans le système immunitaire des insectes, l'antigène O pourrait être important chez les Pseudomonas insecticides pour surmonter les mécanismes de défense de l'hôte. Le système PhoP/PhoQ, connu pour contrôler les modifications du lipide A chez plusieurs bactéries pathogènes, a été identifié chez Pseudomonas chlororaphis PCL1391 et P. protegens CHA0. Pour l'instant, il n'y a pas d'évidence que des modifications du lipide A contribuent à la pathogénicité de cette bactérie envers les insectes. Cependant, le senseur-kinase PhoQ est requis pour une virulence optimale de la souche CHA0, ce qui suggère qu'il régule aussi l'expression des facteurs de virulence de cette bactérie. Les découvertes de cette thèse démontrent que certains Pseudomonas associés aux plantes sont de véritables pathogènes d'insectes et donnent quelques indices sur l'évolution de ces microbes pour survivre dans l'insecte-hôte et éventuellement le tuer. Les résultats suggèrent également qu'une recherche plus approfondie est nécessaire pour comprendre comment ces bactéries sont capables de contourner ou surmonter la réponse immunitaire de l'hôte et de briser les barrières physiques pour envahir l'insecte lors d'une infection orale. Pour cela, les futures études ne devraient pas uniquement se concentrer sur le côté bactérien de l'interaction hôte-microbe, mais aussi étudier l'infection du point de vue de l'hôte. Les connaissances gagnées sur la pathogénicité envers les insectes des Pseudomonas plante-bénéfiques donnent un espoir pour une future application en agriculture, pour protéger les plantes, non seulement contre les maladies, mais aussi contre les insectes ravageurs. -- Pseudomonas bacteria have the astonishing ability to survive within and adapt to different habitats, which has allowed them to conquer a wide range of ecological niches and to interact with different host organisms. Species of the Pseudomonas fluorescens group can readily be isolated from plant roots and are commonly known as plant-beneficial pseudomonads. They are capable of promoting plant growth, inducing systemic resistance in the plant host and antagonizing soil-borne phytopathogens. A defined subgroup of these pseudomonads evolved in addition the ability to infect and kill certain insect species. Profound knowledge about the interaction of these particular bacteria with insects could lead to the development of novel biopesticides for crop protection. This thesis thus aimed at a better understanding of the molecular basis, evolution and regulation of insect pathogenicity in plant-beneficial pseudomonads. More specifically, it was outlined to investigate the production of an insecticidal toxin termed Fit and to identify additional factors contributing to the entomopathogenicity of the bacteria. In the first part of this work, the regulation of Fit toxin production was probed by epifluorescence microscopy using reporter strains of Pseudomonas protegens CHAO that express a fusion between the insecticidal toxin and a red fluorescent protein in place of the native toxin gene. The bacterium was found to express its insecticidal toxin only in insect hemolymph but not on plant roots or in common laboratory media. The host-dependent activation of Fit toxin production is controlled by three local regulatory proteins. The histidine kinase of this regulatory system, FitF, is essential for the tight control of toxin expression and shares a sensing domain with DctB, a sensor kinase regulating carbon uptake in Proteobacteria. It is therefore likely that shuffling of a ubiquitous sensor domain during the evolution of FitF contributed to host- specific production of the Fit toxin. Findings of this study additionally suggest that host-specific expression of the Fit toxin is mainly achieved by repression in the presence of plant-derived compounds rather than by induction upon perceiving an insect-specific signal molecule. In the second part of this thesis, mutant strains were generated that lack factors previously shown to be important for virulence in prominent pathogens. A screening for attenuation in insect virulence suggested that lipopolysaccharide (LPS) O-antigen and the PhoP-PhoQ two-component regulatory system significantly contribute to virulence of P. protegens CHAO. The genetic basis of O-antigen biosynthesis in plant-beneficial pseudomonads displaying insect pathogenicity was elucidated and revealed extensive differences between lineages due to reduction and horizontal acquisition of gene clusters during the evolution of several strains. Specific 0 side chains of LPS were found to be vital for strain CHAO to successfully infect insects by ingestion or upon injection. Insecticidal pseudomonads with plant-beneficial properties were observed to be naturally resistant to polymyxin B, a model antimicrobial peptide. Protection against this particular antimicrobial compound was dependent on the presence of O-antigen and modification of the lipid A portion of LPS with 4-aminoarabinose. Since cationic antimicrobial peptides play a major role in the immune system of insects, O-antigenic polysaccharides could be important for insecticidal pseudomonads to overcome host defense mechanisms. The PhoP-PhoQ system, which is well-known to control lipid A modifications in several pathogenic bacteria, was identified in Pseudomonas chlororaphis PCL1391 and P. protegens CHAO. No evidence was found so far that lipid A modifications contribute to insect pathogenicity in this bacterium. However, the sensor kinase PhoQ was required for full virulence of strain CHAO suggesting that it additionally regulates the expression of virulence factors in this bacterium. The findings of this thesis demonstrate that certain plant-associated pseudomonads are true insect pathogens and give some insights into how these microbes evolved to survive within and eventually kill the insect host. Results however also point out that more in-depth research is needed to know how exactly these fascinating bacteria manage to bypass or overcome host immune responses and to breach physical barriers to invade insects upon oral infection. To achieve this, future studies should not only focus on the bacterial side of the microbe-host interactions but also investigate the infection from a host-oriented view. The knowledge gained about the entomopathogenicity of plant-beneficial pseudomonads gives hope for their future application in agriculture to protect plants not only against plant diseases but also against insect pests.

Public-private sector wage differentials by type of contract: evidence from Spain

The article examines public-private sector wage differentials in Spain using microdata from the Structure of Earnings Survey (Encuesta de Estructura Salarial). When applying various decomposition techniques, we find that it is important to distinguish by gender and type of contract. Our results also highlight the presence of a positive wage premium for public sector workers that can be partially explained by their better endowment of characteristics, in particular by the characteristics of the establishment where they work. The wage premium is greater for female and fixed-term employees and falls across the wage distribution, being negative for more highly skilled workers.

The genetic basis of color-related local adaptation in a ring-like colonization around the Mediterranean.

Uncovering the genetic basis of phenotypic variation and the population history under which it established is key to understand the trajectories along which local adaptation evolves. Here, we investigated the genetic basis and evolutionary history of a clinal plumage color polymorphism in European barn owls (Tyto alba). Our results suggest that barn owls colonized the Western Palearctic in a ring-like manner around the Mediterranean and meet in secondary contact in Greece. Rufous coloration appears to be linked to a recently evolved nonsynonymous-derived variant of the melanocortin 1 receptor (MC1R) gene, which according to quantitative genetic analyses evolved under local adaptation during or following the colonization of Central Europe. Admixture patterns and linkage disequilibrium between the neutral genetic background and color found exclusively within the secondary contact zone suggest limited introgression at secondary contact. These results from a system reminiscent of ring species provide a striking example of how local adaptation can evolve from derived genetic variation.

Contract manufacturing networks structure and supply chain management

The usual objectives that companies have for subcontracting are studied in this thesis. The case company’s objectives for contract manufacturing now and in the future are identified. The main objective of the thesis is to create a focused model for the structure and supply chain management in the contract manufacturing network. This model is made for case company’s certain profit center. The different possibilities and their advantages and disadvantages for the structure and supply chain management are examined trough a theoretical review of literature. The possibilities found are then examined from the case company’s point of view. The case company point of view is established based on the opinions of the case company’s representatives. The outcome of the thesis is that the star shaped structure with supply chain management centralized to case company would be the best choice for the case company to manage the contract manufacture network.

Public-private sector wage differentials by type of contract: evidence from Spain

The article examines public-private sector wage differentials in Spain using microdata from the Structure of Earnings Survey (Encuesta de Estructura Salarial). When applying various decomposition techniques, we find that it is important to distinguish by gender and type of contract. Our results also highlight the presence of a positive wage premium for public sector workers that can be partially explained by their better endowment of characteristics, in particular by the characteristics of the establishment where they work. The wage premium is greater for female and fixed-term employees and falls across the wage distribution, being negative for more highly skilled workers.

Genetic Basis and Diagnostics of Extended-Spectrum Beta-Lactamases among Enterobacteriaceae in Finland

Since the introduction of antibiotic agents, the amount and prevalence of Beta-lactam resistant enterobacteria has become an increasing problem. Many enterobacteria are opportunistic pathogens that easily acquire resistance mechanisms and genes, which make the situation menacing. These bacteria have acquired resistance and can hydrolyse extended spectrum cephalosporins and penicillins by producing enzymes called extended-spectrum Beta-lactamases (ESBLs). ESBL-producing bacteria are most commonly found in the gastro-intestinal tract of colonised patients. These resistant strains can be found in both health-care associated and community-acquired isolates. The detection and treatment of infections caused by bacteria producing ESBLs are problematic. This study investigated the genetic basis of extended-spectrum Beta-lactamases in Enterobacteriaceae, especially in Escherichia coli and Klebsiella pneumoniae isolates. A total of 994 Finnish Enterobacteriaceae strains, collected at 26 hospital laboratories, during 2000 and 2007 were analysed. For the genetic basis studies, PCR, sequencing and pyrosequencing methods were optimised. In addition, international standard methods, the agar dilution and disk diffusion methods were performed for the resistance studies, and the susceptibility of these strains was tested for antimicrobial agents that are used for treating patients. The genetic analysis showed that bla_CTX-M was the most prevalent gene among the E. coli isolates, while blaS_HV-12 was the most common Beta-lactamase gene in K. pneumoniae. The susceptibility testing results showed that about 60% of the strains were multidrug resistant. The prevalence of ESBL-producing isolates in Finland has been increasing since 2000. However, the situation in Finland is still much better than in many other European countries.

Environmental policy instruments concerning industrial end-use energy efficiency

Energy efficiency and saving energy are the main question marks when thinking of reducing carbon dioxide emissions or cutting costs. The objective of thesis is to evaluate policy instruments concerning end-use energy efficiency of heavy industry in European Union. These policy instruments may be divided in various ways, but in this thesis the division is to administrative, financial, informative and voluntary instruments. Administrative instruments introduced in this thesis are Directive on Integrated Pollution Prevention and Control, Directive on Energy End-use Efficiency and Energy Services, and Climate and Energy Package. Financial means include energy and emission taxation, EU Emission Trading Scheme and diverse support systems. Informative instruments consist of horizontal BAT Reference Document for Energy Efficiency, as well as substantial EU documents including Green Paper on Energy Efficiency, Action Plan for Energy Efficiency and An Energy Policy for Europe. And finally, voluntary instruments include environmental managements systems like ISO 14001 and EMAS, energy auditing and benchmarking. The efficiency of different policy instruments vary quite a lot. Informative instruments lack the commitment from industry and are thus almost ineffective, contrary to EU Emission Trading Scheme, which is said to be the solution to climate problems. The efficiency of administrative means can be placed between those mentioned and voluntary instruments are still quite fresh to be examined fruitfully. However, each instrument has their potential and challenges. Cases from corporate world strengthen the results from theoretical part. Cases were written mainly on the basis of interviews. The interviewees praised the energy efficiency contract of Finnish industry, but the EU ETS takes the leading role of policy instruments. However, for industry the reductions do not come easily.

Sopimusvalmistajan tarjouslaskentamallin kehittäminen monituoteympäristöön

Työn tavoitteena oli luoda yhtenäinen, tehokas ja helppokäyttöinen tarjouslaskentamalli sopimusvalmistajan käyttöön. Konsernin käytössä oli aiemmin neljä erilaista tarjouslaskentamallia ja niitä oli erittäin vaikea vertailla keskenään, eikä niillä voitu laskea tarjouksia kaikille tehtaille. Työ jakautuu kirjallisuuskatsaukseen ja empiiriseen osaan. Kirjallisuuskatsauksessa käsitellään erilaisia hinnoitteluvaihtoehtoja, kustannuslaskennan roolia ja kustannuslaskentamenetelmiä. Empiirinen osa koostuu tarjouslaskennan nykytilan analysoinnista, uuden tarjouslaskentamallin rakentamisesta sekä kehitetyn tarjouslaskentamallin toimintakuvauksesta. Tarjouslaskentamallin pohjaksi valittiin kustannusperusteinen hinnoittelu, koska sitä oli käytetty aikaisemminkin ja se oli koettu hyväksi. Kustannusperusteisen tarjouslaskentamallin pohjaksi luotiin yhtenäiset työ – ja konetuntihinnat koko konserniin. Konetuntihinnat laskettiin 28 koneelle. Konetuntihintoja analysoitiin ja ryhmiteltiin siten, että päädyttiin 16 konelisän käyttöön. Tarjouslaskentamalli käsittelee vain valmistuksen kustannuksia eikä ota kantaa hallintokustannusten syntyyn. Yhtenäinen tarjouslaskentamalli helpottaa eri myyntimiesten tekemien tarjousten vertailua ja mahdollistaa tuotteiden myynnin useille tehtaille. Tarjouslaskentamalli toteutettiin taulukkolaskentaohjelmalla.

Improving Cross Direction Basis Weight Profile in a Fourdrinier Paper Machine

The purpose of this study was to improve PM7’s basis weight CD profile in Stora Enso’s Berghuizer mill and to search mechanical defects which affect to the formation of the basis weight CD profile. In the theoretical part PM7’s structure was presented and the formation of the basis weight and caliper CD profiles was examined as well as disturbances which are affecting to the formation. The function of the control system was scrutinised for the side of CD profiles as well as the formation of the measured CD profiles. Tuning of the control system was examined through the response model and filtering. Specification of the response model and filtering was explained and how to determine 2sigma statistical number. In the end of the theoretical part ATPA hardware and a new profile browser were introduced. In the experimental part focus was in the beginning to search and remove mechanical defects which are affecting to CD profiles. The next step was to verify the reliability of the online measurements, to study the stability of the basis weight CD profile and to find out so called fingerprint, a basis weight CD profile which is unique for each paper machine. New response model and filtering value for basis weight CD profile was determined by bump tests. After a follow up period the affect of the new response model and filtering was analysed.

Comparing service contract prices of outsourcing partner

The main objective of this Master’s thesis is to find out which one of the two pricing models is the most cost-effective. In this thesis there are two companies that have made an outsourcing contract, in which they have a possibility to choose between two different pricing models. The first model is so called FTE (Full Time Employee) -based. The total cost will be based on the amount of outsourced person-workyears. The second pricing model is the transaction-based, in which the price will be formed according to the amount of transactions. Changing the pricing model from FTE-based to the transaction-based will also incur other costs. It is very important that these other costs are also taken into consideration, so that it is possible to determine the total costs of the pricing models. These other costs are direct costs, indirect costs and performance related costs of outsourcing. Activity based-costing (ABC) was used in order to find out the trues indirect costs of the outsourced processes. Performance related costs are related to quality, so Pareto-analysis was used to analyse the costs. Based on all of that, a framework for service related cost analysis was developed. Quality costs were almost impossible to quantify, so quality had to be taken into consideration in a qualitative way. Furthermore, considering only the indirect and direct costs in a quantitative way and quality costs in a qualitative way, it was possible to find a conditional solution for the research question.

The Structural Basis for inorganic Pyrophosphatase Catalysis and Regulation

Inorganic pyrophosphatases (PPases) are essential enzymes for every living cell. PPases provide the necessary thermodynamic pull for many biosynthetic reactions by hydrolyzing pyrophosphate. There are two types of PPases: integral membrane-bound and soluble enzymes. The latter type is divided into two non-homologous protein families, I and II. Family I PPases are present in all kingdoms of life, whereas family II PPases are only found in prokaryotes, including archae. Family I PPases, particularly that from Saccharomyces cerevisiae, are among the most extensively characterized phosphoryl transfer enzymes. In the present study, we have solved the structures of wild-type and seven active site variants of S. cerevisiae PPase bound to its natural metal cofactor, magnesium ion. These structures have facilitated derivation of the complete enzyme reaction scheme for PPase, fulfilling structures of all the reaction intermediates. The main focus in this study was on a novel subfamily of family II PPases (CBSPPase) containing a large insert formed by two CBS domains and a DRTGG domain within the catalytic domain. The CBS domain (named after cystathionine beta-synthase in which it was initially identified) usually occurs as tandem pairs with two or four copies in many proteins in all kingdoms of life. The structure formed by a pair of CBS domains is also known as a Bateman domain. CBS domains function as regulatory units, with adenylate ligands as the main effectors. The DRTGG domain (designated based on its most conserved residues) occurs less frequently and only in prokaryotes. Often, the domain co-exists with CBS domains, but its function remains unknown. The key objective of the current study was to explore the structural rearrangements in the CBS domains induced by regulatory adenylate ligands and their functional consequences. Two CBS-PPases were investigated, one from Clostridium perfringens (cpCBS-PPase) containing both CBS and DRTGG domains in its regulatory region and the other from Moorella thermoacetica (mt CBS-PPase) lacking the DRTGG domain. We additionally constructed a separate regulatory region of cpCBS-PPase (cpCBS). Both full-length enzymes and cpCBS formed homodimers. Two structures of the regulatory region of cpCBS-PPase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate, were solved. The structures were significantly different, providing information on the structural pathway from bound adenylates to the interface between the regulatory and catalytic parts. To our knowledge, these are the first reported structures of a regulated CBS enzyme, which reveal large conformational changes upon regulator binding. The activator-bound structure was more open, consistent with the different thermostabilities of the activator- and inhibitor-bound forms of cpCBS-PPase. The results of the functional studies on wild-type and variant CBS-PPases provide support for inferences made on the basis of structural analyses. Moreover, these findings indicate that CBS-PPase activity is highly sensitive to adenine nucleotide distribution between AMP, ADP and ATP, and hence to the energy level of the cell. CBS-PPase activity is markedly inhibited at low energy levels, allowing PPi energy to be used for cell survival instead of being converted into heat.

Teknologiasta liiketoimintaa. Julkisen tutkimuksen kaupallistaminen - Case VTT

Tässä tutkielmassa perehdyttiin julkisen tutkimuksen kaupallistamiseen tutkimusorganisaatio VTT:n tapauksen kautta. Tutkimuksen tavoitteena oli selvittää, kuinka julkista tutkimusta voidaan yleensä kaupallistaa, mitkä ovat kaupallistamisprosessin vaiheet, haasteet sekä kriittiset onnistumistekijät. Tutkimus on luonteeltaan laadullinen ja se toteutettiin henkilöhaastatteluina, joita täydennettiin kirjallisilla lähteillä. Tutkimuksessa ilmeni, että VTT:llä julkisrahoitteisen tutkimuksen tuloksia kaupallistetaan lisensoimalla, toimeksianto- eli sopimustutkimuksen kautta, spin-off toimilla, allianssien ja erilaisten palvelujen, kuten valmistus-, testaus-, ja analyysipalvelujen kautta. VTT:n näkökulmasta katsottuna merkittävin kaupallistamiskanava on sopimus- eli toimeksiantotutkimus, jota seuraavat lisensointi sekä spin-off toiminta. Kaupallistamistavan valinta riippuu lukuisista tekijöistä, kuten organisaation kaupallistamisstrategiasta, innovaatiotyypistä, teknologian kypsyydestä, tiedon luonteesta, toimialojen ja markkinoiden ominaisuuksista, innovaation hyödynnettävyydestä ja suojauksesta, arvonmuodostuspotentiaalista kuin yksittäisten henkilöiden motivaatiostakin.Valinta onkin tehtävä tapauskohtaisesti. Julkisesta tutkimuksesta lähtöisin olevien teknologioiden tie markkinoille voi olla haastava, sillä teknologiat ovat kehitetty ei-kaupallisessa ympäristössä. Tutkimuksessa kävi ilmi, että kriittisimmiksi koetut kaupallistamisen onnistumistekijät liittyivät organisaatioon, teknologiaan, markkina- ja asiakastarpeeseen, aineettoman omaisuuden suojaukseen sekä markkinoille menon nopeuteen.

Changes in motor behavior during pregnancy in rats: the basis for a possible animal model of restless legs syndrome

PURPOSE:Pregnant women have a 2-3 fold higher probability of developing restless legs syndrome (RLS – sleep-related movement disorders) than general population. This study aims to evaluate the behavior and locomotion of rats during pregnancy in order to verify if part of these animals exhibit some RLS-like features.METHODS:We used 14 female 80-day-old Wistar rats that weighed between 200 and 250 g. The rats were distributed into control (CTRL) and pregnant (PN) groups. After a baseline evaluation of their behavior and locomotor activity in an open-field environment, the PN group was inducted into pregnancy, and their behavior and locomotor activity were evaluated on days 3, 10 and 19 of pregnancy and in the post-lactation period in parallel with the CTRL group. The serum iron and transferrin levels in the CTRL and PN groups were analyzed in blood collected after euthanasia by decapitation.RESULTS:There were no significant differences in the total ambulation, grooming events, fecal boli or urine pools between the CTRL and PN groups. However, the PN group exhibited fewer rearing events, increased grooming time and reduced immobilization time than the CTRL group (ANOVA, p<0.05).CONCLUSION:These results suggest that pregnant rats show behavioral and locomotor alterations similar to those observed in animal models of RLS, demonstrating to be a possible animal model of this sleep disorder.