Characterization of the molecular components and function of BARE-1, Hin-Mu and Mu transposition machineries

Transposable elements, transposons, are discrete DNA segments that are able to move or copy themselves from one locus to another within or between their host genome(s) without a requirement for DNA homology. They are abundant residents in virtually all the genomes studied, for instance, the genomic portion of TEs is approximately 3% in Saccharomyces cerevisiae, 45% in humans, and apparently more than 70% in some plant genomes such as maize and barley. Transposons plays essential role in genome evolution, in lateral transfer of antibiotic resistance genes among bacteria and in life cycle of certain viruses such as HIV-1 and bacteriophage Mu. Despite the diversity of transposable elements they all use a fundamentally similar mechanism called transpositional DNA recombination (transposition) for the movement within and between the genomes of their host organisms. The DNA breakage and joining reactions that underlie their transposition are chemically similar in virtually all known transposition systems. The similarity of the reactions is also reflected in the structure and function of the catalyzing enzymes, transposases and integrases. The transposition reactions take place within the context of a transposition machinery, which can be particularly complex, as in the case of the VLP (virus like particle) machinery of retroelements, which in vivo contains RNA or cDNA and a number of element encoded structural and catalytic proteins. Yet, the minimal core machinery required for transposition comprises a multimer of transposase or integrase proteins and their binding sites at the element DNA ends only. Although the chemistry of DNA transposition is fairly well characterized, the components and function of the transposition machinery have been investigated in detail for only a small group of elements. This work focuses on the identification, characterization, and functional studies of the molecular components of the transposition machineries of BARE-1, Hin-Mu and Mu. For BARE-1 and Hin-Mu transpositional activity has not been shown previously, whereas bacteriophage Mu is a general model of transposition. For BARE-1, which is a retroelement of barley (Hordeum vulgare), the protein and DNA components of the functional VLP machinery were identified from cell extracts. In the case of Hin-Mu, which is a Mu-like prophage in Haemophilus influenzae Rd genome, the components of the core machinery (transposase and its binding sites) were characterized and their functionality was studied by using an in vitro methodology developed for Mu. The function of Mu core machinery was studied for its ability to use various DNA substrates: Hin-Mu end specific DNA substrates and Mu end specific hairpin substrates. The hairpin processing reaction by MuA was characterized in detail. New information was gained of all three machineries. The components or their activity required for functional BARE-1 VLP machinery and retrotransposon life cycle were present in vivo and VLP-like structures could be detected. The Hin-Mu core machinery components were identified and shown to be functional. The components of the Mu and Hin-Mu core machineries were partially interchangeable, reflecting both evolutionary conservation and flexibility within the core machineries. The Mu core machinery displayed surprising flexibility in substrate usage, as it was able to utilize Hin-Mu end specific DNA substrates and to process Mu end DNA hairpin substrates. This flexibility may be evolutionarily and mechanistically important.

Production of F4 Fimbrial Adhesin in Plants: a Model for Oral Porcine Vaccine against Enterotoxigenic Escherichia coli

F4 fimbriae of enterotoxigenic Escherichia coli (ETEC) are highly stable multimeric structures with a capacity to evoke mucosal immune responses. With these characters F4 offer a unique model system to study oral vaccination against ETEC-induced porcine postweaning diarrhea. Postweaning diarrhea is a major problem in piggeries worldwide and results in significant economic losses. No vaccine is currently available to protect weaned piglets against ETEC infections. Transgenic plants provide an economically feasible platform for large-scale production of vaccine antigens for animal health. In this study, the capacity of transgenic plants to produce FaeG protein, the major structural subunit and adhesin of F4 fimbria, was evaluated. Using the model plant tobacco, the optimal subcellular location for FaeG accumulation was examined. Targeting of FaeG into chloroplasts offered a superior accumulation level of 1% of total soluble proteins (TSP) over the other investigated subcellular locations, namely, the endoplasmic reticulum and the apoplast. Moreover, we determined whether the FaeG protein, when isolated from its fimbrial background and produced in a plant cell, would retain the key properties of an oral vaccine, i.e. stability in gastrointestinal conditions, binding to porcine intestinal F4 receptors (F4R), and inhibition of the F4-possessing (F4+) ETEC attachment to F4R. The chloroplast-derived FaeG protein did show resistance against low pH and proteolysis in the simulated gastrointestinal conditions and was able to bind to the F4R, subsequently inhibiting the F4+ ETEC binding in a dose-dependent manner. To investigate the oral immunogenicity of FaeG protein, the edible crop plant alfalfa was transformed with the chloroplast-targeting construct and equally to tobacco plants, a high-yield FaeG accumulation of 1% of TSP was obtained. A similar yield was also obtained in the seeds of barley, a valuable crop plant, when the FaeG-encoding gene was expressed under an endosperm-specific promoter and subcellularly targeted into the endoplasmic reticulum. Furthermore, desiccated alfalfa plants and barley grains were shown to have a capacity to store FaeG protein in a stable form for years. When the transgenic alfalfa plants were administred orally to weaned piglets, slight F4-specific systemic and mucosal immune responses were induced. Co-administration of the transgenic alfalfa and the mucosal adjuvant cholera toxin enhanced the F4-specific immune response; the duration and number of F4+ E. coli excretion following F4+ ETEC challenge were significantly reduced as compared with pigs that had received nontransgenic plant material. In conclusion, the results suggest that transgenic plants producing the FaeG subunit protein could be used for production and delivery of oral vaccines against porcine F4+ ETEC infections. The findings here thus present new approaches to develop the vaccination strategy against porcine postweaning diarrhea.

Ultrastructure of Biofilms Formed by Bacteria from Industrial Processes

Microorganisms exist predominantly as sessile multispecies communities in natural habitats. Most bacterial species can form these matrix-enclosed microbial communities called biofilms. Biofilms occur in a wide range of environments, on every surface with sufficient moisture and nutrients, also on surfaces in industrial settings and engineered water systems. This unwanted biofilm formation on equipment surfaces is called biofouling. Biofouling can significantly decrease equipment performance and lifetime and cause contamination and impaired quality of the industrial product. In this thesis we studied bacterial adherence to abiotic surfaces by using coupons of stainless steel coated or not coated with fluoropolymer or diamond like carbon (DLC). As model organisms we used bacterial isolates from paper machines (Meiothermus silvanus, Pseudoxanthomonas taiwanensis and Deinococcus geothermalis) and also well characterised species isolated from medical implants (Staphylococcus epidermidis). We found that coating of steel surface with these materials reduced its tendency towards biofouling: Fluoropolymer and DLC coatings repelled all four biofilm formers on steel. We found great differences between bacterial species in their preference of surfaces to adhere as well as their ultrastructural details, like number and thickness of adhesion organelles they expressed. These details responded differently towards the different surfaces they adhered to. We further found that biofilms of D. geothermalis formed on titanium dioxide coated coupons of glass, steel and titanium, were effectively removed by photocatalytic action in response to irradiation at 360 nm. However, on non-coated glass or steel surfaces irradiation had no detectable effect on the amount of bacterial biomass. We showed that the adhesion organelles of bacteria on illuminated TiO2 coated coupons were complety destroyed whereas on non-coated coupons they looked intact when observed by microscope. Stainless steel is the most widely used material for industrial process equipments and surfaces. The results in this thesis showed that stainless steel is prone to biofouling by phylogenetically distant bacterial species and that coating of the steel may offer a tool for reduced biofouling of industrial equipment. Photocatalysis, on the other hand, is a potential technique for biofilm removal from surfaces in locations where high level of hygiene is required. Our study of natural biofilms on barley kernel surfaces showed that also there the microbes possessed adhesion organelles visible with electronmicroscope both before and after steeping. The microbial community of dry barley kernels turned into a dense biofilm covered with slimy extracellular polymeric substance (EPS) in the kernels after steeping in water. Steeping is the first step in malting. We also presented evidence showing that certain strains of Lactobacillus plantarum and Wickerhamomyces anomalus, when used as starter cultures in the steeping water, could enter the barley kernel and colonise the tissues of the barley kernel. By use of a starter culture it was possible to reduce the extensive production of EPS, which resulted in a faster filtration of the mash.

Climate change and agriculture: theory and application of mitigation policies

Agriculture’s contribution to climate change is controversial as it is a significant source of greenhouse gases but also a sink of carbon. Hence its economic and technological potential to mitigate climate change have been argued to be noteworthy. However, social profitability of emission mitigation is a result from factors among emission reductions such as surface water quality impact or profit from production. Consequently, to value comprehensive results of agricultural climate emission mitigation practices, these co-effects to environment and economics should be taken into account. The objective of this thesis was to develop an integrated economic and ecological model to analyse the social welfare of crop cultivation in Finland on distinctive cultivation technologies, conventional tillage and conservation tillage (no-till). Further, we ask whether it would be privately or socially profitable to allocate some of barley cultivation for alternative land use, such as green set-aside or afforestation, when production costs, GHG’s and water quality impacts are taken into account. In the theoretical framework we depict the optimal input use and land allocation choices in terms of environmental impacts and profit from production and derive the optimal tax and payment policies for climate and water quality friendly land allocation. The empirical application of the model uses Finnish data about production cost and profit structure and environmental impacts. According to our results, given emission mitigation practices are not self-evidently beneficial for farmers or society. On the contrary, in some cases alternative land allocation could even reduce social welfare, profiting conventional crop cultivation. This is the case regarding mineral soils such as clay and silt soils. On organic agricultural soils, climate mitigation practices, in this case afforestation and green fallow give more promising results, decreasing climate emissions and nutrient runoff to water systems. No-till technology does not seem to profit climate mitigation although it does decrease other environmental impacts. Nevertheless, the data behind climate emission mitigation practices impact to production and climate is limited and partly contradictory. More specific experiment studies on interaction of emission mitigation practices and environment would be needed. Further study would be important. Particularly area specific production and environmental factors and also food security and safety and socio-economic impacts should be taken into account.

Aspergillus niger -proliiniendopeptidaasi prolamiinien eliminoinnissa

Tutkielman kirjallisuusosassa perehdyttiin vehnän, rukiin ja ohran, eli Triticeaeprolamiinien erityisasemaan keliakianäkökulmasta tarkasteltuna ja prolamiinien hydrolyysiin proliinispesifeillä entsyymeillä. Lisäksi tarkasteltiin prolamiinien immunologisia määritysmenetelmiä. Keliakiassa haitalliset gluteenipeptidit sisältävät runsaasti proliinia ja ovat hankalia pilkkoa muilla kuin proliinispesifeillä peptidaaseilla. Suurin osa immunologisen reaktion aiheuttavista gluteenilähtöisistä peptideistä voidaan pilkkoa idätetyn viljan endogeenisilla entsyymeillä happamissa olosuhteissa, mutta jäljellejäävä prolamiinipitoisuus ylittää edelleen gluteenittomille tuotteille sallitun rajan. Kokeellisen työn tavoitteena oli eliminoida happamalla mallasinkubaatiolla valmistettujen vehnä-, ohra- ja ruismallasautolysaattien sisältämä jäännösprolamiini Aspergillus niger -homeen tuottamalla proliinispesifillä endopeptidaasilla (AN-PEP) siten, että hydrolysaattia voitaisiin käyttää gluteenittomissa leivontasovelluksissa. Proteiinien hydrolyysiä tarkkailtiin kokoekskluusiokromatografialla (SEC), vapaan aminotypen (FAN) muodostumisena ja SDS-PAGE-elektroforeesilla. Jäännösprolamiinien pilkkoutumista seurattiin immunologisella R5-ELISA-menetelmällä. AN-PEP-inkubaatiolla saatiin aikaan voimakasta prolamiinien pilkkoutumista; mallasautolysaattien jäännösprolamiinista pilkkoutui yli 96 %. SEC- ja FAN-analyysien perusteella inkubaatioaikaa kannatti jatkaa yli 4 h, jolloin polypeptidit pilkkoutuivat edelleen pienemmiksi hydrolyysituotteiksi. Vehnä- ja ruismallashydrolysaattien prolamiinipitoisuuden todettiin laskevan 22 h inkubaation aikana alle tason 100 mg/kg R5-ELISA-menetelmällä määritettynä. Matalimmat prolamiinipitoisuudet saavutettiin AN-PEP-pitoisuudella 35 ?l / g mallasautolysaattia. Codex Alimentarius -komission säädöksen mukaan keliakiaruokavalioon soveltuvat ns. erittäin vähägluteeniset tuotteet saavat sisältää gluteenia enintään 100 mg/kg. Erityisesti AN-PEP-käsiteltyä ruismallasraaka-ainetta voitaisiin mahdollisesti käyttää tuomaan rukiista aromia gluteenittomiin leipiin. Ennen kuin mallashydrolysaatit ovat valmiita kaupallisiin sovelluksiin, on tarkasteltava niiden todellisia mahdollisuuksia parantaa elintarvikkeiden makua ja aromia sekä todettava uuden teknologian turvallisuus keliaakikoille.

Charred seeds in Mikkeli Orijärvi : A study of subsistence strategies of an Iron Age settlement in East Finland

In this study the over 350 macrofossil samples, containing over 2300 charred plant remains from an Iron Age settlement containing fossil fields in Mikkeli Orijärvi Kihlinpelto, were studied archaeobotanically. The aim was to get more information about subsistence strategies, especially agriculture and study differences in the plant combinations in the different structures and use the archaeobotanical theory to interpret these structures. The methodological question was to study the taphonomy of the charred plant material. The results gave a diverse impression of the agriculture and subsistence strategies of the settlement in Orijärvi, where barley was the most important cereal with rye, wheat and oat cultivated as minor crops. The arable weed assemblage indicates that the fields were situated in different kinds of soils and the crops were cultivated when different kind of weather conditions were prevailing. Ergot was found with the cereals, and it was growing on some of the arable crops and it also indicates wet climate. Hemp and flax were cultivated and wild plants were collected. The meadow and wetland plants found in the material derive most probably from animal fodder. Tubers of bulbous oat-grass were interesting, because they are usually found in graves. Comparison with other Iron Age settlements and graves indicates that the plant material found from the ancient field layers derives most probably from dwellings and graves, which were taken into cultivation.

Idén om en karriär - ett kulturellt manuskript för skapandet av det postmoderna självet:

Bakgrunden till denna undersökning utgörs av arbetslivets förändring och dess konsekvenser för konstrueringen av självidentiteten; hur avväger unga konsulter i början av sin karriär mellan olika diskurser för att skapa en förhandlad självidentitet som styrs av idén om en karriär och såvida är hållbar även i ett långtidsperspektiv? Undersökningen tar avstamp i de sociologiska diskussionerna om informationssamhällets framväxt och dess inverkan på arbetets karaktär. Undersökningen utgår från begreppet kunskapsarbete (Alvesson 2001; Blom, Melin & Pyöriä 2001) eftersom konsultyrket kan anses representera denna typ av arbete. Ett genomgående tema är flexibiliteten och det höga tempot som å ena sidan möjliggör självförverkligande men å andra sidan försvårar skapandet av en sammanhängande självidentitet (Sennett 2002). Denna problematik är viktigt att ta i beaktande då förlängandet av arbetskarriärerna och ökandet av arbetslivets attraktivitet blivit centrala mål för den finska samhällspolitiken (Julkunen & Pärnänen 2005) samtidigt som ett generationsskifte håller på att ske på den finländska arbetsmarknaden. Undersökningen utgår från idén om karriären som ett manuskript som ger upphov för identitetsförhandlingar och sammanbinder livets olika domäner till ett personligt liv inte bara i nuläget utan även i ett långtidsperspektiv. Enligt Alvesson & Willmot (2002) framkallas identitetsförhandlingar genom både samhälleliga och organisationsspecifika identitetsreglerande praktiker. Jag utgår från antagandet att självidentiteten fungerar som den sammanbindande knytpunkten mellan karriären och det övriga livet, vilket gör det intressant att koncentrera sig på de identitetsförhandlingar som informanterna utsätts för i förhållande till sitt arbete och idén om en karriär. Begreppet personligt liv ger utrymme för individuell aktivitet och personliga betydelser men behåller ändå föreställningen om individen som inbäddad och sammanbunden i och med det sociala och kulturella (Smart 2007). I bakgrunden finns teorierna om den nya form som makten antagit i organisationer (Foucault; Rose 1999; Grey 1994, Casey 1995) och förvandlat individen till en ett självstyrande subjekt som arbetar som en entreprenör (Rose 1999). Jag poängterar den subjektiva aspekten av karriären som någonting som individen själv konstruerar (Hughes 1997; Barley 1989) och uppfattar i likhet med Julkunen (2003) och Acker (1990; 2006) ålder och kön som sociala kategorier i organisationsstrukturen. Syftet med denna studie är att undersöka hur unga personer utför identitetsförhandlingar i förhållande till arbetet och idén om en karriär i nuläget, samt sammanbinder karriären, framtiden och livet över lag med sin självidentitet i ett karriärperspektiv. Jag har valt unga konsulter som forskningsobjekt, eftersom endast en liten del av den sociologiska arbetslivsforskningen intresserat sig för just unga. Konsultbranschen är ett intressant forskningsobjekt av samma orsak – denna bransch har behandlats i mycket liten skala i finländsk forskning. Det empiriska materialet består av kvalitativa intervjuer med unga, högutbildade män och kvinnor i konsultbranschen. Som analysmetod har jag använt mig av tema-analys inspirerad av diskursanalys. Genom att undersöka individers identitetsförhandlingar i ett karriärperspektiv hoppas jag kunna bidra med nya synpunkter och infallsvinklar samt bättre förståelse för det postindustriella arbetslivet som karakteriseras av kunskapsintensifiering och snabba förändringar i organisationer. Studien visar att idén om en karriär fungerar som ett medel för designandet av det postmoderna självet på samma sätt som till exempel konsumtionen av vissa varumärken. Enligt studien har den flexibla kapitalismen olika inverkan på män och kvinnors idéer om en karriär och betjänar den manliga karriären betydligt bättre än den kvinnliga, åtminstone så länge traditionella könskonstruktioner dominerar. För männen är det naturligt att totalt utesluta faderskapet från karriärkonstruktionen, vilket får dem att kapitulera inför organisationens krav. För kvinnorna är det lika naturligt att justera idén om den framtida karriären enligt möjliga barn i framtiden. Möjligheten att bejaka moderskapets krav verkar beskydda den kvinnliga självidentiteten från att totalt ge efter för organisationens försök att kontrollera subjektiviteten. De unga informanterna konstruerar sitt arbete i mycket annorlunda termer än vad som traditionellt förknippas med arbete; konsultarbetets projektbaserade karaktär konstrueras som äventyrligt, roligt, intressant och omväxlande. Eftersom avsaknad av långsiktighet och rutiner förknippas med den tid i livet då man är ung, underlättades sammanförandet av konsultarbetet med självidentiteten. Samtidigt visar studien hur svårt individen, som fungerar likt en entreprenör i förhållande till sin arbetsgivare, har att skilja mellan arbete och övrigt liv samtidigt som diskursen om balans mellan arbete och fritid dominerar i samhället. Detta ger upphov till en konstruktion av självet som en lycklig och autonom individ som är nöjd över sitt val av start på karriären och tycker om att jobba, medan de långa arbetsdagarna och kraven på ständig effektivitet och prestationer konstrueras som något naturligt. Slutligen efterlyser studien en omdefiniering av begreppet kunskapsarbete. Arbetssociologin i sin helhet har utgått från tanken om arbete som något ofrivilligt eller som tvång, medan informanterna i denna undersökning tvärtom konstruerar sitt arbete i termer som traditionellt förknippas med fritid och frivillighet . Man kan fråga sig ifall kunskapsarbetet håller på att slita sig loss från den traditionella uppfattningen om arbete och skapa en allt skarpare kontrast till det arbete som utförs i traditionell bemärkelse?

Tumorigenesis in celiac disease

Celiac disease is life-long autoimmune disorder of the small intestine, which is caused by a reaction to gliadin found in wheat, rye and barley in genetically predisposed individuals. Proline- and glutamine -rich proteins cause villous atrophy and crypt hyperplasia with extensive inflammation in the epithelium and lamina propria. Symptoms of celiac disease vary considerably and elimination of gluten from diet is the only way to treat disease. In small intestine of celiac disease patient transglutaminase 2 (TG2) modifies gluten peptides, which causes T-cell activation and inflammation in the epithelium of mucosa. T-cell activation induces development of celiac disease specific antibodies. These celiac disease specific antibodies recognise TG2 and interfere in vitro and in vivo in angiogenesis. Abnormal angiogenesis is typical in many disorders, such in cancer, in which TG2 has a crucial role in the development and growth of tumor. Overexpression of TG2 has been shown to correlate with accelerated growth of tumor. TG2-specific antibodies are suggested to inhibit differentation of epithelial cell, increase their proliferation, decrease their barrier-function and increase the permeability of blood vessels. The aims of the pilot study were to establish whether celiac disease TG2 antibodies affect in vivo tumorigenesis and tumorangiogenesis as well as to try to clarify the mechanism behind the phenomenon. Tumor xenograft model was used in severe combined immunodeficient (SCID) mice. Human oesophageal carcinoma (OE-19) cancer cells were incubated with celiacs TG2 miniautoantibody (mini 2.8), non-celiac miniautoantibody (mini 6.2) or PBS before cancer cells were injected to mice subcutaneously. During the experiment mice were weighted and tumor size was measured couple of times per week. To estimate the volumes of tumors the following formula was used: π/6 * L* W* H. Experiment lasted for four weeks after which the mice were euthanized, cardiac blood and tissue samples taken and tumours were excised and weighted. Sections were made from tumors and immunohistochemical stainings were done to compare blood vessel areas and to study general tumors´morphology and other parameters. Western blot -analyse were performed to cancer cells. The masses and volumes were clearly smaller in mini 2.8-group compared to control groups and the necrotic area of tumor in mini 2.8 was smallest as percentage compared to control groups. Blood vessel area were smallest in mini 2.8 group. Results suggest that celiac disease anti-TG2-autoantibodies inhibit tumor growth, but the number of animals is insufficient to give an accurate outcome.

The challenge of LC/MS/MS multi-mycotoxin analysis - Heracles battling the Hydra?

Mycotoxins are secondary metabolites of filamentous fungi. They pose a health risk to humans and animals due to their harmful biological properties and common occurrence in food and feed. Liquid chromatography/mass spectrometry (LC/MS) has gained popularity in the trace analysis of food contaminants. In this study, the applicability of the technique was evaluated in multi-residue methods of mycotoxins aiming at simultaneous detection of chemically diverse compounds. Methods were developed for rapid determination of toxins produced by fungal genera of Aspergillus, Fusarium, Penicillium and Claviceps from cheese, cereal based agar matrices and grains. Analytes were extracted from these matrices with organic solvents. Minimal sample clean-up was carried out before the analysis of the mycotoxins with reversed phase LC coupled to tandem MS (MS/MS). The methods were validated and applied for investigating mycotoxins in cheese and ergot alkaloid occurrence in Finnish grains. Additionally, the toxin production of two Fusarium species predominant in northern Europe was studied. Nine mycotoxins could be determined from cheese with the method developed. The limits of quantification (LOQ) allowed the quantification at concentrations varying from 0.6 to 5.0 µg/kg. The recoveries ranged between 96 and 143 %, and the within-day repeatability (as relative standard deviation, RSDr) between 2.3 and 12.1 %. Roquefortine C and mycophenolic acid could be detected at levels of 300 up to 12000 µg/kg in the mould cheese samples analysed. A total of 29 or 31 toxins could be analysed with the method developed for agar matrices and grains, with the LOQs ranging overall from 0.1 to 1250 µg/kg. The recoveries ranged generally between 44 and 139 %, and the RSDr between 2.0 and 38 %. Type-A trichothecenes and beauvericin were determined from the cereal based agar and grain cultures of F. sporotrichioides and F. langsethiae. T-2 toxin was the main metabolite, the average levels reaching 22000 µg/kg in the grain cultures after 28 days of incubation. The method developed for ten ergot alkaloids from grains allowed their quantification at levels varying from 0.01 to 10 µg/kg. The recoveries ranged from 51 to 139 %, and the RSDr from 0.6 to 13.9 %. Ergot alkaloids were measured in barley and rye at average levels of 59 and 720 µg/kg, respectively. The two most prevalent alkaloids were ergocornine and ergocristine. The LC/MS methods developed enabled rapid detection of mycotoxins in such applications where several toxins co-occurred. Generally, the performance of the methods was good, allowing reliable analysis of the mycotoxins of interest with sufficiently low quantification limits. However, the variation in validation results highlighted the challenges related to optimising this type of multi-residue methods. New data was obtained about the occurrence of mycotoxins in mould cheeses and of ergot alkaloids in Finnish grains. In addition, the study revealed the high mycotoxin-producing potential of two common fungi in Finnish crops. The information can be useful when risks related to fungal and mycotoxin contamination will be assessed.

Immunochemical analysis of prolamins in gluten-free foods

People with coeliac disease have to maintain a gluten-free diet, which means excluding wheat, barley and rye prolamin proteins from their diet. Immunochemical methods are used to analyse the harmful proteins and to control the purity of gluten-free foods. In this thesis, the behaviour of prolamins in immunological gluten assays and with different prolamin-specific antibodies was examined. The immunoassays were also used to detect residual rye prolamins in sourdough systems after enzymatic hydrolysis and wheat prolamins after deamidation. The aim was to characterize the ability of the gluten analysis assays to quantify different prolamins in varying matrices in order to improve the accuracy of the assays. Prolamin groups of cereals consist of a complex mixture of proteins that vary in their size and amino acid sequences. Two common characteristics distinguish prolamins from other cereal proteins. Firstly, they are soluble in aqueous alcohols, and secondly, most of the prolamins are mainly formed from repetitive amino acid sequences containing high amounts of proline and glutamine. The diversity among prolamin proteins sets high requirements for their quantification. In the present study, prolamin contents were evaluated using enzyme-linked immunosorbent assays based on ω- and R5 antibodies. In addition, assays based on A1 and G12 antibodies were used to examine the effect of deamidation on prolamin proteins. The prolamin compositions and the cross-reactivity of antibodies with prolamin groups were evaluated with electrophoretic separation and Western blotting. The results of this thesis research demonstrate that the currently used gluten analysis methods are not able to accurately quantify barley prolamins, especially when hydrolysed or mixed in oats. However, more precise results can be obtained when the standard more closely matches the sample proteins, as demonstrated with barley prolamin standards. The study also revealed that all of the harmful prolamins, i.e. wheat, barley and rye prolamins, are most efficiently extracted with 40% 1-propanol containing 1% dithiothreitol at 50 °C. The extractability of barley and rye prolamins was considerably higher with 40% 1-propanol than with 60% ethanol, which is typically used for prolamin extraction. The prolamin levels of rye were lowered by 99.5% from the original levels when an enzyme-active rye-malt sourdough system was used for prolamin degradation. Such extensive degradation of rye prolamins suggest the use of sourdough as a part of gluten-free baking. Deamidation increases the diversity of prolamins and improves their solubility and ability to form structures such as emulsions and foams. Deamidation changes the protein structure, which has consequences for antibody recognition in gluten analysis. According to the resuts of the present work, the analysis methods were not able to quantify wheat gluten after deamidation except at very high concentrations. Consequently, deamidated gluten peptides can exist in food products and remain undetected, and thus cause a risk for people with gluten intolerance. The results of this thesis demonstrate that current gluten analysis methods cannot accurately quantify prolamins in all food matrices. New information on the prolamins of rye and barley in addition to wheat prolamins is also provided in this thesis, which is essential for improving gluten analysis methods so that they can more accurately quantify prolamins from harmful cereals.

The Transcriptional Regulation of Retrotransposon BARE

The purpose of this research project was to understand the steps of the retrotransposon BARE (BArley REtrotransposon) life cycle, from regulation of transcription to Virus-Like Particle (VLP) formation and ultimate integration back into the genome. Our study concentrates mainly on BARE1 transcriptional regulation because transcription is the crucial first step in the retrotransposon life cycle. The BARE element is a Class I LTR (Long Terminal Repeat) retrotransposon belonging to the Copia superfamily and was originally isolated in our research group. The LTR retrotransposons are transcribed from promoters in the LTRs and encode proteins for packaging of their transcripts, the reverse transcription of the transcripts into cDNA, and integration of the cDNA back into the genome. BARE1 is translated as a single polyprotein and cleaved into the capsid protein (GAG), integrase (IN), and reverse transcriptase-RNaseH (RT-RH) by the integral aspartic proteinase (AP). The BARE retrotransposon family comprises more than 104 copies in the barley (Hordeum vulgare) genome. The element is bound by long terminal repeats (LTRs, 1829 bp) containing promoters required for replication, signals for RNA processing, and motifs necessary for the integration of the cDNA. Members of the BARE1 subfamily are transcribed, translated, and form virus-like particles. Several basic questions concerning transcription are explored in the thesis: BARE1 transcription control, promoter choice in different barley tissues, start and termination sites for BARE transcripts, and BARE1 transcript polyadenylation (I). Polyadenylation is an important step during mRNA maturation, and determines its stability and translatability among other characteristics. Our work has found a novel way used by BARE1 to make extra GAG protein, which is critical for VLP formation. The discovery that BARE1 uses one RNA population for protein synthesis and another RNA population for making cDNA has established the most important step of the BARE1 life cycle (III). The relationship between BARE1 and BARE2 has been investigated. Besides BARE, we have examined the retrotransposon Cassandra (II), which uses a very different transcriptional mechanism and a fully parasitic life cycle. In general, this work is focused on BARE1 promoter activity, transcriptional regulation including differential promoter usage and RNA pools, extra GAG protein production and VLP formation. The results of this study give new insights into transcription regulation of LTR retrotransposons.

The effect of structure on the dilute solution properties of branched polysaccharides studied with SEC and AsFlFFF

Cereal arabinoxylans, guar galactomannans, and dextrans produced by lactic acid bacteria(LAB) are a structurally diverse group of branched polysaccharides with nutritional and industrial functions. In this thesis, the effect of the chemical structure on the dilute solution properties of these polysaccharides was investigated using size-exclusion chromatography(SEC) and asymmetric flow field-flow fractionation (AsFlFFF) with multiple-detection. The chemical structures of arabinoxylans were determined, whereas galactomannan and dextran structures were studied in previous investigations. Characterization of arabinoxylans revealed differences in the chemical structures of cereal arabinoxylans. Although arabinoxylans from wheat, rye, and barley fiber contained similar amounts of arabinose side units, the substitution pattern of arabinoxylans from different cereals varied. Arabinoxylans from barley husks and commercial low-viscosity wheat arabinoxylan contained a lower number of arabinose side units. Structurally different dextrans were obtained from different LAB. The structural effects on the solution properties could be studied in detail by modifying pure wheat and rye arabinoxylans and guar galactomannan with specific enzymes. The solution characterization of arabinoxylans, enzymatically modified galactomannans, and dextrans revealed the presence of aggregates in aqueous polysaccharide solutions. In the case of arabinoxylans and dextrans, the comparison of molar mass data from aqueous and organic SEC analyses was essential in confirming aggregation, which could not be observed only from the peak or molar mass distribution shapes obtained with aqueous SEC. The AsFlFFF analyses gave further evidence of aggregation. Comparison of molar mass and intrinsic viscosity data of unmodified and partially debranched guar galactomannan, on the other hand, revealed the aggregation of native galactomannan. The arabinoxylan and galactomannan samples with low or enzymatically extensively decreased side unit content behaved similarly in aqueous solution: lower molar mass samples stayed in solution but formed large aggregates, whereas the water solubility of the higher-molar-mass samples decreased significantly. Due to the restricted solubility of galactomannans in organic solvents, only aqueous galactomannan solutions were studied. The SEC and AsFlFFF results differed for the wheat arabinoxylan and dextran samples. Column matrix effects and possible differences in the separation parameters are discussed, and a problem related to the non-established relationship between the separation parameters of the two separation techniques is highlighted. This thesis shows that complementary approaches in the solution characterization of chemically heterogeneous polysaccharides are needed to comprehensively investigate macromolecular behavior in solution. These results may also be valuable when characterizing other branched polysaccharides.

Lipeäkäsitellyn vehnän vaikutus lypsylehmien syöntiin ja tuotokseen vapaalla seosrehuruokinnalla

Lipeä on vahva emäs, jonka on havaittu lisäävän hemiselluloosan ja ligniinin hydrolyysiä pötsissä. Näin ollen lipeäkäsittelyllä on mahdollista korvata viljan mekaaninen litistys ja jauhatus. Seosrehuruokinnalla, jonka osana on lipeäkäsitelty vilja, on mahdollista vähentää liiallisesta tärkkelyksestä aiheutuvia metabolisia ongelmia pötsissä. Tämän tutkielman tarkoituksena oli selvittää lipeäkäsitellyn vehnän vaikutusta lypsylehmien syöntiin ja tuotokseen ad libitum seosrehuruokinnoilla. Ruokinnoissa korvattiin kuivaa murskattua vehnää asteittain kokonaisella lipeäkäsitellyllä vehnällä. Kontrollina oli perinteisesti käytetty kuiva, murskattu ohra-kaura seos. Koe tehtiin Ruotsin maatalousyliopiston (SLU) maataloustieteiden laitoksella Uumajassa. Koe alkoi syyskuussa ja päättyi joulukuussa 2010. Kokeessa oli 17 useamman kerran poikinutta lehmää ja 6 ensikkoa (Ruotsin punainen -rotu). Lehmät olivat lämpimässä pihattonavetassa, jossa seosrehun syöntiä mitattiin vaakakuppien avulla. Koekäsittelyt olivat murskattu ohra-kaura seos (1:1), murskattu kuiva vehnä (1:0), murskatun kuivan vehnän ja kokonaisen lipeävehnän seos (1:1) ja kokonainen lipeävehnä (1:0). Ruokintojen kuiva-ainepitoisuudeksi asetettiin 370 g/kg ja raakavalkuaispitoisuudeksi 180 g/kg kuiva-ainetta. Näennäinen ravintoaineen sulavuus määritettiin happoon liukenemattoman tuhkan avulla. Typen hyväksikäyttöä arvioitiin laskennallisen typpitaseen avulla. Koe toteutettiin 4x4 latinalaisen neliön koemallin mukaisesti ja käsittelyjen väliset tilastolliset erot testattiin kontrastien avulla. Kuiva-aineen (PQ=0,02) ja orgaanisen aineen (PQ=0,02) syönnit lisääntyivät, samalla kun niiden sulavuudet paranivat korvattaessa puolet kuivasta vehnästä lipeävehnällä. Ruokintojen välillä ei ollut tilastollisesti merkitsevää eroa maitotuotoksessa eikä energiakorjatussa maitotuotoksessa. Maidon rasvatuotos lisääntyi vähän (PQ=0,04) ja rasvapitoisuus selvästi (PQ=0,004), kun kuivasta vehnästä korvattiin puolet lipeävehnällä. Kun kaikki kuiva vehnä korvattiin lipeävehnällä, maidon valkuaispitoisuus väheni (PL<0,001). Samoin kävi maidon ureapitoisuudelle (PL=0,002). Lipeäkäsittely ei tuottanut tässä kokeessa taloudellisesti kannattavaa tulosta, sillä maidon valkuaispitoisuus väheni ja syönti lisääntyi maitotuotoksen pysyessä samana. Vehnäruokinnoista paras tuotosvaste saatiin kuivan vehnän ja lipeävehnän seoksella.

Mykorritsa fosforin kierrättäjänä

Suomen maatalousmaihin kertynyttä fosforia hyödynnetään tehottomasti, ja samalla muokkauskerroksen suuri fosforimäärä on alttiina huuhtoutumiselle. Arbuskelimykorritsaa (AM) hyödyntämällä on mahdollista tehostaa viljelykasvin fosforinottoa ja kasvua, ja siten vähentää fosforin huuhtoutumista. Tämän tutkielman tavoitteena oli selvittää mykorritsan vaikutus kasvin kasvuun ja fosforinottoon karjanlantalannoituksella mineraalilannoitukseen verrattuna sekä näiden lannoitusten pitkäaikaisvaikutusta AM-sieniyhteisöihin. Jotta lannoituskäytäntöjen vaikutus mykorritsaan voitiin suhteuttaa muihin maan laatutekijöihin, näiden käytäntöjen vaikutus myös satomääriin sekä muihin maan laatumittareihin arvioitiin. Pitkäaikainen kenttäkoe perustettiin kolmelle paikkakunnalle Pohjois-Ruotsissa vuosina 1965–66. Kuusivuotinen viljelykierto koostui joko viisivuotisesta nurmesta ja ohrasta tai ohramonokulttuurista. Lannoituskäsittelyt 32-vuoden ajan olivat suositusten mukainen (NPK) ja edelliseen nähden kaksinkertainen (2NPK) mineraalilannoitus sekä karjanlantalannoitus (KL), jonka ravinnemäärä vastasi NPK -käsittelyä. Kolmen lannoituskäsittelyn vaikutusta mykorritsan tehokkuuteen kasvin kasvun ja fosforiravitsemuksen näkökulmasta tutkittiin astiakokeissa. Mykorritsasieniyhteisöjen toiminnallisten erojen selvittämiseksi tehtiin takaisin- ja ristiinsiirrostuskoe. (5 v-%) steriloitua maanäytettä NPK- ja KL -käsittelyistä siirrostettiin käsittelemättömiin maanäytteisiin, jotka olivat samoista lannoituskäsittelyistä. Mykorritsan positiivinen vaikutus kasvin kasvuun ja fosforiravitsemukseen oli suurin kun käytettiin karjanlantaa. NPK ja 2NPK -käsittelyiden välillä ei havaittu eroja. Takaisin- ja ristiinsiirrostuskokeessa ei ollut tilastollisesti merkitseviä eroja. Nurmi- ja ohrasadot olivat suurimmat kun mineraalilannoitetta annettiin suosituksiin nähden kaksinkertainen määrä. Satomäärät olivat yhtä suuret tai suuremmat kun käytettiin karjanlantaa NPK –lannoituksen sijaan. Karjanlantakäsittely lisäsi maaperän kokonaishiili- ja kokonaistyppipitoisuutta verrattuna NPK -käsittelyyn, joka sisälsi saman määrän ravinteita. Samalla huuhtoutumiselle altis liukoisen fosforin pitoisuus säilyi alhaisella tasolla. Karjanlanta edisti mykorritsan toimintaedellytyksiä, ja siksi mykorritsasta saatua hyötyä fosforinotossa ja kasvuvaikutuksena mineraalilannoitteisiin verrattuna, mutta se ei vaikuttanut mykorritsasieniyhteisön toiminnallisiin ominaisuuksiin. Karjanlantalannoitus paransi mitattuja maan ominaisuuksia kokonaisuudessaan, eikä se vähentänyt satoja.