Functional studies on bacterial nucleotide-regulated inorganic pyrophosphatases

CBS domains are ~60 amino acid tandemly repeated regulatory modules forming a widely distributed domain superfamily. Found in thousands of proteins from all kingdoms of life, CBS domains have adopted a variety of functions during evolution, one of which is regulation of enzyme activity through binding of adenylate-containing compounds in a hydrophobic cavity. Mutations in human CBS domain-containing proteins cause hereditary diseases. Inorganic pyrophosphatases (PPases) are ubiquitous enzymes, which pull pyrophosphate (PPi) producing reactions forward by hydrolyzing PPi into phosphate. Of the two nonhomologous soluble PPases, dimeric family II PPases, belonging to the DHH family of phosphoesterases, require a transition metal and magnesium for maximal activity. A quarter of the almost 500 family II PPases, found in bacteria and archaea, contain a 120-250 amino acid N-terminal insertion, comprised of two CBS domains separated in sequence by a DRTGG domain. These enzymes are thus named CBS-PPases. The function of the DRTGG domain in proteins is unknown. The aim of this PhD thesis was to elucidate the structural and functional differences of CBS-PPases in comparison to family II PPases lacking the regulatory insert. To this end, we expressed, purified and characterized the CBS-PPases from Clostridium perfringens (cpCBS-PPase) and Moorella thermoacetica (mtCBS-PPase), the latter lacking a DRTGG domain. Both enzymes are homodimers in solution and display maximal activity against PPi in the presence of Co2+ and Mg2+. Uniquely, the DRTGG domain was found to enable tripolyphosphate hydrolysis at rates similar to that of PPi. Additionally, we found that AMP and ADP inhibit, while ATP and AP4A activate CBSPPases, thus enabling regulation in response to changes in cellular energy status. We then observed substrate- and nucleotide-induced conformational transitions in mtCBS-PPase and found that the enzyme exists in two differentially active conformations, interconverted through substrate binding and resulting in a 2.5-fold enzyme activation. AMP binding was shown to produce an alternate conformation, which is reached through a different pathway than the substrate-induced conformation. We solved the structure of the regulatory insert from cpCBS-PPase in complex with AMP and AP4A and proposed that conformational changes in the loops connecting the catalytic and regulatory domains enable activity regulation. We examined the effects of mutations in the CBS domains of mtCBS-PPase on catalytic activity, as well as, nucleotide binding and inhibition.

Hybridikomposiittien hyödyntäminen tuulivoimalan rakenteissa

Diplomityön tarkoituksena oli tutkia hybridikomposiittien soveltuvuutta tuulivoimalan osien tai osakokonaisuuksien rakennemateriaaliksi. Lähtökohtana oli selvittää erityisesti luonnonkuitukomposiitin materiaaliteknisten ominaisuuksien, etenkin lujuusominaisuuksi-en soveltuminen tuulivoiman rakenteisiin. Työn johdanto-osuudessa esitellään tuulivoiman rooli tämän päivän energiantuotannossa, yksittäisen tuulivoimalalaitoksen rakenne, rakenteiden suunnittelussa huomioitavat seikat, voimalan eri osien kuten tornin, lapojen ja nasellin yleisimmät valmistusmenetelmät, sekä muovien ja eri lujitteiden ohella puumuovikomposiitin materiaaliominaisuudet, valmistus-menetelmät ja yleisimmät käyttökohteet. Hybridikomposiittien lujuusominaisuuksia tutkittiin Lappeenrannan teknillisen yliopiston puutekniikan laboratoriossa suoritetuissa mittauksissa. Saatuja tuloksia verrattiin referens-situlosten ohella myös tällä hetkellä tuulivoimalan rakenteissa yleisesti käytettävien lasi- ja hiilikuidun, sekä teräksen ominaisuuksiin. Mittaustulosten perusteella bambu- ja lasikuitu-lujitteiset puumuovikomposiitit soveltuvat parhaiten tuulivoimalarakenteisiin, mutta niiden valmistaminen ekstruusiomenetelmällä on melko haasteellista.

Elintarviketehtaan tuotantolinjan kunnonvalvonnan kehittäminen

Tutkimuksen tavoitteena oli kunnonvalvontajärjestelmän kehittäminen nykyaikaiselle elintarviketeollisuuden tuotantolinjalle. Työssä etsittiin olemassa olevia kunnonvalvontamenetelmiä ja -sovelluksia erityisesti jäätelötuotantolaitteiden tarpeisiin. Kirjallisuudesta löydettiin sovelluksia lähinnä yksittäisille komponenteille ja kone-elimille. Tutkimuksen kohteena oleva tuotantolinja käytiin läpi toimintojen ja komponenttien osalta. Tehtaan laitekantaa ja valmiutta kunnonvalvonnan käyttöönotolle käsiteltiin yleisellä tasolla. Kunnonvalvontajärjestelmää kehitettäessä käytettiin hyväksi kirjallisuudesta löytyneitä menetelmiä, joista soveltuvimpia käytettiin kunnonvalvontametodien kartoittamiseen. Kunnonvalvontametodit muodostettiin toiminnanohjausjärjestelmästä ja linjan käyttöönotosta saatujen tietojen avulla. Tietoja käytettiin vika-vaikutusanalyysissä, joka oli pohjana komponentti- ja menetelmäkohtaisessa analyysissa. Kriittisiä ja tuotannon tehokkuuteen vaikuttavia koneenosia ja kokonaisuuksia tarkasteltiin kunnonvalvontamenetelmien soveltuvuuden avulla. Tutkimuksen edetessä kehitettiin kokonaisvaltaiset toiminnanohjausjärjestelmän avulla toimivat eritasoiset kunnonvalvontahypoteesit, joiden avulla linjan yksittäisten komponenttien kuntoa voidaan valvoa. Lisäksi tutkimuksessa kehitettiin uusia kunnonvalvontasovelluksia, joita on mahdollista käyttää myös tehtaan muilla linjoilla. Kunnonvalvontahypoteeseja käytettiin kokonaisen kunnonvalvontajärjestelmän luomiseen. Tehtaalla on mahdollisuus valita käyttöönotettavan kunnonvalvonnan taso hypoteesien sekä kunnossapitostrategian avulla.

Modal testing and numerical modeling of the dynamic properties of layered sheet-steel structure

Recently, due to the increasing total construction and transportation cost and difficulties associated with handling massive structural components or assemblies, there has been increasing financial pressure to reduce structural weight. Furthermore, advances in material technology coupled with continuing advances in design tools and techniques have encouraged engineers to vary and combine materials, offering new opportunities to reduce the weight of mechanical structures. These new lower mass systems, however, are more susceptible to inherent imbalances, a weakness that can result in higher shock and harmonic resonances which leads to poor structural dynamic performances. The objective of this thesis is the modeling of layered sheet steel elements, to accurately predict dynamic performance. During the development of the layered sheet steel model, the numerical modeling approach, the Finite Element Analysis and the Experimental Modal Analysis are applied in building a modal model of the layered sheet steel elements. Furthermore, in view of getting a better understanding of the dynamic behavior of layered sheet steel, several binding methods have been studied to understand and demonstrate how a binding method affects the dynamic behavior of layered sheet steel elements when compared to single homogeneous steel plate. Based on the developed layered sheet steel model, the dynamic behavior of a lightweight wheel structure to be used as the structure for the stator of an outer rotor Direct-Drive Permanent Magnet Synchronous Generator designed for high-power wind turbines is studied.

Development of a Glycocluster Adjuvant Mimicking Natural Yeast beta-(1,2)-Structures

Allergy is characterized by T helper (Th) 2-type immune response after encounter with an allergen leading to subsequent immunoglobulin (Ig) E-mediated hypersensitivity reaction and further allergic inflammation. Allergen-specific immunotherapy (SIT) balances the Th2-biased immunity towards Th1 and T regulatory responses. Adjuvants are used in allergen preparations to intensify and modify SIT. β-(1,2)-oligomannoside constituents present in Candida albicans (C. albicans) cell wall possess Th1-type immunostimulatory properties. The aim of this thesis was to develop a β-(1,2)-linked carbohydrate compound with known structure and anti-allergic properties to be applied as an adjuvant in SIT. First the immunostimulatory properties of various fungal extracts were studied. C. albicans appeared to be the most promising Th1-inducing extract, which led to the synthesis of various mono- or divalent oligomannosides designed on the basis of C. albicans. These carbohydrates did not induce strong cytokine production in human peripheral blood mononuclear cell (PBMC) cultures. In contrast to earlier reports using native oligosaccharides from C. albicans, synthetic -(1,2)-linked mannotetraose did not induce any tumor necrosis factor production in murine macrophages. Next, similarities with synthesized divalent mannosides and the antigenic epitopes of β-(1,2)-linked C. albicans mannan were investigated. Two divalent compounds inhibited specific IgG antibodies binding to below 3 kDa hydrolyzed mannan down to the level of 30–50% showing similar antigenicity to C. albicans. Immunomodulatory properties of synthesized carbohydrate assemblies ranging from mono- to pentavalent were evaluated. A trivalent acetylated dimannose (TADM) induced interleukin-10 (IL-10) and interferon-γ responses. TADM also suppressed birch pollen induced IL-4 and IL-5 responses in allergen (Bet v) stimulated PBMCs of birch pollen allergic subjects. This suppression was stronger with TADM than with other used adjuvants, immunostimulatory oligonucleotides and monophosphoryl lipid A. In a murine model of asthma, the allergen induced inflammatory responses could also be suppressed by TADM on cytokine and antibody levels.

Yrityskohtainen ilmansuojelulaitteiden vaatimustenmukaisuuden arviointi SFS-EN 1090-1 periaatteiden mukaisesti

Rakennustuotteena käytettävien teräskokoonpanojen lisääntyneet CE- merkintävaatimukset ovat aiheuttaneet runsaasti hämmennystä alan toimijoiden keskuudessa. Tämän tutkimuksen tavoitteena oli selvittää teräskokoonpanojen SFS-EN 1090-1 standardin soveltamisala suhteessa konedirektiiviin sekä kartoittaa mahdollisen muutoksen aiheuttamat toimenpiteet ilmansuojelulaitetoimituksissa. Työssä tutustuttiin asetusten teoreettiseen viitekehykseen, standardeihin sekä alan kirjallisuuteen. Teoriaa sovellettiin tulevaisuudentutkimuksen Delfoi- menetelmään, jossa asiantuntijahaastatteluiden perusteella arvioitiin asetuksen soveltamisalaa ja vaatimuksia. Tutkimuksen avaintuloksia ovat standardin SFS-EN 1090-1 vaatimuksenmukaisuuteen vaikuttavien tekijöiden tunnistaminen ja niiden perusteella laaditut ehdotukset yritystoiminnan kehittämiseksi.

Influencing Perceived Agency: A Study into User Experiences in Digital Interactive Storytelling

User experience is a crucial element in interactive storytelling, and as such it is important to recognize the different aspects of a positive user experience in an interactive story. Towards that goal, in the first half of this thesis, we will go through the different elements that make up the user experience, with a strong focus on agency. Agency can be understood as the user’s ability to affect the story or the world in which the story is told with interesting and satisfying choices. The freedoms granted by agency are not completely compatible with traditional storytelling, and as such we will also go through some of the issues of agency-centric design philosophies and explore alternate schools of thought. The core purpose of this thesis is to determine the most important aspects of agency with regards to a positive user experience and attempt to find ways for authors to improve the overall quality of user experience in interactive stories. The latter half of this thesis deals with the research conducted on this matter. This research was carried out by analyzing data from an online survey coupled with data gathered by the interactive storytelling system specifically made for this research (Regicide). The most important aspects of this research deal with influencing perceived agency and facilitating an illusion of agency in different ways, and comparing user experiences in these different test environments. The most important findings based on this research include the importance of context-controlled and focused agency and settings in which the agency takes place and the importance of ensuring user-competency within an interactive storytelling system. Another essential conclusion to this research boils down to communication between the user and the system; the goal of influencing perceived agency should primarily be to ensure that the user is aware of all the theoretical agency they possess.

De novo transcriptome assembly and annotation of the isopod Idotea balthica

The Baltic Sea is a unique environment that contains unique genetic populations. In order to study these populations on a genetic level basic molecular research is needed. The aim of this thesis was to provide a basic genetic resource for population genomic studies by de novo assembling a transcriptome for the Baltic Sea isopod Idotea balthica. RNA was extracted from a whole single adult male isopod and sequenced using Illumina (125bp PE) RNA-Seq. The reads were preprocessed using FASTQC for quality control, TRIMMOMATIC for trimming, and RCORRECTOR for error correction. The preprocessed reads were then assembled with TRINITY, a de Bruijn graph-based assembler, using different k-mer sizes. The different assemblies were combined and clustered using CD-HIT. The assemblies were evaluated using TRANSRATE for quality and filtering, BUSCO for completeness, and TRANSDECODER for annotation potential. The 25-mer assembly was annotated using PANNZER (protein annotation with z-score) and BLASTX. The 25-mer assembly represents the best first draft assembly since it contains the most information. However, this assembly shows high levels of polymorphism, which currently cannot be differentiated as paralogs or allelic variants. Furthermore, this assembly is incomplete, which could be improved by sampling additional developmental stages.