Responses of yield and N use of spring sown crops to N fertilization, with special reference to the use of plant growth regulators

Selostus: Typpilannoituksen ja kasvunsääteiden vaikutukset kevätviljojen ja rypsin satoon sekä typen käyttöön

Timing applications of growth regulators to alter spring cereal development at high latitudes

Selostus: Aikaisen ja tavanomaisen kasvunsäädekäsittelyn vaikutus kevätviljojen kasvustoon ja satoon

Plant growth regulators to manipulate oat stands

Selostus: Kasvunsääteet vaikuttavat kauran kasvuston rakenteeseen ja tasoon

Identification of novel regulators for tumor progression in breast cancer

Breast cancer is the most frequent solid tumor among women and the leading cause of cancer related death in women worldwide. The prognosis of breast cancer patients is tightly correlated with the degree of spread beyond the primary tumor. In this thesis, the aim was to identify novel regulators of tumor progression in breast cancer as well as to get insights into the molecular mechanisms of breast cancer progression and metastasis. First, the role of phospholipid remodeling genes and enzymes important for breast cancer progression was studied in breast cancer samples as well as in cultured breast cancer cells. Tumor samples displayed increased de novo synthesized fatty acids especially in aggressive breast cancer. Furthermore, RNAi mediated cell based assays implicated several target genes critical for breast cancer cell proliferation and survival. Second, the role of arachidonic acid pathway members 15-hydroxyprostaglandin dehydrogenase (HPGD) and phospholipase A2 group VII (PLA2G7) in tumorigenesis associated processes was explored in metastatic breast cancer cells. Both targets were found to contribute to epithelial-mesenchymal transition related processes. Third, a high-throughput RNAi lysate microarray screen was utilized to identify novel vimentin expression regulating genes. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) was found to promote cellular features connected with metastatic disease, thus implicating MTHFD2 as a potential drug target to block breast cancer cell migration and invasion. Taken together, this study identified several putative targets for breast cancer therapy. In addition, these results provide novel information about the mechanisms and factors underlying breast cancer progression.

MicroRNAs as novel regulators of skeletal homeostasis

The human skeleton is composed of bone and cartilage. The differentiation of bone and cartilage cells from their bone marrow progenitors is regulated by an intrinsic network of intracellular and extracellular signaling molecules. In addition, cells coordinate their differentiation and function through reciprocal cell‐to‐cell interactions. MicroRNAs (miRNAs) are small, single‐stranded RNA molecules that inhibit protein translation by binding to messenger RNAs (mRNAs). Recent evidence demonstrates the involvement of miRNAs in multiple biological processes. However, their role in skeletal development and bone remodeling is still poorly understood. The aim of this thesis was to elucidate miRNA‐mediated gene regulation in bone and cartilage cells, namely in osteoblasts, osteoclasts, chondrocytes and bone marrow adipocytes. Comparison of miRNA expression during osteogenic and chondrogenic differentiation of bone marrow‐derived mesenchymal stem cells (MSCs) revealed several miRNAs with substantial difference between bone and cartilage cells. These miRNAs were predicted to target genes essentially involved in MSC differentiation. Three miRNAs, miR‐96, miR‐124 and miR‐199a, showed marked upregulation upon osteogenic, chondrogenic or adipogenic differentiation. Based on functional studies, these miRNAs regulate gene expression in MSCs and may thereby play a role in the commitment and/or differentiation of MSCs. Characterization of miRNA expression during osteoclastogenesis of mouse bone marrow cells revealed a unique expression pattern for several miRNAs. Potential targets of the differentially expressed miRNAs included many molecules essentially involved in osteoclast differentiation. These results provide novel insights into the expression and function of miRNAs during the differentiation of bone and cartilage cells. This information may be useful for the development of novel stem cell‐based treatments for skeletal defects and diseases.

Choosing the target loci : heat shock factors HSF1 and HSF2 as regulators of cell stress and development

Heat shock factors (HSFs) are an evolutionarily well conserved family of transcription factors that coordinate stress-induced gene expression and direct versatile physiological processes in eukaryote organisms. The essentiality of HSFs for cellular homeostasis has been well demonstrated, mainly through HSF1-induced transcription of heat shock protein (HSP) genes. HSFs are important regulators of many fundamental processes such as gametogenesis, metabolic control and aging, and are involved in pathological conditions including cancer progression and neurodegenerative diseases. In each of the HSF-mediated processes, however, the detailed mechanisms of HSF family members and their complete set of target genes have remained unknown. Recently, rapid advances in chromatin studies have enabled genome-wide characterization of protein binding sites in a high resolution and in an unbiased manner. In this PhD thesis, these novel methods that base on chromatin immunoprecipitation (ChIP) are utilized and the genome-wide target loci for HSF1 and HSF2 are identified in cellular stress responses and in developmental processes. The thesis and its original publications characterize the individual and shared target genes of HSF1 and HSF2, describe HSF1 as a potent transactivator, and discover HSF2 as an epigenetic regulator that coordinates gene expression throughout the cell cycle progression. In male gametogenesis, novel physiological functions for HSF1 and HSF2 are revealed and HSFs are demonstrated to control the expression of X- and Y-chromosomal multicopy genes in a silenced chromatin environment. In stressed human cells, HSF1 and HSF2 are shown to coordinate the expression of a wide variety of genes including genes for chaperone machinery, ubiquitin, regulators of cell cycle progression and signaling. These results highlight the importance of cell type and cell cycle phase in transcriptional responses, reveal the myriad of processes that are adjusted in a stressed cell and describe novel mechanisms that maintain transcriptional memory in mitotic cell division.

Filopodia and stromal extracellular matrix as regulators of cancer cell invasion and growth

Bidirectional exchange of information between the cancer cells and their environment is essential for cancer to evolve. Cancer cells lose the ability to regulate their growth, gain the ability to detach from neighboring cells and finally some of the cells disseminate from the primary tumor and invade to the adjacent tissue. During cancer progression, cells acquire features that promote cancer motility and proliferation one of them being increased filopodia number. Filopodia are dynamic actin-rich structures extending from the leading edge of migrating cells and the main function of these structures is to serve as environmental sensors. It is nowadays widely appreciated, that not only the cancer cells, but also the surrounding of the tumor – the tumor microenvironment- contribute to cancer cell dissemination and tumor growth. Activated stromal fibroblasts, also known as cancer-associated fibroblasts (CAFs) actively participate on tumor progression. CAFs are the most abundant cell type surrounding the cancer cells and they are the main cell type producing the extracellular matrix (ECM) within tumor stroma. CAFs secrete growth factors to promote tumor growth, direct cancer cell invasion as well as modify the stromal ECM architecture. The aim of this thesis was to investigate the function of filopodia, particularly the role of filopodia-inducing protein Myosin-X (Myo10), in breast cancer cell invasion and metastasis. We found that Myo10 is an important regulator of basal type breast cancer spreading downstream of mutant p53. In addition, I investigated the role of CAFs and their secreted matrix on tumor growth. According to the results, CAF-derived matrix has altered organization and stiffness which induces the carcinoma cell proliferation via epigenetic mechanisms. I identified histone demethylase enzyme JMJD1a to be regulated by the stiffness and to participate in stiffness induced growth control.

Sähkönjakeluverkon ylläpitopalveluiden ulkoistamisella saavutettavat hyödyt

Sähkönsiirto jakeluverkoissa on alueellista monopoliliiketoimintaa. Tätä monopoliliiketoimintaa säätelee Suomessa energiamarkkinavirasto. Siirtoliiketoiminnalle on asetettu kohtuullisen tuoton rajoitukset siten, että liiketoiminnan täytyy olla myös tehokkaasti toteutettua. Sähköverkkojen ylläpitopalveluiden toteuttamiseksi on markkinoille syntynyt useita näiden palveluiden tuottamiseen erikoistuneita palveluyrityksiä. Ulkoistamalla ylläpitopalvelut ulkopuoliselle palveluyritykselle sähköverkkoyhtiöt pyrkivät hakemaan toiminnalleen tehokkuutta, kustannussäästöjä ja toimintojen selkeytymistä. Yhtiön johto voi näin keskittyä toiminnan ja tuloksen kehittämisen kannalta olennaisiin asioihin. Tässä työssä on selvitetty sähköverkkojen ylläpitotoimintojen ulkoistamisella saavutettavissa olevia hyötyjä. Hyötyjen toteutumista on tutkittu Eltel Networks Pohjoinen Oy:n ja sen asiakasyritysten Kainuun Sähkö Oyj:n, Kajaanin Lämpö Oy:n ja Kajaanin Puhelinosuuskunnan asiakastapauksissa.

Pankkien vakavaraisuussäännöstön uudistus

Tutkielman tavoitteena on kuvata pankkien vakavaraisuusuudistuksen eri osa-alueita. Tarkempi analyysi rajautuu uudistuksen tuomiin muutoksiin luotto- ja operatiivisen riskin pääomavaateissa. Tutkielman empiirisen osuuden tavoitteena on perehtyä vakavaraisuussäännöstön uudistusten vaikutuksiin Nordeassa. Tutkimusmetodologiaksi on valittu normatiivinen tutkimusote. Lisäksi tutkielma sisältää deskriptiivisiä ja positivistisia osia. Lähdeaineisto koostuu Baselin pankkivalvontakomitean ja Suomen Pankin julkaisemista tutkimuksista ja dokumenteista sekä alan julkaisuissa ilmestyneistä artikkeleista. Pankkien vakavaraisuussäännöstöuudistuksen tavoitteena on lisätä rahoitusmarkkinoiden vakautta. Sääntelyn kautta pyritään turvaamaan pankkien varojen riittävyys suhteessa niiden riskien ottoon. Vakavaraisuussäännöstön uudistus muodostuu kolmesta pilarista: (1) minimipääomavaatimuksista, (2) pankkivalvonnan vahvistamisesta ja (3) markkinakurin hyödyntämisestä luottolaitosten toiminnan julkistamisvaatimuksia lisäämällä. Pankkivalvonnan harmonisoinnista vallitsee kansainvälinen yhteisymmärrys, mutta ennen kuin Basel II voi astua voimaan on useita ongelmia ratkaisematta. Baselin vakavaraisuuskehikko ei ole ainut lähitulevaisuudessa pankkitoimialaa koetteleva uudistus. Kansainväliset tilinpäätösstandardit; International Accounting Standards ja erityisesti IAS 39 sekä International Financial Reporting Standards, lyhyemmin IFRS tulevat muuttamaan merkittävästi pankkien tilinpäätöskäyttäytymistä. Epäselvää on vielä kuitenkin tukevatko uudistukset toisiaan ja missä määrin pankkien tulosvolatiliteetin odotetaan kasvavan. Tutkielmassa pohditaan vakavaraisuussäännöstön uudistuksen hyötyjä kansainvälisen kilpailuneutraliteetin osalta, sillä Yhdysvalloissa uudistus koskee vain suurimpia pankkeja. Tutkielmassa paneudutaan lisäksi uudistuksen mahdolliseen talouden syklejä voimistavaan vaikutukseen ja tarkastellaan parannusehdotuksia prosyklisyyden hillitsemiseksi. Yksi vakavaraisuusuudistuksen tärkeimmistä tehtävistä on luoda pankeille kannustin kehittää omia riskienhallinta malleja. Kannustin ongelma on pyritty ratkaisemaan vapaampien sisäisten mallien menetelmien avulla. Ongelmaa ei ole pystytty kuitenkaan ratkaisemaan aivan täysin, sillä luottoriskien osalta pankkien lainaportfolioiden rakenne määrittää sen, hyötyvätkö pankit siirtymisestä sisäisten mallien menetelmän käyttöön. Tutkielma sisältää myös Nordean arvion vakavaraisuusuudistuksen vaikutuksista pankkitoimialaan.

Pankkien tuloksenjärjestelymahdollisuudet siirryttäessä IFRS-normistoon

Tutkielman ensisijaisena tavoitteena oli määrittää pankkien tuloksenjärjestelymahdollisuuksien muutoksia siirryttäessä kansallisesta FAS GAAP -tilinpäätösnormistosta IFRS-normistoon. Tuloksenjärjestelyn ilmiötä käsiteltiin esittelemällä useita koti- ja ulkomaisia tuloksenjärjestelytutkimuksia. Pankeilla on toimialalleen erityisiä kannustimia tuloksenjärjestelyyn. Pankkitoimialalla tuloksenjärjestely on perinteisesti esiintynyt tuloksentasaamisena tilikausien välillä. Tutkimuksessa saatiin selkeä kuva tuloksenjärjestelymahdollisuuksien muutoksesta. IFRS-normiston sisältämä johdon harkintavalta ilmenee tilinpäätöksen laadintaperiaatteiden valinnaisuutena ja standardien laskelmien sisältäminä johdon arvioina ja oletuksina. Harkintavaltaa käyttämällä johto voi vaikuttaa tilinpäätöksen taseeseen ja tulokseen. IFRS-normistossa tilinpäätöksen laadintaperiaatteisiin liittyvä johdon harkintavalta on vähentynyt verrattuna kansalliseen FAS GAAP-tilinpäätöskäytäntöön, mutta IFRS jättää johdolle edelleen harkintavaltaa muutamien kirjaus- ja raportointikäytäntöjen suhteen. Perinteisten tuloksenjärjestelykeinojen rinnalle IFRS:ssä nousevat useiden standardien laskelmien sisältämät johdon arviot ja oletukset tulevaisuuden muuttujista. Omaisuuserien käypään arvoon arvostamisen myötä tilikauden tuloksista saattaa tulla aikaisempaa volatiilimpia. Käypiin arvoihin arvostamisen seurauksena pankit saattavat siirtyä käyttämään sijoitustensa ostoja ja myyntejä tuloksentasauskeinona. FAS GAAP:ssa pankit ovat käyttäneet tuloksentasaamiseen tiettyjä suoriteperusteisia eriä, esimerkiksi luottotappiovarauksia. IFRS tiukentaa varausten tekemisen periaatteita, mutta tuo tilalle lainakannan arvonalentumistestaukseen sisältyvät johdon arviot.

Mitotic regulation by Polo-like kinase 1 and the Chromosomal Passenger Complex

During mitosis, the duplicated genome must be accurately divided between two daughter cells. Polo-like kinase 1 (Plk1) and Aurora B kinase, together with its binding partners Incenp, Survivin and Borealin (chromosomal passenger complex, CPC), have key roles in coordinating mitotic events. The accuracy of cell division is safeguarded by a signaling cascade termed the mitotic spindle checkpoint (SC), which ensures that chromosomes are not physically separated before correct bipolar attachments have been formed between kinetochores and spindle microtubules (MT). An inhibitory “wait anaphase” signal, which delays chromosome separation (anaphase onset), is created at individual kinetochores and broadcasted throughout the cell in response to lack of kinetochore-microtubule (kMT) attachment or proper interkinetochore tension. It is believed that the fast turnover of SC molecules at kinetochores contributes to the cell’s ability to produce this signal and enables rapid responses to changing cellular conditions. Kinetochores that lack MT attachment and tension express a certain phosphoepitope called the 3F3/2 phosphoepitope, which has been linked to SC signaling. In the experimental part, we investigated the regulation of the 3F3/2 phosphoepitope, analyzed whether CPC molecules turn over at centromeres, and dissected the mitotic roles of the CPC using a microinjection technique that allowed precise temporal control over its function. We found that the kinetochore 3F3/2 phosphoepitope is created by Plk1, and that CPC proteins exhibit constant exchange at centromeres. Moreover, we found that CPC function is necessary in the regulation of chromatid movements and spindle morphology in anaphase. In summary, we identified new functions of key mitotic regulators Plk1 and CPC, and provided insighs into the coordination of mitotic events.

Negative Regulation of Receptor Tyrosine Kinases by T-cell Protein Tyrosine Phosphatase

Protein tyrosine phosphorylation controls a wide array of cellular responses such as growth, migration, proliferation, differentiation, metabolism and cytoskeletal organisation. Tyrosine phosphorylation is a dynamic process involving the competing activities of protein tyrosine kinases and protein tyrosine phosphatases. The protein tyrosine kinases are further divided into non-receptor- and receptor tyrosine kinases. The latter are transmembrane glycoproteins activated by the binding of specific ligands, mostly growth factors, to their extracellular domain, transmitting different signals to the cell. Growth factor receptors such as the epidermal growth factor receptor, vascular endothelial growth factor receptor 2 and platelet-derived growth factor receptor β, belong to the receptor tyrosine kinases, the signalling of which is often disturbed in various diseases, including cancer. This has led to the development of receptor tyrosine kinase antagonists for use as anti-cancer drugs. As the receptor tyrosine kinases, also the protein tyrosine phosphatases can be divided into receptor- and non-receptor types. The protein tyrosine phosphatases have attained much less attention than the receptor tyrosine kinases partly because they were identified later. However, accumulating evidence shows that the protein tyrosine phosphatases have important roles as specific and active regulators of tyrosine phosphorylation in cells and of physiological processes. Consequently, the protein tyrosine phosphatases are receiving arising interest as novel drug targets. The aim of this work was to elucidate the negative regulation of receptor tyrosine kinases by one non-receptor protein tyrosine phosphatase, T-cell protein tyrosine phosphatase TCPTP. The results show that TCPTP activated by cell adhesion receptor integrin α1 functions as a negative regulator of the epidermal growth factor receptor. It was also found that TCPTP affects vascular endothelial growth factor receptor 2 signalling and angiogenesis. Lastly, a High-throughput screen with 64,280 compounds was performed to identify novel TCPTP activators, resulting in identification of one small molecule compound capable of exerting similar effects on TCPTP signalling as integrin α1. This compound is shown to downregulate signalling of epidermal growth factor receptor and platelet-derived growth factor receptor β, as well as to inhibit cell proliferation and angiogenesis. Our results suggest that a suitable small-molecule TCPTP activator could be utilized in the development of novel anti-cancer drugs.

β1 integrin regulation

Integrins are heterodimeric adhesion receptors mediating adhesion to extracellular matrix proteins and to other cells. Integrins are important in embryonic development, structural integrity of connective tissue, blood thrombus formation, and immune defense system. Integrins are transmembrane proteins whose ligand binding capacity (activity) is regulated by large conformational changes. Extracellular ligand binding or intracellular effector binding to integrin cytoplasmic face regulate integrin activity. Integrins are thus able to mediate bi-directional signaling. Integrin function is also regulated by intracellular location. Integrins are constantly recycled from endocytic vesicles to plasma membrane, and this has been shown to be important for cell migration and invasion as well. Deregulation of integrin functionality can lead to deleterious illnesses, such as bleeding or inflammatory disorders. It is also evident that integrin deregulation is associated with cancer progression. In this study, a novel Beta1 integrin associating protein, Rab21, was characterized. Rab21 binding to integrin cytoplasmic tail was shown to be important for Beta1 integrin endo- and exocytosis – intracellular trafficking. It was furher shown that this interaction has an important role in cell adhesion, migration, as well as in the final step of cell division, cytokinesis. This work showed that abrogation of Rab21 function or β1 integrin endocytic traffic, can lead to defects in cell division and results in formation of multinucleated cells. Multinucleation and especially tetraploidy can be a transient pathway to aneuploidy and tumorigenesis. This work characterized chromosomal deletions in rab21 locus in ovarian and prostate cancer samples and showed that a cell line with rab21 deletion also had impairment in cell division, which could be rescued by Rab21 re-expression. The work demonstrates an important role for Rab21 and Beta1 integrin traffic regulation in cell adhesion and division, and suggests a probable associaton with tumorigenesis. In this study, Beta1 integrin activity regulation was also addressed. A novel cell array platform for genome-scale RNAi screenings was characterized here. More than 4500 genes were knocked-down in prostate cancer cells using siRNA-mediated silencing. The effects on Beta1 integrin activity were analyzed upon knock-downs. The screen identified more that 400 putative regulators of Beta1 integrin activity in prostate cancer. In conclusion, this work will help us to understand complex regulatory pathways involved in cancer cell adhesion and migration.

New mechanisms regulating human Th1 and Th2 cell differentiation

Selective development of human T helper (Th) cells into functionally distinct Th1 and Th2 subtypes plays an essential role in the host immune response towards pathogens. However, abnormal function or differentiation of these cells can lead to development of various autoimmune diseases as well as asthma and allergy. Therefore, identification of key factors and the molecular mechanisms mediating Th1 and Th2 cell differentiation is important for understanding the molecular mechanisms of these diseases. The goal of this study was to identify novel factors involved in the regulation of Th1 and Th2 differentiation processes. A new method was optimized for enrichment of transiently transfected resting human primary T lymphocytes, that allowed the study of the influence of genes of interest in human Th1/Th2 cell differentiation and other primary Th cell functions. Functional characterization of PRELI, a novel activation-induced protein in human Th cells, identified it as a mitochondrial protein involved in the regulation of Th cell differentiation and apoptosis. By influencing the intracellular redox state, PRELI induces mitochondrial apoptosis pathway and downregulates STAT6 and Th2 differentiation. The data suggested that Calpain, an oxidative stress induced cysteine protease, is involved as a mediator in PRELI-induced downregulation of STAT6. PIM serine/threonine-specific kinases were identified as new regulators of human Th1 cell differentiation. PIM1 and PIM2 kinases were shown to be preferentially expressed in Th1 cells as compared to Th2 cells. RNA interference studies showed that PIM kinases enhance the production of IFN, the hallmark cytokine produced by Th1 cells. They also induce the expression of the key Th1-driving factor T-bet and the IL-12 signaling pathway during early phases of Th1 cell differentiation. Taken together, new regulators of human T helper cell differentiation were identified in this study, which provides new insights into the signaling mechanisms controlling the selective activation of human Th cell subsets.