Novel Proteins Involved in Dynamics of The Photosynthetic Apparatus

During the past few years, a considerable number of research articles have been published relating to the structure and function of the major photosynthetic protein complexes, photosystem (PS) I, PSII, cytochrome (Cyt) b6f, and adenosine triphosphate (ATP) synthase. Sequencing of the Arabidopsis thaliana (Arabidopsis) genome together with several high-quality proteomics studies has, however, revealed that the thylakoid membrane network of plant chloroplasts still contains a number of functionally unknown proteins. These proteins may have a role as auxiliary proteins guiding the assembly, maintenance, and turnover of the thylakoid protein complexes, or they may be as yet unknown subunits of the photosynthetic complexes. Novel subunits are most likely to be found in the NAD(P)H dehydrogenase (NDH) complex, the structure and function of which have remained obscure in the absence of detailed crystallographic data, thus making this thylakoid protein complex a particularly interesting target of investigation. In this thesis, several novel thylakoid-associated proteins were identified by proteomics-based methods. The major goal of characterization of the stroma thylakoid associated polysome-nascent chain complexes was to determine the proteins that guide the dynamic life cycle of PSII. In addition, a large protein complex of ≥ 1,000 kDa, residing in the stroma thylakoid, was characterized in greater depth and it was found to be a supercomplex composed of the PSI and NDH complexes. A set of newly identified proteins from Arabidopsis thylakoids was subjected to detailed characterization using the reverse genetics approach and extensive biochemical and biophysical analysis. The role of the novel proteins, either as auxiliary proteins or subunits of the photosynthetic protein complexes, was revealed. Two novel thylakoid lumen proteins, TLP18.3 and AtCYP38, function as auxiliary proteins assisting specific steps of the assembly/repair of PSII. The role of the 10-kDa thylakoid lumen protein PsbR is related to the optimization of oxygen evolution of PSII by assisting the assembly of the PsbP protein. Two integral thylakoid membrane proteins, NDH45 and NDH48, are novel subunits of the chloroplast NDH complex. Finally, the thylakoid lumen immunophilin AtCYP20-2 is suggested to interact with the NDH complex, instead of PSII as was hypothesized earlier.

Height distributions of Scots pine sapling stands affected by retained tree and edge stand competition

Abstract

The regulation and function of collagenase-3 (MMP-13) in cutaneous wound healing and squamous cell carcinoma

Matrix metalloproteinase-13 (MMP-13) is a potent proteolytic enzyme, whose expression has been previously associated with fetal bone development and postnatal bone remodeling and with adult gingival wound healing. MMP-13 is also known to be involved in the growth and invasion of various cancers including squamous cell carcinoma (SCC) of the skin. The aim of this study was to further elucidate the function and regulation of MMP-13 in wound repair and cancer. In this study, it was shown that fetal skin fibroblasts express MMP-13 in response to transforming growth factor-β in a p38 MAP kinase dependent manner. In addition, MMP-13 was found to be expressed in vivo by wound fibroblasts in human fetal skin grafted on SCID mice. Adenovirally delivered expression of MMP-13 enhanced collagen matrix contraction by fibroblasts in vitro in association with altered cytoskeletal structure, enhanced proliferation and survival. These results indicate that MMP-13 is involved in cell-mediated collagen matrix remodeling and suggest a role for MMP-13 in superior matrix remodeling and scarless healing of fetal skin wounds. Using an MMP-13 deficient mouse strain, it was shown that MMP-13 is essential for the normal development of experimental granulation tissue in mice. MMP-13 was implicated in the regulation of myofibroblast function and angiogenesis and the expression of genes involved in cellular proliferation and movement, immune response, angiogenesis and proteolysis. Finally, epidermal mitogen, keratinocyte growth factor (KGF) was shown to suppress the malignant properties of skin SCC cells by downregulating the expression of several target genes with potential cancer promoting properties, including MMP-13, and by reducing SCC cell invasion. These results provide evidence that MMP-13 potently regulates cell viability, myofibroblast function and angiogenesis associated with wound healing and cancer. In addition, fibroblasts expressing MMP-13 show high collagen reorganization capacity. Moreover, the results suggest that KGF mediates the anti-cancer effects on skin SCC

Therapeutic Properties of Superoxide Dismutase 3 and Mesenchymal Stromal Cells in Peripheral Ischemia

The aim of this study was to characterize the cellular mechanisms leading to the beneficial effect of anti-oxidative gene therapy and pro-angiogenic stem cell therapy in acute peripheral ischemia. Post-ischemic events aim to re-establish tissue blood perfusion, to clear cellular debris, and to regenerate lost tissue by differentiation of satellite cells into myoblasts. Although leukocytes have an essential role in clearing cellular debris and promoting angiogenesis, they also contribute to tissue injury through excessive ROS production. First, we investigated the therapeutic properties of extracellular superoxide dismutase (SOD3) gene transfer. SOD3 was shown to reduce oxidative stress, to normalize glucose metabolism, and to enhance cell proliferation in the ischemic muscle. Analysis of the mitogenic Ras-Erk1/2 pathway showed SOD3 mediated induction offering a plausible explanation for enhanced cell proliferation. In addition, SOD3 reduced NF-κB activity by enhancing IκBα expression thus leading to reduced expression of inflammatory cytokines and adhesion molecules with consequent reduction in macrophage infiltration. Secondly, we sought to determine the fate and the effect of locally transplanted mesenchymal stem/stromal cells (MSCs) in acute ischemia. We showed that a vast majority of the transplanted cells are cleared from the injury site within 24 hours after local transplantation. Despite rapid clearance, transplantation was able to temporarily promote angiogenesis and cell proliferation in the muscle. Lack of graft-derived growth factor expression suggests other than secretory function to mediate this observed effect. In conclusion, both SOD3 and MSCs could be utilized to alleviate peripheral ischemia induced tissue injury. We have described a previously unidentified growth regulatory role for SOD3, and suggest a novel mechanism whereby transplanted MSCs enhance the reparative potential of the recipient tissue through physical contacts.

Superoxide dismutase 3-mediated cell survival and proliferation

This dissertation studies the signaling events mediated by the extracellular superoxide dismutase (SOD3). SOD3 is an antioxidant enzyme which converts the harmful superoxide into hydrogen peroxide. Overproduction of these reactive oxygen species (ROS) in the cellular environment as a result of tissue injury or impaired antioxidant defense system has detrimental effects on tissue integrity and function. However, especially hydrogen peroxide is also an important signaling agent. Ischemic injury in muscle causes acute oxidative stress and inflammation. We investigated the ability of SOD3 to attenuate ischemia induced inflammation and to promote recovery of skeletal muscle tissue. We found that SOD3 can downregulate the expression of several inflammatory cytokines and cell adhesion molecules thus preventing the accumulation of oxidant-producing inflammatory cells. Secondly, SOD3 was able to promote long-term activation of the mitogenic Erk pathway, but increased only briefly the activity of pro-survival Akt pathway at an early stage of ischemic inflammation, thus reducing apoptosis. SOD3 is a prominent antioxidant in the thyroid gland where oxidative stress is constantly present. We investigated the role of SOD3 in normal thyroid follicular cells and the changes in its expression in various hyperproliferative disorders. We first showed that SOD3 is TSH-responsive which indicated its participation in thyroid function. Its principal function seems to be in follicular cell proliferation since knockdown cells were deficient in proliferation. Additionally, it was overexpressed in goiter tissue. However, SOD3 was consistently downregulated in thyroid cancer cell lines and tissues. In conclusion, SOD3 is involved in tissue maintenance, cell proliferation and inflammatory cell migration. Its mechanisms of action are the activation of known proliferation/survival pathways, inhibition of apoptosis and regulation of adhesion molecule expression.

Yliopiston reunalla. Tutkimus suomalaisen avoimen yliopiston muotoutumisesta

Avoin yliopisto on koulutusmuoto, johon kuka tahansa voi osallistua ja jossa voi suorittaa yliopistotasoisia opintoja. Avoimen yliopiston ideassa keskeistä on koulutuksellisen tasa-arvon edistämisen tavoite. Tämän tutkimuksen tehtävänä on kuvata ja analysoida suomalaisen avoimen yliopiston muotoutumista. Tutkimuksella etsitään vastauksia kolmeen kysymykseen: 1. Millaisten vaiheiden kautta avoin yliopisto on muotoutunut? Mitä avoimen yliopiston historiassa on tapahtunut ja millaista keskustelua näistä tapahtumista on käyty? 2. Millaisia diskursseja avointa yliopistoa koskeneessa keskustelussa voidaan tunnistaa ja miten eri toimijatahot ovat näihin puhetapoihin kiinnittyneet? 3. Millaisena koulutuksellisen tasa-arvon toteuttajana avoin yliopisto näyttäytyy tutkimusaineiston valossa? Tutkimuksen aineisto koostuu erilaisista julkisista teksteistä. Aineistossa on mukana useita erilaisia tekstityyppejä: komiteanmietintöjä ja työryhmäraportteja, korkeakoululaitoksen kehittämissuunnitelmia, muita suunnitteluasiakirjoja, tutkimuksia, selvityksiä, puheenvuoroja ja esitelmiä sekä lehtikirjoituksia. Täydentävänä aineistona on lisäksi käytetty tilastoja. Tutkimusaineistoa on analysoitu diskurssianalyysillä. Keskeisenä lähtökohtana analyysissa on, että tekstien avulla tuotetaan avointa yliopistoa koskevia käsityksiä ja merkityksiä. Nämä merkitykset myös muuttuvat ajassa. Aineiston analyysin tuloksena avoimen yliopiston historiassa voidaan erottaa erilaisia vaiheita ja näiden vaiheiden välisiä taitekohtia. Ensimmäiseksi murrokseksi avoimen yliopiston historiassa voidaan paikantaa avoimen yliopiston synty, joka ajoittui 1970-luvun alkupuoliskolle. Avoin yliopisto sai vakiintuneet puitteensa vasta 1980-luvun puolivälissä, jolloin se organisoitiin osaksi yliopistojen täydennyskoulutusta. Tämä voidaan nähdä avoimen yliopiston historian toisena murroksena. Kolmas murros ajoittui 1990-luvulle, jolloin avoimen yliopiston resursointi muuttui ja nuoret tulivat sen näkyväksi opiskelijaryhmäksi. Tämä murros problematisoi avoimen yliopiston ja tutkintokoulutuksen suhteen aiemmasta poikkeavalla tavalla ja avoimen yliopiston tutkintoväylä nousi keskeiseksi keskusteluteemaksi. Tämä jännite purkautui tultaessa 2000-luvulle, ja tutkinnonuudistuksen yhteydessä avoimen yliopiston väylä sai paikkansa suhteessa kahden syklin tutkintoihin. Nyt elämme tutkinnonuudistuksen jälkeistä aikaa, jolloin avoimen yliopiston väylä vertautuu paljolti maisterikoulutuksiin. Kysymys aikuisten asemasta suhteessa tutkintokoulutukseen on kuitenkin edelleen ajankohtainen. Esillä ovat etenkin kysymykset aiemmin opitun tunnustamisesta, aikuisten ohjauksesta sekä avoimen yliopiston suhteesta työ- ja elinkeinoelämään. Keskustelussa avoimesta yliopistosta on paikannettavissa erilaisia positioita, jotka määrittävät avoimen yliopiston merkitystä ja tehtävää. Näitä positioita voidaan nimittää diskursseiksi, jotka konstituoivat avoimen yliopiston paikkaa yliopistokoulutuksen kentällä. Aineistosta on paikannettu neljä eri diskurssia: (1) akateemisia arvoja painottava yliopistollinen diskurssi, (2) osallistumisen tasa-arvoa korostava sivistyksellisen demokratian diskurssi, (3) yksilöllisiä mahdollisuuksia ja innovatiivisuutta korostava joustavuuden diskurssi sekä (4) työelämää, taloudellisuutta ja statusta korostava tehokkuuden diskurssi. Nämä diskurssit käyvät aineiston teksteissä keskinäisiä neuvotteluja ja kantavat merkityksiä suhteessa toisiinsa. Diskurssien välisiä suhteita voidaan kuvata kahden eri dimension kautta. Yhtäältä vastakkaisiksi arvoiksi asettuvat akateeminen eksklusiivisuus ja koulutuksellinen tasa-arvo. Toisena ulottuvuutena on koulutuksen arvottaminen sivistyksen versus hyödyn näkökulmasta. Avoimen yliopiston tehtävä tasa-arvon edistäjänä on eri aikoina mielletty eri tavoin. Avoimen yliopiston historiassa sen merkitystä ja tehtävää on kehystetty erilaisin puhetavoin, ja eri diskurssipositioiden vuoropuhelun kautta myös avoimen yliopiston tasa-arvotehtävästä on eri aikoina keskusteltu eri tavoin. Avoimen yliopiston alkuvaiheessa sen tehtävänä näyttäytyi sivistyksellisen demokratian turvaajana toimiminen. Kun avoimen yliopiston kurssit käynnistyivät, määrittyi toiminta selkeästi aikuisten koulutukseksi. Avoin yliopisto määrittyikin nyt aikuisten toiseksi mahdollisuudeksi hankkia koulutusta, jota vaille he olivat nuorempina jääneet. Avoimen yliopiston puitteiden lukkoonlyömisen jälkeen keskustelussa nousi vahvasti esiin toiminnan ja opetusmuotojen kehittäminen. Avoin yliopisto määrittyikin nyt aikuisten monipuoliseksi ja joustavaksi koulutusmahdollisuudeksi. Tasa-arvoisten mahdollisuuksien luominen näyttäytyi innovatiivisena, dynaamisena ja eteenpäinpyrkivänä toimintana, jossa otettiin huomioon aikuisten erilaiset tarpeet. Relander-ohjelman myötä avoimen yliopiston julkilausuttu tehtävä nimenomaan aikuisten kouluttajana kuitenkin muuttui. Avoin yliopisto näyttäytyi nyt yksilöllisiä tarpeita palvelevana mahdollisuuksien talona, jossa oli sijaa kaikille. Tärkeäksi määrittyi myös opiskelun tavoitteiden ja motiivien moninaisuus. Tutkinnon suorittamisen avoimen yliopiston opintojen kautta tuli olla realistisesti mahdollista. Viimeisimmän murroksen jälkeen avoin yliopisto määrittyy yhä selvemmin työelämän sekä alueellisten tarpeiden kautta. Avoin yliopisto näyttäytyy joustavana ja erilaisia tarpeita palvelevana opiskelufoorumina. Avoin yliopisto palvelee paitsi yksilöiden, myös työelämän ja yritysten tarpeita sekä on osaltaan turvaamassa alueiden kilpailukykyä ja elinvoimaisuutta. Tässä tutkimuksessa tarkastellaan avointa yliopistoa, sen historiaa ja siitä käytyä keskustelua erityisesti tasa-arvon näkökulmasta. Yhtenä keskeisenä tuloksena on, että avoimen yliopiston paikka on ollut aina jollakin tavalla marginaalissa. Tätä ilmentää mm. yliopistollisuuden ja tasa-arvon välinen jännite, jonka ympärille avointa yliopistoa koskeva keskustelu paljolti on järjestynyt. Ylipäätään aikuisten asemaa yliopistossa määrittää tietty epämukavuus ja täyden legitimiteetin puute erityisesti suhteessa tutkintokoulutukseen. Aikuisten koulutuksesta puhutaan yliopiston yhtenä perustehtävänä ja retorisesti voidaan todeta tämän tehtävän tärkeys. Aikuinen opiskelija aiempine osaamisineen ja osaamistarpeineen positioituu kuitenkin yliopistokoulutuksen ja työelämän väliselle rajapinnalle. Aikuisen paikka määrittyykin yliopiston ytimeen nähden selvästi marginaaliin.

The Structure and Function of αI Domains in Collagen Receptor and Leukocyte Integrins

Integrins are heterodimeric, signaling transmembrane adhesion receptors that connect the intracellular actin microfilaments to the extracellular matrix composed of collagens and other matrix molecules. Bidirectional signaling is mediated via drastic conformational changes in integrins. These changes also occur in the integrin αI domains, which are responsible for ligand binding by collagen receptor and leukocyte specific integrins. Like intact integrins, soluble αI domains exist in the closed, low affinity form and in the open, high affinity form, and so it is possible to use isolated αI domains to study the factors and mechanisms involved in integrin activation/deactivation. Integrins are found in all mammalian tissues and cells, where they play crucial roles in growth, migration, defense mechanisms and apoptosis. Integrins are involved in many human diseases, such as inflammatory, cardiovascular and metastatic diseases, and so plenty of effort has been invested into developing integrin specific drugs. Humans have 24 different integrins, four of which are collagen receptor (α1β1, α2β1, α10β1, α11β1) and five leukocyte specific integrins (αLβ2, αMβ2, αXβ2, αDβ2, αEβ7). These two integrin groups are quite unselective having both primary and secondary ligands. This work presents the first systematic studies performed on these integrin groups to find out how integrin activation affects ligand binding and selectivity. These kinds of studies are important not only for understanding the partially overlapping functions of integrins, but also for drug development. In general, our results indicated that selectivity in ligand recognition is greatly reduced upon integrin activation. Interestingly, in some cases the ligand binding properties of integrins have been shown to be cell type specific. The reason for this is not known, but our observations suggest that cell types with a higher integrin activation state have lower ligand selectivity, and vice versa. Furthermore, we solved the three-dimensional structure for the activated form of the collagen receptor α1I domain. This structure revealed a novel intermediate conformation not previously seen with any other integrin αI domain. This is the first 3D structure for an activated collagen receptor αI domain without ligand. Based on the differences between the open and closed conformation of the αI domain we set structural criteria for a search for effective collagen receptor drugs. By docking a large number of molecules into the closed conformation of the α2I domain we discovered two polyketides, which best fulfilled the set structural criteria, and by cell adhesion studies we showed them to be specific inhibitors of the collagen receptor integrins.

Regulation of HSF2 and its function in mitosis

The cell is continuously subjected to various forms of external and intrinsic proteindamaging stresses, including hyperthermia, pathophysiological states, as well as cell differentiation and proliferation. Proteindamaging stresses result in denaturation and improper folding of proteins, leading to the formation of toxic aggregates that are detrimental for various pathological conditions, including Alzheimer’s and Huntington’s diseases. In order to maintain protein homeostasis, cells have developed different cytoprotective mechanisms, one of which is the evolutionary well-conserved heat shock response. The heat shock response results in the expression of heat shock proteins (Hsps), which act as molecular chaperones that bind to misfolded proteins, facilitate their refolding and prevent the formation of protein aggregates. Stress-induced expression of Hsps is mediated by a family of transcription factors, the heat shock factors, HSFs. Of the four HSFs found in vertebrates, HSF1-4, HSF1 is the major stress-responsive factor that is required for the induction of the heat shock response. HSF2 cannot alone induce Hsps, but modulates the heat shock response by forming heterotrimers with HSF1. HSFs are not only involved in the heat shock response, but they have also been found to have a function in development, neurodegenerative disorders, cancer, and longevity. Therefore, insight into how HSFs are regulated is important for the understanding of both normal physiological and disease processes. The activity of HSF1 is mainly regulated by intricate post-translational modifications, whereas the activity of HSF2 is concentrationdependent. However, there is only limited understanding of how the abundance of HSF2 is regulated. This study describes two different means of how HSF2 levels are regulated. In the first study it was shown that microRNA miR-18, a member of the miR-17~92 cluster, directly regulates Hsf2 mRNA stability and thus protein levels. HSF2 has earlier been shown to play a profound role in the regulation of male germ cell maturation during the spermatogenesis. The effect on miR-18 on HSF2 was examined in vivo by transfecting intact seminiferous tubules, and it was found that inhibition of miR-18 resulted in increased HSF2 levels and modified expression of the HSF2 targets Ssty2 and Speer4a. HSF2 has earlier been reported to modulate the heat shock response by forming heterotrimers with HSF1. In the second study, it was shown that HSF2 is cleared off the Hsp70 promoter and degraded by the ubiquitinproteasome pathway upon acute stress. By silencing components of the anaphase promoting complex/cyclosome (APC/C), including the co-activators Cdc20 and Cdh1, it was shown that APC/C mediates the heatinduced ubiquitylation of HSF2. Furthermore, down-regulation of Cdc20 was shown to alter the expression of heat shock-responsive genes. Next, we studied if APC/C-Cdc20, which controls cell cycle progression, also regulates HSF2 during the cell cycle. We found that both HSF2 mRNA and protein levels decreased during mitosis in several but not all human cell lines, indicating that HSF2 has a function in mitotic cells. Interestingly, although transcription is globally repressed during mitosis, mainly due to the displacement of RNA polymerase II and transcription factors, including HSF1, from the mitotic chromatin, HSF2 is capable of binding DNA during mitosis. Thus, during mitosis the heat shock response is impaired, leaving mitotic cells vulnerable to proteotoxic stress. However, in HSF2-deficient mitotic cells the Hsp70 promoter is accessible to both HSF1 and RNA polymerase II, allowing for stress-inducible Hsp expression to occur. As a consequence HSF2-deficient mitotic cells have a survival advantage upon acute heat stress. The results, presented in this thesis contribute to the understanding of the regulatory mechanisms of HSF2 and its function in the heat shock response in both interphase and mitotic cells.