VEGF-C/VEGFR-3 and PDGF-B/PDGFR-b pathways in embryonic lymphangiogenesis

The circulatory system comprises the blood vascular system and the lymphatic vascular system. These two systems function in parallel. Blood vessels form a closed system that delivers oxygen and nutrients to the tissues and removes waste products from the tissues, while lymphatic vessels are blind-ended tubes that collect extravasated fluid and cells from the tissues and return them back to blood circulation. Development of blood and lymphatic vascular systems occurs in series. Blood vessels are formed via vasculogenesis and angiogenesis whereas lymphatic vessels develop via lymphangiogenesis, after the blood vascular system is already functional. Members of the vascular endothelial growth factor (VEGF) family are regulators of both angiogenesis and lymphangiogenesis, while members of the platelet-derived growth factor (PDGF) family are major mitogens for pericytes and smooth muscle cells and regulate formation of blood vessels. Vascular endothelial growth factor C (VEGF-C) is the major lymphatic growth factor and signaling through its receptor vascular endothelial growth factor receptor 3 (VEGFR-3) is sufficient for lymphangiogenesis in adults. We studied the role of VEGF-C in embryonic lymphangiogenesis and showed that VEGF-C is absolutely required for the formation of lymph sacs from embryonic veins. VEGFR-3 is also required for normal development of the blood vascular system during embryogenesis, as Vegfr3 knockout mice die at mid-gestation due to failure in remodeling of the blood vessels. We showed that sufficient VEGFR-3 signaling in the embryo proper is required for embryonic angiogenesis and in a dosage-sensitive manner for embryonic lymphangiogenesis. Importantly, mice deficient in both VEGFR-3 ligands, Vegfc and Vegfd, developed a normal blood vasculature, suggesting VEGF-C- and VEGF-D- independent functions for VEGFR-3 in the early embryo. Platelet-derived growth factor B (PDGF-B) signals via PDGFR-b and regulates formation of blood vessels by recruiting pericytes and smooth muscle cells around nascent endothelial tubes. We showed that PDGF-B fails to induce lymphangiogenesis when overexpressed in adult mouse skin using adenoviral vectors. However, mouse embryos lacking Pdgfb showed abnormal lymphatic vessels, suggesting that PDGF-B plays a role in lymphatic vessel maturation and separation from blood vessels during embryogenesis. Lymphatic vessels play a key role in immune surveillance, fat absorption and maintenance of fluid homeostasis in the body. However, lymphatic vessels are also involved in various diseases, such as lymphedema and tumor metastasis. These studies elucidate the basic mechanisms of embryonic lymphangiogenesis and add to the knowledge of lymphedema and tumor metastasis treatments by giving novel insights into how lymphatic vessel growth could be induced (in lymphedema) or inhibited (in tumor metastasis).

A quantum chemical investigation of the metal centres in cytochrome c oxidase

Quantum effects are often of key importance for the function of biological systems at molecular level. Cellular respiration, where energy is extracted from the reduction of molecular oxygen to water, is no exception. In this work, the end station of the electron transport chain in mitochondria, cytochrome c oxidase, is investigated using quantum chemical methodology. Cytochrome c oxidase contains two haems, haem a and haem a3. Haem a3, with its copper companion, CuB, is involved in the final reduction of oxygen into water. This binuclear centre receives the necessary electrons from haem a. Haem a, in turn, receives its electrons from a copper ion pair in the vicinity, called CuA. Density functional theory (DFT) has been used to clarify the charge and spin distributions of haem a, as well as changes in these during redox activity. Upon reduction, the added electron is shown to be evenly distributed over the entire haem structure, important for the accommodation of the prosthetic group within the protein. At the same time, the spin distribution of the open-shell oxidised state is more localised to the central iron. The exact spin density distribution has been disputed in the literature, however, different experiments indicating different distributions of the unpaired electron. The apparent contradiction is shown to be due to the false assumption of a unit amount of unpaired electron density; in fact, the oxidised state has about 1.3 unpaired electrons. The validity of the DFT results have been corroborated by wave function based coupled cluster calculations. Point charges, for use in classical force field based simulations, have been parameterised for the four metal centres, using a newly developed methodology. In the procedure, the subsystem for which point charges are to be obtained, is surrounded by an outer region, with the purpose of stabilising the inner region, both electronically and structurally. Finally, the possibility of vibrational promotion of the electron transfer step between haem a and a3 has been investigated. Calculating the full vibrational spectra, at DFT level, of a combined model of the two haems, revealed several normal modes that do shift electron density between the haems. The magnitude of the shift was found to be moderate, at most. The proposed mechanism could have an assisting role in the electron transfer, which still seems to be dominated by electron tunnelling.

Kirkko sydämessä : Uskonnon muokkautuminen ja individuaatio inkerinsuomalaisten elämänvaiheissa 1900-luvulla

The Church in one s heart. The formation of religion and individuation in the lives of Ingrian Finns in the 20th century. Sinikka Haapaniemi University of Helsinki, Finland 302 pages The study falls within the sphere of religious views and the problematique of the life trajectory. The target group comprises those Finnish speakers (Ingrian Finns, Ingrians) living in what was historically Ingermanland and who in varying circumstances became scattered. These times were characterized by pressures for change due to societal reasons and reasons of war. In conditions of change external living conditions matters of religious conviction may assume new meaning and form. The examination focuses on sustaining personal faith in difficult life situations and on how crises affected religious views. Another level of scrutiny takes shape through the terminology of the analytic psychology of C.G. Jung. Individuation is deemed to occur as a cumulative process through the stages of life. The basic data for the study comprises interviews with twenty (20) natives of Ingria and their biographical narratives written in standard language. Many biographical accounts and memoirs serve as secondary data. The interviewees, who were largely selected at random, recounted their lives without questions formulated in advance. The study falls within the field of comparative religion and adheres to the principles of qualitative research practice and the case-study method. Effort was made to get to know each interviewee in the situation which his/her narrative presents. The aim is to pay attention to the interpretations given by the narrators of their various experiences and to understand their meanings on a personal level. The years during which the Ingrians were scattered, wandering and returning raise problems of survival. An individual s own initiative assumes individual forms and emphases. Religion was part of the narrators lives as one factor in the quality of life. Their religious thinking was influenced by both their home upbringing and the teaching of the Church. The interviewees took a serious attitude to the informative teaching of confirmation training. When there was no longer a church, it was claimed that the church travelled with them. Changed circumstances tested the validity of the teachings. The message of the Church institution persisted and helped them to preserve their traditions. A striving for unity and for the presence of a community emerged both in the form of ritual behaviour and in a predilection to sociability. Gradually, as they returned, the activity of the Church of Ingria began to revive. At the turn of the millennium the network of parishes was extensive and cultural activity flourished wherever the Ingrians settled in the postwar decades. Religion is part of the process of individuation. Examination of religion and individuation shows that religion remained an individual view, whose factual base was formed by Christianity and the tradition of the Church. Home upbringing served to orientate, but not to bind. With ageing the importance of independent thought is emphasized, for example in relation to confession, it did not pose a threat to individuality. Keywords: Life story, Religiosity, individuation, Ingrian, the Church of Ingria

Pyhä Teresa ja keskiajan mystiikan teoriat

In my dissertation I have studied St Teresa (1515-1582) in the light of medieval mystical theories. I have two main levels in my research: historical and theological. On the historical level I study St Teresa s personal history in the context of her family and the Spanish society. On the theological level I study both St Teresa s mysticism and her religious experience in the light of medieval mysticism. St Teresa wrote a book called Life , which is her narrative autobiography and story about her mystical spiritual formation. She reflected herself through biblical texts interpreting them in the course of the biblical hermeneutics like allegory, typology, tropology and anagogy. In addition to that she read others life stories from her period of time, but reflected herself only slightly through the sociological point of view. She used irony as a means to gain acceptance to her authority and motive to write. Her position has been described as a double bind because of writing at the request of educated men and to the non-educated women as she herself was uneducated. She used irony as a means to achieve valuation to women, to gain negative attributes connected to them and to gain authority to teach them mystical spirituality, the Bible and prayer. In this ironic tendency she was a feminist writer. In order to understand medieval mysticism I have written in the first chapter a review of the main trends in medieval mysticism in connection with the classical emotional theories. Two medieval mystical theories show an important role in St Teresa s mysticism. One is love mysticism and the other is the three partite way of mysticism (purification, illumination and union). The classic-philosophical emotional theories play a role in both patterns. The theory of love mysticism St Teresa interpreted in the traditional way stressing the spiritual meaning of love in connexion with God and neighbors. Love is an emotion, which is bound with other emotions, but all objects of love don t strengthen spiritual love. In the three partite way of mysticism purification means to find biblical values in life and to practice meditative self-knowledge theologically interpreted. In illumination human understanding has to be illuminated by God and united to mystical knowledge from God. St Teresa considered illumination a way to learn things. Illumination has also psychological aspects like recognition of many trials and pains, which come from life on earth. Theologically interpreted in illumination one should die to oneself, let oneself be transformed and renewed by God. I have also written a review of the modern philosophical discussion on personal identity where memory and mental experiences are important creators of personal identity. St Teresa bound medieval mystical teaching together with her personal religious experience. Her personal identity is by its character based on her narrative life story where mental experiences play important role. Previous researchers have labelled St Teresa as an ecstatic person whose experiences produced ecstatic phenomena to the mysticism. These phenomena combined with visions have in one respect made of her a person who has brought physical and visionary tendencies to theology. In spite of that she also represents a modern tendency trying to give words to experiences, which at first seem to be exceptional and extreme and which are easily interpreted as one-sided either physical or sexual or unsaid. In other respect I have stressed the personality of St Teresa that was represented as both strong and weak. The strong personality for her is demonstrated by religious faith and in its practice. The weak personality was for her a natural personal identity. St Teresa saw a unifying aspect in almost all. Firstly, her mysticism was aimed towards union with God and secondly, the unifying aspects and common rules in human relations in community life were central. Union with God is based on the fact that in a soul God is living in its centre, where God is present in the Trinitarian way. The picture of God in ourselves is a mirror but to get to know God better is to recognize his/her presence in us. When the soul recognizes itself as a dwelling place of God, it knows itself as God knows him/herself. There is equality between God and the soul. To be a Christian means to participate in God in his Trinitarian being. The participation to God is a process of divinization that puts a person into transformation, change and renewal. The unitive aspect concludes also knowledge of opposites between experience of community and solitude as well as community and separateness. As a founder of monasteries St Teresa practiced theology of poverty. She renewed the monastic life founding a rule called discalced that stressed ascetic tendencies. Supporters of her work were after the difficulties in the beginning both society and churchly leaders. She wrote about the monasteries including in her description at times seriousness at times humor and irony. Her stories are said to be picaresque histories that contain stories of ordinary laymen and many unexpected occasions. She exercised a kind of Bakhtinian dialogue in her letters. St Teresa stressed the virtues like sacrifice, determination and courage in the monastic life. Most of what she taught of virtues is based on biblical spirituality but there are also psychological tendencies in her writings. The theological pedagogical advice is mixed with psychology, but she herself made no distinction between different aspects in her teaching. To understand St Teresa and her mysticism is to recognize that she mixes her personal religious experience and mysticism, which widens mysticism to religious experience in a new way, although this corresponds also the very definition of mysticism. St Teresa concentrated on mental-spiritual experiences and the aim of her mystical teaching was to produce a human mind well cured like a garden that has God as its gardener.

Aspects of Arminian Soteriology in Methodist-Lutheran Ecumenical Dialogues in 20th and 21st Century

The aim of this thesis is to analyse the key ecumenical dialogues between Methodists and Lutherans from the perspective of Arminian soteriology and Methodist theology in general. The primary research question is defined as: "To what extent do the dialogues under analysis relate to Arminian soteriology?" By seeking an answer to this question, new knowledge is sought on the current soteriological position of the Methodist-Lutheran dialogues, the contemporary Methodist theology and the commonalities between the Lutheran and Arminian understanding of soteriology. This way the soteriological picture of the Methodist-Lutheran discussions is clarified. The dialogues under analysis were selected on the basis of versatility. Firstly, the sole world organisation level dialogue was chosen: The Church – Community of Grace. Additionally, the document World Methodist Council and the Joint Declaration on the Doctrine of Justification is analysed as a supporting document. Secondly, a document concerning the discussions between two main-line churches in the United States of America was selected: Confessing Our Faith Together. Thirdly, two dialogues between non-main-line Methodist churches and main-line Lutheran national churches in Europe were chosen: Fellowship of Grace from Norway and Kristuksesta osalliset from Finland. The theoretical approach to the research conducted in this thesis is systematic analysis. The Remonstrant articles of Arminian soteriology are utilised as an analysis tool to examine the soteriological positions of the dialogues. New knowledge is sought by analysing the stances of the dialogues concerning the doctrines of partial depravity, conditional election, universal atonement, resistible grace and conditional perseverance of saints. This way information is also provided for approaching the Calvinist-Arminian controversy from new perspectives. The results of this thesis show that the current soteriological position of the Methodist-Lutheran dialogues is closer to Arminianism than Calvinism. The dialogues relate to Arminian soteriology especially concerning the doctrines of universal atonement, resistible grace and conditional perseverance of saints. The commonalities between the Lutheran and Arminian understanding of soteriology exist mainly in these three doctrines as they are uniformly favoured in the dialogues. The most discussed area of soteriology is human depravity, in which the largest diversity of stances occurs as well. On the other hand, divine election is the least discussed topic. The overall perspective, which the results of the analysis provide, indicates that the Lutherans could approach the Calvinist churches together with the Methodists with a wider theological perspective and understanding when the soteriological issues are considered as principal. Human depravity is discovered as the area of soteriology which requires most work in future ecumenical dialogues. However, the detected Lutheran hybrid notion on depravity (a Calvinist-Arminian mixture) appears to provide a useful new perspective for Calvinist-Arminian ecumenism and offers potentially fruitful considerations to future ecumenical dialogues.

Lymphatic vessels in health and disease: Role of the VEGF-C/VEGFR-3 pathway and the transcription factor FOXC2

The circulatory system consists of two vessel types, which act in concert but significantly differ from each other in several structural and functional aspects as well as in mechanisms governing their development. The blood vasculature transports oxygen, nutrients and cells to tissues whereas the lymphatic vessels collect extravasated fluid, macromolecules and cells of the immune system and return them back to the blood circulation. Understanding the molecular mechanisms behind the developmental and functional regulation of the lymphatic system long lagged behind that of the blood vasculature. Identification of several markers specific for the lymphatic endothelium, and the discovery of key factors controlling the development and function of the lymphatic vessels have greatly facilitated research in lymphatic biology over the past few years. Recognition of the crucial importance of lymphatic vessels in certain pathological conditions, most importantly in tumor metastasis, lymphedema and inflammation, has increased interest in this vessel type, for so long overshadowed by its blood vascular cousin. VEGF-C (Vascular Endothelial Growth Factor C) and its receptor VEGFR-3 are essential for the development and maintenance of embryonic lymphatic vasculature. Furthermore, VEGF-C has been shown to be upregulated in many tumors and its expression found to positively correlate with lymphatic metastasis. Mutations in the transcription factor FOXC2 result in lymphedema-distichiasis (LD), which suggests a role for FOXC2 in the regulation of lymphatic development or function. This study was undertaken to obtain more information about the role of the VEGF-C/VEGFR-3 pathway and FOXC2 in regulating lymphatic development, growth, function and survival in physiological as well as in pathological conditions. We found that the silk-like carboxyterminal propeptide is not necessary for the lymphangiogenic activity of VEGF-C, but enhances it, and that the aminoterminal propeptide mediates binding of VEGF-C to the neuropilin-2 coreceptor, which we suggest to be involved in VEGF-C signalling via VEGFR-3. Furthermore, we found that overexpression of VEGF-C increases tumor lymphangiogenesis and intralymphatic tumor growth, both of which could be inhibited by a soluble form of VEGFR-3. These results suggest that blocking VEGFR-3 signalling could be used for prevention of lymphatic tumor metastasis. This might prove to be a safe treatment method for human cancer patients, since inhibition of VEGFR-3 activity had no effect on the normal lymphatic vasculature in adult mice, though it did lead to regression of lymphatic vessels in the postnatal period. Interestingly, in contrast to VEGF-C, which induces lymphangiogenesis already during embryonic development, we found that the related VEGF-D promotes lymphatic vessel growth only after birth. These results suggest, that the lymphatic vasculature undergoes postnatal maturation, which renders it independent of ligand induced VEGFR-3 signalling for survival but responsive to VEGF-D for growth. Finally, we show that FOXC2 is necessary for the later stages of lymphatic development by regulating the morphogenesis of lymphatic valves, as well as interactions of the lymphatic endothelium with vascular mural cells, in which it cooperates with VEGFR-3. Furthermore, our study indicates that the absence of lymphatic valves, abnormal association of lymphatic capillaries with mural cells and an increased amount of basement membrane underlie the pathogenesis of LD. These findings have given new insight into the mechanisms of normal lymphatic development, as well as into the pathogenesis of diseases involving the lymphatic vasculature. They also reveal new therapeutic targets for the prevention and treatment of tumor metastasis and lymphatic vascular failure in certain forms of lymphedema. Several interesting questions were posed that still need to be addressed. Most importantly, the mechanism of VEGF-C promoted tumor metastasis and the molecular nature of the postnatal lymphatic vessel maturation remain to be elucidated.

Redox-linked proton transfer by cytochrome c oxidase

The respiratory chain is found in the inner mitochondrial membrane of higher organisms and in the plasma membrane of many bacteria. It consists of several membrane-spanning enzymes, which conserve the energy that is liberated from the degradation of food molecules as an electrochemical proton gradient across the membrane. The proton gradient can later be utilized by the cell for different energy requiring processes, e.g. ATP production, cellular motion or active transport of ions. The difference in proton concentration between the two sides of the membrane is a result of the translocation of protons by the enzymes of the respiratory chain, from the negatively charged (N-side) to the positively charged side (P-side) of the lipid bilayer, against the proton concentration gradient. The endergonic proton transfer is driven by the flow of electrons through the enzymes of the respiratory chain, from low redox-potential electron donors to acceptors of higher potential, and ultimately to oxygen. Cytochrome c oxidase is the last enzyme in the respiratory chain and catalyzes the reduction of dioxygen to water. The redox reaction is coupled to proton transport across the membrane by a yet unresolved mechanism. Cytochrome c oxidase has two proton-conducting pathways through which protons are taken up to the interior part of the enzyme from the N-side of the membrane. The K-pathway transfers merely substrate protons, which are consumed in the process of water formation at the catalytic site. The D-pathway transfers both substrate protons and protons that are pumped to the P-side of the membrane. This thesis focuses on the role of two conserved amino acids in proton translocation by cytochrome c oxidase, glutamate 278 and tryptophan 164. Glu278 is located at the end of the D-pathway and is thought to constitute the branching point for substrate and pumped protons. In this work, it was shown that although Glu278 has an important role in the proton transfer mechanism, its presence is not an obligatory requirement. Alternative structural solutions in the area around Glu278, much like the ones present in some distantly related heme-copper oxidases, could in the absence of Glu278 support the formation of a long hydrogen-bonded water chain through which proton transfer from the D-pathway to the catalytic site is possible. The other studied amino acid, Trp164, is hydrogen bonded to the ∆-propionate of heme a3 of the catalytic site. Mutation of this amino acid showed that it may be involved in regulation of proton access to a proton acceptor, a pump site, from which the proton later is expelled to the P-side of the membrane. The ion pair that is formed by the ∆-propionate of heme a3 and arginine 473 is likely to form a gate-like structure, which regulates proton mobility to the P-side of the membrane. The same gate may also be part of an exit path through which water molecules produced at the catalytically active site are removed towards the external side of the membrane. Time-resolved optical and electrometrical experiments with the Trp164 to phenylalanine mutant revealed a so far undetected step in the proton pumping mechanism. During the A to PR transition of the catalytic cycle, a proton is transferred from Glu278 to the pump site, located somewhere in the vicinity of the ∆-propionate of heme a3. A mechanism for proton pumping by cytochrome c oxidase is proposed on the basis of the presented results and the mechanism is discussed in relation to some relevant experimental data. A common proton pumping mechanism for all members of the heme-copper oxidase family is moreover considered.

Membrane Insertion of C-tail Anchored Proteins

The correct localization of proteins is essential for cell viability. In order to achieve correct protein localization to cellular membranes, conserved membrane targeting and translocation mechanisms have evolved. The focus of this work was membrane targeting and translocation of a group of proteins that circumvent the known targeting and translocation mechanisms, the C-tail anchored protein family. Members of this protein family carry out a wide range of functions, from protein translocation and recognition events preceding membrane fusion, to the regulation of programmed cell death. In this work, the mechanisms of membrane insertion and targeting of two C-tail anchored proteins were studied utilizing in vivo and in vitro methods, in yeast and mammalian cell systems. The proteins studied were cytochrome b(5), a well characterized C-tail anchored model protein, and N-Bak, a novel member of the Bcl-2 family of regulators of programmed cell death. Membrane insertion of cytochrome b(5) into the endoplasmic reticulum membrane was found to occur independently of the known protein conducting channels, through which signal peptide-containing polypeptides are translocated. In fact, the membrane insertion process was independent of any protein components and did not require energy. Instead membrane insertion was observed to be dependent on the lipid composition of the membrane. The targeting of N-Bak was found to depend on the cellular context. Either the mitochondrial or endoplasmic reticulum membranes were targeted, which resulted in morphological changes of the target membranes. These findings indicate the existence of a novel membrane insertion mechanism for C-tail anchored proteins, in which membrane integration of the transmembrane domain, and the translocation of C-terminal fragments, appears to be spontaneous. This mode of membrane insertion is regulated by the target membrane fluidity, which depends on the lipid composition of the bilayer, and the hydrophobicity of the transmembrane domain of the C-tail anchored protein, as well as by the availability of the C-tail for membrane integration. Together these mechanisms enable the cell to achieve spatial and temporal regulation of sub-cellular localization of C-tail anchored proteins.