Novel conditional mouse models for targeted kidney research : Emphasis on the analysis of the biological function of nephrin

Nephrin is a transmembrane protein belonging to the immunoglobulin superfamily and is expressed primarily in the podocytes, which are highly differentiated epithelial cells needed for primary urine formation in the kidney. Mutations leading to nephrin loss abrogate podocyte morphology, and result in massive protein loss into urine and consequent early death in humans carrying specific mutations in this gene. The disease phenotype is closely replicated in respective mouse models. The purpose of this thesis was to generate novel inducible mouse-lines, which allow targeted gene deletion in a time and tissue-specific manner. A proof of principle model for succesful gene therapy for this disease was generated, which allowed podocyte specific transgene replacement to rescue gene deficient mice from perinatal lethality. Furthermore, the phenotypic consequences of nephrin restoration in the kidney and nephrin deficiency in the testis, brain and pancreas in rescued mice were investigated. A novel podocyte-specific construct was achieved by using standard cloning techniques to provide an inducible tool for in vitro and in vivo gene targeting. Using modified constructs and microinjection procedures two novel transgenic mouse-lines were generated. First, a mouse-line with doxycycline inducible expression of Cre recombinase that allows podocyte-specific gene deletion was generated. Second, a mouse-line with doxycycline inducible expression of rat nephrin, which allows podocyte-specific nephrin over-expression was made. Furthermore, it was possible to rescue nephrin deficient mice from perinatal lethality by cross-breeding them with a mouse-line with inducible rat nephrin expression that restored the missing endogenous nephrin only in the kidney after doxycycline treatment. The rescued mice were smaller, infertile, showed genital malformations and developed distinct histological abnormalities in the kidney with an altered molecular composition of the podocytes. Histological changes were also found in the testis, cerebellum and pancreas. The expression of another molecule with limited tissue expression, densin, was localized to the plasma membranes of Sertoli cells in the testis by immunofluorescence staining. Densin may be an essential adherens junction protein between Sertoli cells and developing germ cells and these junctions share similar protein assembly with kidney podocytes. This single, binary conditional construct serves as a cost- and time-efficient tool to increase the understanding of podocyte-specific key proteins in health and disease. The results verified a tightly controlled inducible podocyte-specific transgene expression in vitro and in vivo as expected. These novel mouse-lines with doxycycline inducible Cre recombinase and with rat nephrin expression will be useful for conditional gene targeting of essential podocyte proteins and to study in detail their functions in the adult mice. This is important for future diagnostic and pharmacologic development platforms.

Molecular basis of the kidney filtration barrier : Role of the nephrin protein complex

The kidney filtration barrier consists of fenestrated endothelial cell layer, glomerular basement membrane and slit diaphragm (SD), the specialized junction between glomerular viscelar epithelial cells (podocytes). Podocyte injury is associated with the development of proteinuria, and if not reversed the injury will lead to permanent deterioration of the glomerular filter. The early events are characterized by disruption of the integrity of the SD, but the molecular pathways involved are not fully understood. Congenital nephrotic syndrome of the Finnish type (CNF) is caused by mutations in NPHS1, the gene encoding the SD protein nephrin. Lack of nephrin results in loss of the SD and massive proteinuria beginning before birth. Furthermore, nephrin expression is decreased in acquired human kidney diseases including diabetic nephropathy. This highlights the importance of nephrin and consequently SD in regulating the kidney filtration function. However, the precise molecular mechanism of how nephrin is involved in the formation of the SD is unknown. This thesis work aimed at clarifying the role of nephrin and its interaction partners in the formation of the SD. The purpose was to identify novel proteins that associate with nephrin in order to define the essential molecular complex required for the establishment of the SD. The aim was also to decipher the role of novel nephrin interacting proteins in podocytes. Nephrin binds to nephrin-like proteins Neph1 and Neph2, and to adherens junction protein P-cadherin. These interactions have been suggested to play a role in the formation of the SD. In this thesis work, we identified densin as a novel interaction partner for nephrin. Densin was localized to the SD and it was shown to bind to adherens junction protein beta-catenin. Furthermore, densin was shown to behave in a similar fashion as adherens junction proteins in cell-cell contacts. These results indicate that densin may play a role in cell adhesion and, therefore, may contribute to the formation of the SD together with nephrin and adherens junction proteins. Nephrin was also shown to bind to Neph3, which has been previously localized to the SD. Neph3 and Neph1 were shown to induce cell adhesion alone, whereas nephrin needed to trans-interact with Neph1 or Neph3 from the opposite cell surface in order to make cell-cell contacts. This was associated with the decreased tyrosine phosphorylation of nephrin. These data extend the current knowledge of the molecular composition of the nephrin protein complex at the SD and also provide novel insights of how the SD may be formed. This thesis work also showed that densin was up-regulated in the podocytes of CNF patients. Neph3 was up-regulated in nephrin deficient mouse kidneys, which share similar podocyte alterations and lack of the SD as observed in CNF patients podocytes. These data suggest that densin and Neph3 may have a role in the formation of morphological alterations in podocytes detected in CNF patients. Furthermore, this thesis work showed that deletion of beta-catenin specifically from adult mouse podocytes protected the mice from the development of adriamycin-induced podocyte injury and proteinuria compared to wild-type mice. These results show that beta-catenin play a role in the adriamycin induced podocyte injury. Podocyte injury is a hallmark in many kidney diseases and the changes observed in the podocytes of CNF patient share characteristics with injured podocytes observed in chronic kidney diseases. Therefore, the results obtained in this thesis work suggest that densin, Neph3 and beta-catenin participate in the molecular pathways which result in morphological alterations commonly detected in injured podocytes in kidney diseases.

Nephrin and the Pathogenesis of Nephropathy - Emphasis on Type I Diabetes

End-stage renal disease is an increasingly common pathologic condition, with a current incidence of 87 per million inhabitants in Finland. It is the end point of various nephropathies, most common of which is the diabetic nephropathy. This thesis focuses on exploring the role of nephrin in the pathogenesis of diabetic nephropathy. Nephrin is a protein of the glomerular epithelial cell, or podocyte, and it appears to have a crucial function as a component of the filtration slit diaphragm in the kidney glomeruli. Mutations in the nephrin gene NPHS1 lead to massive proteinuria. Along with the originally described location in the podocyte, nephrin has now been found to be expressed in the brain, testis, placenta and pancreatic beta cells. In type 1 diabetes, the fundamental pathologic event is the autoimmune destruction of the beta cells. Autoantibodies against various beta cell antigens are generated during this process. Due to the location of nephrin in the beta cell, we hypothesized that patients with type 1 diabetes may present with nephrin autoantibodies. We also wanted to test whether such autoantibodies could be involved in the pathogenesis of diabetic nephropathy. The puromycin aminonucleoside nephrosis model in the rat, the streptozotocin model in the rat, and the non-obese diabetic mice were studied by immunochemical techniques, in situ -hybridization and the polymerase chain reaction -based methods to resolve the expression of nephrin mRNA and protein in experimental nephropathies. To test the effect of antiproteinuric therapies, streptozotocin-treated rats were also treated with aminoguanidine or perindopril. To detect nephrin antibodies we developed a radioimmunoprecipitation assay and analyzed follow-up material of 66 patients with type 1 diabetes. In the puromycin aminonucleoside nephrosis model, the nephrin expression level was uniformly decreased together with the appearance of proteinuria. In the streptozotocin-treated rats and in non-obese diabetic mice, the nephrin mRNA and protein expression levels were seen to increase in the early stages of nephropathy. However, as observed in the streptozotocin rats, in prolonged diabetic nephropathy the expression level decreased. We also found out that treatment with perindopril could not only prevent proteinuria but also a decrease in nephrin expression in streptozotocin-treated rats. Aminoguanidine did not have an effect on nephrin expression, although it could attenuate the proteinuria. Circulating antibodies to nephrin in patients with type 1 diabetes were found, although there was no correlation with the development of diabetic nephropathy. At diagnosis, 24% of the patients had these antibodies, while at 2, 5 and 10 years of disease duration the respective proportions were 23%, 14% and 18%. During the total follow-up of 16 to 19 years after diagnosis of diabetes, 14 patients had signs of nephropathy and 29% of them tested positive for nephrin autoantibodies in at least one sample. In conclusion, this thesis work could show changes of nephrin expression along with the development of proteinuria. The autoantibodies against nephrin are likely generated in the autoimmune process leading to type 1 diabetes. However, according to the present work it is unlikely that these autoantibodies are contributing significantly to the development of diabetic nephropathy.

Dengue virus infection : Diagnostics and molecular epidemiology

Dengue is a mosquito-borne viral disease caused by the four dengue virus serotypes (DENV-1-4) and is currently considered as the most important arthropod-borne viral disease in the world. Nearly half of the human population lives in risk areas, and 50-100 million infections occur yearly according to World Health Organization. The disease can vary from a mild febrile disease to severe haemorrhagic fever and shock. A secondary infection with heterologous serotype increases the risk for severe disease outcome. During the last three decades the impact of dengue has dramatically increased in the endemic areas including the tropics and subtropics of the world. The current situation with massive epidemics of severe disease forms has been associated with socio-ecological changes that have increased the transmission and enabled the co-circulation of different serotypes. Consequently, an increase of dengue has also been observed in travelers visiting these areas. Currently approximately 30 cases are diagnosed yearly in Finnish travelers. In travelers dengue is rarely a life-threatening disease, however in the current study, a fatality was documented in a young Finnish patient who experienced a prolonged primary dengue infection. To improve particularly early laboratory diagnostics, a novel real-time RT-PCR method was developed for the detection of DENV-1-4 RNA based on TaqMan chemistry. The method was shown to be sensitive and specific for detecting DENV RNA and suitable for diagnostic use. The newly developed real-time RT-PCR was compared to other available early diagnostic methods including IgM and NS1 antigen detection using a panel of selected patient samples. The results suggest that the best diagnostic rates are achieved by a combination of IgM with RNA or NS1 detection. The dengue virus strains studied here included the first DENV strains isolated from serum samples of Finnish travelers collected in 2000-2005. The results of sequence analysis demonstrated that the 11 isolates included all four DENV serotypes and presented a global sample of DENV strains from different geographical areas including Asia, Africa and South America. In the present study sequence analysis was also carried out for a collection of 23 novel DENV-2 isolates from Venezuelan patients collected in 1999-2005. The Venezuelan DENV-2 exclusively represented the American-Asian genotype, suggesting that no foreign DENV-2 lineages have recently been introduced to the country. The results also suggest that the DENV-2 viruses detected earlier from Venezuela have been maintained in the area where they have evolved into several lineages. This is in contrast to the pattern observed in some other dengue endemic areas, where introductions of novel virus types and lineages are frequently detected.

Microbial life and deposits in paper machine circuits

Väitöskirjani käsittele mikrobien ja erilaisten kemikaalien rooleja saostumien ja biofilmien muodostumisessa paperi- ja kartonkikoneilla. "Saostuma" tässä työssä tarkoittaa kiinteän aineen kertymää konepinnoille tai rajapinnoille konekierroissa, jotka on tarkoitettu massasulppujen, lietteiden, vesien tai ilman kuljetukseen. Saostumasta tulee "biofilmi" silloin kun sen oleellinen rakennekomponentti on mikrobisolut tai niiden tuotteet. Väitöstyöni työhypoteesina oli, että i. tietämys saostumien koostumuksesta, sekä ii. niiden rakenteesta, biologisista, fysikaalis-kemiallisista ja teknisistä ominaisuuksista ohjaavat tutkijaa löytämään ympäristöä säästäviä keinoja estää epätoivottujen saostumien muodostus tai purkaa jo muodostuneita saostumia. Selvittääkseni saostumien koostumista ja rakennetta käytin monia erilaisia analytiikan työkaluja, kuten elektronimikroskopiaa, konfokaali-laser mikroskopiaa (CLSM), energiadispersiivistä röntgenanalyysiä (EDX), pyrolyysi kaasukromatografiaa yhdistettynä massaspektrometriaan (Py-GCMS), joninvaihtokromatografiaa, kaasukromatografiaa ja mikrobiologisia analyysejä. Osallistuin aktiivisesti innovatiivisen, valon takaisinsirontaan perustuvan sensorin kehittämistyöhön, käytettäväksi biofilmin kasvun mittaukseen suoraan koneen vesikierroista ja säiliöistä. Työni osoitti, että monet paperinvalmistuksessa käytetyistä kemikaaleista reagoivat keskenään tuottaen orgaanisia tahmakerroksia konekiertojen teräspinnoille. Löysin myös kerrostumia, jotka valomikroskooppisessa tarkastelussa oli tulkittu mikrobeiksi, mutta jotka elektronimikroskopia paljasti alunasta syntyneiksi, alumiinihydroksidiksi joka saostui pH:ssa 6,8 kiertokuitua käyttävän koneen viiravesistä. Monet paperintekijät käyttävät vieläkin alunaa kiinnitysaineena vaikka prosessiolot ovat muuttuneet happamista neutraaleiksi. Sitä pidetään paperitekijän "aspiriinina", mutta väitöstutkimukseni osoitti sen riskit. Löysin myös orgaanisia saostumia, joiden alkuperä oli aineiden, kuten pihkan, saippuoituminen (kalsium saippuat) niin että muodostui tahmankasvua ylläpitävä alusta monilla paperi- ja kartonkikoneilla. Näin solumuodoiltaan Deinococcus geothermalista muistuttavia bakteereita kasvamassa lujasti teräskoepalojen pintaan kiinnittyneinä pesäkkeinä, kun koepaloja upotettiin paperikoneiden vesikiertoihin. Nämä deinokokkimaiset pesäkkeet voivat toimia jalustana, tarttumisalustana muiden mikrobien massoille, joka selittäisi miksi saostumat yleisesti sisältävät deinokokkeja pienenä, muttei koskaan pääasiallisena rakenneosana. Kun paperikoneiden käyttämien vesien (raakavedet, lämminvesi, biologisesti puhdistettu jätevesi) laatua tutkitaan, mittausmenetelmällä on suuri merkitys. Koepalan upotusmenetelmällä todettu biofilmikasvu ja viljelmenetelmällä mitattu bakteerisaastuneisuus korreloivat toisiinsa huonosti etenkin silloin kun likaantumisessa oli mukana rihmamaiseti kasvavia bakteereja. Huoli ympäristöstä on pakottanut paperi- ja kartonkikoneiden vesikiertojen sulkemiseen. Vesien kierrätys ja prosessivesien uudelleenkäyttö nostavat prosessilämpötilaa ja lisäävät koneella kiertävien kolloidisten ja liuenneiden aineiden määriä. Tutkin kiertovesien pitoisuuksia kolmessa eriasteisesti suljetussa tehtaassa, joiden päästöt olivat 0 m3, 0,5 m3 ja 4 m3 jätevettä tuotetonnia kohden, perustuen puhdistetun jäteveden uudelleen käyttöön. Nollapäästöisellä tehtaalla kiertovesiin kertyi paljon orgaanisesti sidottua hiiltä (> 10 g L-1), etenkin haihtuvina happoina (maito-, etikka-, propioni- ja voi-). Myös sulfaatteja, klorideja, natriumia ja kalsiumia kertyi paljon, > 1 g L-1 kutakin. Pääosa (>40%) kaikista bakteereista oli 16S rRNA geenisekvenssianalyysien tulosten perusteella sukua, joskin etäistä (< 96%) ainoastaan Enterococcus cecorum bakteerille. 4 m3 päästävältä tehtaalta löytyi lisäksi Bacillus thermoamylovorans ja Bacillus coagulans. Tehtaiden saostumat sisälsivät arkkeja suurina pitoisuuksina, ≥ 108 g-1, mutta tunnistukseen riittävää sekvenssisamanlaisuutta löytyi vain yhteen arkkisukuun, Methanothrix. Tutkimustulokset osoittivat että tehtaan vesikiertojen sulkeminen vähensi rajusti mikrobiston monimuotoisuutta, muttei estänyt liuenneen aineen ja kiintoaineen mineralisoitumista.

Quality of ALICE SSD modules revealed by electrical tests

ALICE (A Large Ion Collider Experiment) is an experiment at CERN (European Organization for Nuclear Research), where a heavy-ion detector is dedicated to exploit the unique physics potential of nucleus-nucleus interactions at LHC (Large Hadron Collider) energies. In a part of that project, 716 so-called type V4 modules were assembles in Detector Laboratory of Helsinki Institute of Physics during the years 2004 - 2006. Altogether over a million detector strips has made this project the most massive particle detector project in the science history of Finland. One ALICE SSD module consists of a double-sided silicon sensor, two hybrids containing 12 HAL25 front end readout chips and some passive components, such has resistors and capacitors. The components are connected together by TAB (Tape Automated Bonding) microcables. The components of the modules were tested in every assembly phase with comparable electrical tests to ensure the reliable functioning of the detectors and to plot the possible problems. The components were accepted or rejected by the limits confirmed by ALICE collaboration. This study is concentrating on the test results of framed chips, hybrids and modules. The total yield of the framed chips is 90.8%, hybrids 96.1% and modules 86.2%. The individual test results have been investigated in the light of the known error sources that appeared during the project. After solving the problems appearing during the learning-curve of the project, the material problems, such as defected chip cables and sensors, seemed to induce the most of the assembly rejections. The problems were typically seen in tests as too many individual channel failures. Instead, the bonding failures rarely caused the rejections of any component. One sensor type among three different sensor manufacturers has proven to have lower quality than the others. The sensors of this manufacturer are very noisy and their depletion voltage are usually outside of the specification given to the manufacturers. Reaching 95% assembling yield during the module production demonstrates that the assembly process has been highly successful.

Parity violation in molecular magnetic resonance properties

The Standard Model of particle physics consists of the quantum electrodynamics (QED) and the weak and strong nuclear interactions. The QED is the basis for molecular properties, and thus it defines much of the world we see. The weak nuclear interaction is responsible for decays of nuclei, among other things, and in principle, it should also effects at the molecular scale. The strong nuclear interaction is hidden in interactions inside nuclei. From the high-energy and atomic experiments it is known that the weak interaction does not conserve parity. Consequently, the weak interaction and specifically the exchange of the Z^0 boson between a nucleon and an electron induces small energy shifts of different sign for mirror image molecules. This in turn will make the other enantiomer of a molecule energetically favorable than the other and also shifts the spectral lines of the mirror image pair of molecules into different directions creating a split. Parity violation (PV) in molecules, however, has not been observed. The topic of this thesis is how the weak interaction affects certain molecular magnetic properties, namely certain parameters of nuclear magnetic resonance (NMR) and electron spin resonance (ESR) spectroscopies. The thesis consists of numerical estimates of NMR and ESR spectral parameters and investigations of the effects of different aspects of quantum chemical computations to them. PV contributions to the NMR shielding and spin-spin coupling constants are investigated from the computational point of view. All the aspects of quantum chemical electronic structure computations are found to be very important, which makes accurate computations challenging. Effects of molecular geometry are also investigated using a model system of polysilyene chains. PV contribution to the NMR shielding constant is found to saturate after the chain reaches a certain length, but the effects of local geometry can be large. Rigorous vibrational averaging is also performed for a relatively small and rigid molecule. Vibrational corrections to the PV contribution are found to be only a couple of per cents. PV contributions to the ESR g-tensor are also evaluated using a series of molecules. Unfortunately, all the estimates are below the experimental limits, but PV in some of the heavier molecules comes close to the present day experimental resolution.

Conrad Summenhart's theory of individual rights and its medieval background

This study focuses on the theory of individual rights that the German theologian Conrad Summenhart (1455-1502) explicated in his massive work Opus septipartitum de contractibus pro foro conscientiae et theologico. The central question to be studied is: How does Summenhart understand the concept of an individual right and its immediate implications? The basic premiss of this study is that in Opus septipartitum Summenhart composed a comprehensive theory of individual rights as a contribution to the on-going medieval discourse on rights. With this rationale, the first part of the study concentrates on earlier discussions on rights as the background for Summenhart s theory. Special attention is paid to language in which right was defined in terms of power . In the fourteenth century writers like Hervaeus Natalis and William Ockham maintained that right signifies power by which the right-holder can to use material things licitly. It will also be shown how the attempts to describe what is meant by the term right became more specified and cultivated. Gerson followed the implications that the term power had in natural philosophy and attributed rights to animals and other creatures. To secure right as a normative concept, Gerson utilized the ancient ius suum cuique-principle of justice and introduced a definition in which right was seen as derived from justice. The latter part of this study makes effort to reconstructing Summenhart s theory of individual rights in three sections. The first section clarifies Summenhart s discussion of the right of the individual or the concept of an individual right. Summenhart specified Gerson s description of right as power, taking further use of the language of natural philosophy. In this respect, Summenhart s theory managed to bring an end to a particular continuity of thought that was centered upon a view in which right was understood to signify power to licit action. Perhaps the most significant feature of Summenhart s discussion was the way he explicated the implication of liberty that was present in Gerson s language of rights. Summenhart assimilated libertas with the self-mastery or dominion that in the economic context of discussion took the form of (a moderate) self-ownership. Summenhart discussion also introduced two apparent extensions to Gerson s terminology. First, Summenhart classified right as relation, and second, he equated right with dominion. It is distinctive of Summenhart s view that he took action as the primary determinant of right: Everyone has as much rights or dominion in regard to a thing, as much actions it is licit for him to exercise in regard to the thing. The second section elaborates Summenhart s discussion of the species dominion, which delivered an answer to the question of what kind of rights exist, and clarified thereby the implications of the concept of an individual right. The central feature in Summenhart s discussion was his conscious effort to systematize Gerson s language by combining classifications of dominion into a coherent whole. In this respect, his treatement of the natural dominion is emblematic. Summenhart constructed the concept of natural dominion by making use of the concepts of foundation (founded on a natural gift) and law (according to the natural law). In defining natural dominion as dominion founded on a natural gift, Summenhart attributed natural dominion to animals and even to heavenly bodies. In discussing man s natural dominion, Summenhart pointed out that the natural dominion is not sufficiently identified by its foundation, but requires further specification, which Summenhart finds in the idea that natural dominion is appropriate to the subject according to the natural law. This characterization lead him to treat God s dominion as natural dominion. Partly, this was due to Summenhart s specific understanding of the natural law, which made reasonableness as the primary criterion for the natural dominion at the expense of any metaphysical considerations. The third section clarifies Summenhart s discussion of the property rights defined by the positive human law. By delivering an account on juridical property rights Summenhart connected his philosophical and theological theory on rights to the juridical language of his times, and demonstrated that his own language of rights was compatible with current juridical terminology. Summenhart prepared his discussion of property rights with an account of the justification for private property, which gave private property a direct and strong natural law-based justification. Summenhart s discussion of the four property rights usus, usufructus, proprietas, and possession aimed at delivering a detailed report of the usage of these concepts in juridical discourse. His discussion was characterized by extensive use of the juridical source texts, which was more direct and verbal the more his discussion became entangled with the details of juridical doctrine. At the same time he promoted his own language on rights, especially by applying the idea of right as relation. He also showed recognizable effort towards systematizing juridical language related to property rights.

N-syndecan and HB-GAM in neural migration and differentiation : Modulation of growth factor activity in brain

The juvenile sea squirt wanders through the sea searching for a suitable rock or hunk of coral to cling to and make its home for life. For this task it has a rudimentary nervous system. When it finds its spot and takes root, it doesn't need its brain any more so it eats it. It's rather like getting tenure. Daniel C. Dennett (from Consciousness Explained, 1991) The little sea squirt needs its brain for a task that is very simple and short. When the task is completed, the sea squirt starts a new life in a vegetative state, after having a nourishing meal. The little brain is more tightly structured than our massive primate brains. The number of neurons is exact, no leeway in neural proliferation is tolerated. Each neuroblast migrates exactly to the correct position, and only a certain number of connections with the right companions is allowed. In comparison, growth of a mammalian brain is a merry mess. The reason is obvious: Squirt brain needs to perform only a few, predictable functions, before becoming waste. The more mobile and complex mammals engage their brains in tasks requiring quick adaptation and plasticity in a constantly changing environment. Although the regulation of nervous system development varies between species, many regulatory elements remain the same. For example, all multicellular animals possess a collection of proteoglycans (PG); proteins with attached, complex sugar chains called glycosaminoglycans (GAG). In development, PGs participate in the organization of the animal body, like in the construction of parts of the nervous system. The PGs capture water with their GAG chains, forming a biochemically active gel at the surface of the cell, and in the extracellular matrix (ECM). In the nervous system, this gel traps inside it different molecules: growth factors and ECM-associated proteins. They regulate the proliferation of neural stem cells (NSC), guide the migration of neurons, and coordinate the formation of neuronal connections. In this work I have followed the role of two molecules contributing to the complexity of mammalian brain development. N-syndecan is a transmembrane heparan sulfate proteoglycan (HSPG) with cell signaling functions. Heparin-binding growth-associated molecule (HB-GAM) is an ECM-associated protein with high expression in the perinatal nervous system, and high affinity to HS and heparin. N-syndecan is a receptor for several growth factors and for HB-GAM. HB-GAM induces specific signaling via N-syndecan, activating c-Src, calcium/calmodulin-dependent serine protein kinase (CASK) and cortactin. By studying the gene knockouts of HB-GAM and N-syndecan in mice, I have found that HB-GAM and N-syndecan are involved as a receptor-ligand-pair in neural migration and differentiation. HB-GAM competes with the growth factors fibriblast growth factor (FGF)-2 and heparin-binding epidermal growth factor (HB-EGF) in HS-binding, causing NSCs to stop proliferation and to differentiate, and affects HB-EGF-induced EGF receptor (EGFR) signaling in neural cells during migration. N-syndecan signaling affects the motility of young neurons, by boosting EGFR-mediated cell migration. In addition, these two receptors form a complex at the surface of the neurons, probably creating a motility-regulating structure.

GABAa Receptor Mediated Signalling in the Brain: Inhibition, Shunting and Excitation

Within central nervous system, the simple division of chemical synaptic transmission to depolarizing excitation mediated by glutamate and hyperpolarizing inhibition mediated by γ-amino butyric acid (GABA), is evidently an oversimplification. The GABAa receptor (GABAaR) mediated responses can be of opposite sign within a single resting cell, due to the compartmentalized distribution of cation chloride cotransporters (CCCs). The K+/Cl- cotransporter 2 (KCC2), member of the CCC family, promotes K+ fuelled Cl- extrusion and sets the reversal potential of GABA evoked anion currents typically slightly below the resting membrane potential. The interesting ionic plasticity property of GABAergic signalling emerges from the short-term and long-term alterations in the intraneuronal concentrations of GABAaR permeable anions (Cl- and HCO3-). The short-term effects arise rapidly (in the time scale of hundreds of milliseconds) and are due to the GABAaR activation dependent shifts in anion gradients, whereas the changes in expression, distribution and kinetic regulation of CCCs are underlying the long-term effects, which may take minutes or even hours to develop. In this Thesis, the differences in the reversal potential of GABAaR mediated responses between dopaminergic and GABAergic cell types, located in the substantia nigra, were shown to be attributable to the differences in the chloride extrusion mechanisms. The stronger inhibitory effect of GABA on GABAergic neurons was due to the cell type specific expression of KCC2 whereas the KCC2 was absent from dopaminergic neurons, leading to a less prominent inhibition brought by GABAaR activation. The levels of KCC2 protein exhibited activity dependent alterations in hippocampal pyramidal neurons. Intense neuronal activity, leading to a massive release of brain derived neurotrophic factor (BDNF) in vivo, or applications of tyrosine receptor kinase B (TrkB) agonists BDNF or neurotrophin-4 in vitro, were shown to down-regulate KCC2 protein levels which led to a reduction in the efficacy of Cl- extrusion. The GABAergic transmission is interestingly involved in an increase of extracellular K+ concentration. A substantial increase in interstitial K+ tends to depolarize the cell membrane. The effects that varying ion gradients had on the generation of biphasic GABAaR mediated responses were addressed, with particular emphasis on the novel idea that the K+/Cl- extrusion via KCC2 is accelerated in response to a rapid accumulation of intracellular Cl-. The KCC2 inhibitor furosemide produced a large reduction in the GABAaR dependent extracellular K+ transients. Thus, paradoxically, both the inefficient KCC2 activity (via increased intracellular Cl-) and efficient KCC2 activity (via increased extracellular K+) may promote excitation.

Should I stay or should I go? : Reproductive and dispersal strategies of site-specific liverwort species

The area of intensively managed forests, in which required conditions for several liverwort species are seldom found, has expanded over the forest landscape during the last century. Liverworts are very sensitive to habitat changes, because they demand continuously moist microclimate. Consequently, about third of the forest liverworts have been classified as threatened or near threatened in Finland. The general objective of this thesis is to increase knowledge of the reproductive and dispersal strategies of the substrate-specific forest bryophytes. A further aim was to develop recommendations for conservation measures for species inhabiting unstable and stable habitats in forest landscape. Both population ecological and genetic methods have been applied in the research. Anastrophyllum hellerianum inhabits spatially and temporally limited substrate patches, decaying logs, which can be considered as unstable habitats. The results show that asexual reproduction by gemmae is the dominant mode of reproduction, whereas sexual reproduction is considerably infrequent. Unlike previously assumed, not only spores but also the asexual propagules may contribute to long-distance dispersal. The combination of occasional spore production and practically continuous, massive gemma production facilitates dispersal both on a local scale and over long distances, and it compensates for the great propagule losses that take place preceding successful establishment at suitable sites. However, establishment probability of spores may be restricted because of environmental and biological limitations linked to the low success of sexual reproduction. Long-lasting dry seasons are likely to result in a low success of sexual reproduction and decreased release rate of gemmae from the shoots, and consequent fluctuations in population sizes. In the long term, the substratum limitation is likely to restrict population sizes and cause local extinctions, especially in small-sized remnant populations. Contrastingly, larger forest fragments with more natural disturbance dynamics, to which the species is adapted, are pivotal to species survival. Trichocolea tomentella occupies stable spring and mesic habitats in woodland. The relatively small populations are increasingly fragmented with a high risk for extinction for extrinsic reasons. The results show that T. tomentella mainly invests in population persistence by effective clonal growth via forming independent ramets and in competitive ability, and considerably less in sexuality and dispersal potential. The populations possess relatively high levels of genetic diversity regardless of population size and of degree of isolation. Thus, the small-sized populations inhabiting stable habitats should not be neglected when establishing conservation strategies for the species and when considering the habitat protection of small spring sites. Restricted dispersal capacity, also on a relatively small spatial scale, is likely to prevent successful (re-)colonization in the potential habitat patches of recovering forest landscapes. By contrast, random short-range dispersal of detached vegetative fragments within populations at suitable habitat seems to be frequent. Thus, the restoration actions of spring and streamside habitats close to the populations of T. tomentella may contribute to population expansion. That, in turn, decreases the harmful effects of environmental stochasticity.

Structure, function and intracellular dynamics of alphavirus replication complexes

Intracellular membrane alterations are hallmarks of positive-sense RNA (+RNA) virus replication. Strong evidence indicates that within these exotic compartments, viral replicase proteins engage in RNA genome replication and transcription. To date, fundamental questions such as the origin of altered membranes, mechanisms of membrane deformation and topological distribution and function of viral components, are still waiting for comprehensive answers. This study addressed some of the above mentioned questions for the membrane alterations induced during Semliki Forest virus (SFV) infection of mammalian cells. With the aid of electron and fluorescence microscopy coupled with radioactive labelling and immuno-cytochemistry techniques, our group and others showed that few hours after infection the four non structural proteins (nsP1-4) and newly synthesized RNAs of SFV colocalized in close proximity of small membrane invaginations, designated as spherules . These 50-70 nm structures were mainly detected in the perinuclear area, at the limiting membrane of modified endosomes and lysosomes, named CPV-I (cytopathic vacuoles type I). More rarely, spherules were also found at the plasma membrane (PM). In the first part of this study I present the first three-dimensional reconstruction of the CPV-I and the spherules, obtained by electron tomography after chemical or cryo-fixation. Different approaches for imaging these macromolecular assemblies to obtain better structure preservation and higher resolution are presented as unpublished data. This study provides insights into spherule organization and distribution of viral components. The results of this and other experiments presented in this thesis will challenge currently accepted models for virus replication complex formation and function. In a revisitation of our previous models, the second part of this work provides the first complete description of the biogenesis of the CPV-I. The results demonstrate that these virus-induced vacuoles, where hundreds of spherules accumulate at late stages during infection, represent the final phase of a journey initiated at the PM, which apparently serves as a platform for spherule formation. From the PM spherules were internalized by an endocytic event that required the activity of the class I PI3K, caveolin-1, cellular cholesterol and functional actin-myosin network. The resulting neutral endocytic carrier vesicle delivered the spherules to the membrane of pre-existing acidic endosomes via multiple fusion events. Microtubule based transport supported the vectorial transfer of these intermediates to the pericentriolar area where further fusions generated the CPV-I. A signal for spherule internalization was identified in one of the replicase proteins, nsP3. Infections of cells with viruses harbouring a deletion in a highly phosphorylated region of nsP3 did not result in the formation of CPV-Is. Instead, thousands of spherules remained at the PM throughout the infection cycle. Finally, the role of the replicase protein nsP2 during viral RNA replication and transcription was investigated. Three enzymatic activities, protease, NTPase and RNA-triphosphatase were studied with the aid of temperature sensitive mutants in vitro and, when possible, in vivo. The results highlighted the interplay of the different nsP2 functions during different steps of RNA replication and sub-genomic promoter regulation, and suggest that the protein could have different activities when participating in the replication complex or as a free enzyme.

Co-Signaling by Neurotrophic Factors and the Extracellular Matrix for Axonal Growth and Neuronal Survival

Neurotrophic factors (NTFs) and the extracellular matrix (ECM) are important regulators of axonal growth and neuronal survival in mammalian nervous system. Understanding of the mechanisms of this regulation is crucial for the development of posttraumatic therapies and drug intervention in the injured nervous system. NTFs act as soluble, target-derived extracellular regulatory molecules for a wide range of physiological functions including axonal guidance and the regulation of programmed cell death in the nervous system. The ECM determines cell adhesion and regulates multiple physiological functions via short range cell-matrix interactions. The present work focuses on the mechanisms of the action of NTFs and the ECM on axonal growth and survival of cultured sensory neurons from dorsal root ganglia (DRG). We first examined signaling mechanisms of the action of the glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) on axonal growth. GDNF, neurturin (NRTN) and artemin (ART) but not persephin (PSPN) promoted axonal initiation in cultured DRG neurons from young adult mice. This effect required Src family kinase (SFK) activity. In neurons from GFRalpha2-deficient mice, NRTN did not significantly promote axonal initiation. GDNF and NRTN induced extensive lamellipodia formation on neuronal somata and growth cones. This study suggested that GDNF, NRTN and ARTN may serve as stimulators of nerve regeneration under posttraumatic conditions. Consequently we studied the convergence of signaling pathways induced by NTFs and the ECM molecule laminin in the intracellular signaling network that regulates axonal growth. We demonstrated that co-stimulation of DRG neurons with NTFs (GDNF, NRTN or nerve growth factor (NGF)) and laminin leads to axonal growth that requires activation of SFKs. A different, SFK-independent signaling pathway evoked axonal growth on laminin in the absence of the NTFs. In contrast, axonal branching was regulated by SFKs both in the presence and in the absence of NGF. We proposed and experimentally verified a Boolean model of the signaling network triggered by NTFs and laminin. Our results put forward an approach for predictable, Boolean logics-driven pharmacological manipulation of a complex signaling network. Finally we found that N-syndecan, the receptor for the ECM component HB-GAM was required for the survival of neonatal sensory neurons in vitro. We demonstrated massive cell death of cultured DRG neurons from mice deficient in the N-syndecan gene as compared to wild type controls. Importantly, this cell death could not be prevented by NGF the neurotrophin which activates multiple anti-apoptotic cascades in DRG neurons. The survival deficit was observed during first postnatal week. By contrast, DRG neurons from young adult N-syndecan knock-out mice exhibited normal survival. This study identifies a completely new syndecan-dependent type of signaling that regulates cell death in neurons.

Novel Molecular Mechanisms of Arabidopsis Disease Resistance

Plants are capable of recognizing phytopathogens through the perception of pathogen-derived molecules or plant cell-wall degradation products due to the activities of pathogen-secreted enzymes. Such elicitor recognition events trigger an array of inducible defense responses involving signal transduction networks and massive transcriptional re-programming. The outcome of a pathogen infection relies on the balance between different signaling pathways, which are integrated by regulatory proteins. This thesis characterized two key regulatory components: a damage control enzyme, chlorophyllase 1 (AtCHL1), and a transcription factor, WRKY70. Their roles in defense signaling were then investigated. The Erwinia-derived elicitors rapidly activated the expression of AtCLH1 and WRKY70 through different signaling pathways. The expression of the AtCHL1 gene was up-regulated by jasmonic acid (JA) but down-regulated by salicylic acid (SA), whereas WRKY70 was activated by SA and repressed by JA. In order to elucidate the functions of AtCLH1 and WRKY70 in plant defense, stable transgenic lines were produced where these genes were overexpressed or silenced. Additionally, independent knockout lines were also characterized. Bacterial and fungal pathogens were then used to assess the contribution of these genes to the Arabidopsis disease resistance. The transcriptional modulation of AtCLH1 by either the constitutive over-expression or RNAi silencing caused alterations in the chlorophyll-to-chlorophyllide ratio, supporting the claim that chlorophyllase 1 has a role in the chlorophyll degradation pathway. Silencing of this gene led to light-dependent over-accumulation of the reactive oxygen species (ROS) in response to infection by Erwinia carotovora subsp. carotovora SCC1. This was followed by an enhanced induction of SA-dependent defense genes and an increased resistance to this pathogen. Interestingly, little effect on the pathogen-induced SA accumulation at the early infection was observed, suggesting that action of ROS might potentiate SA signaling. In contrast, the pathogen-induced JA production was significantly reduced in the RNAi silenced plants. Moreover, JA signaling and resistance to Alternaria brassicicola were impaired. These observations provide support for the argument that the ROS generated in chloroplasts might have a negative impact on JA signaling. The over-expression of WRKY70 resulted in an enhanced resistance to E. carotovora subsp. carotovora SCC1, Pseudomonas syringae pv. tomato DC3000 and Erysiphe cichoracearum UCSC1, whilst an antisense suppression or an insertional inactivation of WRKY70 led to a compromised resistance to E. carotovora subsp. carotovora SCC1 and to E. cichoracearum UCSC1 but not to P. syringae pv. tomato DC3000. Gene expression analysis revealed that WRKY70 activated many known defense-related genes associated with the SAR response but suppressed a subset of the JA-responsive genes. In particular, I was able to show that both the basal and the induced expression of AtCLH1 was enhanced by the antisense silencing or the insertional inactivation of WRKY70, whereas a reduction in AtCLH1 expression was observed in the WRKY70 over-expressors following an MeJA application or an A. brassicicola infection. Moreover, the SA-induced suppression of AtCLH1 was relieved in wrky70 mutants. These results indicate that WRKY70 down-regulates AtCLH1. An epistasis analysis suggested that WRKY70 functions downstream of the NPR1 in an SA-dependent signaling pathway. When challenged with A. brassicicola, WRKY70 over-expressing plants exhibited a compromised disease resistance while wrky70 mutants had the opposite effect. These results confirmed the WRKY70-mediated inhibitory effects on JA signaling. Furthermore, the WRKY70-controlled suppression of A. brassicicola resistance was mainly through an NPR1-dependent mechanism. Taking all the data together, I suggest that the pathogen-responsive transcription factor WRKY70 is a common component in both SA- and JA-dependent pathways and plays a crucial role in the SA-mediated suppression of JA signaling.

Postoperative Volume Therapy in Cardiac Surgery : Effects on Hemostatic and Circulatory Variables

Background: Patients may need massive volume-replacement therapy after cardiac surgery because of large fluid transfer perioperatively, and the use of cardiopulmonary bypass. Hemodynamic stability is better maintained with colloids than crystalloids but colloids have more adverse effects such as coagulation disturbances and impairment of renal function than do crystalloids. The present study examined the effects of modern hydroxyethyl starch (HES) and gelatin solutions on blood coagulation and hemodynamics. The mechanism by which colloids disturb blood coagulation was investigated by thromboelastometry (TEM) after cardiac surgery and in vitro by use of experimental hemodilution. Materials and methods: Ninety patients scheduled for elective primary cardiac surgery (Studies I, II, IV, V), and twelve healthy volunteers (Study III) were included in this study. After admission to the cardiac surgical intensive care unit (ICU), patients were randomized to receive different doses of HES 130/0.4, HES 200/0.5, or 4% albumin solutions. Ringer’s acetate or albumin solutions served as controls. Coagulation was assessed by TEM, and hemodynamic measurements were based on thermodilutionally measured cardiac index (CI). Results: HES and gelatin solutions impaired whole blood coagulation similarly as measured by TEM even at a small dose of 7 mL/kg. These solutions reduced clot strength and prolonged clot formation time. These effects were more pronounced with increasing doses of colloids. Neither albumin nor Ringer’s acetate solution disturbed blood coagulation significantly. Coagulation disturbances after infusion of HES or gelatin solutions were clinically slight, and postoperative blood loss was comparable with that of Ringer’s acetate or albumin solutions. Both single and multiple doses of all the colloids increased CI postoperatively, and this effect was dose-dependent. Ringer’s acetate had no effect on CI. At a small dose (7 mL/kg), the effect of gelatin on CI was comparable with that of Ringer’s acetate and significantly less than that of HES 130/0.4 (Study V). However, when the dose was increased to 14 and 21 mL/kg, the hemodynamic effect of gelatin rose and became comparable with that of HES 130/0.4. Conclusions: After cardiac surgery, HES and gelatin solutions impaired clot strength in a dose-dependent manner. The potential mechanisms were interaction with fibrinogen and fibrin formation, resulting in decreased clot strength, and hemodilution. Although the use of HES and gelatin inhibited coagulation, postoperative bleeding on the first postoperative morning in all the study groups was similar. A single dose of HES solutions improved CI postoperatively more than did gelatin, albumin, or Ringer’s acetate. However, when administered in a repeated fashion, (cumulative dose of 14 mL/kg or more), no differences were evident between HES 130/0.4 and gelatin.