Alternate pathways of the early secretory route in yeast

The diversity of functions of eukaryotic cells is preserved by enclosing different enzymatic activities into membrane-bound organelles. Separation of exocytic proteins from those which remain in the endoplasmic reticulum (ER) casts the foundation for correct compartmentalization. The secretory pathway, starting from the ER membrane, operates by the aid of cytosolic coat proteins (COPs). In anterograde transport, polymerization of the COPII coat on the ER membrane is essential for the ER exit of proteins. Polymerization of the COPI coatomer on the cis-Golgi membrane functions for the retrieval of proteins from the Golgi for repeated use in the ER. The COPII coat is formed by essential proteins; Sec13/31p and Sec23/24p have been thought to be indispensable for the ER exit of all exocytic proteins. However, we found that functional Sec13p was not required for the ER exit of yeast endogenous glycoprotein Hsp150 in the yeast Saccharomyces cerevisiae. Hsp150 turned out to be an ATP phosphatase. ATP hydrolysis by a Walker motif located in the C-terminal domain of Hsp150 was an active mediator for the Sec13p and Sec24p independent ER exit. Our results suggest that in yeast cells a fast track transport route operates in parallel with the previously described cisternal maturation route of the Golgi. The fast track is used by Hsp150 with the aid of its C-terminal ATPase activity at the ER-exit. Hsp150 is matured with a half time of less than one minute. The cisternal maturation track is several-fold slower and used by other exocytic proteins studied so far. Operative COPI coat is needed for ER exit by a subset of proteins but not by Hsp150. We located a second active determinant to the Hsp150 polypeptide s N-terminal portion that guided also heterologous fusion proteins out of the ER in COPII coated vesicles under non-functional COPI conditions for several hours. Our data indicate that ER exit is a selective, receptor-mediated event, not a bulk flow. Furthermore, it suggests the existence of another retrieval pathway for essential reusable components, besides the COPI-operated retrotransport route. Additional experiments suggest that activation of the COPI primer, ADP ribosylation factor (ARF), is essential also for Hsp150 transport. Moreover, it seemed that a subset of proteins directly needed activated ARF in the anterograde transport to complete the ER exit. Our results indicate that coat structures and transport routes are more variable than it has been imagined.

Roles of forced nicotine exposure and Comt gene disruption in the development of addiction-related behavioural and neurochemical changes in mice

Activation of midbrain dopamine systems is thought to be critically involved in the addictive properties of abused substances. Drugs of abuse increase dopamine release in the nucleus accumbens and dorsal striatum, which are the target areas of mesolimbic and nigrostriatal dopamine pathways, respectively. Dopamine release in the nucleus accumbens is thought to mediate the attribution of incentive salience to rewards, and dorsal striatal dopamine release is involved in habit formation. In addition, changes in the function of prefrontal cortex (PFC), the target area of mesocortical dopamine pathway, may skew information processing and memory formation such that the addict pays an abnormal amount of attention to drug-related cues. In this study, we wanted to explore how long-term forced oral nicotine exposure or the lack of catechol-O-methyltransferase (COMT), one of the dopamine metabolizing enzymes, would affect the functioning of these pathways. We also wanted to find out how the forced nicotine exposure or the lack of COMT would affect the consumption of nicotine, alcohol, or cocaine. First, we studied the effect of forced chronic nicotine exposure on the sensitivity of dopamine D2-like autoreceptors in microdialysis and locomotor activity experiments. We found that the sensitivity of these receptors was unchanged after forced oral nicotine exposure, although an increase in the sensitivity was observed in mice treated with intermittent nicotine injections twice daily for 10 days. Thus, the effect of nicotine treatment on dopamine autoreceptor sensitivity depends on the route, frequency, and time course of drug administration. Second, we investigated whether the forced oral nicotine exposure would affect the reinforcing properties of nicotine injections. The chronic nicotine exposure did not significantly affect the development of conditioned place preference to nicotine. In the intravenous self-administration paradigm, however, the nicotine-exposed animals self-administered nicotine at a lower unit dose than the control animals, indicating that their sensitivity to the reinforcing effects of nicotine was enhanced. Next, we wanted to study whether the Comt gene knock-out animals would be a suitable model to study alcohol and cocaine consumption or addiction. Although previous work had shown male Comt knock-out mice to be less sensitive to the locomotor-activating effects of cocaine, the present study found that the lack of COMT did not affect the consumption of cocaine solutions or the development of cocaine-induced place preference. However, the present work did find that male Comt knock-out mice, but not female knock-out mice, consumed ethanol more avidly than their wild-type littermates. This finding suggests that COMT may be one of the factors, albeit not a primary one, contributing to the risk of alcoholism. Last, we explored the effect of COMT deficiency on dorsal striatal, accumbal, and prefrontal cortical dopamine metabolism under no-net-flux conditions and under levodopa load in freely-moving mice. The lack of COMT did not affect the extracellular dopamine concentrations under baseline conditions in any of the brain areas studied. In the prefrontal cortex, the dopamine levels remained high for a prolonged time after levodopa treatment in male, but not female, Comt knock-out mice. COMT deficiency induced accumulation of 3,4-dihydroxyphenylacetic acid, which increased further under levodopa load. Homovanillic acid was not detectable in Comt knock-out animals either under baseline conditions or after levodopa treatment. Taken together, the present results show that although forced chronic oral nicotine exposure affects the reinforcing properties of self-administered nicotine, it is not an addiction model itself. COMT seems to play a minor role in dopamine metabolism and in the development of addiction under baseline conditions, indicating that dopamine function in the brain is well-protected from perturbation. However, the role of COMT becomes more important when the dopaminergic system is challenged, such as by pharmacological manipulation.

Truth, Proof and Gödelian Arguments : A Defence of Tarskian Truth in Mathematics

One of the most fundamental questions in the philosophy of mathematics concerns the relation between truth and formal proof. The position according to which the two concepts are the same is called deflationism, and the opposing viewpoint substantialism. In an important result of mathematical logic, Kurt Gödel proved in his first incompleteness theorem that all consistent formal systems containing arithmetic include sentences that can neither be proved nor disproved within that system. However, such undecidable Gödel sentences can be established to be true once we expand the formal system with Alfred Tarski s semantical theory of truth, as shown by Stewart Shapiro and Jeffrey Ketland in their semantical arguments for the substantiality of truth. According to them, in Gödel sentences we have an explicit case of true but unprovable sentences, and hence deflationism is refuted. Against that, Neil Tennant has shown that instead of Tarskian truth we can expand the formal system with a soundness principle, according to which all provable sentences are assertable, and the assertability of Gödel sentences follows. This way, the relevant question is not whether we can establish the truth of Gödel sentences, but whether Tarskian truth is a more plausible expansion than a soundness principle. In this work I will argue that this problem is best approached once we think of mathematics as the full human phenomenon, and not just consisting of formal systems. When pre-formal mathematical thinking is included in our account, we see that Tarskian truth is in fact not an expansion at all. I claim that what proof is to formal mathematics, truth is to pre-formal thinking, and the Tarskian account of semantical truth mirrors this relation accurately. However, the introduction of pre-formal mathematics is vulnerable to the deflationist counterargument that while existing in practice, pre-formal thinking could still be philosophically superfluous if it does not refer to anything objective. Against this, I argue that all truly deflationist philosophical theories lead to arbitrariness of mathematics. In all other philosophical accounts of mathematics there is room for a reference of the pre-formal mathematics, and the expansion of Tarkian truth can be made naturally. Hence, if we reject the arbitrariness of mathematics, I argue in this work, we must accept the substantiality of truth. Related subjects such as neo-Fregeanism will also be covered, and shown not to change the need for Tarskian truth. The only remaining route for the deflationist is to change the underlying logic so that our formal languages can include their own truth predicates, which Tarski showed to be impossible for classical first-order languages. With such logics we would have no need to expand the formal systems, and the above argument would fail. From the alternative approaches, in this work I focus mostly on the Independence Friendly (IF) logic of Jaakko Hintikka and Gabriel Sandu. Hintikka has claimed that an IF language can include its own adequate truth predicate. I argue that while this is indeed the case, we cannot recognize the truth predicate as such within the same IF language, and the need for Tarskian truth remains. In addition to IF logic, also second-order logic and Saul Kripke s approach using Kleenean logic will be shown to fail in a similar fashion.

The Walls of Agios Donatos Zervochoriou : A case study of a fortress in Thesprotia, northwestern Greece

The study deals with the dating and the function of the fortress of Agios Donatos, which is located in the Kokytos river valley in Thesprotia, northwestern Greece. To solve the dating problem one had to use parallels found in research literature, preferably within as close range as possible. As most of the fortresses within a close proximity had not been adequately published, one had to use parallels within a larger area, throughout the Hellenistic world. Archaeological material found in trial trenches on site was used when possible. To think of the function of the site one had to study the site itself and its relation to the environment, and to compare the situation with the parallels found in research literature, mostly different archaeological survey projects in Greece and Asia Minor. The fortress was built during a period ranging from the final decades of the fourth century down to the mid-third century BC. Most likely it was built during the first quarter of the third century, that is the reign of Pyrrhus of Epirus when the area experienced a boom in fortress building activity. The function of the site was most likely to protect a trade route from the major port in the area to the central areas of the valley and on to Kalamas river, which was the major route on to Macedon. In the end an appendix dealing with the other fortified sites in Thesprotia has been compiled.

Constructing skilled images

When experts construct mental images, they do not rely only on perceptual features; they also access domain-specific knowledge and skills in long-term memory, which enables them to exceed the capacity limitations of the short-term working memory system. The central question of the present dissertation was whether the facilitating effect of long-term memory knowledge on working memory imagery tasks is primarily based on perceptual chunking or whether it relies on higher-level conceptual knowledge. Three domains of expertise were studied: chess, music, and taxi driving. The effects of skill level, stimulus surface features, and the stimulus structure on incremental construction of mental images were investigated. A method was developed to capture the chunking mechanisms that experts use in constructing images: chess pieces, street names, and visual notes were presented in a piecemeal fashion for later recall. Over 150 experts and non-experts participated in a total of 13 experiments, as reported in five publications. The results showed skill effects in all of the studied domains when experts performed memory and problem solving tasks that required mental imagery. Furthermore, only experts' construction of mental images benefited from meaningful stimuli. Manipulation of the stimulus surface features, such as replacing chess pieces with dots, did not significantly affect experts' performance in the imagery tasks. In contrast, the structure of the stimuli had a significant effect on experts' performance in every task domain. For example, taxi drivers recalled more street names from lists that formed a spatially continuous route than from alphabetically organised lists. The results suggest that the mechanisms of conceptual chunking rather than automatic perceptual pattern matching underlie expert performance, even though the tasks of the present studies required perception-like mental representations. The results show that experts are able to construct skilled images that surpass working memory capacity, and that their images are conceptually organised and interpreted rather than merely depictive.

Minimalistinen yhdyssanateoria ja käsitteiden yhdistely

The thesis aims to link the biolinguistic research program and the results of studies in comceptual combination from cognitive psychology. The thesis derives a theory of syntactic structure of noun and adjectival compounds from the Empty Lexicon Hypothesis. Two compound-forming operations are described: root-compounding and word-compounding. The aptness of theory is tested with finnish and greek compounds. From the syntactic theory semantic requirements for conceptual system are derived, especially requirements for handling morphosyntactic features. These requirements are compared to three formidable theories of conceptual combination: relation theory CARIN, Dual-Process theory and C3-theory. The claims of explanatory power of relational distributions of modifier in CARIN-theory ared discarded, as the method for sampling and building relational distributions is not reliable and the algorithmic instantiation of theory does not compute what it claims to compute. From relational theory there still remains results supporting existence of 'easy' relations for certain concepts. Dual-Process theory is found to provide results that cannot in theory be affected by linguistic system, but the basic idea of property compounds is kept. C3-theory is found to be not computationally realistic, but the basic results of diagnosticity and local properties (domains) of conceptual system are solid. The three conceptual combination models are rethought as a problem of finding the shortest route between the two concepts. The new basis for modeling is suggested to be bare conceptual landscape with morphosyntactiic or semantic features working as guidance and structural features of landscape basically unknown, but such as they react to features from linguistic system. Minimalistic principles to conceptual modeling are suggested.

Keltaista ja kimaltavaa : kodin ja koulun yhteistyöstä koulun ja kodin yhteyteen

Through this study I aim to portray connections between home and school through the patterns of thought and action shared in everyday life in a certain community. My observations are primarily based upon interviews, writings and artwork by people from home (N=32) and school (N=13) contexts. Through the stories told, I depict the characters and characteristic features of the home-school interaction by generations. According to the material, in the school days of the grandparents the focus was on discipline and order. For the parents, the focus had shifted towards knowledge, while for the pupils today, the focus lies on evaluation, through which the upbringing of the child is steered towards favourable outcomes. Teachers and those people at home hold partially different understandings of home-school interaction, both of its manifested forms and potentials. The forms of contact in use today are largely seen as one-sided. Yearning for openness and regularity is shared by both sides, yet understood differently. Common causes for failure are said to lie in plain human difficulties in communication and social interaction, but deeply rooted traditions regarding forms of contact also cast a shadow on the route to successful co-operation. This study started around the idea, that home-school interaction should be steered towards the ex-change of constructive ideas between both the home and school environments. Combining the dif-ferent views gives to something to build upon. To test this idea, I drafted a practice period, which was implemented in a small pre-school environment in the fall of 1997. My focus of interest in this project was on the handling of ordinary life information in the schools. So I combined individual views, patterns of knowledge and understanding of the world into the process of teaching. Works of art and writings by the informants worked as tools for information processing and as practical forms of building home-school interaction. Experiences from the pre-school environ-ment were later on echoed in constructing home-school interaction in five other schools. In both these projects, the teaching in the school was based on stories, thoughts and performances put to-gether by the parents, grandparents and children at home. During these processes, the material used in this study, consisting of artwork, writings and interviews (N=501), was collected. The data shows that information originating from the home environments was both a motivating and interesting addition to the teaching. There even was a sense of pride when assessing the seeds of knowledge from one’s own roots. In most cases and subjects, the homegrown information content was seamlessly connected to the functions of school and the curriculum. This project initiated thought processes between pupils and teachers, adults, children and parents, teachers and parents, and also between generations. It appeared that many of the subjects covered had not been raised before between the various participant groups. I have a special interest here in visual expression and its various contextual meanings. There art material portrays how content matter and characteristic features of the adult and parent contexts reflect in the works of the children. Another clearly noticeable factor in the art material is the impact of time-related traditions and functions on the means of visual expression. Comparing the visual material to the written material reveals variances of meaning and possibilities between these forms of expression. The visual material appears to be related especially to portraying objects, action and usage. Processing through that making of images was noted to bring back memories of concrete structures, details and also emotions. This process offered the child an intensive social connection with the adults. In some cases, with children and adults alike, this project brought forth an ongoing relation to visual expression. During this study I end up changing the concept to ‘home-school collaboration’. This widely used concept guides and outlines the interaction between schools and homes. In order to broaden the field of possibilities, I choose to use the concept ‘school-home interconnection’. This concept forms better grounds for forming varying impressions and practices when building interactive contexts. This concept places the responsibility of bridging the connection-gap in the schools. Through the experiences and innovations of thought gained from these projects, I form a model of pedagogy that embraces the idea of school-home interconnection and builds on the various impres-sions and expressions contained in it. In this model, school makes use of the experiences, thoughts and conceptions from the home environment. Various forms of expression are used to portray and process this information. This joint evaluation and observation evolves thought patterns both in school and at home. Keywords: percieving, visuality, visual culture, art and text, visual expression, art education, growth in interaction, home-school collaboration, school-home interconnection, school-home interaction model.

Effects of oral calcium sensitizers on experimental heart failure

Suun kautta annosteltava kalsiumherkistäjä parantaa sydämen vajaatoimintaan liittyvää pumppausvajetta kokeellisissa sydämen vajaatoimintamalleissa Huolimatta viime vuosikymmenien lääketieteellisestä kehityksestä krooninen sydämen vajaatoiminta on silti edelleen vakava, elämänlaatua voimakkaasti rajoittava sairaus. Kalsiumherkistäjät ovat uusi, sydämen pumppausvoimaa lisäävä lääkeryhmä. Levosimendaani, kotimaista alkuperää oleva kalsiumherkistäjä, on kliinisessä käytössä akuutin vajaatoiminnan hoitoon suonensisäisesti ja lyhytaikaisesti annosteltavana valmisteena. Levosimendaanilla on aktiivinen metaboliitti, OR-1896, jonka oletetaan olevan vuorokauden mittaisen levosimendaani-infuusion jälkeen havaittujen useita päiviä kestävien hyödyllisisten vaikutuksisten takana. Levosimendaanin kroonisen, suun kautta tapahtuvan annostelun vaikutuksista tieto on vähäisempää, mutta sillä näyttää olevan positiivisia vaikutuksia potilaiden raportoimana. FM Marjut Louhelainen on selvittänyt väitöskirjassaan suun kautta annosteltavan levosimendaanin ja sen pitkäkestoisen aktiivisen metaboliitin vaikutuksia kroonisen vajaatoiminnan hoidossa käyttämällä sekä hypertensiivisen sydäntaudin että 2 tyypin diabeteksen komplisoimaan sydäninfarktin kokeellisia malleja. Tutkimuksessa selvitettiin lisäksi vajaatoimintaan johtavia molekyylitason tapahtumia sydänlihaksessa. Tutkimuksessa osoitettiin, että krooninen suun kautta annosteltu hoito sekä kalsiumherkistäjä levosimendaanilla että sen aktiivisella metaboliitilla estää hypertensiiviseen sydämen vajaatoiminnan aikaasaamaa sydämen uudelleenmuovaantumista ja siihen liittyvää kuolleisuutta. Nämä vaikutukset välittyivät vähentyneen sydänlihassoluhypertrofian, solukuolleisuuden ja neurohumaraalisen aktivaation kautta. Levosimendaanin ja OR-1896:n osoitettiin myös parantavan sydämen pumppausfunktiota tyyppi 2 diabeteksen komplisoimassa sydäninfarktissa. Ei-diabeettiseen tilanteeseen verrattuna diabetekseen liittyvä infarktin jälkeinen vajaatoiminnan kehitys oli yhteydessä lisääntyneeseen tulehdukseen, fibroosiin, solukuolemaan, neurohumoraaliseen aktivaatioon ja ennenaikaiseen kudoksen vanhenemiseen. Sekä levosimendaani, että OR-1869 vähensivät tulehduksen, fibroosin ja solukuoleman merkkejä ja vaimensi neurohumoraalista aktivaatiota. OR-1896 myös vähensi solujen vanhenemiseen liittyvien merkkiaineiden ilmentymistä. Väitöskirjassa todettiin, että suun kautta annosteltuna sekä levosimendaani, että sen aktiivinen metaboliitti OR-1896, omaavat terapeuttista potentiaalia sekä hypertensiivisen sydäntaudin hoitoon että sydäninfarktin jälkeisen vajaatoiminnan estoon. FM Marjut Louhelaisen farmakologian alaan kuuluva väitöskirja Effects of oral calcium sensitizers on experimental heart failure tarkastetaan Helsingin yliopiston Lääketieteellisessä tiedekunnassa perjantaina 29.01.2010 klo 12 (Biomedicum Helsinki, luentosali 2, Haartmaninkatu 8, Helsinki). Vastaväittäjänä toimii professori Raimo Tuominen, Helsingin yliopiston Farmasian tiedekunnasta ja kustoksena professori Eero Mervaala Helsingin yliopiston Lääketieteellisestä tiedekunnasta.

Rab8 and Rab8-interacting proteins as players in cell polarization

Rab8 and its interacting proteins as regulators of cell polarization During the development of a multi-cellular organism, progenitor cells have to divide and migrate appropriately as well as organize their differentiation with one another, in order to produce a viable embryo. To divide, differentiate and migrate cells have to undergo polarization, a process where internal and external components such as actin, microtubules and adhesion receptors are reorganized to produce a cell that is asymmetric, with functionally different surfaces. Also in the adult organism there is a continuous need for these processes, as cells need to migrate in response to tissue damage and to fight infection. Improper regulation of cell proliferation and migration can conversely lead to disease such as cancer. GTP-binding proteins function as molecular switches by cycling between a GTP-bound (active) conformation and a GDP-bound (inactive) conformation. The Ras super-family of small GTPases are found in all eukaryotic cells. They can be functionally divided into five subfamilies. The Ras family members mainly regulate gene expression, controlling cell proliferation and differentiation. Ras was in fact the first human oncogene to be characterized, and as much as 30% of all human tumors may be directly or indirectly caused by mutations of Ras molecules The Rho family members mainly regulate cytoskeletal reorganization. Arf proteins are known to regulate vesicle budding and Rab proteins regulate vesicular transport. Ran regulates nuclear transport as well as microtubule organization during mitosis. The focus of the thesis of Katarina Hattula, is on Rab8, a small GTPase of the Rab family. Activated Rab8 has previously been shown to induce the formation of new surface extensions, reorganizing both actin and microtubules, and to have a role in directed membrane transport to cell surfaces. However, the exact membrane route it regulates has remained elusive. In the thesis three novel interactors of Rab8 are presented. Rabin8 is a Rab8-specific GEF that localizes to vesicles where it presumably recruits and activates its target Rab8. Its expression in cells leads to remodelling of actin and the formation of polarized cell surface domains. Optineurin, known to be associated with a leading cause of blindness in humans (open-angle glaucoma), is shown to interact specifically with GTP-bound Rab8. Rab8 binds to an amino-terminal region and interestingly, the Huntingtin protein binds a carboxy-terminal region of optineurin. (Aberrant Huntingtin protein is known to be the cause Huntington s disease in humans.) Co-expression of Huntingtin and optineurin enhanced the recruitment of Huntingtin to Rab8-positive vesicular structures. Furthermore, optineurin promoted cell polarization in a similar way to Rab8. A third novel interactor of Rab8 presented in this thesis is JFC1, a member of the synaptogamin-like protein (Slp) family. JFC1 interacts with Rab8 specifically in its GTP-bound form, co-localizes with endogenous Rab8 on tubular and vesicular structures, and is probably involved in controlling Rab8 membrane dynamics. Rab8 is in this thesis work clearly shown to have a strong effect on cell shape. Blocking Rab8 activity by expression of Rab8 RNAi, or by expressing the dominant negative Rab8 (T22N) mutant leads to loss of cell polarity. Conversely, cells expressing the constitutively active Rab8 (Q67L) mutant exhibit a strongly polarized phenotype. Experiments in live cells show that Rab8 is associated with macropinosomes generated at ruffling areas of the membrane. These macropinosomes fuse with or transform into tubules that move toward the cell centre, from where they are recycled back to the leading edge to participate in protrusion formation. The biogenesis of these tubules is shown to be dependent on both actin and microtubule dynamics. The Rab8-specific membrane route studied contained several markers known to be internalized and recycled (1 integrin, transferrin, transferrin receptor, cholera toxin B subunit (CTxB), and major histocompatibility complex class I protein (MHCI)). Co-expression studies revealed that Rab8 localization overlaps with that of Rab11 and Arf6. Rab8 is furthermore clearly functionally linked to Arf6. The data presented in this thesis strongly suggests a role for Rab8 as a regulator for a recycling compartment, which is involved in providing structural and regulatory components to the leading edge to participate in protrusion formation.

Regulation of tumor suppressor protein p53 in cellular stress and tumorigenesis

Functional loss of tumor suppressor protein p53 is a common feature in diverse human cancers. The ability of this protein to sense cellular damage and halt the progression of the cell cycle or direct the cells to apoptosis is essential in preventing tumorigenesis. Tumors having wild-type p53 also respond better to current chemotherapies. The loss of p53 function may arise from TP53 mutations or dysregulation of factors controlling its levels and activity. Probably the most significant inhibitor of p53 function is Mdm2, a protein mediating its degradation and inactivation. Clearly, the maintenance of a strictly controlled p53-Mdm2 route is of great importance in preventing neoplastic transformation. Moreover, impairing Mdm2 function could be a nongenotoxic way to increase p53 levels and activity. Understanding the precise molecular mechanisms behind p53-Mdm2 relationship is thus essential from a therapeutic point of view. The aim of this thesis study was to discover factors affecting the negative regulation of p53 by Mdm2, causing activation of p53 in stressed cells. As a model of cellular damage, we used UVC radiation, inducing a complex cellular stress pathway. Exposure to UVC, as well as to several chemotherapeutic drugs, causes robust transcriptional stress in the cells and leads to activation of p53. By using this model of cellular stress, our goal was to understand how and by which proteins p53 is regulated. Furthermore, we wanted to address whether these pathways affecting p53 function could be altered in human cancers. In the study, two different p53 pathway proteins, nucleophosmin (NPM) and promyelocytic leukemia protein (PML), were found to participate in the p53 stress response following UV stress. Subcellular translocations of these proteins were discovered rapidly after exposure to UV. The alterations in the cellular localizations were connected to transient interactions with p53 and Mdm2, implicating their significance in the regulation of p53 stress response. NPM was shown to control Mdm2-p53 interface and mediate p53 stabilization by blocking the ability of Mdm2 to promote p53 degradation. Furthermore, NPM mediated p53 stabilization upon viral insult. We further detected a connection between cellular pathways of NPM and PML, as PML was found to associate with NPM in UV-radiated cells. The observed temporal UV-induced interactions strongly imply existence of a multiprotein complex participating in the p53 response. In addition, PML controlled the UV response of NPM, its localization and complex formation with chromatin associated factors. The relevance of the UV-promoted interactions was demonstrated in studies in a human leukemia cell line, being under abnormal transcriptional repression due to expression of oncogenic PML-RARa fusion protein. Reversing the leukemic phenotype with a therapeutically significant drug was associated with similar complex formation between p53 and its partners as following UV. In conclusion, this thesis study identifies novel p53 pathway interactions associated with the recovery from UV-promoted as well as oncogenic transcriptional repression.

Rhizoctonia solani as a potato pathogen : Variation of isolates in Finland and host response

Rhizoctonia solani is a soil inhabiting basidiomycetous fungus able to induce a wide range of symptoms in many plant species. This genetically complex species is divided to 13 anastomosis groups (AG), of which AG-3 is specialized to infect potato. However, also a few other AGs are able to infect or live in close contact with potato. On potato, R. solani infection causes two main types of diseases including stem canker observed as a dark brown lesions on developing stems and stolons, and black scurf that develops on new tubers close to the time of harvest. These disease symptoms are collectively called a ‘Rhizoctonia disease complex’. Between the growing seasons R. solani survives in soil and plant debri as sclerotia or as the sclerotia called black scurf on potato tubers which when used as seed offer the main route for dispersal of the fungus to new areas. The reasons for the dominance of AG-3 on potato seem to be attributable to its highly specialization to potato and its ability to infect and form sclerotia efficiently at low temperatures. In this study, a large nationwide survey of R. solani isolates was made in potato crops in Finland. Almost all characterized isolates belonged to AG-3. Additionally, three other AGs (AG-2-1, AG-4 and AG-5) were found associated with symptoms on potato plants but they were weaker pathogens on potato than AG-3 as less prone to form black scurf. According to phylogenetic analysis of the internal transcribed sequences (ITS) of the ribosomal RNA genes the Finnish AG-3 isolates are closely related to each other even though a wide variation of physiological features was observed between them. Detailed analysis of the ITS regions revealed single nucleotide polymorphism in 14 nucleotide positions of ITS-1 and ITS-2. Additionally, compensatory base changes on ITS-2 were detected which suggests that potato-infecting R. solani AG-3 could be considered as a separate species instead of an AG of R. solani. For the first time, molecular defence responses were studied and detected during the early phases of interaction between R. solani AG-3 and potato. Extensive systemic signalling for defence exploiting several known defence pathways was activated as soon as R. solani came into close contact with the base of a sprout. The defence response was strong enough to protect vulnerable sprout tips from new attacks by the pathogen. These results at least partly explain why potato emergence is eventually successful even under heavy infection pressure by R. solani.

L-rhamnose-1-dehydrogenase gene and L-rhamnose catabolism in the yeast Pichia stipitis

The purpose of this work was to identify some of the genes of the catabolic route of L-rhamnose in the yeast Pichia stipitis. There are at least two distinctly different pathways for L-rhamnose catabolism. The one described in bacteria has phosphorylated intermediates and the enzymes and the genes of this route have been described. The pathway described in yeast does not have phosphorylated intermediates. The intermediates and the enzymes of this pathway are known but none of the genes have been identified. The work was started by purifying the L-rhamnose dehydrogenase, which oxidates L-rhamnose to rhamnonic acid-gamma-lactone. NAD is used as a cofactor in this reaction. A DEAE ion exchange column was used for purification. The active fraction was further purified using a non-denaturing PAGE and the active protein identified by zymogram staining. In the last step the protein was separated in a SDS-PAGE, the protein band trypsinated and analysed by MALDI-TOF MS. This resulted in the identification of the corresponding gene, RHA1, which was then, after a codon change, expressed in Saccharomyces cerevisiae. Also C- or N-terminal histidine tags were added but as the activity of the enzyme was lost or strongly reduced these were not used. The kinetic properties of the protein were analysed in the cell extract. Substrate specifity was tested with different sugars; L-rhamnose, L-lyxose and L-mannose were oxidated by the enzyme. Vmax values were 180 nkat/mg, 160 nkat/mg and 72 nkat/mg, respectively. The highest affinity was towards L-rhamnose, the Km value being 0.9 mM. Lower affinities were obtained with L-lyxose, Km 4.3 mM, and L-mannose Km 25 mM. Northern analysis was done to study the transcription of RHA1 with different carbon sources. Transcription was observed only on L-rhamnose suggesting that RHA1 expression is L-rhamnose induced. A RHA1 deletion cassette for P. stipitis was constructed but the cassette had integrated randomly and not targeted to delete the RHA1 gene. Enzyme assays for L-lactaldehyde dehydrogenase were done similarly to L-rhamnose dehydrogenase assays. NAD is used as a cofactor also in this reaction where L-lactaldehyde is oxidised to L-lactate. The observed enzyme activities were very low and the activity was lost during the purification procedures.

Components of productivity growth in Finnish agriculture

The objective was to measure productivity growth and its components in Finnish agriculture, especially in dairy farming. The objective was also to compare different methods and models - both parametric (stochastic frontier analysis) and non-parametric (data envelopment analysis) - in estimating the components of productivity growth and the sensitivity of results with respect to different approaches. The parametric approach was also applied in the investigation of various aspects of heterogeneity. A common feature of the first three of five articles is that they concentrate empirically on technical change, technical efficiency change and the scale effect, mainly on the basis of the decompositions of Malmquist productivity index. The last two articles explore an intermediate route between the Fisher and Malmquist productivity indices and develop a detailed but meaningful decomposition for the Fisher index, including also empirical applications. Distance functions play a central role in the decomposition of Malmquist and Fisher productivity indices. Three panel data sets from 1990s have been applied in the study. The common feature of all data used is that they cover the periods before and after Finnish EU accession. Another common feature is that the analysis mainly concentrates on dairy farms or their roughage production systems. Productivity growth on Finnish dairy farms was relatively slow in the 1990s: approximately one percent per year, independent of the method used. Despite considerable annual variation, productivity growth seems to have accelerated towards the end of the period. There was a slowdown in the mid-1990s at the time of EU accession. No clear immediate effects of EU accession with respect to technical efficiency could be observed. Technical change has been the main contributor to productivity growth on dairy farms. However, average technical efficiency often showed a declining trend, meaning that the deviations from the best practice frontier are increasing over time. This suggests different paths of adjustment at the farm level. However, different methods to some extent provide different results, especially for the sub-components of productivity growth. In most analyses on dairy farms the scale effect on productivity growth was minor. A positive scale effect would be important for improving the competitiveness of Finnish agriculture through increasing farm size. This small effect may also be related to the structure of agriculture and to the allocation of investments to specific groups of farms during the research period. The result may also indicate that the utilization of scale economies faces special constraints in Finnish conditions. However, the analysis of a sample of all types of farms suggested a more considerable scale effect than the analysis on dairy farms.

Synthesis, Reactivity and Biological Activity of 17β-HSD1 Inhibitors Based on a Thieno[2,3-d]pryrimidin-4(3H)-one Core

Breast cancer is the most common cancer in women in Western countries. In the early stages of development most breast cancers are hormone-dependent, and estrogens, especially estradiol, have a pivotal role in their development and progression. One approach to the treatment of hormone-dependent breast cancers is to block the formation of the active estrogens by inhibiting the action of the steroid metabolising enzymes. 17beta-Hydroxysteroid dehydrogenase type 1 (17beta-HSD1) is a key enzyme in the biosynthesis of estradiol, the most potent female sex hormone. The 17beta-HSD1 enzyme catalyses the final step and converts estrone into the biologically active estradiol. Blocking 17beta-HSD1 activity with a specific enzyme inhibitor could provide a means to reduce circulating and tumour estradiol levels and thus promote tumour regression. In recent years 17beta-HSD1 has been recognised as an important drug target. Some inhibitors of 17beta-HSD1 have been reported, however, there are no inhibitors on the market nor have clinical trials been announced. The majority of known 17beta-HSD1 inhibitors are based on steroidal structures, while relatively little has been reported on non-steroidal inhibitors. As compared with 17beta-HSD1 inhibitors based on steroidal structures, non-steroidal compounds could have advantages of synthetic accessibility, drug-likeness, selectivity and non-estrogenicity. This study describes the synthesis of large group of novel 17beta-HSD1 inhibitors based on a non-steroidal thieno[2,3-d]pyrimidin-4(3H)-one core. An efficient synthesis route was developed for the lead compound and subsequently employed in the synthesis of thieno[2,3-d]pyrimidin-4(3H)-one based molecule library. The biological activities and binding of these inhibitors to 17beta-HSD1 and, finally, the quantitative structure activity relationship (QSAR) model are also reported. In this study, several potent and selective 17beta-HSD1 inhibitors without estrogenic activity were identified. This establishment of a novel class of inhibitors is a progressive achievement in 17beta-HSD1 inhibitor development. Furthermore, the 3D-QSAR model, constructed on the basis of this study, offers a powerful tool for future 17beta-HSD1 inhibitor development. As part of the fundamental science underpinning this research, the chemical reactivity of fused (di)cycloalkeno thieno[2,3-d]pyrimidin-4(3H)-ones with electrophilic reagents, i.e. Vilsmeier reagent and dimethylformamide dimethylacetal, was investigated. These findings resulted in a revision of the reaction mechanism of Vilsmeier haloformylation and further contributed to understanding the chemical reactivity of this compound class. This study revealed that the reactivity is dependent upon a stereoelectronic effect arising from different ring conformations.

Regio- and stereoselectivity of oxidative coupling reactions of phenols : Spirodienones as construction units in lignin

Dimeric phenolic compounds lignans and dilignols form in the so-called oxidative coupling reaction of phenols. Enzymes such as peroxidases and lac-cases catalyze the reaction using hydrogen peroxide or oxygen respectively as oxidant generating phenoxy radicals which couple together according to certain rules. In this thesis, the effects of the structures of starting materials mono-lignols and the effects of reaction conditions such as pH and solvent system on this coupling mechanism and on its regio- and stereoselectivity have been studied. After the primary coupling of two phenoxy radicals a very reactive quinone me-thide intermediate is formed. This intermediate reacts quickly with a suitable nucleophile which can be, for example, an intramolecular hydroxyl group or another nucleophile such as water, methanol, or a phenolic compound in the reaction system. This reaction is catalyzed by acids. After the nucleophilic addi-tion to the quinone methide, other hydrolytic reactions, rearrangements, and elimination reactions occur leading finally to stable dimeric structures called lignans or dilignols. Similar reactions occur also in the so-called lignification process when monolignol (or dilignol) reacts with the growing lignin polymer. New kinds of structures have been observed in this thesis. The dimeric com-pounds with so-called spirodienone structure have been observed to form both in the dehydrodimerization of methyl sinapate and in the beta-1-type cross-coupling reaction of two different monolignols. This beta-1-type dilignol with a spirodienone structure was the first synthetized and published dilignol model compound, and at present, it has been observed to exist as a fundamental construction unit in lignins. The enantioselectivity of the oxidative coupling reaction was also studied for obtaining enantiopure lignans and dilignols. A rather good enantioselectivity was obtained in the oxidative coupling reaction of two monolignols with chiral auxiliary substituents using peroxidase/H2O2 as an oxidation system. This observation was published as one of the first enantioselective oxidative coupling reaction of phenols. Pure enantiomers of lignans were also obtained by using chiral cryogenic chromatography as a chiral resolution technique. This technique was shown to be an alternative route to prepare enantiopure lignans or lignin model compounds in a preparative scale.