Function and regulation of Cdk7

Understanding the process of cell division is crucial for modern cancer medicine due to the central role of uncontrolled cell division in this disease. Cancer involves unrestrained proliferation as a result of cells loosing normal control and being driven through the cell cycle, where they normally would be non-dividing or quiescent. Progression through the cell cycle is thought to be dependent on the sequential activation of cyclin-dependent kinases (Cdks). The full activation of Cdks requires the phosphorylation of a conserved residue (threonine-160 on human Cdk2) on the T-loop of the kinase domain. In metazoan species, a trimeric complex consisting of Cdk7, cyclin H and Mat1 has been suggested to be the T-loop kinase of several Cdks. In addition, Cdk7 have also been implicated in the regulation of transcription. Cdk7, cyclin H, and Mat1 can be found as subunits of general transcription factor TFIIH. Cdk7, in this context, phosphorylates the Carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (RNA pol II), specifically on serine-5 residues of the CTD repeat. The regulation of Cdk7 in these and other functions is not well known and the unambiguous characterization of the in vivo role of Cdk7 in both T-loop activation and CTD serine-5 phosphorylation has proved challenging. In this study, the fission yeast Cdk7-cyclin H homologous complex, Mcs6-Mcs2, is identified as the in vivo T-loop kinase of Cdk1(Cdc2). It also identifies multiple levels of regulation of Mcs6 kinase activity, i.e. association with Pmh1, a novel fission yeast protein that is the apparent homolog of metazoan Mat1, and T-loop phosphorylation of Mcs6, mediated by Csk1, a monomeric T-loop kinase with similarity to Cak1 of budding yeast. In addition, Skp1, a component of the SCF (Skp1-Cullin-F box protein) ubiquitin ligase is identified by its interactions with Mcs2 and Pmh1. The Skp1 association with Mcs2 and Pmh1 is however SCF independent and does not involve proteolytic degradation but may reflect a novel mechanism to modulate the activity or complex assembly of Mcs6. In addition to Cdk7, also Cdk8 has been shown to have CTD serine-5 kinase activity in vitro. Cdk8 is not essential in yeast but has been shown to function as a transcriptional regulator. The function of Cdk8 is unknown in flies and mammals. This prompted the investigation of murine Cdk8 and its potential role as a redundant CTD serine-5 kinase. We find that Cdk8 is required for development prior to implantation, at a time that is co-incident with a burst of Cdk8 expression during normal development. The results does not support a role of Cdk8 as a serine-5 CTD kinase in vivo but rather shows an unexpected requirement for Cdk8, early in mammalian development. The results presented in this thesis extends our current knowledge of the regulation of the cell cycle by characterizing the function of two distinct cell cycle regulating T-loop kinases, including the unambiguous identification of Mcs6, the fission yeast Cdk7 homolog, as the T-loop kinase of Cdk1. The results also indicate that the function of Mcs6 is conserved from fission yeast to human Cdk7 and suggests novel mechanisms by which the distinct functions of Cdk7 and Mcs6 could be regulated. These findings are important for our understanding of how progression of the cell cycle and proper transcription is controlled, during normal development and tissue homeostasis but also under condition where cells have escaped these control mechanisms e.g. cancer.

Molecular mechanisms of cancer predisposition in HNPCC/Lynch syndrome

Hereditary non-polyposis colorectal carcinoma (HNPCC; Lynch syndrome) is among the most common hereditary cancers in man and a model of cancers arising through deficient DNA mismatch repair (MMR). It is inherited in a dominant manner with predisposing germline mutations in the MMR genes, mainly MLH1, MSH2, MSH6 and PMS2. Both copies of the MMR gene need to be inactivated for cancer development. Since Lynch syndrome family members are born with one defective copy of one of the MMR genes in their germline, they only need to acquire a so called second hit to inactivate the MMR gene. Hence, they usually develop cancer at an early age. MMR gene inactivation leads to accumulation of mutations particularly in short repeat tracts, known as microsatellites, causing microsatellite instability (MSI). MSI is the hallmark of Lynch syndrome tumors, but is present in approximately 15% of sporadic tumors as well. There are several possible mechanisms of somatic inactivation (i.e. the second hit ) of MMR genes, for instance deletion of the wild-type copy, leading to loss of heterozygosity (LOH), methylation of promoter regions necessary for gene transcription, or mitotic recombination or gene conversion. In the Lynch syndrome tumors carrying germline mutations in the MMR gene, LOH was found to be the most frequent mechanism of somatic inactivation in the present study. We also studied MLH1/MSH2 deletion carriers and found that somatic mutations identical to the ones in the germline occurred frequently in colorectal cancers and were also present in extracolonic Lynch syndrome-associated tumors. Chromosome-specific marker analysis implied that gene conversion, rather than mitotic recombination or deletion of the respective gene locus accounted for wild-type inactivation. Lynch syndrome patients are predisposed to certain types of cancers, the most common ones being colorectal, endometrial and gastric cancer. Gastric cancer and uroepithelial tumors of bladder and ureter were observed to be true Lynch syndrome tumors with MMR deficiency as the driving force of tumorigenesis. Brain tumors and kidney carcinoma, on the other hand, were mostly MSS, implying the possibility of alternative routes of tumor development. These results present possible implications in clinical cancer surveillance. In about one-third of families suspected of Lynch syndrome, mutations in MMR genes are not found, and we therefore looked for alternative mechanisms of predisposition. According to our results, large genomic deletions, mainly in MSH2, and germline epimutations in MLH1, together explain a significant fraction of point mutation-negative families suspected of Lynch syndrome and are associated with characteristic clinical and family features. Our findings have important implications in the diagnosis and management of Lynch syndrome families.

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.

Adult-type hypolactasia: Genotype-phenotype correlation

Adult-type hypolactasia (primary lactose malabsorption, lactase non-persistence) is the most common enzyme deficiency worldwide, and manifests with symptoms of lactose intolerance such as abdominal pain, gas formation and diarrhea. In humans with adult-type hypolactasia, lactase activity is high at birth, but declines during childhood to about one-tenth of the activity at birth. In 2002, a one base polymorphism C/T-13910, located 14 kilobases from the starting codon of the lactase-phlorizin hydrolase (LPH) gene was observed to be associated with the persistence of lactase activity. The T-13910 allele (C/T-13910 and T/T-13910 genotypes) associates with persistence of lactase activity throughout life, whereas the C/C-13910 genotype associates with adult-type hypolactasia. In this thesis work, the timing and mechanism of decline of lactase enzyme activity during development was studied using the C/T-13910 polymorphism as a molecular marker. We observed an excellent correlation between low lactase activity and the C/C-13910 genotype in all subjects > 12 years of age, irrespective their ethnicity. In children of African origin, the lactase activity declined somewhat earlier than among Finnish children. Furthermore, we observed an increasing imbalance in the relative lactase mRNA expression from the C-13910 and T-13910 alleles in Finnish children beginning from five years of age. The genetic test for adult-type hypolactasia showed a sensitivity of 93% and a specificity of 100% in the Finnish children and adolescents > 12 years of age. The relation of milk consumption and the milk-related abdominal complaints to the C/T-13910 genotypes associated with lactase persistence/non-persistence was studied by a questionnaire-based approach in > 2100 Finns. Both Finnish children and adults with the C/C-13910 genotype consumed significantly less dairy products compared to those with the C/T-13910 and T/T-13910 genotypes. Flatulence was the only of the abdominal symptoms of lactose intolerance that subjects with the C/C-13910 genotype reported significantly more often than those with the C/T-13910 and T/T-13910 genotypes. A minor proportion (<10%) of subjects with the C/C-13910 genotype, nevertheless, reported drinking milk without any symptoms afterwards. There was no association between cow's milk allergy starting as a newborn and adult-type hypolactasia. In an association study an increased risk of colorectal cancer was observed among those with molecular diagnosis of adult-type hypolactasia. It warrants further studies to clarify whether the increased risk observed in the Finnish population is associated with lactose or decreased intake of dairy products in these subjects.

Molecular pathogenesis of human parechovirus 1 infection

The Parechoviruses (HPEV) belong to the family Picornaviridae of positive-stranded RNA viruses. Although the parechovirus genome shares the general properties of other picornaviruses, the genus has several unique features when compared to other family members. We found that HPEV1 attaches to αv integrins on the cell surface and is internalized through the clathrin-mediated endocytic pathway. During he course of the infection, the Golgi was found to disintegrate and the ER membranes to swell and loose their ribosomes. The replication of HPEV1 was found to take place on small clusters of vesicles which contained the trans-Golgi marker GalT as well as the viral non-structural 2C protein. 2C was additionally found on stretches of modified ER-membranes, seemingly not involved in RNA replication. The viral non-structural 2A and 2C proteins were studied in further detail and were found to display several interesting features. The 2A protein was found to be a RNA-binding protein that preferably binds to positive sense 3 UTR RNA. It was found to bind also duplex RNA containing 3 UTR(+)-3 UTR(-), but not other dsRNA molecules studied. Mutagenesis revealed that the N-terminal basic-rich region as well as the C-terminus, are important for RNA-binding. The 2C protein on the other hand, was found to have both ATP-diphosphohydrolase and AMP kinase activities. Neither dATP nor other NTP:s were suitable substrates. Furthermore, we found that as a result of theses activities the protein is autophosphorylated. The intracellular changes brought about by the individual HPEV1 non-structural proteins were studied through the expression of fusion proteins. None of the proteins expressed were able to induce membrane changes similar to those seen during HPEV1 infection. However, the 2C protein, which could be found on the surface of lipid droplets but also on diverse intracellular membranes, was partly relocated to viral replication complexes in transfected, superinfected cells. Although Golgi to ER traffic was arrested in HPEV1-infected cells, none of the individually expressed non-structural proteins had any visible effect on the anterograde membrane traffic. Our results suggest that the HPEV1 replication strategy is different from that of many other picornaviruses. Furthermore, this study shows how relatively small differences in genome sequence result in very different intracellular pathology.

Campylobacter Infections of Domestic Origin in Finland

Campylobacter jejuni and C. coli are the leading causes of human bacterial gastroenteritis in developed countries. Most human Campylobacter infections are sporadic and a seasonal peak in the distribution of infections can be seen in the summer months in several countries, including Finland. A variety of risk factors for Campylobacter infections have been identified; handling and eating poultry, drinking unpasteurized milk, contact with domestic animals, and travelling abroad. However, the relative importance of the different risk factors in sporadic cases of Campylobacter infection remains unknown. In most cases, the infection is self-limiting and no specific treatment is required. Campylobacter enteritis can cause a wide range of complications, including reactive arthritis (ReA) that is reported in 1-5% of the cases. Seven clinical microbiology laboratories serving different geographical areas of Finland, participated in this multi-centre study, conducted during a seasonal peak in 2002. In a matched case-control study, domestically-acquired sporadic Campylobacter infections from three geographical areas were collected. The final study comprised 100 cases and 137 controls. Risk factors for sporadic domestically-acquired Campylobacter infections were identified on the basis of a questionnaire; swimming in natural waters was found to be a novel risk factor for Campylobacter infection. Other independent risk factors were tasting or eating raw or undercooked meat and drinking untreated water from a dug well. The role of bacterial strain and host characteristics are not fully understood in Campylobacter infections. Exposure factors, demographical characteristics, and the serotype of the Campylobacter isolate may affect the severity of the enteritis. This cross-sectional study comprised 114 patients with C. jejuni enteritis, diagnosed in three clinical microbiology laboratories; most of the patients had participated in the previous case-control study. Swimming was associated with age ≤ 5 years and serotype Pen 6,7 was found significantly more often among patients reporting swimming. The geographical distribution among serotypes varied; serotype Pen 4-complex appeared more often in patients from urban areas and serotype Pen 21 among patients from more rural areas. Thus, risk factors and sources of infection for C. jejuni infection may vary among individuals depending on age and geographical location. The in vitro susceptibilities of C. jejuni and C. coli strains isolated from patients infected abroad (85 strains) or domestically (393 strains) revealed that susceptibility to erythromycin is still high, even among isolates of foreign origin. However, the novel antimicrobial agent telithromycin did not offer any advantage over erythromycin; isolates with high minimal inhibitory concentrations (MICs) for erythromycin also showed reduced susceptibility to telithromycin. Reduced susceptibility to fluoroquinolones was detected almost exclusively among isolates of foreign origin and half of these isolates with high MICs for fluoroquinolones also showed elevated MICs for doxycycline. Questionnaires concerning complications associated with C. jejuni enteritis were sent to patients two months after becoming ill; 201 patients from seven different geographical areas were included in the study. Musculoskeletal complications after C. jejuni infection were commonly reported by patients (39%). The incidence of classical ReA was 4% and that of Achilles enthesopathy and/or heel pain 9%. Other C. jejuni-associated reactive joint symptoms were commonly reported, however, due to their milder nature seldom seen and diagnosed by a physician. The severity of the enteritis may predict further complications; stomach ache during enteritis was connected to the development of later joint pain. Early antimicrobial treatment, within two days from the start of symptoms, shortened the duration of diarrhoea by two days but did not prevent later musculoskeletal complications. Campylobacter is an important human enteropathogen and causes a significant burden of illness. As the incidence of Campylobacter infections is high, the importance of the infection and the occurrence of complications will increase. This stresses the importance of understanding the risk factors for acquiring Campylobacter infection and how bacterial strain and host characteristics may affect the risk for infection. The role of antimicrobial treatment for acute Campylobacter enteritis seems to be marginal and should be used restrictively.

Stanniocalcin-1 in cell stress and differentiation

Stanniocalcin-1 (STC-1) is a 56 kD homodimeric protein which was originally identified in bony fish, where it regulates calcium/phosphate homeostasis and protects against toxic hypercalcemia. STC-1 was considered unique to fish until the cloning of cDNA for human STC-1 in 1995 and mouse Stc-1 in 1996. STC-1 is conserved through evolution with human and salmon STC-1 sharing 60% identity and 80% similarity. The surprisingly high homology between mammalian and fish STC-1 and the protective actions of STC-1 in terminally differentiated neurons, originally reported by my colleagues, prompted me to further study the role of STC-1 in cell stress and differentiation. One purpose was to determine whether there is an inter-relationship between terminally differentiated cells and STC-1 expression. The study revealed an accumulation of STC-1 in mature megakaryocytes and adipocytes, i.e. postmitotic cells with limited or lost proliferative capacity. Still proliferating uninduced cells were negative for STC-1 mRNA and protein, whereas differentiating cells accumulated STC-1 in their cytoplasm. Interestingly, in liposarcomas the grade inversely correlated with STC-1 expression. Another aim was to study how STC-1 gene expression is regulated. Given that IL-6 is a cytokine with neuroprotective actions, by unknown mechanisms, we examined whether IL-6 regulates STC-1 gene expression. Treatment of human neural Paju cells with IL-6 induced a dose-dependent upregulation of STC-1 mRNA levels. This induction of STC-1 expression by IL-6 occurred mainly through the MAPK signaling pathway. Furthermore, I studied the role of IL-6-mediated STC-1 expression as a mechanism of cytoprotection conferred by hypoxic preconditioning (HOPC) in brain and heart. My findings show that Stc-1 was upregulated in brain after hypoxia treatment. In the brain of IL-6 deficient mice, however, no upregulation of Stc-1 expression was evident. After induced brain injury the STC-1 response in brains of IL-6 transgenic mice, with IL-6 overexpression in astroglial cells, was stronger than in brains of WT mice. These results indicate that IL-6-mediated expression of STC-1 is one molecular mechanism of HOPC-induced tolerance to brain ischemia. The protection conferred by HOPC in heart occurs during a bimodal time course comprising early and delayed preconditioning. Interestingly, my results showed that the expression of Stc-1 in heart was upregulated in a biphasic manner during HOPC. IL-6 deficient mice did not, however, show a similar biphasic manner of Stc-1 upregulation as did WT mice. Instead, only an early upregulation of Stc-1 expression was evident. The results suggest that the upregulation of Stc-1 during the delayed preconditioning is IL-6-dependent. The upregulated expression of Stc-1 during the early preconditioning, however, is only partly IL-6-dependent and possibly also directly mediated by HIF-1. These findings suggest that STC-1 is a pro-survival protein for terminally differentiated cells and that STC-1 expression may in fact be regulated by stress. In addition, I show that STC-1 gene upregulation, mediated in part by IL-6, is a new mechanism of protection conferred by HOPC in brain and heart. Because of its importance for fundamental biological processes, such as differentiation and cytoprotection, STC-1 may have therapeutic implications for management of stroke, neurodegenerative diseases, cancer, and obesity.

Molecular biology and functions of epilysin (MMP-28)

Epilysin (MMP-28) is the most recently identified member of the matrix metalloproteinase (MMP) family of extracellular proteases. Together these enzymes are capable of degrading almost all components of the extracellular matrix (ECM) and are thus involved in important biological processes such as development, wound healing and immune functions, but also in pathological processes such as tumor invasion, metastasis and arthritis. MMPs do not act solely by degrading the ECM. They also regulate cell behavior by releasing growth factors and biologically active peptides from the ECM, by modulating cell surface receptors and adhesion molecules and by regulating the activity of many important mediators in inflammatory pathways. The aim of this study was to define the unique role of epilysin within the MMP-family, to elucidate how and when it is expressed and how its catalytic activity is regulated. To gain information on its essential functions and substrates, the specific aim was to characterize how epilysin affects the phenotype of epithelial cells, where it is biologically expressed. During the course of the study we found that the epilysin promoter contains a well conserved GT-box that is essential for the basic expression of this gene. Transcription factors Sp1 and Sp3 bind this sequence and could hence regulate both the basic and cell type and differentiation stage specific expression of epilysin. We cloned mouse epilysin cDNA and found that epilysin is well conserved between human and mouse genomes and that epilysin is glycosylated and activated by furin. Similarly to in human tissues, epilysin is normally expressed in a number of mouse tissues. The expression pattern differs from most other MMPs, which are expressed only in response to injury or inflammation and in pathological processes like cancer. These findings implicate that epilysin could be involved in tissue homeostasis, perhaps fine-tuning the phenotype of epithelial cells according to signals from the ECM. In view of these results, it was unexpected to find that epilysin can induce a stable epithelial to mesenchymal transition (EMT) when overexpressed in epithelial lung carcinoma cells. Transforming growth factor b (TGF-b) was recognized as a crucial mediator of this process, which was characterized by the loss of E-cadherin mediated cell-cell adhesion, elevated expression of gelatinase B and MT1-MMP and increased cell migration and invasion into collagen I gels. We also observed that epilysin is bound to the surface of epithelial cells and that this interaction is lost upon cell transformation and is susceptible to degradation by membrane type-1-MMP (MT1-MMP). The wide expression of epilysin under physiological conditions implicates that its effects on epithelial cell phenotype in vivo are not as dramatic as seen in our in vitro cell system. Nevertheless, current results indicate a possible interaction between epilysin and TGF-b also under physiological circumstances, where epilysin activity may not induce EMT but, instead, trigger less permanent changes in TGF-b signaling and cell motility. Epilysin may thus play an important role in TGF-b regulated events such as wound healing and inflammation, processes where involvement of epilysin has been indicated.

Skogsbränslets elementära sammansättning med tanke på förbränningsprocessen och för näringsämnesförluster i ekosystemet

There are several reasons for increasing the usage of forest biomass for energy in Finland. Apart from the fact that forest biomass is a CO2 -neutral energy source, it is also a domestic resource distributed throughout the country. Usage of forest biomass in the form of logging residues decreases Finland’s dependence of energy import and increases both incomes and employment. Wood chips are mainly made from logging residues, which constitute 64 % of the raw material. A large-scale use of forest biomass requires heed also to the potential negative aspects. Forest bioenergy is used extensively, but its impacts on the forests soil nutrition and carbon balance has not been studied much. Nor have there been many studies on the heavy metal or chlorine content of logging residues. The goal of this study was to examine the content of carbon, macronutrients, heavy metals and other for the combustion harmful substances in Scots pine and Norway spruce wood chips, and to estimate the effect of harvesting of logging residues on the forests carbon and nutrient balance. Another goal was to examine the energy content of the clear cut remains. The Wood chips for this study were gathered from pine and spruce dominated clear cut sites in southern Finland, in the costal forests between Hankoo and Siuntio. The number of sample locations were 29, and the average area was 3,15 ha and the average timber volume 212,6 m3 ha -1. The average logged timber volume was for Scots pine timber 70 m3 ha -1 and for Norway spruce timber 124 m3 ha -1 and for deciduous timber (birch and alder) 18,5 m3 ha -1. The proportion of spruce in the logging residues and the stand-volume were relevant for how much nutrients were taken from the forest ecosystem when harvesting logging residues. In this study it was noted that the nutrient content of the logging residues clearly increased when the percentage of spruce in the timber volume increased. The S, K, Na and Cl -contents in the logging residues in this study increased with an increasing percentage of spruce, which is probably due to the fact that the spruce is an effective collector of atmospheric dry-deposition. The amounts of nutrients that were lost when harvesting logging residues were less than those referred to in the literature. Within a circulation period (100 years), the forest soil gets substantially more nutrients from atmospheric deposition, litter fall and weathering than is lost through harvesting of logging residues after a clear cut. Harvesting of the logging residues makes for a relatively modest increase of the quantity of carbon that is removed from the forest compared to traditional forestry. Due to the fact that the clear cut remains in my study showed a high content of chlorine, there is a risk of corrosion in connection to the incineration of the logging residues in power plants especially at coastal areas/forests. The risk of sulphur -related corrosion is probably rather small, because S concentrations are relatively low in woodchips. The clear cut remains showed rather high heavy metal contents. If the heavy metal contents in this study are representative for the clear cut remains in the coastal forests generally, there might be reason to exert some caution when using the ash for forest fertilizing purposes.

Agronomic challenges for organic crop husbandry

With respect to resource management and environmental impact, organic farming offers rationales for agricultural sustainability. However, agronomic productivity is usually higher with conventional farming. This work aimed at investigating two factors of major importance for the agronomic productivity of organic crop husbandry, nitrogen (N) supply through symbiotic N fixation (SNF) and weed occurrence. Perennial red clover-grass leys and spring cereal crops subjected to regular agricultural practices were studied on 34 organic farms located in the southern and the north-western coastal regions of Finland. Herbage growth, clover content as a proportion of the ley and extent of SNF in perennial leys, and the occurrence of weed species and weed-crop competition in spring cereal stands were related to climate conditions, soil properties, and management measures. The herbage accumulated from the first and the second cut of one- and two-year-old leys averaged 7.5 t DM ha-1 (SD ± 1.7 t DM ha-1); the clover content averaged 43.9% (SD ± 18.8%). Along with the clover content, herbage production decreased with ley age. Radiation use efficiency (RUE) correlated positively with clover proportion but despite low clover contents, three-year-old leys were still productive with regard to RUE. SNF in the accumulated annual growth of one- and two-year-old leys averaged 247.5 kg N ha-1 yr-1 (SD ± 114.4 kg N ha-1 yr-1). It was supposed that if red clover-grass leys constituted 40% of the rotation, then the mean N supply by SNF would be able to sustain two or three succeeding cereal crops (green manure and forage ley, respectively), yielding 3.0 to 4.0 t grain ha-1. Being a function of clover biomass, the SNF increased from the first to the second cut and thereafter declined with ley age. Coefficients of variation of clover contents (and SNF) between and within fields were around 50%, which was about twice as high as those of herbage production. The lower were the clover contents, the higher were the within-field variations of clover as a proportion of the ley. Low clover contents in one-year-old leys and increasing variability with ley age suggested that red clover growth was limited by poor establishment and poor overwintering. The proportions of clover in leys were lower and their variability was higher in the northwest than in the south. Soil properties, primarily texture and structure, had a major impact on clover proportion and herbage production, which largely explained regional differences in ley growth. Within-field variability of soil properties can be amended through site-specific measures, including drainage, liming, and applications of organic manures and mineral fertilizers. Overwintering and the persistence of leys can be improved by the choice of winter-hardy varieties, careful establishment and the appropriate harvest regime. Mean grain yields of spring cereal crops amounted to 3.2 t ha-1 in the south and 3.6 t ha-1 in the northwest. At 570 and 565 m-2 for the south and northwest respectively, mean weed densities did not differ between the regions, whereas the respective mean weed biomass of 697 and 1594 kg dry weight ha-1, respectively did differ. Weed abundance varied remarkably between single fields. The number of weed species was higher in the south than in the northwest. For example, Fumaria officinalis and Lamium spp. were found only in the south. Frequencies and abundances of Lapsana communis, Myosotis arvensis, Polygonum aviculare, Tripleurospermum inodorum, and Vicia spp. were higher in the south, whereas those of Elymus repens, Persicaria spp. and Spergula arvensis were higher in the northwest. The number of years since conversion to organic farming, i.e. long-term management, was one of the variables that explained the abundance of single weed species. E. repens was the weed species whose biomass increased most with the duration of organic farming. Another significant variable was crop biomass, which was affected by short-term management. The presence of different weed species was related to the duration of organic farming and to low crop yield. This finding demonstrated that it was not the organic farming regime per se, which resulted in high weed infestation and low yielding crops, but failures in the understanding and the management of organic farming systems. Successful weed control relies on farm- and field-specific long- and short-term management approaches. The agronomic productivity of ley and spring cereal crops managed by full-time farmers with an interest in organic farming was on the same level as of the mean for conventional farming. Given the many options for further improvements of the agronomic performance of organic arable systems, organic farming offers foundations for the development of sustainable agriculture. The main threat to the sustainability of farming in Finland, both conventional and organic, is the spatial separation of crop production and animal husbandry by region, along with the simplification of associated crop rotations.

Syntheses and Self-Assembling Characteristics of Amphiphilic Star Diblock Copolymers

Uusien polymeeripohjaisten teknologioiden ja materiaalien myötä räätälöityjen polymeerien tarve on kasvanut. Viime vuosituhannen lopussa kehitetyt kontrolloidut polymerointimenetelmät ovat avanneet uusia mahdollisuuksia paitsi monimutkaisten polymeerien synteesiin, myös itsejärjestyvyyteen perustuvien funktionaalisten nanorakenteiden suunnitteluun ja valmistukseen. Nämä voivat jäljitellä luonnossa esiintyviä rakenteita, joita muodostavat esimerkiksi lipidit ja proteiinit. Itsejärjestyvät molekyylit ovat usein amfifiilisiä eli ne koostuvat hydrofiilisistä ja hydrofobisista osista ja polymeereissä nämä osat voivat olla omina lohkoinaan, jolloin puhutaan amfifiilisistä lohko- tai blokkikopolymeereistä. Riippuen järjestyneiden rakenteiden koostumuksesta ja muodosta, amfifiilisiä blokkikopolymeerejä on tutkittu tai jo käytetty nanoteknologiassa, elastomeereissä, voiteluaineissa, pinta-aktiivisina aineina, lääkkeenannostelussa, maaleissa, sekä elektroniikka-, kosmetiikka- ja elintarviketeollisuudessa. Tavallisimmin käytetyt amfifiiliset blokkikopolymeerit ovat olleet lineaarisia, mutta viime aikoina tutkimus on suuntautunut kohti monimutkaisempia rakenteita. Tällaisia ovat esimerkiksi tähtipolymeerit. Tähtimäisissä polymeereissä miselleille tyypillinen ydin-kuori-rakenne säilyy hyvin alhaisissakin polymeerikonsentraatioissa, koska polymeeriketjut ovat kiinni toisissaan yhdessä pisteessä. Siten ne ovat erityisen kiinnostavia tutkimuskohteita erilaisten hydrofobisten orgaanisten yhdisteiden sitomiseksi ja vapauttamiseksi. Tässä työssä on tarkasteltu amfifiilisten tähtipolymeerien itsejärjestymistä vesiliuoksissa sekä kokeellisesti ja tietokonesimulaatioin. Työ koostuu kahdesta osasta: tähtipolymeerien synteesistä makrosyklisillä initiaattoreilla ja amfifiilisten tähtimäisten blokkikopolymeerien ominaisuuksien tutkimisesta.

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

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

Stabilization of Phospholipid Coatings in Capillary Electrophoresis

The development of a simple method of coating a semi-permanent phospholipid layer onto a capillary for electrochromatography use was the focus of this study. The work involved finding good coating conditions, stabilizing the phospholipid coating, and examining the effect of adding divalent cations, cetyltrimethylammonium bromide, and polyethylene glycol (PEG)-lipids on the stability of the coating. Since a further purpose was to move toward more biological membrane coatings, the capillaries were also coated with cholesterol-containing liposomes and liposomes of red blood cell ghost lipids. Liposomes were prepared by extrusion, and large unilamellar vesicles with a diameter of about 100 nm were obtained. Zwitterionic phosphatidylcholine (PC) was used as a basic component, mainly 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) but also eggPC and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Different amounts of sphingomyelin, bovine brain phosphatidylserine, and cholesterol were added to the PC. The stability of the coating in 40 mM N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES) solution at pH 7.4 was studied by measuring the electroosmotic flow and by separating neutral steroids, basic proteins, and low-molar-mass drugs. The presence of PC in the coating solution was found to be essential to achieving a coating. The stability of the coating was improved by the addition of negative phosphatidylserine, cholesterol, divalent cations, or PEGylated lipids, and by working in the gel-state region of the phospholipid. Study of the effect on the PC coating of divalent metal ions calcium, magnesium, and zinc showed a molar ratio of 1:3 PC/Ca2+ or PC/Mg2+ to give increased rigidity to the membrane and the best coating stability. The PEGylated lipids used in the study were sterically stabilized commercial lipids with covalently attached PEG chains. The vesicle size generally decreased when PEGylated lipids of higher molar mass were present in the vesicle. The predominance of discoidal micelles over liposomes increased PEG chain length and the average size of the vesicles thus decreased. In the capillary electrophoresis (CE) measurements a highly stable electroosmotic flow was achieved with 20% PEGylated lipid in the POPC coating dispersion, the best results being obtained for disteroyl PEG (3000) conjugates. The results suggest that smaller particles (discoidal micelles) result in tighter packing and better shielding of silanol groups on the silica wall. The effect of temperature on the coating stability was investigated by using DPPC liposomes at temperatures above (45 C) and below (25 C) the main phase transition temperature. Better results were obtained with DPPC in the more rigid gel state than in the fluid state: the electroosmotic flow was heavily suppressed and the PC coating was stabilized. Also dispersions of DPPC with 0−30 mol% of cholesterol and sphingomyelin in different ratios, which more closely resemble natural membranes, resulted in stable coatings. Finally, the CE measurements revealed that a stable coating is formed when capillaries are coated with liposomes of red blood cell ghost lipids.

Radikalcyklisering vid syntes av polycykliska indoler och pyrroler

I de senaste två decennierna har radikalcyklisering, intramolekylär radikaladdition, utvecklats till en viktig syntesmetod för polycykliska indoler och pyrroler. De erhållna produktmolekylerna eller deras derivat är ofta naturliga eller syntetiska alkaloider som väckt biologiskt eller medicinskt intresse. Avhandlingen behandlar både intramolekylära radikaladditioner av pyrrolyl-, indolyl- eller indolylacylradikaler till -bindningar och intramolekylära additioner av flera olika radikaler till indolens eller pyrrolens -system. Också cykliseringar som delreaktioner i radikalkaskader behandlas. Radikalreaktioner kan släckas både oxidativt och reduktivt. För att bibehålla heteroarenens aromaticitet måste cykliseringar till pyrrol- eller indolringen släckas oxidativt. Oxidativa radikaladditioner till aromater benämns homolytiska aromatiska substitutioner. Det finns olika sätt att erhålla en reaktantradikal från en radikalprekursor. I vissa fall har radikalprekursorn en mycket labil bindning som kan brytas fotokemiskt eller med hjälp av en initiator. Till exempel kol-svavelbindningen av en O-etyl-S-alkylxanthat kan brytas på detta sätt. Ofta används dock en radikalmediator för att bilda en reaktantradikal från dess prekursor. Mediatorer är ofta föreningar, som under reaktionsförhållandena själv bildar radikaler med en stor affinitet för en prekursors specifika atom eller atomgrupp vilken abstraheras. Således fungerar mediatorn som mellanhand vid bildning av reaktantradikalen. Exempel på mediatorer av detta slag som använts vid syntes av polycykliska pyrroler och indoler är tributyltennhydrid, hexabutylditenn, tris(trimetylsilyl)silan, tributylgermaniumhydrid, dicumylperoxid, trietylboran, natriumarensulfinater (med ättiksyra) och Se-fenyl-p-toluenselenosulfonat. Också dimetylsulfoxid kan ses som mediator då den bildar metylradikaler vid Fentonreaktion i lösningsmedlet. Övergångsmetallsalter kan även bilda reaktantradikalen från prekursorn genom enelektronoxidationer eller -reduktioner. Vid syntes av polycykliska pyrroler och indoler har reaktantradikalen bildats genom enelektronoxidationer med Mn(OAc)3 eller Ag2+ (Miniscireaktion) och elektronreduktioner med ett Ni(I)-komplex eller SmI2. Avhandlingen är indelad enligt reagenset eller reagensen, som åstadkommer bildning av reaktantradikalen vid syntes av polycykliska indoler och pyrroler. Cirka hälften av avhandlingen behandlar tributyltennhydridmedierade cykliseringar då reagenset trots dess toxicitet är det överlägset mest använda. Avhandlingen diskuterar mekanismen för bildning av reaktantradikalen från prekursorn, cykliseringen och dess möjliga regioselektivitet, andra radikalreaktioner vid radikalkaskader och hur produktradikalen släcks.

Lätta stabila isotopfraktioner som verktyg i biologisk forskning

Det finns en mätbar entitet i all levande materia, nämligen förhållandet mellan ett atomslags lätta och tunga isotop. Denna entitet divideras med en definierad referens och ett värde erhålls som brukar anges i promille ( ). Värdet är ett mått på isotopfraktionering. Genom att studera ekologi, taxonomi, kretslopp, isotopeffekter, dokumenterad data och metabola effekter kan man skissera över vad isotopfraktionen betyder. Migrationsbeteende, trofinätverk, dieter, miljö- och klimatdata kan rekonstrueras i tid och rum. Vid rekonstruktioner av gången tid har de museala samlingarna en nyckelposition. I litteraturöversikten tas teori, praktik och forskning upp för att påvisa metodens faror och användningsområden.