Engineering the pentose phosphate pathway of Saccharomyces cerevisiae for production of ethanol and xylitol

The baker s yeast Saccharomyces cerevisiae has a long tradition in alcohol production from D-glucose of e.g. starch. However, without genetic modifications it is unable to utilise the 5-carbon sugars D-xylose and L arabinose present in plant biomass. In this study, one key metabolic step of the catabolic D-xylose pathway in recombinant D-xylose-utilising S. cerevisiae strains was studied. This step, carried out by xylulokinase (XK), was shown to be rate-limiting, because overexpression of the xylulokinase-encoding gene XKS1 increased both the specific ethanol production rate and the yield from D xylose. In addition, less of the unwanted side product xylitol was produced. Recombinant D-xylose-utilizing S. cerevisiae strains have been constructed by expressing the genes coding for the first two enzymes of the pathway, D-xylose reductase (XR) and xylitol dehydrogenase (XDH) from the D-xylose-utilising yeast Pichia stipitis. In this study, the ability of endogenous genes of S. cerevisiae to enable D-xylose utilisation was evaluated. Overexpression of the GRE3 gene coding for an unspecific aldose reductase and the ScXYL2 gene coding for a xylitol dehydrogenase homologue enabled growth on D-xylose in aerobic conditions. However, the strain with GRE3 and ScXYL2 had a lower growth rate and accumulated more xylitol compared to the strain with the corresponding enzymes from P. stipitis. Use of the strictly NADPH-dependent Gre3p instead of the P. stipitis XR able to utilise both NADH and NADPH leads to a more severe redox imbalance. In a S. cerevisiae strain not engineered for D-xylose utilisation the presence of D-xylose increased xylitol dehydrogenase activity and the expression of the genes SOR1 or SOR2 coding for sorbitol dehydrogenase. Thus, D-xylose utilisation by S. cerevisiae with activities encoded by ScXYL2 or possibly SOR1 or SOR2, and GRE3 is feasible, but requires efficient redox balance engineering. Compared to D-xylose, D-glucose is a cheap and readily available substrate and thus an attractive alternative for xylitol manufacture. In this study, the pentose phosphate pathway (PPP) of S. cerevisiae was engineered for production of xylitol from D-glucose. Xylitol was formed from D-xylulose 5-phosphate in strains lacking transketolase activity and expressing the gene coding for XDH from P. stipitis. In addition to xylitol, ribitol, D-ribose and D-ribulose were also formed. Deletion of the xylulokinase-encoding gene increased xylitol production, whereas the expression of DOG1 coding for sugar phosphate phosphatase increased ribitol, D-ribose and D-ribulose production. Strains lacking phosphoglucose isomerase (Pgi1p) activity were shown to produce 5 carbon compounds through PPP when DOG1 was overexpressed. Expression of genes encoding glyceraldehyde 3-phosphate dehydrogenase of Bacillus subtilis, GapB, or NAD-dependent glutamate dehydrogenase Gdh2p of S. cerevisiae, altered the cellular redox balance and enhanced growth of pgi1 strains on D glucose, but co-expression with DOG1 reduced growth on higher D-glucose concentrations. Strains lacking both transketolase and phosphoglucose isomerase activities tolerated only low D-glucose concentrations, but the yield of 5-carbon sugars and sugar alcohols on D-glucose was about 50% (w/w).

Structural and Functional Studies on Trimeric Autotransporters

Trimeric autotransporters are a family of secreted outer membrane proteins in Gram-negative bacteria. These obligate homotrimeric proteins share a conserved C-terminal region, termed the translocation unit. This domain consists of an integral membrane β-barrel anchor and associated α-helices which pass through the pore of the barrel. The α-helices link to the extracellular portion of the protein, the passenger domain. Autotransportation refers to the way in which the passenger domain is secreted into the extracellular space. It appears that the translocation unit mediates the transport of the passenger domain across the outer membrane, and no external factors, such as ATP, ion gradients nor other proteins, are required. The passenger domain of autotransporters contains the specific activities of each protein. These are usually related to virulence. In trimeric autotransporters, the main function of the proteins is to act as adhesins. One such protein is the Yersinia adhesin YadA, found in enteropathogenic species of Yersinia. The main activity of YadA from Y. enterocolitica is to bind collagen, and it also mediates adhesion to other molecules of the extracellular matrix. In addition, YadA is involved in serum resistance, phagocytosis resistance, binding to epithelial cells and autoagglutination. YadA is an essential virulence factor of Y. enterocolitica, and removal of this protein from the bacteria leads to avirulence. In this study, I investigated the YadA-collagen interaction by studying the binding of YadA to collagen-mimicking peptides by several biochemical and biophysical methods. YadA bound as tightly to the triple-helical model peptide (Pro-Hyp-Gly)10 as to native collagen type I. However, YadA failed to bind a similar peptide that does not form a collagenous triple helix. As (Pro-Hyp-Gly)10 does not contain a specific sequence, we concluded that a triple-helical conformation is necessary for YadA binding, but no specific sequence is required. To further investigate binding determinants for YadA in collagens, I examined the binding of YadA to a library of collagen-mimicking peptides that span the entire triple-helical sequences of human collagens type II and type III. YadA bound promiscuously to many but not all peptides, indicating that a triple-helical conformation alone is not sufficient for binding. The high-binding peptides did not share a clear binding motif, but these peptides were rich in hydroxyproline residues and contained a low number of charged residues. YadA thus binds collagens without sequence specificity. This strategy of promiscuous binding may be advantageous for pathogenic bacteria. The Eib proteins from Escherichia coli are immunoglobulin (Ig)-binding homologues of YadA. I showed conclusively that recombinant EibA, EibC, EibD and EibF bind to IgG Fc. I crystallised a fragment of the passenger domain of EibD, which binds IgA in addition to IgG. The structure has a YadA-like head domain and an extended coiled-coil stalk. The top half of the coiled-coil is right-handed with hendecad periodicity, whereas the lower half is a canonical left-handed coiled-coil. At the transition from right- to left-handedness, a small β-sheet protrudes from each monomer. I was able to map the binding regions for IgG and IgA using truncations and site-directed mutagenesis to the coiled-coil stalk and identified residues critical for Ig binding.

Dispersal and related life history traits in the Glanville fritillary butterfly

Most studies of life history evolution are based on the assumption that species exist at equilibrium and spatially distinct separated populations. In reality, this is rarely the case, as populations are often spatially structured with ephemeral local populations. Therefore, the characteristics of metapopulations should be considered while studying factors affecting life history evolution. Theoretical studies have examined spatial processes shaping the evolution of life history traits to some extent, but there is little empirical data and evidence to investigate model predictions. In my thesis I have tried to bridge the gap between theoretical and empirical studies by using the well-known Glanville fritillary (Melitaea cinxia) metapopulation as a model system. The long-term persistence of classic metapopulations requires sufficient dispersal to establish new local populations to compensate for local extinctions. Previous studies on the Glanville fritillary have shown that females establishing new populations are not a random sample from the metapopulation, but they are in fact more dispersive than females in old populations. Many other life-history traits, such as body size, fecundity and lifespan, may be related to dispersal rate. Therefore, I examined a range of correlated traits for their evolutionary and ecological consequences. I was particularly interested in how the traits vary under natural environmental conditions, hence all studies were conducted in a large (32 x 26 m) outdoor population cage built upon a natural habitat patch. Individuals for the experiments were sampled from newly-established and old populations within a large metapopulation. Results show that females originating from newly-established populations had higher within-habitat patch mobility than females from old populations. I showed that dispersal rate is heritable and that flight activity is related to variation in a gene encoding the glycolytic enzyme phosphoglucose isomerase. Both among-individual and among-population variation in dispersal are correlated with the reproductive performance of females, though I found no evidence for a trade-off between dispersal and fecundity in terms of lifetime egg production or clutch size. Instead, the results suggest that highly dispersive females from newly-established populations have a shorter lifespan than females from old populations, and that dispersive females may pay a cost in terms of reduced lifetime reproductive success due to increased time spent outside habitat patches. In summary, the results of this thesis show that genotype-dependent dispersal rate correlates with other life history traits in the Glanville fritillary, and that the rapid turnover of local populations (extinctions and re-colonisations) is likely to be the mechanism that maintains phenotypic variation in many life history traits at the metapopulation level.

Maintaining plant species richness by cattle grazing - mesic semi-natural grasslands as focal habitats

Semi-natural grasslands are the most important agricultural areas for biodiversity. The present study investigates the effects of traditional livestock grazing and mowing on plant species richness, the main emphasis being on cattle grazing in mesic semi-natural grasslands. The two reviews provide a thorough assessment of the multifaceted impacts and importance of grazing and mowing management to plant species richness. It is emphasized that livestock grazing and mowing have partially compensated the suppression of major natural disturbances by humans and mitigated the negative effects of eutrophication. This hypothesis has important consequences for nature conservation: A large proportion of European species originally adapted to natural disturbances may be at present dependent on livestock grazing and / or mowing. Furthermore, grazing and mowing are key management methods to mitigate effects of nutrient-enrichment. The species composition and richness in old (continuously grazed), new (grazing restarting 3-8 years ago) and abandoned (over 10 years) pastures differed consistently across a range of spatial scales, and was intermediate in new pastures compared to old and abandoned pastures. In mesic grasslands most plant species were shown to benefit from cattle grazing. Indicator species of biologically valuable grasslands and rare species were more abundant in grazed than in abandoned grasslands. Steep S-SW-facing slopes are the most suitable sites for many grassland plants and should be prioritized in grassland restoration. The proportion of species trait groups benefiting from grazing was higher in mesic semi-natural grasslands than in dry and wet grasslands. Consequently, species trait responses to grazing and the effectiveness of the natural factors limiting plant growth may be intimately linked High plant species richness of traditionally mowed and grazed areas is explained by numerous factors which operate on different spatial scales. Particularly important for maintaining large scale plant species richness are evolutionary and mitigation factors. Grazing and mowing cause a shift towards the conditions that have occurred during the evolutionary history of European plant species by modifying key ecological factors (nutrients, pH and light). The results of this Dissertation suggest that restoration of semi-natural grasslands by private farmers is potentially a useful method to manage biodiversity in the agricultural landscape. However, the quality of management is commonly improper, particularly due to financial constraints. For enhanced success of restoration, management regulations in the agri-environment scheme need to be defined more explicitly and the scheme should be revised to encourage management of biodiversity.

Short- and long-term consequences of food resources on Ural owl Strix uralensis reproduction

Life-history theory states that although natural selection would favour a maximisation of both reproductive output and life-span, such a combination can not be achieved in any living organism. According to life-history theory the reason for the fact that not all traits can be maximised simultaneously is that different traits compete with each other for resources. These relationships between traits that constrain the simultaneous evolution of two or more traits are called trade-offs. Therefore, during different life-stages an individual needs to optimise its allocation of resources to life-history components such as growth, reproduction and survival. Resource limitation acts on these traits and therefore investment in one trait, e.g. reproduction, reduces the resources available for investment in another trait, e.g. residual reproduction or survival. In this thesis I study how food resources during different stages of the breeding event affect reproductive decisions in the Ural owl (Strix uralensis) and the consequences of these decisions on parents and offspring. The Ural owl is a suitable study species for such studies in natural populations since they are long-lived, site-tenacious, and feed on voles. The vole populations in Fennoscandia fluctuate in three- to four-year cycles, which create a variable food environment for the Ural owls to cope with. The thesis gives new insight in reproductive costs and their consequences in natural animal populations with emphasis on underlying physiological mechanisms. I found that supplementary fed Ural owl parents invest supplemented food resources during breeding in own self-maintenance instead of allocating those resources to offspring growth. This investment in own maintenance instead of improving current reproduction had carry-over effects to the following year in terms of increased reproductive output. Therefore, I found evidence that reduced reproductive costs improves future reproductive performance. Furthermore, I found evidence for the underlying mechanism behind this carry-over effect of supplementary food on fecundity. The supplementary-fed parents reduced their feeding investment in the offspring compared to controls, which enabled the fed female parents to invest the surplus resources in parasite resistance. Fed female parents had lower blood parasite loads than control females and this effect lasted until the following year when also reproductive output was increased. Hence, increased investment in parasite resistance when resources are plentiful has the potential to mediate positive carry-over effects on future reproduction. I further found that this carry-over effect was only present when potentials for future reproduction were good. The thesis also provides new knowledge on resource limitation on maternal effects. I found that increased resources prior to egg laying improve the condition and health of Ural owl females and enable them to allocate more resources to reproduction than control females. These additional resources are not allocated to increase the number of offspring, but instead to improve the quality of each offspring. Fed Ural owl females increased the size of their eggs and allocated more health improving immunological components into the eggs. Furthermore, the increased egg size had long-lasting effects on offspring growth, as offspring from larger eggs were heavier at fledging. Limiting resources can have different short- and long-term consequences on reproductive decisions that affect both offspring number and quality. In long-lived organisms, such as the Ural owl, it appears to be beneficial in terms of fitness to invest in long breeding life-span instead of additional investment in current reproduction. In Ural owls, females can influence the phenotypic quality of the offspring by transferring additional resources to the eggs that can have long-lasting effects on growth.

Theoretical Studies on Coupled Electron and Proton Transfer in Cytochrome c Oxidase

The complexity of life is based on an effective energy transduction machinery, which has evolved during the last 3.5 billion years. In aerobic life, the utilization of the high oxidizing potential of molecular oxygen powers this machinery. Oxygen is safely reduced by a membrane bound enzyme, cytochrome c oxidase (CcO), to produce an electrochemical proton gradient over the mitochondrial or bacterial membrane. This gradient is used for energy-requiring reactions such as synthesis of ATP by F0F1-ATPase and active transport. In this thesis, the molecular mechanism by which CcO couples the oxygen reduction chemistry to proton-pumping has been studied by theoretical computer simulations. By building both classical and quantum mechanical model systems based on the X-ray structure of CcO from Bos taurus, the dynamics and energetics of the system were studied in different intermediate states of the enzyme. As a result of this work, a mechanism was suggested by which CcO can prevent protons from leaking backwards in proton-pumping. The use and activation of two proton conducting channels were also enlightened together with a mechanism by which CcO sorts the chemical protons from pumped protons. The latter problem is referred to as the gating mechanism of CcO, and has remained a challenge in the bioenergetics field for more than three decades. Furthermore, a new method for deriving charge parameters for classical simulations of complex metalloenzymes was developed.

Phylogeography and hybrid swarms: history of brackish water bivalve diversity in North European marginal seas

This study addressed the large-scale molecular zoogeography in two brackish water bivalve molluscs, Macoma balthica and Cerastoderma glaucum, and genetic signatures of the postglacial colonization of Northern Europe by them. The traditional view poses that M. balthica in the Baltic, White and Barents seas (i.e. marginal seas) represent direct postglacial descendants of the adjacent Northeast Atlantic populations, but this has recently been challenged by observations of close genetic affinities between these marginal populations and those of the Northeast Pacific. The primary aim of the thesis was to verify, quantify and characterize the Pacific genetic contribution across North European populations of M. balthica and to resolve the phylogeographic histories of the two bivalve taxa in range-wide studies using information from mitochondrial DNA (mtDNA) and nuclear allozyme polymorphisms. The presence of recent Pacific genetic influence in M. balthica of the Baltic, White and Barents seas, along with an Atlantic element, was confirmed by mtDNA sequence data. On a broader temporal and geographical scale, altogether four independent trans-Arctic invasions of Macoma from the Pacific since the Miocene seem to have been involved in generating the current North Atlantic lineage diversity. The latest trans-Arctic invasion that affected the current Baltic, White and Barents Sea populations probably took place in the early post-glacial. The nuclear genetic compositions of these marginal sea populations are intermediate between those of pure Pacific and Atlantic subspecies. In the marginal sea populations of mixed ancestry (Barents, White and Northern Baltic seas), the Pacific and Atlantic components are now randomly associated in the genomes of individual clams, which indicates both pervasive historical interbreeding between the previously long-isolated lineages (subspecies), and current isolation of these populations from the adjacent pure Atlantic populations. These mixed populations can be characterized as self-supporting hybrid swarms, and they arguably represent the most extensive marine animal hybrid swarms so far documented. Each of the three swarms still has a distinct genetic composition, and the relative Pacific contributions vary from 30 to 90 % in local populations. This diversity highlights the potential of introgressive hybridization to rapidly give rise to new evolutionarily and ecologically significant units in the marine realm. In the south of the Danish straits and in the Southern Baltic Sea, a broad genetic transition zone links the pure North Sea subspecies M. balthica rubra to the inner Baltic hybrid swarm, which has about 60 % of Pacific contribution in its genome. This transition zone has no regular smooth clinal structure, but its populations show strong genotypic disequilibria typical of a hybrid zone maintained by the interplay of selection and gene flow by dispersing pelagic larvae. The structure of the genetic transition is partly in line with features of Baltic water circulation and salinity stratification, with greater penetration of Atlantic genes on the Baltic south coast and in deeper water populations. In all, the scenarios of historical isolation and secondary contact that arise from the phylogeographic studies of both Macoma and Cerastoderma shed light to the more general but enigmatic patterns seen in marine phylogeography, where deep genetic breaks are often seen in species with high dispersal potential.

Genetic aspects of outcome in kidney transplantation: cytokine and thrombosis associated candidate genes and gene expression biomarkers

Kidney transplantation (Tx) is the treatment of choice for end stage renal disease. Immunosuppressive medications are given to prevent an immunological rejection of the transplant. However, immunosuppressive drugs increase e.g. the risk of infection, cancer or nephrotoxicity. A major genetic contributors to immunological acceptance of the graft are human leukocyte antigen (HLA) genes. Also other non-HLA gene polymorphisms may predict the future risk of complications before Tx, possibly enabling individualised immunotherapy. Graft function after Tx is monitored using non-specific clinical symptoms and laboratory markers. The definitive diagnosis of graft rejection however relies on a biopsy of the graft. In the acute rejection (AR) diagnostics there is a need for an alternative to biopsy that would be an easily repeatable and simple method for regular use. Frequent surveillance of acute or subclinical rejection (SCR) may improve long-term function. In this thesis, associations between cytokine and thrombosis associated candidate genes and the outcome of kidney Tx were studied. Cytotoxic and co-stimulatory T lymphocyte molecule gene expression biomarkers for the diagnosis of the AR and the SCR were also investigated. We found that polymorphisms in the cytokine genes tumor necrosis factor and interleukin 10 (IL10) of the recipients were associated with AR. In addition, certain IL10 gene polymorphisms of the donors were associated with the incidence of cytomegalovirus infection and occurrence of later infection in a subpopulation of recipients. Further, polymorphisms in genes related to the risk of thrombosis and those of certain cytokines were not associated with the occurrence of thrombosis, infarction, AR or graft survival. In the study of biomarkers for AR, whole blood samples were prospectively collected from adult kidney Tx patients. With real-time quantitative PCR (RT-QPCR) gene expression quantities of CD154 and ICOS differentiated the patients with AR from those without, but not from the patients with other causes of graft dysfunction. Biomarkers for SCR were studied in paediatric kidney Tx patients. We used RT-QPCR to quantify the gene expression of immunological candidate genes in a low-density array format. In addition, we used RT-QPCR to validate the results of the microarray analysis. No gene marker differentiated patients with SCR from those without SCR. This research demonstrates the lack of robust markers among polymorphisms or biomarkers in investigated genes that could be included in routine analysis in a clinical laboratory. In genetic studies, kidney Tx can be regarded as a complex trait, i.e. several environmental and genetic factors may determine its outcome. A number of currently unknown genetic factors probably influence the results of Tx.

Mutations of mitochondrial DNA polymerase gamma: an important cause of neurological disorders

Thesis focuses on mutations of POLG1 gene encoding catalytic subunit polγ-α of mitochondrial DNA polymerase gamma holoenzyme (polG) and the association of mutations with different clinical phenotypes. In addition, particular defective mutant variants of the protein were characterized biochemically in vitro. PolG-holoenzyme is the sole DNA polymerase found in mitochondria. It is involved in replication and repair of the mitochondrial genome, mtDNA. Holoenzyme also includes the accessory subunit polγ-β, which is required for the enhanced processivity of polγ-α. Defective polγ-α causes accumulation of secondary mutations on mtDNA, which leads to a defective oxidative phosphorylation system. The clinical consequences of such mutations are variable, affecting nervous system, skeletal muscles, liver and other post-mitotic tissues. The aims of the studies included: 1) Determination of the role of POLG1 mutations in neurological syndromes with features of mitochondrial dysfunction and an unknown molecular cause. 2) Development and set up of diagnostic tests for routine clinical purposes. 3) Biochemical characterization of the functional consequences of the identified polγ-α variants. Studies describe new neurological phenotypes in addition to PEO caused by POLG1 mutations, including parkinsonism, premature amenorrhea, ataxia and Parkinson s disease (PD). POLG1 mutations and polymorphisms are both common and/or potential genetic risk factors at least among the Finnish population. The major findings and applications reported here are: 1) POLG1 mutations cause parkinsonism and premature menopause in PEO families in either a recessive or a dominant manner. 2) A common recessive POLG1 mutations (A467T and W748S) in the homozygous state causes severe adult or juvenile-onset ataxia without muscular symptoms or histological or mtDNA abnormalities in muscles. 3) A common recessive pathogenic change A467T can also cause a mild dominant disease in heterozygote carriers. 4) The A467T variant shows reduced polymerase activity due to defective template binding. 5) Rare polyglutamine tract length variants of POLG1 are significantly enriched in Finnish idiopathic Parkinson s disease patients. 6) Dominant mutations are clearly restricted to the highly conserved polymerase domain motifs, whereas recessive ones are more evenly distributed along the protein. The present results highlight and confirm the new role of mitochondria in parkinsonism/Parkinson s disease and describe a new mitochondrial ataxia. Based on these results, a POLG1 diagnostic routine has been set up in Helsinki University Central Hospital (HUSLAB).

Postmenopausal hot flushes, vascular health and hormone therapy

Vasomotor hot flushes are complained of by approximately 75% of postmenopausal women, but their frequency and severity show great individual variation. Hot flushes have been present in women attending observational studies showing cardiovascular benefit associated with hormone therapy use, whereas they have been absent or very mild in randomized hormone therapy trials showing cardiovascular harm. Therefore, if hot flushes are a factor connected with vascular health, they could perhaps be one explanation for the divergence of cardiovascular data in observational versus randomized studies. For the present study 150 healthy, recently postmenopausal women showing a large variation in hot flushes were studied in regard to cardiovascular health by way of pulse wave analysis, ambulatory blood pressure and several biochemical vascular markers. In addition, the possible impact of hot flushes on outcomes of hormone therapy was studied. This study shows that women with severe hot flushes exhibit a greater vasodilatory reactivity as assessed by pulse wave analysis than do women without vasomotor symptoms. This can be seen as a hot flush-related vascular benefit. Although severe night-time hot flushes seem to be accompanied by transient increases in blood pressure and heart rate, the diurnal blood pressure and heart rate profiles show no significant differences between women without and with mild, moderate or severe hot flushes. The levels of vascular markers, such as lipids, lipoproteins, C-reactive protein and sex hormone-binding globulin show no association with hot flush status. In the 6-month hormone therapy trial the women were classified as having either tolerable or intolerable hot flushes. These groups were treated in a randomized order with transdermal estradiol gel, oral estradiol alone or in combination with medroxyprogesterone acetate, or with placebo. In women with only tolerable hot flushes, oral estradiol leads to a reduced vasodilatory response and increases in 24-hour and daytime blood pressures as compared to women with intolerable hot flushes receiving the same therapy. No such effects were observed with the other treatment regimes or in women with intolerable hot flushes. The responses of vascular biomarkers to hormone therapy are unaffected by hot flush status. In conclusion, hot flush status contributes to cardiovascular health before and during hormone therapy. Severe hot flushes are associated with an increased vasodilatory, and thus, a beneficial vascular status. Oral estradiol leads to vasoconstrictive changes and increases in blood pressure, and thus to possible vascular harm, but only in women whose hot flushes are so mild that they would probably not lead to the initiation of hormone therapy in clinical practice. Healthy, recently postmenopausal women with moderate to severe hot flushes should be given the opportunity to use hormone therapy alleviate hot flushes, and if estrogen is prescribed for indications other than for the control of hot flushes, transdermal route of administration should be favored.

The incidence and outcome of severe sepsis in Finland : The Finnsepsis Study

Severe sepsis is associated with common occurrence, high costs of care and significant mortality. The incidence of severe sepsis has been reported to vary between 0.5/1000 and 3/1000 in different studies. The worldwide Severe Sepsis Campaign, guidelines and treatment protocols aim at decreasing severe sepsis associated high morbidity and mortality. Various mediators of inflammation, such as high mobility group box-1 protein (HMGB1) and vascular endothelial growth factor (VEGF), have been tested for severity of illness and outcome in severe sepsis. Long-term survival with quality of life (QOL) assessment is important outcome after severe sepsis. The objective of this study was to evaluate the incidence, severity of organ dysfunction and outcome of severe sepsis in intensive care treated patients in Finland (study I)). HMGB1 and VEGF were studied in predicting severity of illness, development and type of organ dysfunction and hospital mortality (studies II and III). The long-term outcome and quality of life were assessed and quality-adjusted life years and cost per one QALY were estimated (study IV). A total of 470 patients with severe sepsis were included in the Finnsepsis Study. Patients were treated in 24 Finnish intensive care units in a 4-month period from 1 November 2004 to 28 February 2005. The incidence of severe sepsis was 0.38 /1,000 in the adult population (95% confidence interval 0.34-0.41). Septic shock (77%), severe oxygenation impairment (71.4%) and acute renal failure (23.2%) were the most common organ failures. The ICU, hospital, one-year and two-year mortalities were 15.5%, 28.3%, 40.9% and 44.9% respectively. HMGB1 and VEGF were elevated in patients with severe sepsis. VEGF concentrations were lower in non-survivors than in survivors, but HMGB1 levels did not differ between patients. Neither HMGB1 nor VEGF were predictive of hospital mortality. The QOL was measured median 17 months after severe sepsis and QOL was lower than in reference population. The mean QALY was 15.2 years for a surviving patient and the cost for one QALY was 2,139 . The study showed that the incidence of severe sepsis is lower in Finland than in other countries. The short-term outcome is comparable with that in other countries, but long-term outcome is poor. HMGB1 and VEGF are not useful in predicting mortality in severe sepsis. The mean QALY for a surviving patient is 15.2 and as the cost for one QALY is reasonably low, the intensive care is cost-effective in patients with severe sepsis.

Genetic studies on Finnish lupus erythematosus patients with cutaneous manifestations

Lupus erythematosus (LE) is a chronic, heterogeneous autoimmune disorder with abnormal immune responses, including production of autoantibodies and immune complexes. Clinical presentations of the disease range from mild cutaneous manifestations to a more generalised systemic involvement of internal organs. Cutaneous (CLE) forms are further subclassified into discoid LE (DLE), subacute cutaneous LE (SCLE) and acute cutaneous lupus erythematosus (ACLE), and may later progress to systemic disease (SLE). Both genetic and environmental factors contribute to the disease, although the precise aetiology is still elusive. Furthermore, complex gene-gene or gene-environment interactions may result in different subphenotypes of lupus. The genetic background of CLE is poorly known and only a few genes are confirmed, while the number of robust genetic associations in SLE exceeds 30. The aim of this thesis was to characterise the recruited patients clinically, and identify genetic variants conferring susceptibility to cutaneous variants of LE. Given that cutaneous and systemic disease may share underlying genetic factors, putative CLE candidate genes for genotyping were selected among those showing strong evidence of association in SLE. The correlation between relevant clinical manifestations and risk genotypes was investigated in order to find specific subphenotype associations. In addition, epistatic interactions in SLE were studied. Finally, the role of tissue degrading matrix metalloproteinases (MMP) in LE tissue injury was explored. These studies were conducted in Finnish case-control and family cohort, and Swedish case-control cohort. The clinical picture of the patients in terms of cutaneous, haematological and immunological findings resembled that described in the contemporary literature. However, the proportion of daily smokers was very high supporting the role of smoking in disease aetiology. The results confirmed that, even though clinically distinct entities, CLE and SLE share predisposing genetic factors. For the first time it was shown that known SLE susceptibility genes IRF5 and TYK2 also increase the risk of CLE. A tendency toward gene-gene interaction between these genes was found in SLE. As a remarkable novel finding, it was observed that ITGAM polymorphisms associated even more strongly to DLE than SLE, and the risk estimates were substantially higher than those reported for SLE. Several other recently identified SLE susceptibility genes showed signs of good or modest association especially in DLE. Subphenotype analyses indicated possible associations to clinical features, but marginally significant results reflected lack of sufficient power for these studies. Thorough immunohistochemical analyses of several MMPs demonstrated a role in epidermal changes and dermal tissue remodelling in diseased skin, and suggested that targeted action using selective MMP inhibitors may reduce lupus-induced damage in inflamed tissues. In conclusion, the results provide an insight into the genetics of CLE and demonstrate that genetic predisposition is at least in part shared between cutaneous and systemic variants of LE. This doctoral study has contributed IRF5, TYK2, ITGAM and several other novel genes to the so far short list of genes implicated in CLE susceptibility. Detailed examination of the function of these genes in CLE pathogenesis warrants further studies. Furthermore, the results support the need of subphenotype analysis with sample sizes large enough to reveal possible specific disease associations in order to better understand the heterogeneous nature and clinical specificities of the disease. Comprehensive analysis of clinical data suggests that smoking is an environmental triggering factor.

Depressive Disorders in Primary Health Care

The Vantaa Primary Care Depression Study (PC-VDS) is a naturalistic and prospective cohort study concerning primary care patients with depressive disorders. It forms a collaborative research project between the Department of Mental and Alcohol Research of the National Public Health Institute, and the Primary Health Care Organization of the City of Vantaa. The aim is to obtain a comprehensive view on clinically significant depression in primary care, and to compare depressive patients in primary care and in secondary level psychiatric care in terms of clinical characteristics. Consecutive patients (N=1111) in three primary care health centres were screened for depression with the PRIME-MD, and positive cases interviewed by telephone. Cases with current depressive symptoms were diagnosed face-to-face with the Structured Clinical Interview for DSM-IV Axis I Disorders (SCID-I/P). A cohort of 137 patients with unipolar depressive disorders, comprising all patients with at least two depressive symptoms and clinically significant distress or disability, was recruited. The Structured Clinical Interview for DSM-IV Axis II Disorders (SCID-II), medical records, rating scales, interview and a retrospective life-chart were used to obtain comprehensive cross-sectional and retrospective longitudinal information. For investigation of suicidal behaviour the Scale for Suicidal Ideation (SSI), patient records and the interview were used. The methodology was designed to be comparable to The Vantaa Depression Study (VDS) conducted in secondary level psychiatric care. Comparison of major depressive disorder (MDD) patients aged 20-59 from primary care in PC-VDS (N=79) was conducted with new psychiatric outpatients (N =223) and inpatients (N =46) in VDS. The PC-VDS cohort was prospectively followed up at 3, 6 and 18 months. Altogether 123 patients (90%) completed the follow-up. Duration of the index episode and the timing of relapses or recurrences were examined using a life-chart. The retrospective investigation revealed current MDD in most (66%), and lifetime MDD in nearly all (90%) cases of clinically significant depressive syndromes. Two thirds of the “subsyndromal” cases had a history of major depressive episode (MDE), although they were currently either in partial remission or a potential prodromal phase. Recurrences and chronicity were common. The picture of depression was complicated by Axis I co-morbidity in 59%, Axis II in 52% and chronic Axis III disorders in 47%; only 12% had no co-morbidity. Within their lifetimes, one third (37%) had seriously considered suicide, and one sixth (17%) had attempted it. Suicidal behaviour clustered in patients with moderate to severe MDD, co-morbidity with personality disorders, and a history of treatment in psychiatric care. The majority had received treatment for depression, but suicidal ideation had mostly remained unrecognised. The comparison of patients with MDD in primary care to those in psychiatric care revealed that the majority of suicidal or psychotic patients were receiving psychiatric treatment, and the patients with the most severe symptoms and functional limitations were hospitalized. In other clinical aspects, patients with MDD in primary care were surprisingly similar to psychiatric outpatients. Mental health contacts earlier in the current MDE were common among primary care patients. The 18-month prospective investigation with a life-chart methodology verified the chronic and recurrent nature of depression in primary care. Only one-quarter of patients with MDD achieved and maintained full remission during the follow-up, while another quarter failed to remit at all. The remaining patients suffered either from residual symptoms or recurrences. While severity of depression was the strongest predictor of recovery, presence of co-morbid substance use disorders, chronic medical illness and cluster C personality disorders all contributed to an adverse outcome. In clinical decision making, beside severity of depression and co-morbidity, history of previous MDD should not be ignored by primary care doctors while depression there is usually severe enough to indicate at least follow-up, and concerning those with residual symptoms, evaluation of their current treatment. Moreover, recognition of suicidal behaviour among depressed patients should also be improved. In order to improve outcome of depression in primary care, the often chronic and recurrent nature of depression should be taken into account in organizing the care. According to literature management programs of a chronic disease, with enhancement of the role of case managers and greater integration of primary and specialist care, have been successful. Optimum ways of allocating resources between treatment providers as well as within health centres should be found.

Carotid Stenosis, Endarterectomy, and the Brain : Brain microcirculation, diffusion, and cognitive function before and after carotid endarterectomy in patients with a high-grade carotid stenosis

Carotid atherosclerotic disease is a major cause of stroke, but it may remain clinically asymptomatic. The factors that turn the asymptomatic plaque into a symptomatic one are not fully understood, neither are the subtle effects that a high-grade carotid stenosis may have on the brain. The purpose of this study was to evaluate brain microcirculation, diffusion, and cognitive performance in patients with a high-grade stenosis in carotid artery, clinically either symptomatic or asymptomatic, undergoing carotid endarterectomy (CEA). We wanted to find out whether the stenoses are associated with diffusion or perfusion abnormalities of the brain or variation in the cognitive functioning of the patients, and to what extent the potential findings are affected by CEA, and compare the clinically symptomatic and asymptomatic subjects as well as strictly healthy controls. Coagulation and fibrinolytic parameters were compared with the rate microembolic signals (MES) in transcranial Doppler (TCD) and the macroscopic appearance of stenosing plaques in surgery. Patients (n=92) underwent CEA within the study. Blood samples pertaining to coagulation and fibrinolysis were collected before CEA, and the subjects underwent repeated TCD monitoring for MES. A subpopulation (n= 46) underwent MR imaging and repeated neuropsychological examination (preoperative, as well 4 and 100 days after CEA). In MRI, the average apparent diffusion coefficients were higher in the ipsilateral white matter (WM), and altough the interhemispheric difference was abolished by CEA, the levels remained higher than in controls. Symptomatic stenoses were associated with more sluggish perfusion especially in WM, and lower pulsatility of flow in TCD. All patients had poorer cognitive performance than healthy controls. Cognitive functions improved as expected by learning effect despite transient postoperative worsening in a few subjects. Improvement was greater in patients with deepest hypoperfusion, primarily in executive functions. Symptomatic stenoses were associated with higher hematocrit and tissue plasminogen activator antigen levels, as well as higher rate of MES and ulcerated plaques, and better postoperative improvement of vasoreactivity and pulsatility. In light of the findings, carotid stenosis is associated with differences in brain diffusion, perfusion, and cognition. The effect on diffusion in the ipsilateral WM, partially reversible by CEA, may be associated with WM degeneration. Asymptomatic and symptomatic subpopulations differ from each other in terms of hemodynamic adaptation and in their vascular physiological response to removal of stenosis. Although CEA may be associated with a transient cognitive decline, a true improvement of cognitive performance by CEA is possible in patients with the most pronounced perfusion deficits. Mediators of fibrinolysis and unfavourable hemorheology may contribute to the development of a symptomatic disease in patients with a high-grade stenosis.

Cancer incidence, mortality, and pregnancy outcome among women treated for cervical intraepithelial neoplasia

Cervical cancer develops through precursor lesions, i.e. cervical intraepithelialneoplasms (CIN). These can be detected and treated before progression to invasive cancer. The major risk factor for developing cervical cancer or CIN is persistent or recurrent infection with high-risk human papilloma virus (hrHPV). Other associated risk factors include low socioeconomic status, smoking, sexually transmitted infections, and high number of sexual partners, and these risk factors can predispose to some other cancers, excess mortality, and reproductive health complications as well. The aim was to study long-term cancer incidence, mortality, and reproductive health outcomes among women treated for CIN. Based on the results, we could evaluate the efficacy and safety of CIN treatment practices and estimate the role of the risk factors of CIN patients for cancer incidence, mortality, and reproductive health. We collected a cohort of 7 599 women treated for CIN at Helsinki University Central Hospital from 1974 to 2001. Information about their cancer incidence, cause of death, birth of children and other reproductive endpoints, and socio-economic status were gathered through registerlinkages to the Finnish Cancer Registry, Finnish Population Registry, and Statistics Finland. Depending on the endpoints in question, the women treated were compared to the general population, to themselves, or to an age- and municipality-matched reference cohort. Cervical cancer incidence was increased after treatment of CIN for at least 20 years, regardless of the grade of histology at treatment. Compared to all of the colposcopically guided methods, cold knife conization (CKC) was the least effective method of treatment in terms of later CIN 3 or cervical cancer incidence. In addition to cervical cancer, incidence of other HPV-related anogenital cancers was increased among those treated, as was the incidence of lung cancer and other smoking-related cancers. Mortality from cervical cancer among the women treated was not statistically significantly elevated, and after adjustment for socio-economic status, the hazard ratio (HR) was 1.0. In fact, the excess mortality among those treated was mainly due to increased mortality from other cancers, especially from lung cancer. In terms of post-treatment fertility, the CIN treatments seem to be safe: The women had more deliveries, and their incidence of pregnancy was similar before and after treatment. Incidence of extra-uterine pregnancies and induced abortions was elevated among the treated both before and after treatment. Thus this elevation did not occur because they were treated rather to a great extent was due to the other known risk factors these women had in excess, i.e. sexually transmitted infections. The purpose of any cancer preventive activity is to reduce cancer incidence and mortality. In Finland, cervical cancer is a rare disease and death from it even rarer, mostly due to the effective screening program. Despite this, the women treated are at increased risk for cancer; not just for cervical cancer. They must be followed up carefully and for a long period of time; general health education, especially cessation of smoking, is crucial in the management process, as well as interventions towards proper use of birth control such as condoms.