Living on the edge : Population genetics of Finno-Ugric-speaking humans in North Eurasia

The major aim of this thesis was to examine the origins and distribution of uniparental and autosomal genetic variation among the Finno-Ugric-speaking human populations living in Boreal and Arctic regions of North Eurasia. In more detail, I aimed to disentangle the underlying molecular and population genetic factors which have produced the patterns of uniparental and autosomal genetic diversity in these populations. Among Finno-Ugrics the genetic amalgamation and clinal distribution of West and East Eurasian gene pools were observed within uniparental markers. This admixture indicates that North Eurasia was colonized through Central Asia/ South Siberia by human groups already carrying both West and East Eurasian lineages. The complex combination of founder effects, gene flow and genetic drift underlying the genetic diversity of the Finno-Ugric- speaking populations were emphasized by low haplotype diversity within and among uniparental and biparental markers. A high prevalence of lactase persistence allele among the North Eurasian Finno- Ugric agriculturalist populations was also shown indicating a local adaptation to subsistence change with lactose rich diet. Moreover, the haplotype background of lactase persistence allele among the Finno- Ugric-speakers strongly suggested that the lactase persistence T-13910 mutation was introduced independently more than once to the North Eurasian gene pool. A significant difference in genetic diversity, haplotype structure and LD distribution within the cytochrome P450 CYP2C and CYP2D regions revealed the unique gene pool of the Finno-Ugric Saami created mainly by population genetic processes compared to other Europeans and sub-Saharan Mandenka population. From all studied populations the Saami showed also significantly the highest allele frequency of a CYP2C19 gene mutation causing variable drug reactions. The diversity patterns observed within CYP2C and CYP2D regions emphasize the strong effect of demographic history shaping genetic diversity and LD especially among such small and constant size populations as the Finno-Ugric-speaking Saami. Moreover, the increased LD in Saami due to genetic drift and/or admixture was shown to offer an advantage for further attempts to identify alleles associated to common complex pharmacogenetic traits.

Nutrition and bone metabolism : In vivo effects of inorganic phosphate on rats and in vitro effects of bioactive tripeptides on human osteoblasts

Nutrition affects bone health throughout life. To optimize peak bone mass development and maintenance, it is important to pay attention to the dietary factors that enhance and impair bone metabolism. In this study, the in vivo effects of inorganic dietary phosphate and the in vitro effects of bioactive tripeptides, IPP, VPP and LKP were investigated. Dietary phosphate intake is increased through the use of convenience foods and soft drinks rich in phosphate-containing food additives. Our results show that increased dietary phosphate intake hinders mineral deposition in cortical bone and diminishes bone mineral density (BMD) in the aged skeleton in a rodent model (Study I). In the growing skeleton (Study II), increased phosphate intake was observed to reduce bone material and structural properties, leading to diminished bone strength. Studies I and II revealed that a low Ca:P ratio has negative effects on the mature and growing rat skeleton even when calcium intake is sufficient. High dietary protein intake is beneficial for bone health. Protein is essential for bone turnover and matrix formation. In addition, hydrolysis of proteins in the gastrointestinal tract produces short peptides that possess a biological function beyond that of being tissue building blocks. The effects of three bioactive tripeptides, IPP, VPP and LKP, were assessed in short- and long-term in vitro experiments. Short-term treatment (24 h) with tripeptide IPP, VPP or LKP influenced osteoblast gene expression (Study III). IPP in particular, regulates genes associated with cell differentiation, cell growth and cell signal transduction. The upregulation of these genes indicates that IPP enhances osteoblast proliferation and differentiation. Long-term treatment with IPP enhanced osteoblast gene expression in favour of bone formation and increased mineralization (Study IV). The in vivo effects of IPP on osteoblast differentiation might differ since eating frequency drives food consumption, and protein degradation products, such as bioactive peptides, are available periodically, not continuously as in this study. To sum up, Studies I and II raise concern about the appropriate amount of dietary phosphate to support bone health as excess is harmful. Studies III and IV in turn, support findings of the beneficial effects of dietary protein on bone and provide a mechanistic explanation since cell proliferation and osteoblast function were improved by treatment with bioactive tripeptide IPP.

Pentitol phosphate dehydrogenases: Discovery, characterization and use in D-arabitol and xylitol production by metabolically engineered Bacillus subtilis

The ultimate goal of this study has been to construct metabolically engineered microbial strains capable of fermenting glucose into pentitols D-arabitol and, especially, xylitol. The path that was chosen to achieve this goal required discovery, isolation and sequencing of at least two pentitol phosphate dehydrogenases of different specificity, followed by cloning and expression of their genes and characterization of recombinant arabitol and xylitol phosphate dehydrogenases. An enzyme of a previously unknown specificity, D-arabitol phosphate dehydrogenase (APDH), was discovered in Enterococcus avium. The enzyme was purified to homogenity from E. avium strain ATCC 33665. SDS/PAGE revealed that the enzyme has a molecular mass of 41 ± 2 kDa, whereas a molecular mass of 160 ± 5 kDa was observed under non-denaturing conditions implying that the APDH may exist as a tetramer with identical subunits. Purified APDH was found to have narrow substrate specificity, converting only D-arabitol 1-phosphate and D-arabitol 5-phosphate into D-xylulose 5-phosphate and D-ribulose 5-phosphate, respectively, in the oxidative reaction. Both NAD+ and NADP+ were accepted as co-factors. Based on the partial protein sequences, the gene encoding APDH was cloned. Homology comparisons place APDH within the medium chain dehydrogenase family. Unlike most members of this family, APDH requires Mn2+ but no Zn2+ for enzymatic activity. The DNA sequence surrounding the gene suggests that it belongs to an operon that also contains several components of phosphotransferase system (PTS). The apparent role of the enzyme is to participate in arabitol catabolism via the arabitol phosphate route similar to the ribitol and xylitol catabolic routes described previously. Xylitol phosphate dehydrogenase (XPDH) was isolated from Lactobacillus rhamnosus strain ATCC 15820. The enzyme was partially sequenced. Amino acid sequences were used to isolate the gene encoding the enzyme. The homology comparisons of the deduced amino acid sequence of L. rhamnosus XPDH revealed several similar enzymes in genomes of various species of Gram-positive bacteria. Two enzymes of Clostridium difficile and an enzyme of Bacillus halodurans were cloned and their substrate specificities together with the substrate specificity of L. rhamnosus XPDH were compared. It was found that one of the XPDH enzymes of C. difficile and the XPDH of L. rhamnosus had the highest selectivity towards D-xylulose 5-phosphate. A known transketolase-deficient and D-ribose-producing mutant of Bacillus subtilis (ATCC 31094) was further modified by disrupting its rpi (D-ribose phosphate isomerase) gene to create D-ribulose- and D-xylulose-producing strain. Expression of APDH of E. avium and XPDH of L. rhamnosus and C. difficile in D-ribulose- and D-xylulose-producing strain of B. subtilis resulted in strains capable of converting D-glucose into D-arabitol and xylitol, respectively. The D-arabitol yield on D-glucose was 38 % (w/w). Xylitol production was accompanied by co-production of ribitol limiting xylitol yield to 23 %.

Population dynamics of blue mussels in a variable environment at the edge of their range

Spatial and temporal variation in the abundance of species can often be ascribed to spatial and temporal variation in the surrounding environment. Knowledge of how biotic and abiotic factors operate over different spatial and temporal scales in determining distribution, abundance, and structure of populations lies at the heart of ecology. The major part of the current ecological theory stems from studies carried out in central parts of the distributional range of species, whereas knowledge of how marginal populations function is inadequate. Understanding how marginal populations, living at the edge of their range, function is however in a key position to advance ecology and evolutionary biology as scientific disciplines. My thesis focuses on the factors affecting dynamics of marginal populations of blue mussels (Mytilus edulis) living close to their tolerance limits with regard to salinity. The thesis aims to highlight the dynamics at the edge of the range and contrast these with dynamics in more central parts of the range in order to understand the potential interplay between the central and the marginal part in the focal system. The objectives of the thesis are approached by studies on: (1) factors affecting regional patterns of the species, (2) long-term temporal dynamics of the focal species spaced along a regional salinity gradient, (3) selective predation by increasing populations of roach (Rutilus rutilus) when feeding on their main food item, the blue mussel, (4) the primary and secondary effects of local wave exposure gradients and (5) the role of small-scale habitat heterogeneity as determinants of large-scale pattern. The thesis shows that populations of blue mussels are largely determined by large scale changes in sea water salinity, affecting mainly recruitment success and longevity of local populations. In opposite to the traditional view, the thesis strongly indicate that vertebrate predators strongly affect abundance and size structure of blue mussel populations, and that the role of these predators increases towards the margin where populations are increasingly top-down controlled. The thesis also indicates that the positive role of biogenic habitat modifiers increases towards the marginal areas, where populations of blue mussels are largely recruitment limited. Finally, the thesis shows that local blue mussel populations are strongly dependent on high water turbulence, and therefore, dense populations are constrained to offshore habitats. Finally, the thesis suggests that ongoing sedimentation of rocky shores is detrimental for the species, affecting recruitment success and post-recruit survival, pushing stable mussel beds towards offshore areas. Ongoing large scale changes in the Baltic Sea, especially dilution processes with attendant effects, are predicted to substantially contract the distributional range of the mussel, but also affect more central populations. The thesis shows that in order to understand the functioning of marginal populations, research should (1) strive for multi-scale approaches in order to link ecosystem patterns with ecosystem processes, and (2) challenge the prevailing tenets that origin from research carried out in central areas that may not be valid at the edge.

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).

Primary polyestradiol phosphate versus orchiectomy for advanced prostate cancer : Studies on the efficacy, cardiovascular complications and prognostic factors

Paikallisesti levinnyttä (T3-4 M0) ja luustoon levinnyttä (T1-4 M1) eturauhassyöpää sairastaneet potilaat satunnaistettiin kirurgiseen kastraatioon (orkiektomia) tai lääkkeelliseen kastraatioon lihaksensisäisellä polyestradiolifosfaatilla (PEP) annoksella 240 mg/kk. Verrattiin hoitojen kliinistä tehoa sekä sydän- ja verisuonikomplikaatioita (SV-komplikaatioita). Verrattiin myös hoitoa edeltäviä plasman testosteroni (T) ja estradioli (E2) pitoisuuksia T3-4 M0 ja T1-4 M1 potilaiden välillä sekä selvitettiin potilaiden yleistilan vaikutusta näihin hormonitasoihin. Lopuksi luotiin T1-4 M1 potilaille eturauhassyövän aiheuttaman kuoleman ennusteellinen riskiluokittelu kolmeen riskiryhmään käyttämällä hoitoa edeltäviä ennustetekijöitä. Kliinisessä tehossa ei orkiektomian ja PEP-hoidon välillä todettu tilastollisesti merkitsevää eroa. Odotetusti T1-4 M1 potilaiden ennuste oli huonompi kuin T3-4 M0 potilaiden. T1-4 M1 potilailla ei ollut SV-kuolemissa hoitoryhmien välillä tilastollista eroa, mutta ei-tappavia SV-komplikaatioita oli PEP ryhmässä (5.9%) enemmän kuin orkiektomia ryhmässä (2.0%). T3-4 M0 potilailla PEP-hoitoon liittyi tilastollisesti merkitsevä SV-kuolleisuus riski orkiektomiaan verrattuna (p = 0.001). PEP ryhmässä 67% kuolemista oli akuutteja sydäninfarkteja. Tämä PEP hoitoon liittyvä sydäninfarktiriski (mukaan lukien myös ei-tappavat sydäninfarktit) oli merkitsevästi pienempi potilailla, joiden hoitoa edeltävä E2 taso oli vähintään 93 pmol/l (p = 0.022). E2 taso oli merkitsevästi matalampi T1-4 M1 potilailla (74.7 pmol/l) kuin T3-4 M0 potilailla (87.9 pmol/l), mutta vastaavaa eroa ei ollut T tasoissa. Sekä T3-4 M0 että T1-4 M1 potilailla yleistilan lasku osittain selitti yksilöllisen T ja E2 tasojen laskun. Eturauhassyövän aiheuttaman kuoleman riskiryhmäluokittelu (Rg) kolmeen ryhmään luotiin käyttämällä alkalista fosfataasia (AFOS), prostata spesifistä antigeenia (PSA), laskoa (La) ja potilaan ikää. Yksi riskipiste annettiin, jos AFOS > 180 U/l (tällä hetkellä käytössä olevalla menetelmällä AFOS > 83 U/l), PSA > 35 µg/l, La > 80 mm/h ja ikä < 60 vuotta. Lopuksi pisteet laskettiin yhteen. Muodostettiin seuraavat ryhmät: Rg-a (0 -1 riskipistettä), Rg-b (2 riskipistettä) ja Rg-c (3 – 4 riskipistettä). Eturauhassyövän aiheuttama kuoleman riski lisääntyi merkitsevästi siirryttäessä riskiryhmästä seuraavaan (p < 0.001). Rg-luokittelu oli kliinisesti käytännöllinen ja hyvä havaitsemaan huonon ennusteen potilaat.

Distributed algorithms for edge dominating sets

An edge dominating set for a graph G is a set D of edges such that each edge of G is in D or adjacent to at least one edge in D. This work studies deterministic distributed approximation algorithms for finding minimum-size edge dominating sets. The focus is on anonymous port-numbered networks: there are no unique identifiers, but a node of degree d can refer to its neighbours by integers 1, 2, ..., d. The present work shows that in the port-numbering model, edge dominating sets can be approximated as follows: in d-regular graphs, to within 4 − 6/(d + 1) for an odd d and to within 4 − 2/d for an even d; and in graphs with maximum degree Δ, to within 4 − 2/(Δ − 1) for an odd Δ and to within 4 − 2/Δ for an even Δ. These approximation ratios are tight for all values of d and Δ: there are matching lower bounds.