Glycogen debranching enzyme activity in the muscles of meat producing animals

Muscle glycogen exists in two forms: low molecular weight pro-glycogen and high molecular weight macro-glycogen. The degradation of glycogen to glucose 1 phosphate and free glucose is catalysed by glycogen phosphorylase together with glycogen debranching enzyme (GDE). The process in which glycogen is broken down via anaerobic pathways to lactate, results in the acidification of the muscles and has a great influence on meat quality. Thus, the overall aim of this thesis was to characterise the post mortem action of GDE in muscles of meat production animals (pigs, cattle and chickens). Interest was focused on the differences in GDE activity between fast twitch glycolytic muscles and slow twitch oxidative muscles. The effects of pH, temperature, RN genotype (PRKAG3 gene), and of time post mortem on GDE activity were also investigated. This thesis showed that there are differences in GDE activity between animal species and between different muscles of an animal. It was shown that in pigs and cattle, higher GDE activity and phosphorylase activity exists in the fast twitch glycolytic muscles than in slow twitch oxidative muscles of the same animal. Thus, the high activity of these enzymes enables a faster rate of glycogenolysis in glycolytic M. longissimus dorsi compared to oxidative M. masseter. In chicken muscles, the GDE activity was low compared to pig or cattle muscles. Furthermore, the GDE activity in the glycolytic M. pectoralis superficialis was lower than in more oxidative M. quadriceps femoris despite the high phosphorylase activity in the former. The relative ratios between phosphorylase and GDE activity were higher in fast twitch glycolytic muscles than in slow twitch oxidative muscles of all studied animals. This suggests that the relatively low GDE activity compared to the phosphorylase activity in fast twitch glycolytic muscles may be a protection mechanism in living muscle against a very fast pH decrease. Chilling significantly decreased GDE activity and below 15 C porcine GDE was almost inactive. The effect of pH on GDE activity was only minor at the range normally found in post mortem muscles (pH 7.4 to 5.0). The GDE activity remained level for several hours after slaughter. During the first hours post mortem, GDE activity was similar in RN- carrier pigs and in wild type pigs. However, the GDE activity declined faster in M. longissimus dorsi from wild type pigs than in the RN carrier pigs, the difference between genotypes was significant after 24 h post mortem. Pro-glycogen and macro-glycogen contents were higher, pH decrease was faster and ultimate pH was lower in RN- carrier pigs than in wild type pigs. In the RN- carriers, the prolonged high GDE activity level may enable an extended pH decrease and lower ultimate pH in their muscles. In conclusion, GDE is not the main factor determining the rate or the extent of post mortem glycogenolysis, but under certain conditions, such as in very fast chilling, the inhibition of GDE activity in meat may reduce the rate of pH decrease and result in higher ultimate pH. The rate and extent of pH decrease affects several meat quality traits.

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

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

Forest inventory-based large-scale forest biomass and carbon budget assessment: new enhanced methods and use of remote sensing for verification

In recent years, concern has arisen over the effects of increasing carbon dioxide (CO2) in the earth's atmosphere due to the burning of fossil fuels. One way to mitigate increase in atmospheric CO2 concentration and climate change is carbon sequestration to forest vegeta-tion through photosynthesis. Comparable regional scale estimates for the carbon balance of forests are therefore needed for scientific and political purposes. The aim of the present dissertation was to improve methods for quantifying and verifying inventory-based carbon pool estimates of the boreal forests in the mineral soils. Ongoing forest inventories provide a data based on statistically sounded sampling for estimating the level of carbon stocks and stock changes, but improved modelling tools and comparison of methods are still needed. In this dissertation, the entire inventory-based large-scale forest carbon stock assessment method was presented together with some separate methods for enhancing and comparing it. The enhancement methods presented here include ways to quantify the biomass of understorey vegetation as well as to estimate the litter production of needles and branches. In addition, the optical remote sensing method illustrated in this dis-sertation can be used to compare with independent data. The forest inventory-based large-scale carbon stock assessment method demonstrated here provided reliable carbon estimates when compared with independent data. Future ac-tivity to improve the accuracy of this method could consist of reducing the uncertainties regarding belowground biomass and litter production as well as the soil compartment. The methods developed will serve the needs for UNFCCC reporting and the reporting under the Kyoto Protocol. This method is principally intended for analysts or planners interested in quantifying carbon over extensive forest areas.

Reinventing Nordic Openness : Transparency and State Information in Finland

The study explores new ideational changes in the information strategy of the Finnish state between 1998 and 2007, after a juncture in Finnish governing in the early 1990s. The study scrutinizes the economic reframing of institutional openness in Finland that comes with significant and often unintended institutional consequences of transparency. Most notably, the constitutional principle of publicity (julkisuusperiaate), a Nordic institutional peculiarity allowing public access to state information, is now becoming an instrument of economic performance and accountability through results. Finland has a long institutional history in the publicity of government information, acknowledged by law since 1951. Nevertheless, access to government information became a policy concern in the mid-1990s, involving a historical narrative of openness as a Nordic tradition of Finnish governing Nordic openness (pohjoismainen avoimuus). International interest in transparency of governance has also marked an opening for institutional re-descriptions in Nordic context. The essential added value, or contradictory term, that transparency has on the Finnish conceptualisation of governing is the innovation that public acts of governing can be economically efficient. This is most apparent in the new attempts at providing standardised information on government and expressing it in numbers. In Finland, the publicity of government information has been a concept of democratic connotations, but new internationally diffusing ideas of performance and national economic competitiveness are discussed under the notion of transparency and its peer concepts openness and public (sector) information, which are also newcomers to Finnish vocabulary of governing. The above concepts often conflict with one another, paving the way to unintended consequences for the reforms conducted in their name. Moreover, the study argues that the policy concerns over openness and public sector information are linked to the new drive for transparency. Drawing on theories of new institutionalism, political economy, and conceptual history, the study argues for a reinvention of Nordic openness in two senses. First, in referring to institutional history, the policy discourse of Nordic openness discovers an administrative tradition in response to new dilemmas of public governance. Moreover, this normatively appealing discourse also legitimizes the new ideational changes. Second, a former mechanism of democratic accountability is being reframed with market and performance ideas, mostly originating from the sphere of transnational governance and governance indices. Mobilizing different research techniques and data (public documents of the Finnish government and international organizations, some 30 interviews of Finnish civil servants, and statistical time series), the study asks how the above ideational changes have been possible, pointing to the importance of nationalistically appealing historical narratives and normative concepts of governing. Concerning institutional developments, the study analyses the ideational changes in central steering mechanisms (political, normative and financial steering) and the introduction of budget transparency and performance management in two cases: census data (Population Register Centre) and foreign political information (Ministry for Foreign Affairs). The new policy domain of governance indices is also explored as a type of transparency. The study further asks what institutional transformations are to be observed in the above cases and in the accountability system. The study concludes that while the information rights of citizens have been reinforced and recalibrated during the period under scrutiny, there has also been a conversion of institutional practices towards economic performance. As the discourse of Nordic openness has been rather unquestioned, the new internationally circulating ideas of transparency and the knowledge economy have entered this discourse without public notice. Since the mid 1990s, state registry data has been perceived as an exploitable economic resource in Finland and in the EU public sector information. This is a parallel development to the new drive for budget transparency in organisations as vital to the state as the Population Register Centre, which has led to marketization of census data in Finland, an international exceptionality. In the Finnish Ministry for Foreign Affairs, the post-Cold War rhetorical shift from secrecy to performance-driven openness marked a conversion in institutional practices that now see information services with high regards. But this has not necessarily led to the increased publicity of foreign political information. In this context, openness is also defined as sharing information with select actors, as a trust based non-public activity, deemed necessary amid the global economic competition. Regarding accountability system, deliberation and performance now overlap, making it increasingly difficult to identify to whom and for what the public administration is accountable. These evolving institutional practices are characterised by unintended consequences and paradoxes. History is a paradoxical component in the above institutional change, as long-term institutional developments now justify short-term reforms.

Role of Temperature in the Biological Activity of a Boreal Forest

In northern latitudes, temperature is the key factor driving the temporal scales of biological activity, namely the length of the growing season and the seasonal efficiency of photosynthesis. The formation of atmospheric concentrations of biogenic volatile organic compounds (BVOCs) are linked to the intensity of biological activity. However, interdisciplinary knowledge of the role of temperature in the biological processes related to the annual cycle and photosynthesis and atmospheric chemistry is not fully understood. The aim of this study was to improve understanding of the role of temperature in these three interlinked areas: 1) onset of growing season, 2) photosynthetic efficiency and 3) BVOC air concentrations in a boreal forest. The results present a cross-section of the role of temperature on different spatial (southern northern boreal), structural (tree forest stand - forest) and temporal (day-season- year) scales. The fundamental status of the Thermal Time model in predicting the onset of spring recovery was confirmed. However, it was recommended that sequential models would be more appropriate tools when the onset of the growing season is estimated under a warmer climate. A similar type of relationship between photosynthetic efficiency and temperature history was found in both southern and northern boreal forest stands. This result draws attention to the critical question of the seasonal efficiency of coniferous species to emit organic compounds under a warmer climate. New knowledge about the temperature dependence of the concentrations of biogenic volatile organic compounds in a boreal forest stand was obtained. The seasonal progress and the inter-correlation of BVOC concentrations in ambient air indicated a link to biological activity. Temperature was found to be the main driving factor for the concentrations. However, in addition to temperature, other factors may play a significant role here, especially when the peak concentrations are studied. There is strong evidence that the spring recovery and phenological events of many plant species have already advanced in Europe. This study does not fully support this observation. In a boreal forest, changes in the annual cycle, especially the temperature requirement in winter, would have an impact on the atmospheric BVOC composition. According to this study, more joint phenological and BVOC field observations and laboratory experiments are still needed to improve these scenarios.

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

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

Heparin-binding growth-associated molecule (HB-GAM) in activity-dependent neuronal plasticity in hippocampus

Cell adhesion and extracellular matrix (ECM) molecules play a significant role in neuronal plasticity both during development and in the adult. Plastic changes in which ECM components are implicated may underlie important nervous system functions, such as memory formation and learning. Heparin-binding growthassociated molecule (HB-GAM, also known as pleiotrophin), is an ECM protein involved in neurite outgrowth, axonal guidance and synaptogenesis during perinatal period. In the adult brain HB-GAM expression is restricted to the regions which display pronounced synaptic plasticity (e.g., hippocampal CA3-CA1 areas, cerebral cortex laminae II-IV, olfactory bulb). Expression of HB-GAM is regulated in an activity-dependent manner and is also induced in response to neuronal injury. In this work mutant mice were used to study the in vivo function of HB-GAM and its receptor syndecan-3 in hippocampal synaptic plasticity and in hippocampus-dependent behavioral tasks. Phenotypic analysis of HBGAM null mutants and mice overexpressing HB-GAM revealed that opposite genetic manipulations result in reverse changes in synaptic plasticity as well as behavior in the mutants. Electrophysiological recordings showed that mice lacking HB-GAM have an increased level of long-term potentiation (LTP) in the area CA1 of hippocampus and impaired spatial learning, whereas animals with enhanced level of HB-GAM expression have attenuated LTP, but outperformed their wild-type controls in spatial learning. It was also found that GABA(A) receptor-mediated synaptic transmission is altered in the transgenic mice overexpressing HB-GAM. The results suggest that these animals have accentuated hippocampal GABAergic inhibition, which may contribute to the altered glutamatergic synaptic plasticity. Structural studies of HB-GAM demonstrated that this protein belongs to the thrombospondin type I repeat (TSR) superfamily and contains two β-sheet domains connected by a flexible linker. It was found that didomain structure is necessary for biological activity of HB-GAM and electrophysiological phenotype displayed by the HB-GAM mutants. The individual domains displayed weaker binding to heparan sulfate and failed to promote neurite outgrowth as well as affect hippocampal LTP. Effects of HB-GAM on hippocampal synaptic plasticity are believed to be mediated by one of its (co-)receptor molecules, namely syndecan-3. In support of that, HB-GAM did not attenuate LTP in mice deficient in syndecan-3 as it did in wild-type controls. In addition, syndecan-3 knockout mice displayed electrophysiological and behavioral phenotype similar to that of HB-GAM knockouts (i.e. enhanced LTP and impaired learning in Morris water-maze). Thus HB-GAM and syndecan-3 are important modulators of synaptic plasticity in hippocampus and play a role in regulation of learning-related behavior.

Synthesis of neoglycoconjugates and oligosaccharides with potential anti-Helicobacter pylori activity

The significance of carbohydrate-protein interactions in many biological phenomena is now widely acknowledged and carbohydrate based pharmaceuticals are under intensive development. The interactions between monomeric carbohydrate ligands and their receptors are usually of low affinity. To overcome this limitation natural carbohydrate ligands are often organized as multivalent structures. Therefore, artificial carbohydrate pharmaceuticals should be constructed on the same concept, as multivalent carbohydrates or glycoclusters. Infections of specific host tissues by bacteria, viruses, and fungi are among the unfavorable disease processes for which suitably designed carbohydrate inhibitors represent worthy targets. The bacterium Helicobacter pylori colonizes more than half of all people worldwide, causing gastritis, gastric ulcer, and conferring a greater risk of stomach cancer. The present medication therapy for H. pylori includes the use of antibiotics, which is associated with increasing incidence of bacterial resistance to traditional antibiotics. Therefore, the need for an alternative treatment method is urgent. In this study, four novel synthesis procedures of multivalent glycoconjugates were created. Three different scaffolds representing linear (chondroitin oligomer), cyclic (γ-cyclodextrin), and globular (dendrimer) molecules were used. Multivalent conjugates were produced using the human milk type oligosaccharides LNDFH I (Lewis-b hexasaccharide), LNnT (Galβ1-4GlcNAcβ1-3Galβ1-4Glc), and GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc all representing analogues of the tissue binding epitopes for H. pylori. The first synthetic method included the reductive amination of scaffold molecules modified to express primary amine groups, and in the case of dendrimer direct amination to scaffold molecule presenting 64 primary amine groups. The second method described a direct procedure for amidation of glycosylamine modified oligosaccharides to scaffold molecules presenting carboxyl groups. The final two methods that were created both included an oxime-linkage on linkers of different length. All the new synthetic procedures synthesized had the advantage of using unmodified reducing sugars as starting material making it easy to synthesize glycoconjugates of different specificity. In addition, the binding activity of an array of neoglycolipids to H. pylori was studied. Consequently, two new neolacto-based structures, Glcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-Cer and GlcAβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-Cer, with binding activity toward H. pylori were discovered. Interestingly, N-methyl and N-ethyl amide modification of the GlcAβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-Cer glucuronic acid residue resulted in more effective H. pylori binding epitopes than the parent molecule.

The high- and low-activity forms of human plasma phospholipid transfer protein (PLTP)

Plasma phospholipid transfer protein (PLTP) plays a crucial role in high-density lipoprotein (HDL) metabolism and reverse cholesterol transport (RCT). It mediates the generation of pre-beta-HDL particles, enhances the cholesterol efflux from peripheral cells to pre-beta-HDL, and metabolically maintains the plasma HDL levels by facilitating the transfer of post-lipolytic surface remnants of triglyceride-rich lipoproteins to HDL. In addition to the antiatherogenic properties, recent findings indicate that PLTP has also proatherogenic characteristics, and that these opposite characteristics of PLTP are dependent on the site of PLTP expression and action. In human plasma, PLTP exists in a high-activity (HA-PLTP) and a low-activity form (LA-PLTP), which are associated with macromolecular complexes of different size and composition. The aims of this thesis were to isolate the two PLTP forms from human plasma, to characterize the molecular complexes in which the HA- and LA-PLTP reside, and to study the interactions of the PLTP forms with apolipoproteins (apo) and the ability of apolipoproteins to regulate PLTP activity. In addition, we aimed to study the distribution of the two PLTP forms in a Finnish population sample as well as to find possible regulatory factors for PLTP by investigating the influence of lipid and glucose metabolism on the balance between the HA- and LA-PLTP. For these purposes, an enzyme-linked immunosorbent assay (ELISA) capable of determining the serum total PLTP concentration and quantitating the two PLTP forms separately was developed. In this thesis, it was demonstrated that the HA-PLTP isolated from human plasma copurified with apoE, whereas the LA-PLTP formed a complex with apoA-I. The separation of these two PLTP forms was carried out by a dextran sulfate (DxSO4)-CaCl2 precipitation of plasma samples before the mass determination. A similar immunoreactivity of the two PLTP forms in the ELISA could be reached after a partial sample denaturation by SDS. Among normolipidemic Finnish individuals, the mean PLTP mass was 6.6 +/- 1.5 mg/l and the mean PLTP activity 6.6 +/- 1.7 umol/ml/h. Of the serum PLTP concentration, almost 50% represented HA-PLTP. The results indicate that plasma HDL levels could regulate PLTP concentration, while PLTP activity could be regulated by plasma triglyceride-rich very low-density lipoprotein (VLDL) concentration. Furthermore, new evidence is presented that PLTP could also play a role in glucose metabolism. Finally, both PLTP forms were found to interact with apoA-I, apoA-IV, and apoE. In addition, both apoE and apoA-IV, but not apoA-I, were capable of activating the LA-PLTP. These findings suggest that the distribution of the HA- and LA-PLTP in human plasma is subject to dynamic regulation by apolipoproteins.