Campylobacter jejuni and C.coli in Finnish poultry production

Campylobacter, mainly Campylobacter jejuni and C. coli, are worldwide recognized as a major cause of bacterial food-borne gastroenteritis (World Health Organization 2010). Epidemiological studies have shown handling or eating of poultry to be significant risk factors for human infections. Campylobacter contamination can occur at all stages of a poultry meat production cycle. In summer 1999, every broiler flock from all three major Finnish poultry slaughterhouses was studied during a five month period. Caecal samples were taken in the slaughterhouses from five birds per flock. A total of 1 132 broiler flocks were tested and 33 (2.9%) of those were Campylobacter-positive. Thirty-one isolates were identified as C. jejuni and two isolates were C. coli. The isolates were serotyped for heat-stable antigens (HS) and genotyped by pulsed-field gel electrophoresis (PFGE). The most common serotypes found were HS 6,7, 12 and 4-complex. Using a combination of SmaI and KpnI patterns, 18 different PFGE types were identified. Thirty-five Finnish C. jejuni strains with five SmaI/SacII PFGE types selected among human and chicken isolates from 1997 and 1998 were used for comparison of their PFGE patterns, amplified fragment length polymorphism (AFLP) patterns, HaeIII ribotypes, and HS serotypes. The discriminatory power of PFGE, AFLP and ribotyping with HaeIII were shown to be at the same level for this selected set of strains, and these methods assigned the strains into the same groups. The PFGE and AFLP patterns within a genotype were highly similar, indicating genetic relatedness. An HS serotype was distributed among different genotypes, and different serotypes were identified within one genotype. From one turkey parent flock, the hatchery, six different commercial turkey farms (together 12 flocks) and from 11 stages at the slaughterhouse a total of 456 samples were collected during one and the half year. For the detection of Campylobacter both conventional culture and a PCR method were used. No Campylobacter were detected in either of the samples from the turkey parent flock or from the hatchery samples using the culture method. Instead PCR detected DNA of Campylobacter in five faecal samples from the turkey parent flock and in one fluff and an eggshell sample. Six out of 12 commercial turkey flocks were found negative at the farm level but only two of those were negative at slaughter. Campylobacter-positive samples within the flock at slaughter were detected between 0% and 94%, with evisceration and chilling water being the most critical stages for contamination. All of a total of 121 Campylobacter isolates were shown to be C. jejuni using a multiplex PCR assay. PFGE analysis of all isolates with KpnI restriction enzyme resulted in 11 PFGE types (I-XI) and flaA-SVR typing yielded nine flaA-SVR alleles. Three Campylobacter-positive turkey flocks were colonized by a limited number of Campylobacter genotypes both at the farm and slaughter level.In conclusion, in our first study in 1999 a low prevalence of Campylobacter in Finnish broiler flocks was detected and it has remained at a low level during the study period until the present. In the turkey meat production, we found that flocks which were negative at the farm became contaminated with Campylobacter at the slaughter process. These results suggest that proper and efficient cleaning and disinfection of slaughter and processing premises are needed to avoid cross-contamination. Prevention of colonization at the farm by a high level of biosecurity control and hygiene may be one of the most efficient ways to reduce the amount of Campylobacter-positive poultry meat in Finland. In Finland, with a persistent low level of Campylobacter-positive flocks, it could be speculated that the use of logistic slaughtering, according to Campylobacter status at farm, might have be advantageous in reducing Campylobacter contamination of retail poultry products. However, the significance of the domestic poultry meat for human campylobacteriosis in Finland should be evaluated.

Contamination routes and control of Listeria monocytogenes in Food Production

Listeria monocytogenes is the causative agent of the severe foodborne infection listeriosis. The number of listeriosis cases in recent years has increased in many European countries, including Finland. Contamination of the pathogen needs to be minimized and growth to high numbers in foods prevented in order to reduce the incidence of human cases. The aim of this study was to evaluate contamination routes of L. monocytogenes in the food chain and to investigate methods for control of the pathogen in food processing. L. monocytogenes was commonly found in wild birds, the pig production chain and in pork production plants. It was found most frequently in birds feeding at landfill site, organic farms, tonsil samples, and sites associated with brining. L. monococytogenes in birds, farms, food processing plant or foods did not form distinct genetic groups, but populations overlapped. The majority of genotypes recovered from birds were also detected in foods, food processing environments and other animal species and birds may disseminate L. monocytogenes into food chain. Similar genotypes were found in different pigs on the same farm, as well as in pigs on farms and later in the slaughterhouse. L. monocytogenes contamination spreads at farm level and may be a contamination source into slaughterhouses and further into meat. Incoming raw pork in the processing plant was frequently contaminated with L. monocytogenes and genotypes in raw meat were also found in processing environment and in RTE products. Thus, raw material seems to be a considerable source of contamination into processing facilities. In the pork processing plant, the prevalence of L. monocytogenes increased in the brining area, showing that the brining was an important contamination site. Recovery of the inoculated L. monocytogenes strains showed that there were strain-specific differences in the ability to survive in lettuce and dry sausage. The ability of some L. monocytogenes strains to survive well in food production raises a challenge for industry, because these strains can be especially difficult to remove from the products and raises a need to use an appropriate hurdle concept to control most resistant strains. Control of L. monocytogenes can be implemented throughout the food chain. Farm-specific factors affected the prevalence of L. monocytogenes and good farm-level practices can therefore be utilized to reduce the prevalence of this pathogen on the farm and possibly further in the food chain. Well separated areas in a pork production plant had low prevalences of L. monocytogenes, thus showing that compartmentalization controls the pathogen in the processing line. The food processing plant, especially the brining area, should be subjected to disassembling, extensive cleaning and disinfection to eliminate persistent contamination by L. monocytogenes, and replacing brining with dry-salting should be considered. All of the evaluated washing solutions decreased the populations of L. monocytogenes on precut lettuce, but did not eliminate the pathogen. Thus, the safety of fresh-cut produce cannot rely on washing with disinfectants, and high-quality raw material and good manufacturing practices remain important. L. monocytogenes was detected in higher levels in sausages without the protective culture than in sausages with this protective strain, although numbers of L. monocytogenes by the end of the ripening decreased to the level of < 100 MPN/g in all sausages. Protective starter cultures provide an appealing hurdle in dry sausage processing and assist in the control of L. monocytogenes.

Learning Challenges in Biogas Production for Sustainability : An activity theoretical study of a network from a swine industry chain

This study is about the challenges of learning in the creation and implementation of new sustainable technologies. The system of biogas production in the Programme of Sustainable Swine Production (3S Programme) conducted by the Sadia food processing company in Santa Catarina State, Brazil, is used as a case example for exploring the challenges, possibilities and obstacles of learning in the use of biogas production as a way to increase the environmental sustainability of swine production. The aim is to contribute to the discussion about the possibilities of developing systems of biogas production for sustainability (BPfS). In the study I develop hypotheses concerning the central challenges and possibilities for developing systems of BPfS in three phases. First, I construct a model of the network of activities involved in the BP for sustainability in the case study. Next, I construct a) an idealised model of the historically evolved concepts of BPfS through an analysis of the development of forms of BP and b) a hypothesis of the current central contradictions within and between the activity systems involved in BP for sustainability in the case study. This hypothesis is further developed through two actual empirical analyses: an analysis of the actors senses in taking part in the system, and an analysis of the disturbance processes in the implementation and operation of the BP system in the 3S Programme. The historical analysis shows that BP for sustainability in the 3S Programme emerged as a feasible solution for the contradiction between environmental protection and concentration, intensification and specialisation in swine production. This contradiction created a threat to the supply of swine to the food processing company. In the food production activity, the contradiction was expressed as a contradiction between the desire of the company to become a sustainable company and the situation in the outsourced farms. For the swine producers the contradiction was expressed between the contradictory rules in which the market exerted pressure which pushed for continual increases in scale, specialisation and concentration to keep the production economically viable, while the environmental rules imposed a limit to this expansion. Although the observed disturbances in the biogas system seemed to be merely technical and localised within the farms, the analysis proposed that these disturbances were formed in and between the activity systems involved in the network of BPfS during the implementation. The disturbances observed could be explained by four contradictions: a) contradictions between the new, more expanded activity of sustainable swine production and the old activity, b) a contradiction between the concept of BP for carbon credits and BP for local use in the BPfS that was implemented, c) contradictions between the new UNFCCC1 methodology for applying for carbon credits and the small size of the farms, and d) between the technologies of biogas use and burning available in the market and the small size of the farms. The main finding of this study relates to the zone of proximal development (ZPD) of the BPfS in Sadia food production chain. The model is first developed as a general model of concepts of BPfS and further developed here to the specific case of the BPfS in the 3S Programme. The model is composed of two developmental dimensions: societal and functional integration. The dimension of societal integration refers to the level of integration with other activities outside the farm. At one extreme, biogas production is self-sufficient and highly independent and the products of BP are consumed within the farm, while at the other extreme BP is highly integrated in markets and networks of collaboration, and BP products are exchanged within the markets. The dimension of functional integration refers to the level of integration between products and production processes so that economies of scope can be achieved by combining several functions using the same utility. At one extreme, BP is specialised in only one product, which allows achieving economies of scale, while at the other extreme there is an integrated production in which several biogas products are produced in order to maximise the outcomes from the BP system. The analysis suggests that BP is moving towards a societal integration, towards the market and towards a functional integration in which several biogas products are combined. The model is a hypothesis to be further tested through interventions by collectively constructing the new proposed concept of BPfS. Another important contribution of this study refers to the concept of the learning challenge. Three central learning challenges for developing a sustainable system of BP in the 3S Programme were identified: 1) the development of cheaper and more practical technologies of burning and measuring the gas, as well as the reduction of costs of the process of certification, 2) the development of new ways of using biogas within farms, and 3) the creation of new local markets and networks for selling BP products. One general learning challenge is to find more varied and synergic ways of using BP products than solely for the production of carbon credits. Both the model of the ZPD of BPfS and the identified learning challenges could be used as learning tools to facilitate the development of biogas production systems. The proposed model of the ZPD could be used to analyse different types of agricultural activities that face a similar contradiction. The findings could be used in interventions to help actors to find their own expansive actions and developmental projects for change. Rather than proposing a standardised best concept of BPfS, the idea of these learning tools is to facilitate the analysis of local situations and to help actors to make their activities more sustainable.

Diastereomeric Drug Glucuronides: Enzymatic Glucuronidation and Analysis by Capillary Electrophoresis and Liquid Chromatography-Mass Spectrometry

Foreign compounds, such as drugs are metabolised in the body in numerous reactions. Metabolic reactions are divided into phase I (functionalisation) and phase II (conjugation) reactions. Uridine diphosphoglucuronosyltransferase enzymes (UGTs) are important catalysts of phase II metabolic system. They catalyse the transfer of glucuronic acid to small lipophilic molecules and convert them to hydrophilic and polar glucuronides that are readily excreted from the body. Liver is the main site of drug metabolism. Many drugs are racemic mixtures of two enantiomers. Glucuronidation of a racemic compound yields a pair of diastereomeric glucuronides. Stereoisomers are interesting substrates in glucuronidation studies since some UGTs display stereoselectivity. Diastereomeric glucuronides of O-desmethyltramadol (M1) and entacapone were selected as model compounds in this work. The investigations of the thesis deal with enzymatic glucuronidation and the development of analytical methods for drug metabolites, particularly diastereomeric glucuronides. The glucuronides were analysed from complex biological matrices, such as urine or from in vitro incubation matrices. Various pretreatment techniques were needed to purify, concentrate and isolate the analytes of interest. Analyses were carried out by liquid chromatography (LC) with ultraviolet (UV) or mass spectrometric (MS) detection or with capillary electromigration techniques. Commercial glucuronide standards were not available for the studies. Enzyme-assisted synthesis with rat liver microsomes was therefore used to produce M1 glucuronides as reference compounds. The glucuronides were isolated by LC/UV and ultra performance liquid chromatography (UPLC)/MS, while tandem mass spectrometry (MS/MS) and nuclear magnetic resonance (NMR) spectroscopy were employed in structural characterisation. The glucuronides were identified as phenolic O-glucuronides of M1. To identify the active UGT enzymes in (±)-M1 glucuronidation recombinant human UGTs and human tissue microsomes were incubated with (±)-M1. The study revealed that several UGTs can catalyse (±)-M1 glucuronidation. Glucuronidation in human liver microsomes like in rat liver microsomes is stereoselective. The results of the studies showed that UGT2B7, most probably, is the main UGT responsible for (±)-M1 glucuronidation in human liver. Large variation in stereoselectivity of UGTs toward (±)-M1 enantiomers was observed. Formation of M1 glucuronides was monitored with a fast and selective UPLC/MS method. Capillary electromigration techniques are known for their high resolution power. A method that relied on capillary electrophoresis (CE) with UV detection was developed for the separation of tramadol and its free and glucuronidated metabolites. The suitability of the method to identify tramadol metabolites in an authentic urine samples was tested. Unaltered tramadol and four of its main metabolites were detected in the electropherogram. A micellar electrokinetic chromatography (MEKC) /UV method was developed for the separation of the glucuronides of entacapone in human urine. The validated method was tested in the analysis of urine samples of patients. The glucuronides of entacapone could be quantified after oral entacapone dosing.

Production process of Scots pine.

The accompanying collective research report is the result of the research project in 1986­90 between The Finnish Academy and the former Soviet Academy of Sciences. The project was organized around common field work in Finland and in the former Soviet Union and theoretical analyses of tree growth determining processes. Based on theoretical analyses, dynamic stand growth models were made and their parameters were determined utilizing the field results. Annual cycle affects the tree growth. Our theoretical approach was based on adaptation to local climate conditions from Lapland to South Russia. The initiation of growth was described as a simple low and high temperature accumulation driven model. Linking the theoretical model with long term temperature data allowed us to analyze what type of temperature response produced favorable outcome in different climates. Initiation of growth consumes the carbohydrate reserves in plants. We measured the dynamics of insoluble and soluble sugars in the very northern and Karelian conditions. Clear cyclical pattern was observed but the differences between locations were surprisingly small. Analysis of field measurements of CO2 exchange showed that irradiance is the dominating factor causing variation in photosynthetic rate in natural conditions during summer. The effect of other factors is so small that they can be omitted without any considerable loss of accuracy. A special experiment carried out in Hyytiälä showed that the needle living space, defined as the ratio between the shoot cylindric volume and needle surface area, correlates with the shoot photosynthesis. The penetration of irradiance into Scots pine canopy is a complicated phenomenon because of the movement of the sun on the sky and the complicated structure of branches and needles. A moderately simple but balanced forest radiation regime submodel was constructed. It consists of the tree crown and forest structure, the gap probability calculation and the consideration of spatial and temporal variation of radiation inside the forest. The common field excursions in different geographical regions resulted in a lot of experimental data of regularities of woody structures. The water transport seems to be a good common factor to analyse these properties of tree structure. There are evident regressions between cross-sectional areas measured at different locations along the water pathway from fine roots to needles. The observed regressions have clear geographical trends. For example, the same cross-sectional area can support three times higher needle mass in South Russia than in Lapland. Geographical trends can also be seen in shoot and needle structure. Analysis of data published by several Russian authors show, that one ton of needles transpire 42 ton of water a year. This annual amount of transpiration seems to be independent of geographical location, year and site conditions. The produced theoretical and experimental material is utilised in the development of stand growth model that describes the growth and development of Scots pine stands in Finland and the former Soviet Union. The core of the model is carbon and nutrient balances. This means that carbon obtained in photosynthesis is consumed for growth and maintenance and nutrients are taken according to the metabolic needs. The annual photosynthetic production by trees in the stand is determined as a function of irradiance and shading during the active period. The utilisation of the annual photosynthetic production to the growth of different components of trees is based on structural regularities. Since the fundamental metabolic processes are the same in all locations the same growth model structure can be applied in the large range of Scots pine. The annual photosynthetic production and structural regularities determining the allocation of resources have geographical features. The common field measurements enable the application of the model to the analysis of growth and development of stands growing on the five locations of experiments. The model enables the analysis of geographical differences in the growth of Scots pine. For example, the annual photosynthetic production of a 100-year-old stand at Voronez is 3.5 times higher than in Lapland. The share consumed to needle growth (30 %) and to growth of branches (5 %) seems to be the same in all locations. In contrast, the share of fine roots is decreasing when moving from north to south. It is 20 % in Lapland, 15 % in Hyytiälä Central Finland and Kentjärvi Karelia and 15 % in Voronez South Russia. The stem masses (115­113 ton/ha) are rather similar in Hyytiälä, Kentjärvi and Voronez, but rather low (50 ton/ha) in Lapland. In Voronez the height of the trees reach 29 m being in Hyytiälä and Kentjärvi 22 m and in Lapland only 14 m. The present approach enables utilization of structural and functional knowledge, gained in places of intensive research, in the analysis of growth and development of any stand. This opens new possibilities for growth research and also for applications in forestry practice.

Development of radiosynthesis methods for 18F-labelled radiopharmaceuticals : General considerations and production of a dopamine transporter radioligand

Positron emission tomography (PET) is a molecular imaging technique that utilises radiopharmaceuticals (radiotracers) labelled with a positron-emitting radionuclide, such as fluorine-18 (18F). Development of a new radiotracer requires an appropriate radiosynthesis method: the most common of which with 18F is nucleophilic substitution with [18F]fluoride ion. The success of the labelling reaction is dependent on various factors such as the reactivity of [18F]fluoride, the structure of the target compound in addition to the chosen solvent. The overall radiosynthesis procedure must be optimised in terms of radiochemical yield and quality of the final product. Therefore, both quantitative and qualitative radioanalytical methods are essential in developing radiosynthesis methods. Furthermore, biological properties of the tracer candidate need to be evaluated by various pre-clinical studies in animal models. In this work, the feasibility of various nucleophilic 18F-fluorination strategies were studied and a labelling method for a novel radiotracer, N-3-[18F]fluoropropyl-2beta-carbomethoxy-3beta-4-fluorophenyl)nortropane ([18F]beta-CFT-FP), was optimised. The effect of solvent was studied by labelling a series of model compounds, 4-(R1-methyl)benzyl R2-benzoates. 18F-Fluorination reactions were carried out both in polar aprotic and protic solvents (tertiary alcohols). Assessment of the 18F-fluorinated products was studied by mass spectrometry (MS) in addition to conventional radiochromatographic methods, using radiosynthesis of 4-[18F]fluoro-N-[2-[1-(2-methoxyphenyl)-1-piperazinyl]ethyl-N-2-pyridinyl-benzamide (p-[18F]MPPF) as a model reaction. Labelling of [18F]beta-CFT-FP was studied using two 18F-fluoroalkylation reagents, [18F]fluoropropyl bromide and [18F]fluoropropyl tosylate, as well as by direct 18F-fluorination of sulfonate ester precursor. Subsequently, the suitability of [18F]beta-CFT-FP for imaging dopamine transporter (DAT) was evaluated by determining its biodistribution in rats. The results showed that protic solvents can be useful co-solvents in aliphatic 18F-fluorinations, especially in the labelling of sulfonate esters. Aromatic 18F-fluorination was not promoted in tert-alcohols. Sensitivity of the ion trap MS was sufficient for the qualitative analysis of the 18F-labelled products; p-[18F]MPPF was identified from the isolated product fraction with a mass-to-charge (m/z) ratio of 435 (i.e. protonated molecule [M+H]+). [18F]beta-CFT-FP was produced most efficiently via [18F]fluoropropyl tosylate, leading to sufficient radiochemical yield and specific radioactivity for PET studies. The ex vivo studies in rats showed fast kinetics as well as the specific uptake of [18F]beta-CFT-FP to the DAT rich brain regions. Thus, it was concluded that [18F]beta-CFT-FP has potential as a radiotracer for imaging DAT by PET.

Microfinance, Efficiency and Agricultural Production in Bangladesh

The objectives of this study were to make a detailed and systematic empirical analysis of microfinance borrowers and non-borrowers in Bangladesh and also examine how efficiency measures are influenced by the access to agricultural microfinance. In the empirical analysis, this study used both parametric and non-parametric frontier approaches to investigate differences in efficiency estimates between microfinance borrowers and non-borrowers. This thesis, based on five articles, applied data obtained from a survey of 360 farm households from north-central and north-western regions in Bangladesh. The methods used in this investigation involve stochastic frontier (SFA) and data envelopment analysis (DEA) in addition to sample selectivity and limited dependent variable models. In article I, technical efficiency (TE) estimation and identification of its determinants were performed by applying an extended Cobb-Douglas stochastic frontier production function. The results show that farm households had a mean TE of 83% with lower TE scores for the non-borrowers of agricultural microfinance. Addressing institutional policies regarding the consolidation of individual plots into farm units, ensuring access to microfinance, extension education for the farmers with longer farming experience are suggested to improve the TE of the farmers. In article II, the objective was to assess the effects of access to microfinance on household production and cost efficiency (CE) and to determine the efficiency differences between the microfinance participating and non-participating farms. In addition, a non-discretionary DEA model was applied to capture directly the influence of microfinance on farm households production and CE. The results suggested that under both pooled DEA models and non-discretionary DEA models, farmers with access to microfinance were significantly more efficient than their non-borrowing counterparts. Results also revealed that land fragmentation, family size, household wealth, on farm-training and off farm income share are the main determinants of inefficiency after effectively correcting for sample selection bias. In article III, the TE of traditional variety (TV) and high-yielding-variety (HYV) rice producers were estimated in addition to investigating the determinants of adoption rate of HYV rice. Furthermore, the role of TE as a potential determinant to explain the differences of adoption rate of HYV rice among the farmers was assessed. The results indicated that in spite of its much higher yield potential, HYV rice production was associated with lower TE and had a greater variability in yield. It was also found that TE had a significant positive influence on the adoption rates of HYV rice. In article IV, we estimated profit efficiency (PE) and profit-loss between microfinance borrowers and non-borrowers by a sample selection framework, which provided a general framework for testing and taking into account the sample selection in the stochastic (profit) frontier function analysis. After effectively correcting for selectivity bias, the mean PE of the microfinance borrowers and non-borrowers were estimated at 68% and 52% respectively. This suggested that a considerable share of profits were lost due to profit inefficiencies in rice production. The results also demonstrated that access to microfinance contributes significantly to increasing PE and reducing profit-loss per hectare land. In article V, the effects of credit constraints on TE, allocative efficiency (AE) and CE were assessed while adequately controlling for sample selection bias. The confidence intervals were determined by the bootstrap method for both samples. The results indicated that differences in average efficiency scores of credit constrained and unconstrained farms were not statistically significant although the average efficiencies tended to be higher in the group of unconstrained farms. After effectively correcting for selectivity bias, household experience, number of dependents, off-farm income, farm size, access to on farm training and yearly savings were found to be the main determinants of inefficiencies. In general, the results of the study revealed the existence substantial technical, allocative, economic inefficiencies and also considerable profit inefficiencies. The results of the study suggested the need to streamline agricultural microfinance by the microfinance institutions (MFIs), donor agencies and government at all tiers. Moreover, formulating policies that ensure greater access to agricultural microfinance to the smallholder farmers on a sustainable basis in the study areas to enhance productivity and efficiency has been recommended. Key Words: Technical, allocative, economic efficiency, DEA, Non-discretionary DEA, selection bias, bootstrapping, microfinance, Bangladesh.

Effects of gender, and SLCO1B1 and CYP7A1 polymorphisms on plasma bile acids in humans and the pharmacokinetics of therapeutic ursodeoxycholic acid

Bile acids are important steroid-derived molecules essential for fat absorption in the small intestine. They are produced in the liver and secreted into the bile. Bile acids are transported by bile flow to the small intestine, where they aid the digestion of lipids. Most bile acids are reabsorbed in the small intestine and return to the liver through the portal vein. The whole recycling process is referred to as the enterohepatic circulation, during which only a small amount of bile acids are removed from the body via faeces. The enterohepatic circulation of bile acids involves the delicate coordination of a number of bile acid transporters expressed in the liver and the small intestine. Organic anion transporting polypeptide 1B1 (OATP1B1), encoded by the solute carrier organic anion transporter family, member 1B1 (SLCO1B1) gene, mediates the sodium independent hepatocellular uptake of bile acids. Two common SNPs in the SLCO1B1 gene are well known to affect the transport activity of OATP1B1. Moreover, bile acid synthesis is an important elimination route for cholesterol. Cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme of bile acid production. The aim of this thesis was to investigate the effects of SLCO1B1 polymorphism on the fasting plasma levels of individual endogenous bile acids and a bile acid synthesis marker, and the pharmacokinetics of exogenously administered ursodeoxycholic acid (UDCA). Furthermore, the effects of CYP7A1 genetic polymorphism and gender on the fasting plasma concentrations of individual endogenous bile acids and the bile acid synthesis marker were evaluated. Firstly, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the determination of bile acids was developed (Study I). A retrospective study examined the effects of SLCO1B1 genetic polymorphism on the fasting plasma concentrations of individual bile acids and a bile acid synthesis marker in 65 healthy subjects (Study II). In another retrospective study with 143 healthy individuals, the effects of CYP7A1 genetic polymorphism and gender as well as SLCO1B1 polymorphism on the fasting plasma levels of individual bile acids and the bile acid synthesis marker were investigated (Study III). The effects of SLCO1B1 polymorphism on the pharmacokinetics of exogenously administered UDCA were evaluated in a prospective genotype panel study including 27 healthy volunteers (Study IV). A robust, sensitive and simple HPLC-MS/MS method was developed for the simultaneous determination of 16 individual bile acids in human plasma. The method validation parameters for all the analytes met the requirements of the FDA (Food and Drug Administration) bioanalytical guidelines. This HPLC-MS/MS method was applied in Studies II-IV. In Study II, the fasting plasma concentrations of several bile acids and the bile acid synthesis marker seemed to be affected by SLCO1B1 genetic polymorphism, but these findings were not replicated in Study III with a larger sample size. Moreover, SLCO1B1 polymorphism had no effect on the pharmacokinetic parameters of exogenously administered UDCA. Furthermore, no consistent association was observed between CYP7A1 genetic polymorphism and the fasting plasma concentrations of individual bile acids or the bile acid synthesis marker. In contrast, gender had a major effect on the fasting plasma concentrations of several bile acids and also total bile acids. In conclusion, gender, but not SLCO1B1 or CYP7A1 polymorphisms, has a major effect on the fasting plasma concentrations of individual bile acids. Moreover, the common genetic polymorphism of CYP7A1 is unlikely to influence the activity of CYP7A1 under normal physiological conditions. OATP1B1 does not play an important role in the in vivo disposition of exogenously administered UDCA.

Harvesting undelimbed Scots pine (Pinus sylvestris L.) from first thinnings for integrated production of kraft pulp and energy

The present study evaluates the feasibility of undelimbed Scots pine (Pinus sylvestris L.) for integrated production of pulp and energy in a kraft pulp mill from the technical, economic and environmental points of view, focusing on the potential of bundle harvesting. The feasibility of tree sections for pulp production was tested by conducting an industrial wood-handling experiment, laboratory cooking and bleaching trials, using conventional small-diameter Scots pine pulpwood as a reference. These trials showed that undelimbed Scots pine sections can be processed in favourable conditions as a blend with conventional small-diameter pulpwood without reducing the pulp quality. However, fibre losses at various phases of the process may increase when using undelimbed material. In the economic evaluation, both pulp production and wood procurement costs were considered, using the relative wood paying capability of a kraft pulp mill as a determinant. The calculations were made for three Scots pine first-thinning stands with the breast-height diameter of the removal (6 12 cm) as the main distinctive factor. The supply chains included in the comparison were based on cut-to-length harvesting, whole-tree harvesting and bundle harvesting (whole-tree bundling). With the current ratio of pulp and energy prices, the wood paying capability declines with an increase in the proportion of the energy fraction of the raw material. The supply system based on the cut-to-length method was the most efficient option, resulting in the highest residual value at stump in most cases. A decline in the pulp price and an increase in the energy price improved the competitiveness of the whole-tree systems. With short truck transportation distances and low pulp prices, however, the harvesting of loose whole trees can result in higher residual value at stump in small-diameter stands. While savings in transportation costs did not compensate for the high cutting and compaction costs by the second prototype of the bundle harvester, an increase in transportation distances improved its competitiveness. Since harvesting undelimbed assortments increases nutrient export from the site, which can affect soil productivity, the whole-tree alternatives included in the present study cannot be recommended on infertile peatlands and mineral soils. The harvesting of loose whole trees or bundled whole trees implies a reduction in protective logging residues and an increase in site traffic or payloads. These factors increase the risk of soil damage, especially on peat soils with poor bearing capacity. Within the wood procurement parameters which were examined, the CO2 emissions of the supply systems varied from 13 27 kg m3. Compaction of whole trees into bundles reduced emissions from transportation by 30 39%, but these reductions were insufficient to compensate for the increased emissions from cutting and compaction.

Literature Review: Investigating Drug Transport at the Blood-Brain Barrier and the Effects of P-glycoprotein on the Brain Distribution of Drugs.

The blood-brain barrier (BBB) is a unique barrier that strictly regulates the entry of endogenous substrates and xenobiotics into the brain. This is due to its tight junctions and the array of transporters and metabolic enzymes that are expressed. The determination of brain concentrations in vivo is difficult, laborious and expensive which means that there is interest in developing predictive tools of brain distribution. Predicting brain concentrations is important even in early drug development to ensure efficacy of central nervous system (CNS) targeted drugs and safety of non-CNS drugs. The literature review covers the most common current in vitro, in vivo and in silico methods of studying transport into the brain, concentrating on transporter effects. The consequences of efflux mediated by p-glycoprotein, the most widely characterized transporter expressed at the BBB, is also discussed. The aim of the experimental study was to build a pharmacokinetic (PK) model to describe p-glycoprotein substrate drug concentrations in the brain using commonly measured in vivo parameters of brain distribution. The possibility of replacing in vivo parameter values with their in vitro counterparts was also studied. All data for the study was taken from the literature. A simple 2-compartment PK model was built using the Stella™ software. Brain concentrations of morphine, loperamide and quinidine were simulated and compared with published studies. Correlation of in vitro measured efflux ratio (ER) from different studies was evaluated in addition to studying correlation between in vitro and in vivo measured ER. A Stella™ model was also constructed to simulate an in vitro transcellular monolayer experiment, to study the sensitivity of measured ER to changes in passive permeability and Michaelis-Menten kinetic parameter values. Interspecies differences in rats and mice were investigated with regards to brain permeability and drug binding in brain tissue. Although the PK brain model was able to capture the concentration-time profiles for all 3 compounds in both brain and plasma and performed fairly well for morphine, for quinidine it underestimated and for loperamide it overestimated brain concentrations. Because the ratio of concentrations in brain and blood is dependent on the ER, it is suggested that the variable values cited for this parameter and its inaccuracy could be one explanation for the failure of predictions. Validation of the model with more compounds is needed to draw further conclusions. In vitro ER showed variable correlation between studies, indicating variability due to experimental factors such as test concentration, but overall differences were small. Good correlation between in vitro and in vivo ER at low concentrations supports the possibility of using of in vitro ER in the PK model. The in vitro simulation illustrated that in the simulation setting, efflux is significant only with low passive permeability, which highlights the fact that the cell model used to measure ER must have low enough paracellular permeability to correctly mimic the in vivo situation.

Production of Carcinogenic Acetaldehyde by Oral Microbiome

Oral cancer is the seventh most common cancer worldwide and its incidence is increasing. The most important risk factors for oral cancer are chronic alcohol consumption and tobacco smoking, up to 80 % of oral carcinomas are estimated to be caused by alcohol and tobacco. They both trigger an increased level of salivary acetaldehyde, during and after consumption, which is believed to lead to carcinogenesis. Acetaldehyde has multiple mutagenic features and it has recently been classified as a Group 1 carcinogen for humans by the International Agency for Research on Cancer. Acetaldehyde is metabolized from ethanol by microbes of oral microbiota. Some oral microbes possess alcohol dehydrogenase enzyme (ADH) activity, which is the main enzyme in acetaldehyde production. Many microbes are also capable of acetaldehyde production via alcohol fermentation from glucose. However, metabolism of ethanol into acetaldehyde leads to production of high levels of this carcinogen. Acetaldehyde is found in saliva during and after alcohol consumption. In fact, rather low ethanol concentrations (2-20mM) derived from blood to saliva are enough for microbial acetaldehyde production. The high acetaldehyde levels in saliva after alcohol challenge are explained by the lack of oral microbiota and mucosa to detoxify acetaldehyde by metabolizing it into acetate and acetyl coenzymeA. The aim of this thesis project was to specify the role of oral microbes in the in vitro production of acetaldehyde in the presence of ethanol. In addition, it was sought to establish whether microbial metabolism could also produce acetaldehyde from glucose. Furthermore, the potential of xylitol to inhibit ethanol metabolism and acetaldehyde production was explored. Isolates of oral microbes were used in the first three studies. Acetaldehyde production was analyzed after ethanol, glucose and fructose incubation with gas chromatography measurement. In studies I and III, the ADH enzyme activity of some microbes was measured by fluorescence. The effect of xylitol was analyzed by incubating microbes with ethanol and xylitol. The fourth study was made ex vivo and microbial samples obtained from different patient groups were analyzed. This work has demonstrated that isolates of oral microbiota are able to produce acetaldehyde in the presence of clinically relevant ethanol and glucose concentrations. Significant differences were found between microbial species and isolates from different patient groups. In particular, the ability of candidal isolates from APECED patients to produce significantly more acetaldehyde in glucose incubation compared to healthy and cancer patient isolates is an interesting observation. Moreover, xylitol was found to reduce their acetaldehyde production significantly. Significant ADH enzyme activity was found in the analyzed high acetaldehyde producing streptococci and candida isolates. In addition, xylitol was found to reduce the ADH enzyme activity of C. albicans. Some results from the ex vivo study were controversial, since acetaldehyde production did not correlate as expected with the amount of microbes in the samples. Nevertheless, the samples isolated from patients did produce significant amounts of acetaldehyde with a clinically relevant ethanol concentration.