Parameters Affecting the Extraction of Polycyclic Aromatic Hydrocarbons with Pressurised Hot Water

Pressurised hot water extraction (PHWE) exploits the unique temperature-dependent solvent properties of water minimising the use of harmful organic solvents. Water is environmentally friendly, cheap and easily available extraction medium. The effects of temperature, pressure and extraction time in PHWE have often been studied, but here the emphasis was on other parameters important for the extraction, most notably the dimensions of the extraction vessel and the stability and solubility of the analytes to be extracted. Non-linear data analysis and self-organising maps were employed in the data analysis to obtain correlations between the parameters studied, recoveries and relative errors. First, pressurised hot water extraction (PHWE) was combined on-line with liquid chromatography-gas chromatography (LC-GC), and the system was applied to the extraction and analysis of polycyclic aromatic hydrocarbons (PAHs) in sediment. The method is of superior sensitivity compared with the traditional methods, and only a small 10 mg sample was required for analysis. The commercial extraction vessels were replaced by laboratory-made stainless steel vessels because of some problems that arose. The performance of the laboratory-made vessels was comparable to that of the commercial ones. In an investigation of the effect of thermal desorption in PHWE, it was found that at lower temperatures (200ºC and 250ºC) the effect of thermal desorption is smaller than the effect of the solvating property of hot water. At 300ºC, however, thermal desorption is the main mechanism. The effect of the geometry of the extraction vessel on recoveries was studied with five specially constructed extraction vessels. In addition to the extraction vessel geometry, the sediment packing style and the direction of water flow through the vessel were investigated. The geometry of the vessel was found to have only minor effect on the recoveries, and the same was true of the sediment packing style and the direction of water flow through the vessel. These are good results because these parameters do not have to be carefully optimised before the start of extractions. Liquid-liquid extraction (LLE) and solid-phase extraction (SPE) were compared as trapping techniques for PHWE. LLE was more robust than SPE and it provided better recoveries and repeatabilities than did SPE. Problems related to blocking of the Tenax trap and unrepeatable trapping of the analytes were encountered in SPE. Thus, although LLE is more labour intensive, it can be recommended over SPE. The stabilities of the PAHs in aqueous solutions were measured using a batch-type reaction vessel. Degradation was observed at 300ºC even with the shortest heating time. Ketones and quinones and other oxidation products were observed. Although the conditions of the stability studies differed considerably from the extraction conditions in PHWE, the results indicate that the risk of analyte degradation must be taken into account in PHWE. The aqueous solubilities of acenaphthene, anthracene and pyrene were measured, first below and then above the melting point of the analytes. Measurements below the melting point were made to check that the equipment was working, and the results were compared with those obtained earlier. Good agreement was found between the measured and literature values. A new saturation cell was constructed for the solubility measurements above the melting point of the analytes because the flow-through saturation cell could not be used above the melting point. An exponential relationship was found between the solubilities measured for pyrene and anthracene and temperature.

Lymphatic vascular maturation : Roles of FOXC2, NFATc1 and liprin ß1

The blood vascular system is a closed circulatory system, responsible for delivering oxygen and nutrients to the tissues. In contrast, the lymphatic vascular system is a blind-ended transport system that collects the extravasated tissue fluid from the capillary beds, and transports it back to the blood circulation. Failure in collecting or transporting the lymph, due to defects in the lymphatic vasculature, leads to accumulation of extra fluid in the tissues, and consequently to tissue swelling lymphedema. The two vascular systems function in concert. They are structurally related, but their development is regulated by separate, however overlapping, molecular mechanisms. During embryonic development, blood vessels are formed by vasculogenesis and angiogenesis, processes largely mediated by members of the vascular endothelial growth factor (VEGF) family and their tyrosine kinase receptors. The lymphatic vessels are formed after the cardiovascular system is already functional. This process, called lymphangiogenesis, is controlled by distinct members of the VEGF family, together with the transcription factors Prox1 and Sox18. After the primary formation of the vessels, the vasculature needs to mature and remodel into a functional network of hierarchically organized vessels: the blood vasculature into arteries, capillaries and veins; and the lymphatic vasculature into lymphatic capillaries, responsible for the uptake of the extravasated fluid from the tissues, and collecting vessels, responsible for the transport of the lymph back to the blood circulation. A major event in the maturation of the lymphatic vasculature is the formation of collecting lymphatic vessels. These vessels are characterized by the presence of intraluminal valves, preventing backflow of the lymph, and a sparse coverage of smooth muscle cells, which help in pumping the lymph forward. In our study, we have characterized the molecular and morphological events leading to formation of collecting lymphatic vessels. We found that this process is regulated cooperatively by the transcription factors Foxc2 and NFATc1. Mice lacking either Foxc2 or active NFATc1 fail to remodel the primary lymphatic plexus into functional lymphatic capillaries and collecting vessels. The resulting vessels lack valves, display abnormal expression of lymphatic molecules, and are hyperplastic. Moreover, the lymphatic capillaries show aberrant sprouting, and are abnormally covered with smooth muscle cells. In humans, mutations in FOXC2 lead to Lymphedema-Distichiasis (LD), a disabling disease characterized by swelling of the limbs due to insufficient lymphatic function. Our results from Foxc2 mutant mice and LD patients indicate that the underlying cause for lymphatic failure in LD is agenesis of collecting lymphatic valves and aberrant recruitment of periendothelial cells and basal lamina components to lymphatic capillaries. Furthermore, we show that liprin β1, a poorly characterized member of the liprin family of cytoplasmic proteins, is highly expressed in lymphatic endothelial cells in vivo, and is required for lymphatic vessel integrity. These data highlight the important role of FOXC2, NFATc1 and liprin β1 in the regulation of lymphatic development, specifically in the maturation and formation of the collecting lymphatic vessels. As damage to collecting vessels is a major cause of lymphatic dysfunction in humans, our results also suggest that FOXC2 and NFATc1 are potential targets for therapeutic intervention.

Genetic variation in the LDL receptor-related protein 5 (LRP5) gene : Association with bone health and metabolic parameters

Bone mass accrual and maintenance are regulated by a complex interplay between genetic and environmental factors. Recent studies have revealed an important role for the low-density lipoprotein receptor-related protein 5 (LRP5) in this process. The aim of this thesis study was to identify novel variants in the LRP5 gene and to further elucidate the association of LRP5 and its variants with various bone health related clinical characteristics. The results of our studies show that loss-of-function mutations in LRP5 cause severe osteoporosis not only in homozygous subjects but also in the carriers of these mutations, who have significantly reduced bone mineral density (BMD) and increased susceptibility to fractures. In addition, we demonstrated for the first time that a common polymorphic LRP5 variant (p.A1330V) was associated with reduced peak bone mass, an important determinant of BMD and osteoporosis in later life. The results from these two studies are concordant with results seen in other studies on LRP5 mutations and in association studies linking genetic variation in LRP5 with BMD and osteoporosis. Several rare LRP5 variants were identified in children with recurrent fractures. Sequencing and multiplex ligation-dependent probe amplification (MLPA) analyses revealed no disease-causing mutations or whole-exon deletions. Our findings from clinical assessments and family-based genotype-phenotype studies suggested that the rare LRP5 variants identified are not the definite cause of fractures in these children. Clinical assessments of our study subjects with LPR5 mutations revealed an unexpectedly high prevalence of impaired glucose tolerance and dyslipidaemia. Moreover, in subsequent studies we discovered that common polymorphic LRP5 variants are associated with unfavorable metabolic characteristics. Changes in lipid profile were already apparent in pre-pubertal children. These results, together with the findings from other studies, suggest an important role for LRP5 also in glucose and lipid metabolism. Our results underscore the important role of LRP5 not only in bone mass accrual and maintenance of skeletal health but also in glucose and lipid metabolism. The role of LRP5 in bone metabolism has long been studied, but further studies with larger study cohorts are still needed to evaluate the specific role of LRP5 variants as metabolic risk factors.

The connection between on-farm assessed welfare (A-index) and production parameters of the sows

Tämän tutkimuksen tavoitteena oli selvittää tilalla määritetyn hyvinvoinnin yhteyttä emakoiden tuotantotuloksiin. Hyvinvointia arvioitiin suomalaisen hyvinvointi-indeksin, A-indeksi, avulla. Tuotantotuloksina käytettiin kahta erilaista tuotosaineistoa, jotka molemmat pohjautuivat kansalliseen tuotosseuranta aineistoon. Hyvinvointimääritykset tehtiin 30 porsastuotantosikalassa maaliskuun 2007 aikana. A-indeksi koostuu kuudesta kategoriasta ’liikkumismahdollisuudet’, ’alustan ominaisuudet’, ’sosiaaliset kontaktit’, ’valo, ilma ja melu’, ’ruokinta ja veden saanti’ sekä ’eläinten terveys ja hoidon taso’. Jokaisessa kategoriassa on 3-10 pääosin ympäristöperäistä muuttujaa, jotka vaihtelevat osastoittain. Maksimipistemäärä osastolle on 100. Hyvinvointimittaukset tehtiin porsitus-, tiineytys- ja joutilasosastoilla. Erillisten tiineytysosastojen pienen lukumäärän takia (n=7) tilakohtaiset tiineytys- ja joutilasosastopisteet yhdistettiin ja keskiarvoja käytettiin analyyseissä. Yhteyksiä tuotokseen tutkittiin kahden eri aineiston avulla 1) Tilaraportti aineisto (n=29) muodostuu muokkaamattomista tila- ja tuotostuloksista tilavierailua edeltävän vuoden ajalta, 2) POTSIaineisto (n=30) muodostuu POTSI-ohjelmalla (MTT) muokatusta tuotantoaineistosta, joka sisältää managementtiryhmän (tila, vuosi, vuodenaika) vaikutuksen ensikoiden ja emakoiden pahnuekohtaiseen tuotokseen. Yhteyksiä analysointiin korrelaatio- ja regressioanalyysien avulla. Vaikka osallistuminen tutkimukseen oli vapaaehtoista, molempien tuotantoaineistojen perusteella tutkimustilat edustavat keskituottoista suomalaista sikatilaa. A-indeksin kokonaispisteet vaihtelivat välillä 37,5–64,0 porsitusosastolla ja 39,5–83,5 joutilasosastolla. Tilaraporttiaineistoa käytettäessä paremmat pisteet porsitusosaston ’eläinten terveys ja hoidon taso’ -kategoriasta lyhensivät eläinten lisääntymissykliä, lisäsivät syntyvien pahnueiden ja porsaiden määrää sekä alensivat kuolleena syntyneiden lukumäärää. Regressiomallin mukaan ’eläinten terveys ja hoidon taso’ -kategoria selitti syntyvien porsaiden lukumäärän, porsimisvälin pituuden sekä keskiporsimiskerran vaihtelua. Paremmat pisteet joutilasosaston ’liikkumismahdollisuudet’ kategoriasta alensivat syntyneiden pahnueiden sekä syntyneiden että vieroitettujen porsaiden lukumäärää. Regressiomallin mukaan ensikkopahnueiden osuus ja ”liikkumismahdollisuudet” kategorian pisteet selittivät vieroitettujen porsaiden lukumäärän vaihtelua. POTSI-aineiston yhteydessä kuolleena syntyneiden porsaiden lukumäärän aleneminen oli ensikoilla yhteydessä parempiin porsitusosaston ’sosiaalisiin kontakteihin’ ja emakoilla puolestaan joutilasosaston parempiin ’eläinten terveys ja hoidon taso’ pisteisiin. Kahden eri tuotantoaineiston avulla saadut tulokset erosivat toisistaan. Seuraavissa tutkimuksissa onkin suositeltavampaa käyttää Tilaraporttiaineistoja, joissa tuotokset ilmoitetaan vuosikohtaisina. Tämän tutkimuksen perusteella hyvinvoinnilla ja tuotoksella on yhteyksiä, joilla on myös merkittävää taloudellista vaikutusta. Erityisesti hyvä eläinten hoito ja eläinten terveys lisäävät tuotettujen porsaiden määrää ja lyhentävät lisääntymiskiertoa. Erityishuomiota tulee kiinnittää vapaana olevien joutilaiden emakoiden sosiaaliseen stressiin ja rehunsaannin varmistamiseen kaikille yksilöille.

Electrocardiographic parameters of ventricular repolarization : modifiers and the prognostic value

Electric activity of the heart consists of repeated cardiomyocyte depolarizations and repolarizations. Abnormalities in repolarization predispose to ventricular arrhythmias. In body surface electrocardiogram, ventricular repolarization generates the T wave. Several electrocardiographic measures have been developed both for clinical and research purposes to detect repolarization abnormalities. The study aim was to investigate modifiers of ventricular repolarization with the focus on the relationship of the left ventricular mass, antihypertensive drugs, and common gene variants, to electrocardiographic repolarization parameters. The prognostic value of repolarization parameters was also assessed. The study subjects originated from a population of more than 200 middle-aged hypertensive men attending the GENRES hypertension study, and from an epidemiological survey, the Health 2000 Study, including more than 6000 participants. Ventricular repolarization was analysed from digital standard 12-lead resting electrocardiograms with two QT-interval based repolarization parameters (QT interval, T-wave peak to T-wave end interval) and with a set of four T-wave morphology parameters. The results showed that in hypertensive men, a linear change in repolarization parameters is present even in the normal range of left ventricular mass, and that even mild left ventricular hypertrophy is associated with potentially adverse electrocardiographic repolarization changes. In addition, treatments with losartan, bisoprolol, amlodipine, and hydrochlorothiazide have divergent short-term effects on repolarization parameters in hypertensive men. Analyses of the general population sample showed that single nucleotide polymorphisms in KCNH2, KCNE1, and NOS1AP genes are associated with changes in QT-interval based repolarization parameters but not consistently with T-wave morphology parameters. T-wave morphology parameters, but not QT interval or T-wave peak to T-wave end interval, provided independent prognostic information on mortality. The prognostic value was specifically related to cardiovascular mortality. The results indicate that, in hypertension, altered ventricular repolarization is already present in mild left ventricular mass increase, and that commonly used antihypertensive drugs may relatively rapidly and treatment-specifically modify electrocardiographic repolarization parameters. Common variants in cardiac ion channel genes and NOS1AP gene may also modify repolarization-related arrhythmia vulnerability. In the general population, T-wave morphology parameters may be useful in the risk assessment of cardiovascular mortality.

Functional expression and subcellular localization of the Cl- cotransporters KCC2 and NKCC1 in rodent hippocampal and neocortical neurons

The work presented here has focused on the role of cation-chloride cotransporters (CCCs) in (1) the regulation of intracellular chloride concentration within postsynaptic neurons and (2) on the consequent effects on the actions of the neurotransmitter gamma-aminobutyric acid (GABA) mediated by GABAA receptors (GABAARs) during development and in pathophysiological conditions such as epilepsy. In addition, (3) we found that a member of the CCC family, the K-Cl cotransporter isoform 2 (KCC2), has a structural role in the development of dendritic spines during the differentiation of pyramidal neurons. Despite the large number of publications dedicated to regulation of intracellular Cl-, our understanding of the underlying mechanisms is not complete. Experiments on GABA actions under resting steady-state have shown that the effect of GABA shifts from depolarizing to hyperpolarizing during maturation of cortical neurons. However, it remains unclear, whether conclusions from these steady-state measurements can be extrapolated to the highly dynamic situation within an intact and active neuronal network. Indeed, GABAergic signaling in active neuronal networks results in a continuous Cl- load, which must be constantly removed by efficient Cl- extrusion mechanisms. Therefore, it seems plausible to suggest that key parameters are the efficacy and subcellular distribution of Cl- transporters rather than the polarity of steady-state GABA actions. A further related question is: what are the mechanisms of Cl- regulation and homeostasis during pathophysiological conditions such as epilepsy in adults and neonates? Here I present results that were obtained by means of a newly developed method of measurements of the efficacy of a K-Cl cotransport. In Study I, the developmental profile of KCC2 functionality during development was analyzed both in dissociated neuronal cultures and in acute hippocampal slices. A novel method of photolysis of caged GABA in combination with Cl- loading to the somata was used in this study to assess the extrusion efficacy of KCC2. We demonstrated that these two preparations exhibit a different temporal profile of functional KCC2 upregulation. In Study II, we reported an observation of highly distorted dendritic spines in neurons cultured from KCC2-/- embryos. During their development in the culture dish, KCC2-lacking neurons failed to develop mature, mushroom-shaped dendritic spines but instead maintained an immature phenotype of long, branching and extremely motile protrusions. It was shown that the role of KCC2 in spine maturation is not based on its transport activity, but is mediated by interactions with cytoskeletal proteins. Another important player in Cl- regulation, NKCC1 and its role in the induction and maintenance of native Cl- gradients between the axon initial segment (AIS) and soma was the subject of Study III. There we demonstrated that this transporter mediates accumulation of Cl- in the axon initial segment of neocortical and hippocampal principal neurons. The results suggest that the reversal potential of the GABAA response triggered by distinct populations of interneurons show large subcellular variations. Finally, a novel mechanism of fast post-translational upregulation of the membrane-inserted, functionally active KCC2 pool during in-vivo neonatal seizures and epileptiform-like activity in vitro was identified and characterized in Study IV. The seizure-induced KCC2 upregulation may act as an intrinsic antiepileptogenic mechanism.