Interactions of natural products with β-lactoglobulins, members of the lipocalin family

Natural products constitute an important source of new drugs. The bioavailability of the drugs depends on their absorption, distribution, metabolism and elimination. To achieve good bioavailability, the drug must be soluble in water, stable in the gastrointestinal tract and palatable. Binding proteins may improve the solubility of drug compounds, masking unwanted properties, such as bad taste, bitterness or toxicity, transporting or protecting these compounds during processing and storage. The focus of this thesis was to study the interactions, including ligand binding and the effect of pH and temperature, of bovine and reindeer β-lactoglobulin (βLG) with such compounds as retinoids, phenolic compounds as well as with compounds from plant extracts, and to investigate the transport properties of the βLG-ligand complex. To examine the binding interactions of different ligands to βLG, new methods were developed. The fluorescence binding method for the evaluation of ligand binding to βLG was miniaturized from a quartz cell to a 96-well plate. A method of ultrafiltration sampling combined with high-performance liquid chromatography was developed to assess the binding of compounds from extracts. The interactions of phenolic compounds or retinoids and βLG were investigated using the 96-well plate method. The majority of flavones, flavonols, flavanones and isoflavones and all of the retinoids included were shown to bind to bovine and reindeer βLG. Phenolic compounds, contrary to retinol, were not released at acidic pH. Those results suggest that βLG may have more binding sites, probably also on the surface of βLG. An extract from Camellia sinensis (L.) O. Kunze (black tea), Urtica dioica L. (nettle) and Piper nigrum (black pepper) were used to evaluate whether βLG could bind compounds from plant extracts. Piperine from P. nigrum was found to bind tightly and rutin from U. dioica weakly to βLG. No components from C. sinensis bound to βLG in our experiment. The uptake and membrane permeation of bovine and reindeer βLG, free and bound with retinol, palmitic acid and cholesterol, were investigated using Caco-2 cell monolayers. Both bovine and reindeer βLG were able to cross the Caco-2 cell membrane. Free and βLG-bound retinol and palmitic acid were transported equally, whereas cholesterol could not cross the Caco-2 cell monolayer free or bound to βLG. Our results showed that βLG can bind different natural product compounds, but cannot enhance transport of retinol, palmitic acid or cholesterol through Caco-2 cells. Despite this, βLG, as a water-soluble binding protein, may improve the solubility of natural compounds, possibly protecting them from early degradation and transporting some of them through the stomach. Furthermore, it may decrease their bad or bitter taste during oral administration of drugs or in food preparations. βLG can also enhance or decrease the health benefits of herbal teas and food preparations by binding compounds from extracts.

The decline of northern malaria and population dynamics of Plamodium vivax

Europe was declared malaria free in 1975. The disappearance of malaria has traditionally been attributed to numerous deliberate actions like vector control, the screening of houses, more efficient medication etc. Malaria, however, disappeared from many countries like Finland before any counter measures had even started. The aim of this thesis is to study the population ecology of P. vivax and its interaction with the human host and the vector. By finding the factors that attributed to the extinction of vivax malaria it might be possible to improve the modern strategy against P. vivax. The parasite was studied with data from Finland, which provides the longest time series (1749-2008) of malaria statistics in the world. The malaria vectors, Anopheles messeae and A. beklemishevi are still common species in the country. The eradication of vivax malaria is difficult because the parasite has a dormant stage that can cause a relapse long after a primary infection. It was now shown that P. vivax is able to detect the presence of a potential vector. A dormant stage is triggered even from a bite of an uninfected Anopheles mosquito. This optimizes the chances for the Plasmodium to reach a mosquito vector for sexual reproduction. The longevity of the dormant stage could be shown to be at least nine years. The parasite spends several years in its human host and the behaviour of the human carrier had a profound impact on the decline of the disease in Finland. Malaria spring epidemics could be explained by a previous warm summer. Neither annual nor summer mean temperature had any impact on the long term malaria trend. Malaria disappeared slowly from Finland without mosquito control. The sociological change from extended families to nuclear families led to decreased household size. The decreased household size correlated strongly with the decline of malaria. That led to an increased isolation of the subpopulations of P. vivax. Their habitat consisted of the bedrooms in which human carriers slept together with the overwintering vectors. The isolation of the parasite ultimately led to the extinction of vivax malaria. Metapopulation models adapted to local conditions should therefore be implemented as a tool for settlement planning and socio-economic development and become an integrated part of the fight against malaria.

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.

The kinetics and reactivity of several polyatomic free radicals in reactions with NO2, O2, Cl2, and HCl

In this thesis, the kinetics of several alkyl, halogenated alkyl, and alkenyl free radical reactions with NO2, O2, Cl2, and HCl reactants were studied over a wide temperature range in time resolved conditions. Laser photolysis photoionisation mass spectrometer coupled to a flow reactor was the experimental method employed and this thesis present the first measurements performed with the experimental system constructed. During this thesis a great amount of work was devoted to the designing, building, testing, and improving the experimental apparatus. Carbon-centred free radicals were generated by the pulsed 193 or 248 nm photolysis of suitable precursors along the tubular reactor. The kinetics was studied under pseudo-first-order conditions using either He or N2 buffer gas. The temperature and pressure ranges employed were between 190 and 500 K, and 0.5 45 torr, respectively. The possible role of heterogeneous wall reactions was investigated employing reactor tubes with different sizes, i.e. to significantly vary the surface to volume ratio. In this thesis, significant new contributions to the kinetics of carbon-centred free radical reactions with nitrogen dioxide were obtained. Altogether eight substituted alkyl (CH2Cl, CHCl2, CCl3, CH2I, CH2Br, CHBr2, CHBrCl, and CHBrCH3) and two alkenyl (C2H3, C3H3) free radical reactions with NO2 were investigated as a function of temperature. The bimolecular rate coefficients of all these reactions were observed to possess negative temperature dependencies, while pressure dependencies were not noticed for any of these reactions. Halogen substitution was observed to moderately reduce the reactivity of substituted alkyl radicals in the reaction with NO2, while the resonance stabilisation of the alkenyl radical lowers its reactivity with respect to NO2 only slightly. Two reactions relevant to atmospheric chemistry, CH2Br + O2 and CH2I + O2, were also investigated. It was noticed that while CH2Br + O2 reaction shows pronounced pressure dependence, characteristic of peroxy radical formation, no such dependence was observed for the CH2I + O2 reaction. Observed primary products of the CH2I + O2 reaction were the I-atom and the IO radical. Kinetics of CH3 + HCl, CD3 + HCl, CH3 + DCl, and CD3 + DCl reactions were also studied. While all these reactions possess positive activation energies, in contrast to the other systems investigated in this thesis, the CH3 + HCl and CD3 + HCl reactions show a non-linear temperature dependency on the Arrhenius plot. The reactivity of substituted methyl radicals toward NO2 was observed to increase with decreasing electron affinity of the radical. The same trend was observed for the reactions of substituted methyl radicals with Cl2. It is proposed that interactions of frontier orbitals are responsible to these observations and Frontier Orbital Theory could be used to explain the observed reactivity trends of these highly exothermic reactions having reactant-like transition states.

Measuring and tracking quality factors in Free and Open Source Software projects

Free and Open Source Software (FOSS) has gained increased interest in the computer software industry, but assessing its quality remains a challenge. FOSS development is frequently carried out by globally distributed development teams, and all stages of development are publicly visible. Several product and process-level quality factors can be measured using the public data. This thesis presents a theoretical background for software quality and metrics and their application in a FOSS environment. Information available from FOSS projects in three information spaces are presented, and a quality model suitable for use in a FOSS context is constructed. The model includes both process and product quality metrics, and takes into account the tools and working methods commonly used in FOSS projects. A subset of the constructed quality model is applied to three FOSS projects, highlighting both theoretical and practical concerns in implementing automatic metric collection and analysis. The experiment shows that useful quality information can be extracted from the vast amount of data available. In particular, projects vary in their growth rate, complexity, modularity and team structure.

Free/Open Source Software Development : Results and Research Methods

Free and open source software development is an alternative to traditional software engineering as an approach to the development of complex software systems. It is a way of developing software based on geographically distributed teams of volunteers without apparent central plan or traditional mechanisms of coordination. The purpose of this thesis is to summarize the current knowledge about free and open source software development and explore the ways on which further understanding on it could be gained. The results of research on the field as well as the research methods are introduced and discussed. Also adapting software process metrics to the context of free and open source software development is illustrated and the possibilities to utilize them as tools to validate other research are discussed.

Posted Workers and Free Movement of Services in the European Union – the Impact on National Employment and Immigration Law

This thesis examines posting of workers within the free movement of services in the European Union. The emphasis is on the case law of the European Court of Justice and in the role it has played in the liberalisation of the service sector in respect of posting of workers. The case law is examined from two different viewpoints: firstly, that of employment law and secondly, immigration law. The aim is to find out how active a role the Court has taken with regard these two fields of law and what are the implications of the Court’s judgments for the regulation on a national level. The first part of the thesis provides a general review of the Community law principles governing the freedom to provide services in the EU. The second part presents the Posted Workers’ Directive and the case law of the European Court of Justice before and after the enactment of the Directive from the viewpoint of employment law. Special attention is paid to a recent judgment in which the Court has taken a restrictive position with regard to a trade union’s right to take collective action against a service provider established in another Member State. The third part of the thesis concentrates, firstly, on the legal status of non-EU nationals lawfully resident in the EU. Secondly, it looks into the question of how the Court’s case law has affected the possibilities to use non-EU nationals as posted workers within the freedom to provide services. The final chapter includes a critical analysis of the Court’s case law on posted workers. The judgments of the European Court of Justice are the principal source of law for this thesis. In the primary legislation the focus is on Articles 49 EC and 50 EC that lay down the rules concerning the free movement of services. Within the secondary legislation, the present work principally concentrates on the Posted Workers’ Directive. It also examines proposals of the European Commission and directives that have been adopted in the field of immigration. The conclusions of the case study are twofold: while in the field of employment law, the European Court of Justice has based its judgments on a very literal interpretation of the Posted Workers’ Directive, in the field of immigration its conclusions have been much more innovative. In both fields of regulation the Court’s judgments have far-reaching implications for the rules concerning posting of workers leaving very little discretion for the Member States’ authorities.

Structure, function and intracellular dynamics of alphavirus replication complexes

Intracellular membrane alterations are hallmarks of positive-sense RNA (+RNA) virus replication. Strong evidence indicates that within these exotic compartments, viral replicase proteins engage in RNA genome replication and transcription. To date, fundamental questions such as the origin of altered membranes, mechanisms of membrane deformation and topological distribution and function of viral components, are still waiting for comprehensive answers. This study addressed some of the above mentioned questions for the membrane alterations induced during Semliki Forest virus (SFV) infection of mammalian cells. With the aid of electron and fluorescence microscopy coupled with radioactive labelling and immuno-cytochemistry techniques, our group and others showed that few hours after infection the four non structural proteins (nsP1-4) and newly synthesized RNAs of SFV colocalized in close proximity of small membrane invaginations, designated as spherules . These 50-70 nm structures were mainly detected in the perinuclear area, at the limiting membrane of modified endosomes and lysosomes, named CPV-I (cytopathic vacuoles type I). More rarely, spherules were also found at the plasma membrane (PM). In the first part of this study I present the first three-dimensional reconstruction of the CPV-I and the spherules, obtained by electron tomography after chemical or cryo-fixation. Different approaches for imaging these macromolecular assemblies to obtain better structure preservation and higher resolution are presented as unpublished data. This study provides insights into spherule organization and distribution of viral components. The results of this and other experiments presented in this thesis will challenge currently accepted models for virus replication complex formation and function. In a revisitation of our previous models, the second part of this work provides the first complete description of the biogenesis of the CPV-I. The results demonstrate that these virus-induced vacuoles, where hundreds of spherules accumulate at late stages during infection, represent the final phase of a journey initiated at the PM, which apparently serves as a platform for spherule formation. From the PM spherules were internalized by an endocytic event that required the activity of the class I PI3K, caveolin-1, cellular cholesterol and functional actin-myosin network. The resulting neutral endocytic carrier vesicle delivered the spherules to the membrane of pre-existing acidic endosomes via multiple fusion events. Microtubule based transport supported the vectorial transfer of these intermediates to the pericentriolar area where further fusions generated the CPV-I. A signal for spherule internalization was identified in one of the replicase proteins, nsP3. Infections of cells with viruses harbouring a deletion in a highly phosphorylated region of nsP3 did not result in the formation of CPV-Is. Instead, thousands of spherules remained at the PM throughout the infection cycle. Finally, the role of the replicase protein nsP2 during viral RNA replication and transcription was investigated. Three enzymatic activities, protease, NTPase and RNA-triphosphatase were studied with the aid of temperature sensitive mutants in vitro and, when possible, in vivo. The results highlighted the interplay of the different nsP2 functions during different steps of RNA replication and sub-genomic promoter regulation, and suggest that the protein could have different activities when participating in the replication complex or as a free enzyme.

Microneurovascular free muscle transfer with cross-over nerve grafts in facial reanimation

Microneurovascular free muscle transfer with cross-over nerve grafts in facial reanimation Loss of facial symmetry and mimetic function as seen in facial paralysis has an enormous impact on the psychosocial conditions of the patients. Patients with severe long-term facial paralysis are often reanimated with a two-stage procedure combining cross-facial nerve grafting, and 6 to 8 months later with microneurovascular (MNV) muscle transfer. In this thesis, we recorded the long-term results of MNV surgery in facial paralysis and observed the possible contributing factors to final functional and aesthetic outcome after this procedure. Twenty-seven out of forty patients operated on were interviewed, and the functional outcome was graded. Magnetic resonance imaging (MRI) of MNV muscle flaps was done, and nerve graft samples (n=37) were obtained in second stage of the operation and muscle biopsies (n=18) were taken during secondary operations.. The structure of MNV muscles and nerve grafts was evaluated using histological and immunohistochemical methods ( Ki-67, anti-myosin fast, S-100, NF-200, CD-31, p75NGFR, VEGF, Flt-1, Flk-1). Statistical analysis was performed. In our studies, we found that almost two-thirds of the patients achieved good result in facial reanimation. The longer the follow-up time after muscle transfer the weaker was the muscle function. A majority of the patients (78%) defined their quality of life improved after surgery. In MRI study, the free MNV flaps were significantly smaller than originally. A correlation was found between good functional outcome and normal muscle structure in MRI. In muscle biopsies, the mean muscle fiber diameter was diminished to 40% compared to control values. Proliferative activity of satellite cells was seen in 60% of the samples and it tended to decline with an increase of follow-up time. All samples showed intramuscular innervation. Severe muscle atrophy correlated with prolonged intraoperative ischaemia. The good long-term functional outcome correlated with dominance of fast fibers in muscle grafts. In nerve grafts, the mean number of viable axons amounted to 38% of that in control samples. The grafted nerves characterized by fibrosis and regenerated axons were thinner than in control samples although they were well vascularized. A longer time between cross facial nerve grafting and biopsy sampling correlated with a higher number of viable axons. P75Nerve Growth Factor Receptor (p75NGFR) was expressed in every nerve graft sample. The expression of p75NGFR was lower in older than in younger patients. A high expression of p75NGFR was often seen with better function of the transplanted muscle. In grafted nerve Vascular Endothelial Growth Factor (VEGF) and its receptors were expressed in nervous tissue. In conclusion, most of the patients achieved good result in facial reanimation and were satisfied with the functional outcome. The mimic function was poorer in patients with longer follow-up time. MRI can be used to evaluate the structure of the microneurovascular muscle flaps. Regeneration of the muscle flaps was still going on many years after the transplantation and reinnervation was seen in all muscle samples. Grafted nerves were characterized by fibrosis and fewer, thinner axons compared to control nerves although they were well vascularized. P75NGFR and VEGF were expressed in human nerve grafts with higher intensity than in control nerves which is described for the first time.

Functional Outcome after Free Flap Reconstructions in Oral and Pharyngeal Cancer

Oral cancer ranks among the 10 most common cancers worldwide. Since it is commonly diagnosed at locally advanced stage, curing the cancer demands extensive tissue resection. The emergent defect is reconstructed generally with a free flap transfer. Repair of the upper aerodigestive track with maintenance of its multiform activities is challenging. The aim of the study was to extract comprehensive treatment outcomes for patients having undergone microvascular free flap transfer because of large oral cavity or pharyngeal cancer. Ninety-four patients were analyzed for postoperative survival and complications. Forty-four patients were followed-up and analyzed for functional outcome, which was determined in terms of quality of life, speech, swallowing, and intraoral sensation. Quality of life was assessed using the University of Washington Head and Neck Questionnaire. Speech was analyzed for aerodynamic parameters and for nasal acoustic energy, as well as perceptually for articulatory proficiency, voice quality, and intelligibility. Videofluorography was performed to determine the swallowing ability. Intraoral sensation was measured by moving 2-point discrimination. The 3-year overall survival was over 40%. The 1-year disease-free survival was 43%. Postoperative complications arose in over half of the patients. Flap success rate was high. Perioperative mortality varied between 2% and 11%. Unemployment and heavy drinking were the strongest predictors of survival. Sociodemographic factors were found to associate with quality of life. The global quality of life score deteriorated and did not return to the preoperative level. Significant reduction was detectable in the domains measuring chewing and speech, and in appearance and shoulder function. The basic elements necessary for normal speech were maintained. Speech intelligibility reduced and was related to the misarticulations of the /r/ and /s/ phonemes. Deviant /r/ and /s/ persisted in most patients. Hoarseness and hypernasality occurred infrequently. One year postoperatively, 98% of the patients had achieved oral nutrition and half of them were on a regular masticated diet. Overt and silent aspiration was encountered throughout the follow-up. At 12-month swallow test, 44% of the patients aspirated, 70% of whom silently. Of these patients, 15% presented with pulmonary changes referring to aspiration. Intraoral sensation weakened but was unrelated to oral functions. The results provide new data for oral reconstructions and highlight the importance of the functional outcome of the treatment for an oral cancer patient. The mouth and the pharynx encompass a unit of utmost functional complexity. Surgery should continue to make progress in this area, and methods that lead to good function should be developed. Operational outcome should always be evaluated in terms of function.

Cereal alkylresorcinols as dietary biomarkers-absorption and occurrence in biological membranes

Several studies link the consumption of whole-grain products to a lowered risk of chronic diseases, such as certain types of cancer, type II diabetes, and cardiovascular diseases. However, the final conclusions of the exact protective mechanisms remain unclear, partly due to a lack of a suitable biomarker for the whole-grain cereals intake. Alkylresorcinols (AR) are phenolic lipids abundant in the outer parts of wheat and rye grains usually with homologues of C15:0- C25:0 alkyl chains, and are suggested to function as whole-grain biomarkers. Mammalian lignan enterolactone has also previously been studied as a potential whole-grain biomarker. In the present work a quantified gas chromatography-mass spectrometry method for the analysis of AR in plasma, erythrocytes, and lipoproteins was developed. The method was used to determine human and pig plasma AR concentrations after the intake of whole-grain wheat and rye products compared to low-fibre wheat bread diets to assess the usability of AR as biomarkers of whole-grain intake. AR plasma concentrations were compared to serum ENL concentrations. AR absorption and elimination kinetics were investigated in a pig model. AR occurrence in human erythrocyte membranes and plasma lipoproteins were determined, and the distribution of AR in blood was evaluated. Plasma AR seem to be absorbed via the lymphatic system from the small intestine, like many other lipophilic compounds. Their apparent elimination half-life is relatively short and is similar to that of tocopherols, which have a similar chemical structure. Plasma AR concentrations increased significantly after a one- to eight-week intake of whole-grain wheat and further on with whole-grain rye bread. The concentrations were also higher after habitual Finnish diet compared to diet with low-fibre bread. Inter-individual variation after a one-week intake of the same amount of bread was high, but the mean plasma AR concentrations increased with increasing AR intake. AR are incorporated into erythrocyte membranes and plasma lipoproteins, and VLDL and HDL were the main AR carriers in human plasma. Based on these studies, plasma AR could function as specific biomarkers of dietary whole-grain products. AR are exclusively found in whole-grains and are more suitable as specific biomarkers of whole-grain intake than previously investigated mammalian lignan enterolactone, that is formed from several plants other than cereals in the diet. Plasma AR C17:0/C21:0 -ratio could distinguish whether whole-grain products in the diet are mainly wheat or rye. AR could be used in epidemiological studies to determine whole-grain intake and to better assess the role of whole-grains in disease prevention.