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  • Hotti, Anneli (Helsingin yliopisto, 2000)
  • Koivistoinen, Outi (Helsingin yliopisto, 2013)
    The use of metabolic engineering as a tool for production of biochemicals and biofuels requires profound understanding of cell metabolism. The pathways for the most abundant and most important hexoses have already been studied quite extensively but it is also important to get a more complete picture of sugar catabolism. In this thesis, catabolic pathways of L-rhamnose and D-galactose were studied in fungi. Both of these hexoses are present in plant biomass, such as in hemicellulose and pectin. Galactoglucomannan, a type of hemicellulose that is especially rich in softwood, is an abundant source of D-galactose. As biotechnology is moving from the usage of edible and easily metabolisable carbon sources towards the increased use of lignocellulosic biomass, it is important to understand how the different sugars can be efficiently turned into valuable biobased products. Identification of the first fungal L-rhamnose 1-dehydrogenase gene, which codes for the first enzyme of the fungal catabolic L-rhamnose pathway, showed that the protein belongs to a protein family of short-chain alcohol dehydrogenases. Sugar dehydrogenases oxidising a sugar to a sugar acid are not very common in fungi and thus the identification of the L-rhamnose dehydrogenase gene provides more understanding of oxidative sugar catabolism in eukaryotic microbes. Further studies characterising the L-rhamnose cluster in the yeast Scheffersomyces stipitis including the expression of the L-rhamnonate dehydratase in Saccharomyces cerevisiae finalised the biochemical characterisation of the enzymes acting on the pathway. In addition, more understanding of the regulation and evolution of the pathway was gained. D-Galactose catabolism was studied in the filamentous fungus Aspergillus niger. Two genes coding for the enzymes of the oxido-reductive pathway were identified. Galactitol dehydrogenase is the second enzyme of the pathway converting galactitol to L-xylo-3-hexulose. The galactitol dehydrogenase encoding gene ladB was identified and the deletion of the gene resulted in growth arrest on galactitol indicating that the enzyme is an essential part of the oxido-reductive galactose pathway in fungi. The last step of this pathway converts D-sorbitol to D-fructose by sorbitol dehydrogenase encoded by sdhA gene. Sorbitol dehydrogenase was found to be a medium chain dehydrogenase and transcription analysis suggested that the enzyme is involved in D-galactose and D-sorbitol catabolism.   The thesis also demonstrates how the understanding of cell metabolism can be used to engineer yeast to produce glycolic acid. Glycolic acid is a chemical, which can be used for example in the cosmetic industry and as a precursor for biopolymers. Currently, glycolic acid is produced by chemical synthesis in a process requiring toxic formaldehyde and fossil fuels. Thus, a biochemical production route would be preferable from a sustainability point of view. Yeasts do not produce glycolic acid under normal conditions but it is a desired production host for acid production because of its natural tolerance to low pH conditions. As a proof of concept, pure model substrates, e.g. D-xylose and ethanol, were used as starting materials for glycolic acid production but the knowledge can be further applied to an expanded substrate range such as biomass derived sugars. Already the introduction of a heterologous glyoxylate reductase gene resulted in glycolic acid production in the yeasts S. cerevisiae and Kluyveromyces lactis. Further modifications of the glyoxylate cycle increased the production of glycolic acid and it was successfully produced in bioreactor cultivation. The challenge of biotechnology is to produce high value products from cheap raw materials in an economically feasible way. This thesis gives more basic understanding to the topic in the form of new information regarding L-rhamnose and D-galactose metabolism in eukaryotic microbes as well as provides an example on how cell metabolism can be engineered in order to turn the cell into a cell factory that is able to produce a useful chemical.
  • Sibaouih, Ahlam (Helsingin yliopisto, 2015)
    Catalytic transformation of carbon dioxide into useful organic compounds has attracted much attention due to its economic and environmental benefits. In addition, other reasons are also taken into account, such as the possible utilization of CO2 as a renewable source chemical and the growing concern of the greenhouse effect. CO2 is an abundant, cheap, and safe C1 building block in organic synthesis. However, due to the inert nature of CO2, efficient catalytic processes of its chemical fixation remain a significant challenge. In this work, we have studied a possible pathway for practical utilization of CO2. The reaction of CO2 with epoxides giving cyclic carbonates, has been investigated. New catalyst systems based on cobalt capable of catalyzing the chemical transformation of carbon dioxide are described in detail. Oxygen is a cheap, readily available and environmentally friendly natural oxidant. The catalytic activation of molecular oxygen has great potential in a variety of applications. Catalysis and reactions, which are based on molecular oxygen, can also be considered to be ecologically benign processes. Moreover, catalytic reactions in water are highly desirable in terms of green chemistry. In this context, our purpose was to develop an environmentally friendly catalytic systems, suitable for oxidation of alcohols with molecular oxygen in water solution. In this part of the work, efficient catalysts, based on copper complexes have been synthesized and studied in the presence of TEMPO for the oxidation of benzyl and aliphatic alcohols with molecular oxygen in aqueous and nonaqueous medium.
  • Hakola, Maija (Helsingin yliopisto, 2013)
    There is nowadays a strong concern about decreasing oil supplies and global warming leading to ever increasing interest in biobased fuels and chemical production. The utilization of lignocellulosic raw materials for liquid biofuels and chemicals is a challenging task due to raw materials rigid structure which is resistant towards any actions to break it. Thus the raw materials should be pretreated to reach an economically vital process. Catalytic and alkaline oxidation presented here are novel, highly selective and effective methods to separate carbohydrates from lignin in different lignocellulosic materials. Both pretreatment methods are carried out at 120 140 ˚C for 4 20 hours under 10 bar oxygen pressure and in alkaline water solution. In catalytic oxidation also copper(II) phenanthroline catalyst is used. The obtained carbohydrate rich fraction is remarkably easy to hydrolyze with enzymes to corresponding sugars. The carbohydrate rich material from catalytic oxidation is nearly quantitatively converted into corresponding monohydrates in 24 hours by enzymatic hydrolysis. The enzyme loading can also be significantly decreased compared to standard methods, which helps to decrease the total costs of the process. Catalytic and alkaline oxidation pretreatment methods can be applied also to chromated copper arsenite treated waste wood, which is considered as hazardous waste. When combined with very mild sulfuric acid extraction and enzymatic hydrolysis 90 % sugar yield is obtained from the carbohydrate material. The sugars can be further utilized in chemical industry, which makes this method an interesting way to recycle hazardous waste. In a process turning lignocellulosic materials into sugars hydrolysis plays the key role. The hydrolysis is usually carried out enzymatically or with acids. They both have their disadvantages, such as long reaction times, poor yields or corrosive effect. As an alternative to traditional acid hydrolysis a microwave assisted mild acid hydrolysis is presented here. It can be applied to xylan, which is converted to xylose quantitatively in just one minute with 2 wt-% hydrochloric acid. When applied to milled birch wood 70 % yield of xylose is obtained. Subjecting alkaline oxidation pretreated birch to a three-step reaction, all the xylan and 66 % of the cellulose is hydrolyzed to corresponding sugars.
  • Nurttila, Sandra (2013)
    Lignocellulosic biomass has received widespread attention as environmentally benign feedstock for fuels. Biomass consists of cellulose, hemicellulose and lignin and has rather high oxygen content. Different techniques for the conversion of lignocellulose to liquid fuels have been suggested in the literature. In this thesis the emphasis is on the utilization of biomass-derived platform molecules. Platform molecules include eg. ketones, alcohols and carboxylic acids. In the literary section different deoxygenation and C-C coupling reactions for the conversion of biomass-derived platform molecules to larger hydrocarbons have been reviewed. Reaction routes for upgrading of the platform molecules 5-hydroxymethylfurfural, 2-furaldehyde, levulinic acid and some monofunctional compounds have been presented. These paths comprise mainly dehydration, hydrogenation, aldol condensation and ketonization. Heterogeneous catalysis, particularly bifunctional supported catalysts, dominates in this field. The selectivity that may be achieved with homogeneous catalysts is seen as highly desirable and served as the main incentive for the experimental work. Furthermore, the lack of publications in the area of homogeneously catalyzed C-C coupling of biomass-derived compounds also motivated for the work. Herein, 1st row transition metal acetates were utilized as catalysts for the ketonization of biomass-derived levulinic acid and other carboxylic acids. Some experiments were conducted with lignin as well. Reactions were performed under microwave heating or reflux conditions. Products were analyzed by GC-MS, GC-FID, NMR and FTIR. Combinatorial chemistry made it possible to conduct up to twelve reactions simultaneously. More than 160 reactions were performed in less than two months' time. The main product in many of the reactions was aromatic phthalic acid mono 2-ethylhexyl ester. Some other interesting products included hexadecanoic acid, 2,6-dimethyl-2,5-heptadien-4-one, diisooctyl and dibutyl phthalate. Despite small amounts of the products, their presence proves that various compounds may be produced from biomass by tailoring the catalyst and reaction conditions.
  • Kambur, Oleg (Helsingin yliopisto, 2013)
    Acute pain is an important warning signal, however, neuropathic pain and often chronic pain,lack a physiological function. Pain is a major clinical challenge and especially chronic and neuropathic pain are difficult to treat. On individual level, pain causes occupational and functional disability, suffering, and impairs quality of life. On a macro level pain and its direct and indirect consequences cause multi-billion expenses. Genetic factors and mechanisms underlying susceptibility to chronic pain have recently raised significant scientific interest. COMT-gene, which codes for catechol-O-methyltransferase (COMT), is subject for genetic polymorphic variation and COMT polymorphisms modulate pain and opioid analgesia in humans. The effects of COMT on pain and opioid responses were studied in rodents and humans. In mice, COMT deficiency was associated with altered stress- and morphine-induced analgesia reflecting weakened capacity of endogenous pain modulation and changes in opioidergic transmission. In normal mice, COMT inhibitors reduced thresholds of mechanical nociception and shortened thermal nociceptive latencies and thus increased nociceptive sensitivity in models of acute and inflammatory pain. Pronociceptive effects were COMT-dependent. In the spinal nerve ligation model of neuropathic pain in rats nitecapone decreased nociceptive symptoms - cold and mechanical hyperalgesia and allodynia. In humans, genetic variation of COMT gene was associated with pain phenotypes. The associations were strongest for the experimental pain phenotypes but also clinical pain phenotypes, such as acute postoperative pain, showed associations (uncorrected p=0.006-0.007)with three single nucleotide polymorphisms (SNPs). The strongest effect was observed in the SNP located in the 3´UTR-region of COMT, rs887200, pointing to importance of this region in regulation of nociceptive phenotypes and confirming the results in rodents. Together, these results confirm the role of COMT in pain and opioid responses. The antiallodynic effects of COMT inhibitors should be further studied in neuropathic pain since the safety and efficacy of current therapies are not satisfactory. In humans, mutations in COMT gene affect pain. The predictive value of individual SNPs, however, is limited and several SNPs of COMT as well as other genetic factors should be included in the same analysis or treatment algorithms possibly utilizing a haplotypic approach. Finally, the effects of most SNPs associated with pain phenotypes on COMT expression and activity are not known and should be explored in further studies.
  • Hyvärinen, Otto (Helsingin yliopisto, 2011)
    In meteorology, observations and forecasts of a wide range of phenomena for example, snow, clouds, hail, fog, and tornados can be categorical, that is, they can only have discrete values (e.g., "snow" and "no snow"). Concentrating on satellite-based snow and cloud analyses, this thesis explores methods that have been developed for evaluation of categorical products and analyses. Different algorithms for satellite products generate different results; sometimes the differences are subtle, sometimes all too visible. In addition to differences between algorithms, the satellite products are influenced by physical processes and conditions, such as diurnal and seasonal variation in solar radiation, topography, and land use. The analysis of satellite-based snow cover analyses from NOAA, NASA, and EUMETSAT, and snow analyses for numerical weather prediction models from FMI and ECMWF was complicated by the fact that we did not have the true knowledge of snow extent, and we were forced simply to measure the agreement between different products. The Sammon mapping, a multidimensional scaling method, was then used to visualize the differences between different products. The trustworthiness of the results for cloud analyses [EUMETSAT Meteorological Products Extraction Facility cloud mask (MPEF), together with the Nowcasting Satellite Application Facility (SAFNWC) cloud masks provided by Météo-France (SAFNWC/MSG) and the Swedish Meteorological and Hydrological Institute (SAFNWC/PPS)] compared with ceilometers of the Helsinki Testbed was estimated by constructing confidence intervals (CIs). Bootstrapping, a statistical resampling method, was used to construct CIs, especially in the presence of spatial and temporal correlation. The reference data for validation are constantly in short supply. In general, the needs of a particular project drive the requirements for evaluation, for example, for the accuracy and the timeliness of the particular data and methods. In this vein, we discuss tentatively how data provided by general public, e.g., photos shared on the Internet photo-sharing service Flickr, can be used as a new source for validation. Results show that they are of reasonable quality and their use for case studies can be warmly recommended. Last, the use of cluster analysis on meteorological in-situ measurements was explored. The Autoclass algorithm was used to construct compact representations of synoptic conditions of fog at Finnish airports.
  • Partanen, Sanna (Helsingin yliopisto, 2006)
    Cathepsin D (CTSD) is a lysosomal protease, the deficiency of which is fatal and associated with neurodegeneration. CTSD knock-out mice, which die at the age of four weeks, show intestinal necrosis, loss of lymphoid cells and moderate pathological changes in the brain. An active-site mutation in the CTSD gene underlies a neurodegenerative disease in newborn sheep, characterized by brain atrophy without any changes to visceral tissues. The CTSD deficiences belong to the group of neuronal ceroid-lipofuscinoses (NCLs), severe neurodegenerative lysosomal storage disorders. The aim of this thesis was to examine the molecular and cellular mechanisms behind neurodegeneration in CTSD deficiency. We found the developmental expression pattern of CTSD to resemble that of synaptophysin and the increasing expression of CTSD to coincide with the active period of myelination in the rat brain, suggesting a role for CTSD in early rat brain development. An active-site mutation underlying the congenital ovine NCL not only affected enzymatic activity, but also changed the stability, processing and transport of the mutant protein, possibly contributing to the disease pathogenesis. We also provide CTSD deficiency as a first molecular explanation for human congenital NCL, a lysosomal storage disorder, characterized by neuronal loss and demyelination in the central nervous system. Finally, we show the first evidence for synaptic abnormalities and thalamocortical changes in CTSD-deficient mice at the molecular and ultrastructural levels. Keywords: cathepsin D, congenital, cortex, lysosomal storage disorder, lysosome, mutation, neurodegeneration, neuronal ceroid-lipofuscinosis, overexpression, synapse, thalamus
  • Kivikero, Hanna (Helsingin yliopisto, 2011)
    The nature of a burial is always ritualistic. This is often forgotten when dealing with Finnish inhumation burials containing animal bones. Only the animal bones found close to the deceased have traditionally been thought to have a ritualistic purpose. The animal bones found in the filling of the grave, which is still part of the burial, has on the other hand, often been neglected in the previous research. In this Master s thesis I will discuss the function and interpretation of animal bones in graves. The base of this study is six sites, all of different nature, from Finland. Luistari in Eura is from the western coast and is dated to Late Iron Age (and possibly Medieval period), the Medieval hamlet of Finno is situated in Espoo which is situated on the southern coast. Two town burials, Turku and Porvoo, are also included in this study. The graves from Turku are dated to Late Medieval period and Early Renaissance, whereas the cemetery in Porvoo is from the 18th century. Visulahti in Mikkeli is from the Late Iron Age and represents Eastern Finnish burial tradition, the same as Suotniemi from Käkisalmi parish, which is nowadays part of Russia. While parts of the animal bones had already been analysed before, the author also analysed animal bones for the purpose of the present Master´s thesis. The bones were compared to the burial contexts, when possible. Based on the comparisons I have made interpretations which might explain the existence of animal bones in the graves. The interpretations are among others sacrifice, commemoration meals and animal burials. The site could also have been a settlement site prior to the graves, thus the bones in the graves would belong to the settlement phase. When comparing the date of the studied sites, the town burials are later and the animal bones are probably related to previous or contemporary use of the sites as graveyards. On top of this there does not seem to be much difference in burial tradition between Eastern and Western Finland, although at least from the hamlet burials of Finno there are aspects that could be linked to Eastern burials. In making the interpretations I have taken into consideration the aspects of belief during different time periods when they could be accounted as relevant. Also the problems with bone preservation were relevant and challenging for the study. Often only the hardest substance of the skeleton, namely teeth, has been preserved. For this reason the quality of the archaeological documentation was a key issue in this study. In producing quality interpretations of the animal bones in graves, the bones, contexts and their relationship to the surrounding site should be documented with care.
  • Entner, Doris (Helsingin yliopisto, 2013)
    In many fields of science, researchers are keen to learn causal connections among quantities of interest. For instance, in medical studies doctors want to infer the effect of a new drug on the recovery from a particular disease, or economists may be interested in the effect of education on income. The preferred approach to causal inference is to carry out controlled experiments. However, such experiments are not always possible due to ethical, financial or technical restrictions. An important problem is thus the development of methods to infer cause-effect relationships from passive observational data. While this is a rather old problem, in the late 1980s research on this issue gained significant momentum, and much attention has been devoted to this problem ever since. One rather recently introduced framework for causal discovery is given by linear non-Gaussian acyclic models (LiNGAM). In this thesis, we apply and extend this model in several directions, also considering extensions to non-parametric acyclic models. We address the problem of causal structure learning from time series data, and apply a recently developed method using the LiNGAM approach to two economic time series data sets. As an extension of this algorithm, in order to allow for non-linear relationships and latent variables in time series models, we adapt the well-known Fast Causal Inference (FCI) algorithm to such models. We are also concerned with non-temporal data, generalizing the LiNGAM model in several ways: We introduce an algorithm to learn the causal structure among multidimensional variables, and provide a method to find pairwise causal relationships in LiNGAM models with latent variables. Finally, we address the problem of inferring the causal effect of one given variable on another in the presence of latent variables. We first suggest an algorithm in the setting of LiNGAM models, and then introduce a procedure for models without parametric restrictions. Overall, this work provides practitioners with a set of new tools for discovering causal information from passive observational data in a variety of settings.
  • Salo, Pauli (Helsingin yliopisto, 2003)
  • Vitikainen, Emma (Helsingin yliopisto, 2010)
    Human actions cause destruction and fragmentation of natural habitats, predisposing populations to loss of genetic diversity and inbreeding, which may further decrease their fitness and survival. Understanding these processes is a main concern in conservation genetics. Yet data from natural populations is scarce, particularly on invertebrates, owing to difficulties in measuring both fitness and inbreeding in the wild. Ants are social insects, and a prime example of an ecologically important group for which the effects of inbreeding remain largely unstudied. Social insects serve key roles in all terrestrial ecosystems, and the division of labor between the females in the colonies queens reproduce, workers tend to the developing brood probably is central to their ecological success. Sociality also has important implications for the effects of inbreeding. Despite their relative abundance, the effective population sizes of social insects tend to be small, owing to the low numbers of reproductive individuals relative to the numbers of sterile workers. This may subject social insects to loss of genetic diversity and subsequent inbreeding depression. Moreover, both the workers and queens can be inbred, with different and possibly multiplicative consequences. The aim of this study was to investigate causes and consequences of inbreeding in a natural population of ants. I used a combination of long-term field and genetic data from colonies of the narrow-headed ant Formica exsecta to examine dispersal, mating behavior and the occurrence of inbreeding, and its consequences on individual and colony traits. Mating in this species takes place in nuptial flights that have been assumed to be population-wide and panmictic. My results, however, show that dispersal is local, with queens establishing new colonies as close as 60 meters from their natal colony. Even though actual sib-mating was rare, individuals from different but related colonies pair, which causes the population to be inbred. Furthermore, multiple mates of queens were related to each other, which also indicates localized mating flights. Hence, known mechanisms of inbreeding avoidance, dispersal and multiple mating, were not effective in this population, as neither reduced inbreeding level of the future colony. Inbreeding had negative consequences both at the individual and colony level. A queen that has mated with a related male produces inbred workers, which impairs the colony s reproductive success. The inbred colonies were less productive and, specifically, produced fewer new queens, possibly owing to effects of inbreeding on the caste determination of female larvae. A striking finding was that males raised in colonies with inbred workers were smaller, which reflects an effect of the social environment as males, being haploid, cannot be inbred themselves. The queens produced in the inbred colonies, in contrast, were not smaller, but their immune response was up-regulated. Inbreeding had no effect on queen dispersal, but inbred queens had a lower probability of successfully founding a new colony. Ultimately, queens that survived through the colony founding phase had a shorter lifespan. This supports the idea that inbreeding imposes a genetic stress, leading to inbreeding depression on both the queen and the colony level. My results show that inbreeding can have profound consequences on insects in the wild, and that in social species the effects of inbreeding may be multiplicative and mediated through the diversity of the social environment, as well as the genetic makeup of the individuals themselves. This emphasizes the need to take into account all levels of organization when assessing the effects of genetic diversity in social animals.
  • Pitala, Natalia (Helsingin yliopisto, 2007)
    Defence against pathogens is a vital need of all living organisms that has led to the evolution of complex immune mechanisms. However, although immunocompetence the ability to resist pathogens and control infection has in recent decades become a focus for research in evolutionary ecology, the variation in immune function observed in natural populations is relatively little understood. This thesis examines sources of this variation (environmental, genetic and maternal effects) during the nestling stage and its fitness consequences in wild populations of passerines: the blue tit (Cyanistes caeruleus) and the collared flycatcher (Ficedula albicollis). A developing organism may face a dilemma as to whether to allocate limited resources to growth or to immune defences. The optimal level of investment in immunity is shaped inherently by specific requirements of the environment. If the probability of contracting infection is low, maintaining high growth rates even at the expense of immune function may be advantageous for nestlings, as body mass is usually a good predictor of post-fledging survival. In experiments with blue tits and haematophagous hen fleas (Ceratophyllus gallinae) using two methods, methionine supplementation (to manipulate nestlings resource allocation to cellular immune function) and food supplementation (to increase resource availability), I confirmed that there is a trade-off between growth and immunity and that the abundance of ectoparasites is an environmental factor affecting allocation of resources to immune function. A cross-fostering experiment also revealed that environmental heterogeneity in terms of abundance of ectoparasites may contribute to maintaining additive genetic variation in immunity and other traits. Animal model analysis of extensive data collected from the population of collared flycatchers on Gotland (Sweden) allowed examination of the narrow-sense heritability of PHA-response the most commonly used index of cellular immunocompetence in avian studies. PHA-response is not heritable in this population, but is subject to a non-heritable origin (presumably maternal) effect. However, experimental manipulation of yolk androgen levels indicates that the mechanism of the maternal effect in PHA-response is not in ovo deposition of androgens. The relationship between PHA-response and recruitment was studied for over 1300 collared flycatcher nestlings. Multivariate selection analysis shows that it is body mass, not PHA-response, that is under direct selection. PHA-response appears to be related to recruitment because of its positive relationship with body mass. These results imply that either PHA-response fails to capture the immune mechanisms that are relevant for defence against pathogens encountered by fledglings or that the selection pressure from parasites is not as strong as commonly assumed.
  • Koivuniemi, Riitta (Helsingin yliopisto, 2009)
    Rheumatoid arthritis (RA) patients have premature mortality. Contrary to the general population, mortality in RA has not declined over time. This study aimed to evaluate determinants of mortality in RA by examining causes of death (CoDs) over time, accuracy of CoD diagnoses, and contribution of RA medication to CoDs. This study further evaluated detection rate of reactive systemic amyloid A amyloidosis, which is an important contributor to RA mortality. CoDs were examined in 960 RA patients between 1971 and 1991 (Study population A) and in 369 RA patients autopsied from 1952 to 1991, with non-RA patients serving as the reference cases (Study population B). In Study population B, CoDs by the clinician before autopsy were compared to those by the pathologist at autopsy to study accuracy of CoD diagnoses. In Study population B, autopsy tissue samples were re-examined systematically for amyloidosis (90% of patients) and clinical data for RA patients was studied from 1973. RA patients died most frequently of cardiovascular diseases (CVDs), infections, and RA. RA deaths declined over time. Coronary deaths showed no major change in Study population A, but, in Study population B, coronary deaths in RA patients increased from 1952 to 1991, while non-RA cases had a decrease in coronary deaths starting in the 1970s. Between CoD diagnoses by the clinician and those by the pathologist, RA patients had lower agreement than non-RA cases regarding cardiovascular (Kappa reliability measure: 0.31 vs. 0.51) and coronary deaths (0.33 vs. 0.46). Use of disease modifying anti-rheumatic drugs was not associated with any CoD. In RA patients, re-examination of autopsy tissue samples doubled the prevalence of amyloid compared with the original autopsy: from 18% to 30%. In the amyloid-positive RA patients, amyloidosis was diagnosed before autopsy in only 37%; and they had higher inflammatory levels and longer duration of RA than amyloid-negative RA patients. Of the RA patients with amyloid, only half had renal failure or proteinuria during lifetime. In RA, most important determinants of mortality were CVDs, RA, and infections. In RA patients, RA deaths decreased over time, but this was not true for coronary deaths. Coronary death being less accurately diagnosed in RA may indicate that coronary heart disease (CHD) often goes unrecognized during lifetime. Thus, active search for CHD and its effective treatment is important to reduce cardiovascular mortality. Reactive amyloidosis may often go undetected. In RA patients with proteinuria or renal failure, as well as with active and long-lasting RA, a systematic search for amyloid is important to enable early diagnosis and early enhancement of therapy. This is essential to prevent clinical manifestations of amyloidosis such as renal failure, which has a poor prognosis.
  • Metsälä, Markus (Helsingin yliopisto, 2006)
    This thesis contains five experimental spectroscopic studies that probe the vibration-rotation energy level structure of acetylene and some of its isotopologues. The emphasis is on the development of laser spectroscopic methods for high-resolution molecular spectroscopy. Three of the experiments use cavity ringdown spectroscopy. One is a standard setup that employs a non-frequency stabilised continuous wave laser as a source. In the other two experiments, the same laser is actively frequency stabilised to the ringdown cavity. This development allows for increased repetition rate of the experimental signal and thus the spectroscopic sensitivity of the method is improved. These setups are applied to the recording of several vibration-rotation overtone bands of both H(12)C(12)CH and H(13)C(13)CH. An intra-cavity laser absorption spectroscopy setup that uses a commercial continuous wave ring laser and a Fourier transform interferometer is presented. The configuration of the laser is found to be sub-optimal for high-sensitivity work but the spectroscopic results are good and show the viability of this type of approach. Several ro-vibrational bands of carbon-13 substituted acetylenes are recorded and analysed. Compared with earlier work, the signal-to-noise ratio of a laser-induced dispersed infrared fluorescence experiment is enhanced by more than one order of magnitude by exploiting the geometric characteristics of the setup. The higher sensitivity of the spectrometer leads to the observation of two new symmetric vibrational states of H(12)C(12)CH. The precision of the spectroscopic parameters of some previously published symmetric states is also improved. An interesting collisional energy transfer process is observed for the excited vibrational states and this phenomenon is explained by a simple step-down model.
  • Väärikkälä, Sofia (2007)
    Clostridium botulinum on anaerobinen itiöitä muodostava bakteeri, jonka tuottama neurotoksiini aiheuttaa ihmisille ja eläimille hengenvaarallisen botulismin. Tavallisimmin neurotoksiinia saadaan ruoan mukana, sillä raa’at elintarvikkeet kontaminoituvat luonnossa esiintyvillä itiöillä. C. botulinum kannat jaetaan neljään fenotyyppisesti erilaiseen ryhmään (I-IV). C. botulinum muodostaa elintarviketurvallisuusriskin, sillä ihmisille myrkytyksen aiheuttavien ryhmän I kantojen itiömuodot kestävät erittäin korkeita lämpötiloja ja ryhmän II kannat pystyvät lisääntymään jääkaappilämpötiloissa. Selvittämällä C. botulinumin kuuman- ja kylmänsietoon liittyviä mekanismeja elintarviketurvallisuus-riskejä voitaisiin hallita paremmin. Aistikinaasien ja vastesäätelijöiden väliseen fosforinsiirtoon perustuvien kaksikomponenttijärjestelmien tiedetään olevan bakteerien eniten käyttämä mekanismi ympäristön muutoksiin sopeutumisessa, mutta niiden merkitys C. botulinumilla on epäselvä. Tutkimuksen tarkoituksena oli selvittää, mitä merkitystä kaksikomponenttijärjestelmään kuuluvalla aistikinaasia koodaavalla CBO0366-geenillä on C. botulinuminin fysiologiassa sekä kylmän- ja kuumansiedossa. Tutkimuksessa käytettiin ClosTron-menetelmää tutkittavan.C. botulinumin ATCC 3502 -kannan CBO0366-geenin inaktivoimiseksi. Menetelmä perustuu pMTL007-plasmidin liikkuvan ryhmän II intronin kohdespesifisyyden muuttamiseen Splicing by Overlap Extension PCR:ää (SOE-PCR) käyttäen siten, että introni saatiin kiinnittymään spesifisesti C. botulinumin CBO0366-geeniin. Intronin integroituminen genomiin aiheutti kyseisen kohdan geenin lukukehyksen häiriintymisen. Mutatoidun kannan fysiologiaa ja kylmän- ja kuumansietoa tutkittiin käyttämällä ATCC 3502 -kannan villityyppiä kontrollina. Mutatoidun kannan pesäkemorfologia veri- ja munankeltuaislevyllä, metabolinen aktiivisuus 37 °C:ssa ja kasvukäyrät 37 °C:ssa sekä 45 °C:ssa olivat lähes identtiset villityypin kanssa. Mutatoitu kanta kasvoi kuitenkin selvästi villityypin kantaa hitaammin 15 °C:ssa ja 20 °C:ssa. CBO0366-geenillä on siis keskeinen merkitys C. botulinumin ATCC 3502 –kannan kylmänsietokyvyssä. Tulosten perusteella voidaan olettaa, että CBO0366-geenin koodaama aistikinaasi on osa kaksikomponenttijärjestelmää, joka on osallisena alhaisempiin lämpötiloihin sopeutumiseen tarvittavien vasteiden aikaansaamisessa. Geenin inaktivointi todennäköisesti estää kylmästä kertovan signaalin kuljettamisen solun sisälle ja edelleen spesifisen vasteen syntymisen. Siten bakteerin sopeutuminen kylmään heikkenee. Koska mutatoitu kanta kuitenkin kasvoi 15 °C:ssa, on oletettavaa, että geeni ei ole elintärkeä selviytymiseen viileämmässä.
  • Jha, Sawan (Helsingin yliopisto, 2014)
    Lymphangiogenesis is the process that leads to the formation of lymphatic vessels from pre-existing vessels. Vascular endothelial growth factor C (VEGF-C), the ma- jor lymphangiogenic growth factor, is produced as an inactive precursor and needs to be proteolytically processed into a mature form in order to activate its receptors VEGFR-3 and VEGFR-2. A deficiency of VEGF-C during embryonic lymphangiogenesis results in embryonic lethality due to the lack of lymphatic vasculature. Hennekam lymphangiectasia-lymphedema syndrome (OMIM 235510) is in a subset of patients associated with mutations in the collagen- and calcium-binding EGF domains 1 (CCBE1 ) gene. CCBE1 and VEGF-C act at the same stage during embryonic lymphangiogenesis and their deficiency results in similar lymphatic defects. The mechanism behind the lymphatic phenotype caused by CCBE1 mutations is un- known. The aim of this study was to investigate the potential link between VEGF-C and CCBE1 that could contribute to the lymphatic phenotype. In this study, 293T cells were used to observe the effect of CCBE1 on VEGF-C pro- cessing. The co-transfection of constructs coding for CCBE1 and VEGF-C showed processing of the inactive pro-VEGF-C into the active, mature form. However, this processing was efficient only in 293T cells. When CCBE1 from 293T supernatant was purified, A disintegrin and metalloproteinase with thrombospondin type 1 motif 3 (ADAMTS3) co-purified with CCBE1. The levels of pro-VEGF-C and active VEGF-C were monitored by immunoblotting or immunoprecipitating metabolically labeled supernatant with specific antibodies or receptors followed by autoradiography. The activity of the processed VEGF-C was verified by proliferation of Ba/F3 cells stably expressing VEGFR-3/EpoR or VEGFR-2/EpoR chimeras. Furthermore, a VEGFR-3 phosphorylation assay was performed in PAE (Porcine Aortic Endotheial) cells to study details of the CCBE1-mediated regulation of VEGF-C. We found that CCBE1 increases the proteolytic processing of pro-VEGF-C, thereby resulting in increased activity of VEGF-C. CCBE1 itself has no effect on VEGF-C activity but regulates VEGF-C by modulating the activity of the ADAMTS3 protease. We also found that both pro- and mature- VEGF-C can bind to VEGFR-3 but only mature form is able to induce VEGFR-3-mediated signaling. In addition to cleaving VEGF-C, ADAMTS3 was found to directly or indirectly mediate CCBE1 cleavage. The N-terminal amino acid sequence of the ADAMTS3-processed VEGF-C confirmed that ADAMTS3 is the protease responsible for the activation of VEGF-C by 293 cells. Hence, we have identified a mechanism that regulates VEGF-C activity. This mechanism suggests the possible use of CCBE1 as a therapeutic means to treat diseases that involve the lymphatic system.