Browsing by Title

Sort by: Order: Results:

Now showing items 13172-13191 of 25618
  • Kavakka, Jari (Helsingin yliopisto, 2010)
    Photosynthesis is a chemical process in which the energy of the light quanta is transformed into chemical energy. Chlorophyll (Chl) molecules play a key role in photosynthesis; they function in the antennae systems and in the photosynthetic reaction center where the primary charge separation (CS) takes place. Bio-inspired mimicry of the CS is an essential unit in dye-sensitized solar cells. Aim of this study was to design and develop electron donor-acceptor (EDA) pairs from Chls and fullerenes (C60) or carbon nanotubes (CNT). The supramolecular approach was chosen, as long synthetic sequences required by the covalent approach lead to long reaction schemes and low yields. Here, a π-interaction between soluble CNTs and Chl was used in EDA construction. Also, a beta-face selective two-point bound Chl-C60 EDA was introduced. In addition, the photophysical properties of the supramolecular EDA dyads were analyzed. In organic chemistry, nuclear magnetic resonance (NMR) spectroscopy is the most vital analytical technique in use. Multi-dimensional NMR experiments have enabled a structural analysis of complex natural products and proteins. However, in mixture analysis NMR is still facing difficulties. In many cases overlapping signals can t be resolved even with the help of multi-dimensional experiments. In this work, an NMR tool based on simple host-guest chemistry between analytes and macromolecules was developed. Diffusion ordered NMR spectroscopy (DOSY) measures the mobilities of compounds in an NMR sample. In a liquid state NMR sample, each of the analytes has a characteristic diffusion coefficient, which is proportional to the size of the analyte. With normal DOSY experiment, provided that the diffusion coefficients of the analytes differ enough, individual spectra of analytes can be extracted. When similar sized analytes differ chemically, an additive can be introduced into the sample. Since macromolecules in a liquid state NMR sample can be considered practically stationary, even faint supramolecular interaction can change the diffusion coefficient of the analyte sufficiently for a successful resolution in DOSY. In this thesis, polyvinylpyrrolidone and polyethyleneglycol enhanced DOSY NMR techniques, which enable mixture analysis of similar in size but chemically differing natural products, are introduced.
  • Uusi-Rauva, Kristiina (2012)
    Neuronal ceroid lipofuscinoses (NCLs) are inherited severe neurodegenerative diseases of childhood. NCLs are characterised by progressive, selective neuronal death and lysosomal accumulation of autofluorescent storage material. Due to the poor knowledge on the functions of the proteins primarily defective in NCLs, the intracellular changes critical to the pathogenesis of NCLs are not known. In this thesis study, the primary functions of CLN3, a protein defective in classic juvenile onset form of NCLs (juvenile CLN3 disease), was studied in terms of its protein interactions. CLN3 was determined to interact with Na+, K+ ATPase and associated fodrin cytoskeleton and 78 kDa glucose-regulated protein/immunoglobulin heavy chain binding protein (GRP78/BiP). Plasma membrane association and ouabain-induced endocytosis of Na+, K+ ATPase were found to be impaired in CLN3-deficient neurons. This suggests that CLN3 may play a role in the ouabain-regulated non-pumping functions of Na+, K+ ATPase, including intracellular signalling, apoptosis and calcium oscillations. Furthermore, putative structural changes in the fodrin cytoskeleton were observed in CLN3-deficient mouse brain sections and patient cells implying that fodrin-associated events in axonal and synaptic intracellular trafficking, synaptic transmission, and neuritogenesis may also be compromised in early stage of juvenile CLN3 disease. CLN3 was also found to interact with Hook1 involved in late endosomal/lysosomal maturation and with microtubular motor protein complexes, dynein-dynactin and kinesin-2-dynactin. CLN3 was shown to associate with the motor proteins most likely through direct interactions with Rab7 GTPase and its effector Rab7-interacting lysosomal protein (RILP). Rab7 and its effectors have been reported to regulate both minus and plus end-directed microtubular membrane trafficking. Interestingly, membrane trafficking was found to be affected in CLN3 deficiency demonstrated by abnormal intracellular position of late endosomes/lysosomes and unbalanced functional GTP/GDP cycle of Rab7 as well defects in the late endosomal targeting of endocytosed cargo and kinesin-dependent movement of late endosomes/lysosomes in CLN3-deficient cells. These results suggest that Rab7-guided motor protein functions in neurons, such as axonal retrograde trafficking of neurotrophins, neurite outgrowth and maturation, and transportation of neuronal autophagic vesicles could also be affected in CLN3 disease. This thesis work has provided important novel data on the functions of CLN3 and the primary intracellular defects possibly resulting in CLN3 disease. This study also contributes to the determination of the pathogenesis of other NCLs and general processes of neurodegeneration.
  • Lyly, Annina (Helsingin yliopisto, 2008)
    Disorders resulting from degenerative changes in the nervous system are progressive and incurable. Both environmental and inherited factors affect neuron function, and neurodegenerative diseases are often the sum of both factors. The cellular events leading to neuronal death are still mostly unknown. Monogenic diseases can offer a model for studying the mechanisms of neurodegeneration. Neuronal ceroid lipofuscinoses, or NCLs, are a group of monogenic, recessively inherited diseases affecting mostly children. NCLs cause severe and specific loss of neurons in the central nervous system, resulting in the deterioration of motor and mental skills and leading to premature death. In this thesis, the focus has been on two forms of NCL, the infantile NCL (INCL, CLN1) and the Finnish variant of late infantile NCL (vLINCLFin, CLN5). INCL is caused by mutations in the CLN1 gene encoding for the PPT1 (palmitoyl protein thioesterase 1) enzyme. PPT1 removes a palmitate moiety from proteins in experimental conditions, but its substrates in vivo are not known. In the Finnish variant of late infantile NCL (vLINCLFin), the CLN5 gene is defective, but the function of the encoded CLN5 has remained unknown. The aim of this thesis was to elucidate the disease mechanisms of these two NCL diseases by focusing on the molecular interactions of the defective proteins. In this work, the first interaction partner for PPT1, the mitochondrial F1-ATP synthase, was described. This protein has been linked to HDL metabolism in addition to its well-known role in the mitochondrial energy production. The connection between PPT1 and the F1-ATP synthase was studied utilizing the INCL-disease model, the genetically modified Ppt1-deficient mice. The levels of F1-ATP synthase subunits were increased on the surface of Ppt1-deficient neurons when compared to controls. We also detected several changes in lipid metabolism both at the cellular and systemic levels in Ppt1-deficient mice when compared to controls. The interactions between different NCL proteins were also elucidated. We were able to detect novel interactions between CLN5 and other NCL proteins, and to replicate the previously reported interactions. Some of the novel interactions influenced the intracellular trafficking of the proteins. The multiple interactions between CLN5 and other NCL proteins suggest a connection between the NCL subtypes at the cellular level. The main results of this thesis elicit information about the neuronal function of PPT1. The connection between INCL and neuronal lipid metabolism introduces a new perspective to this rather poorly characterized subject. The evidence of the interactions between NCL proteins provides the basis for future research trying to untangle the NCL disease mechanisms and to develop strategies for therapies.
  • Miettinen, Oskari (Helsingin yliopisto, 2010)
    New stars form in dense interstellar clouds of gas and dust called molecular clouds. The actual sites where the process of star formation takes place are the dense clumps and cores deeply embedded in molecular clouds. The details of the star formation process are complex and not completely understood. Thus, determining the physical and chemical properties of molecular cloud cores is necessary for a better understanding of how stars are formed. Some of the main features of the origin of low-mass stars, like the Sun, are already relatively well-known, though many details of the process are still under debate. The mechanism through which high-mass stars form, on the other hand, is poorly understood. Although it is likely that the formation of high-mass stars shares many properties similar to those of low-mass stars, the very first steps of the evolutionary sequence are unclear. Observational studies of star formation are carried out particularly at infrared, submillimetre, millimetre, and radio wavelengths. Much of our knowledge about the early stages of star formation in our Milky Way galaxy is obtained through molecular spectral line and dust continuum observations. The continuum emission of cold dust is one of the best tracers of the column density of molecular hydrogen, the main constituent of molecular clouds. Consequently, dust continuum observations provide a powerful tool to map large portions across molecular clouds, and to identify the dense star-forming sites within them. Molecular line observations, on the other hand, provide information on the gas kinematics and temperature. Together, these two observational tools provide an efficient way to study the dense interstellar gas and the associated dust that form new stars. The properties of highly obscured young stars can be further examined through radio continuum observations at centimetre wavelengths. For example, radio continuum emission carries useful information on conditions in the protostar+disk interaction region where protostellar jets are launched. In this PhD thesis, we study the physical and chemical properties of dense clumps and cores in both low- and high-mass star-forming regions. The sources are mainly studied in a statistical sense, but also in more detail. In this way, we are able to examine the general characteristics of the early stages of star formation, cloud properties on large scales (such as fragmentation), and some of the initial conditions of the collapse process that leads to the formation of a star. The studies presented in this thesis are mainly based on molecular line and dust continuum observations. These are combined with archival observations at infrared wavelengths in order to study the protostellar content of the cloud cores. In addition, centimetre radio continuum emission from young stellar objects (YSOs; i.e., protostars and pre-main sequence stars) is studied in this thesis to determine their evolutionary stages. The main results of this thesis are as follows: i) filamentary and sheet-like molecular cloud structures, such as infrared dark clouds (IRDCs), are likely to be caused by supersonic turbulence but their fragmentation at the scale of cores could be due to gravo-thermal instability; ii) the core evolution in the Orion B9 star-forming region appears to be dynamic and the role played by slow ambipolar diffusion in the formation and collapse of the cores may not be significant; iii) the study of the R CrA star-forming region suggests that the centimetre radio emission properties of a YSO are likely to change with its evolutionary stage; iv) the IRDC G304.74+01.32 contains candidate high-mass starless cores which may represent the very first steps of high-mass star and star cluster formation; v) SiO outflow signatures are seen in several high-mass star-forming regions which suggest that high-mass stars form in a similar way as their low-mass counterparts, i.e., via disk accretion. The results presented in this thesis provide constraints on the initial conditions and early stages of both low- and high-mass star formation. In particular, this thesis presents several observational results on the early stages of clustered star formation, which is the dominant mode of star formation in our Galaxy.
  • Laurila, Pirkka-Pekka (Helsingin yliopisto, 2015)
    Cardiovascular disease is the leading cause of death worldwide, and is characterized by disturbances in lipid metabolism. High blood LDL cholesterol is the most important risk factors for atherosclerosis, a pathological state in which the circulating lipid molecules accumulate in the blood vessel wall. Blood levels of HDL cholesterol are inversely associated with cardiovascular risk, and low HDL-cholesterol is a significant risk factor for cardiovascular disease. In this thesis, new genes predisposing to low HDL-cholesterol level were searched using genome-wide association analysis in Finnish individuals with extremely low or high HDL-cholesterol. Several new genes predisposing to low HDL-cholesterol were discovered, of which many were known to be associated with immune system and inflammatory reaction of the body, but their role in cholesterol metabolism has not previously been characterized. Some of the subjects appeared to be genetically more prone to inflammation than others, especially in the blood and adipose tissue. The more inflammation inducing genetic variants one had, the stronger was the inflammatory state of the body, especially in blood and adipose tissue, and the lower the HDL-cholesterol level. The inflammation may block the transport of cholesterol from vessel walls to circulation leading to lower HDL-cholesterol levels in the circulation. This thesis also examined the composition of HDL particles by determining the concentration of all know lipid species in HDL particles. The quality of HDL particles appeared to vary considerably. In individuals, whose HDL-cholesterol levels were low, the quality of HDL particles was also impaired; they contained smaller amounts of plasmalogens which are fat molecules known to be antioxidant and thus protective to arteries. In individuals with high HDL-cholesterol levels in the circulation, the lipid composition of the HDL particle was more beneficial regarding heart disease risk. This thesis further demonstrates that not only the quantity but also the quality of HDL particles is genetically regulated. The genetic variants regulating the level of HDL-cholesterol also govern its quality, highlighted by inflammation-increasing genetic variants also impairing HDL quality. This thesis work provides novel insights about the molecular background of HDL cholesterol and validates the strong link between inflammation and low HDL-cholesterol levels. The studies show how genetically induced inflammation reduces blood HDL-cholesterol levels and impairs HDL particle quality, potentially predisposing to cardiovascular disease.
  • Rice, David (Helsingin yliopisto, 1999)
  • Pihlajamaa, Päivi (Helsingin yliopisto, 2014)
    Androgens are steroid hormones that regulate the development and function of male reproductive organs as well as physiology of many non-reproductive tissues, such as muscle, bone, liver, and kidney. Moreover, androgen signaling is involved in several pathological conditions, most common of which is prostate cancer. In its target cells, testosterone or its more potent metabolite 5α-dihydrotestosterone regulates cellular processes by modulating gene expression through the androgen receptor (AR). Ligand-activated AR translocates to nucleus and binds to specific DNA sequences, called androgen response elements (AREs), at the regulatory regions of its target genes. AR cistromes, i.e., global maps of genomic AR occupancy, comprise thousands of AR-binding events primarily located at distal enhancers. AR-binding sites are characterized by distinct histone modifications, and AR recruitment is primed by pioneer factors capable of binding to compact chromatin. AR interacts with a plethora of coregulatory proteins that modify the local chromatin environment and interact with basal transcription machinery. These elements create the complex cellular landscape for androgen action. The purpose of this thesis was to study molecular determinants of context-specific AR functions in vivo in murine androgen-responsive tissues. The advantage of in vivo studies is that unlike in cancer cell models androgen target cells reside within their physiological environment with an intact AR pathway. In the first part of this work, an androgen reporter mouse line with the luciferase gene under androgenic control was created. In this model, luciferase activity is a measure of AR function, and it can be used for assessing in vivo effects of chemical compounds on AR signaling. The androgen reporter mice were treated with genistein, a phytoestrogenic compound to which people consuming soy products are also exposed. Previous studies have implied that genistein plays a potential role in prostate cancer prevention. The results in this thesis work showed that genistein exhibits tissue-specific effects on AR signaling in vivo. Furthermore, genistein modulates endogenous AR-mediated gene expression in prostate, supporting its potentially beneficial role in prostate carcinogenesis. In the second part of this work, genomic AR occupancy was examined using chromatin immunoprecipitation (ChIP) coupled with massively parallel sequencing (ChIP-seq). Distinct AR cistromes were identified in three androgen-responsive tissues: prostate, kidney, and epididymis. AR-binding events associate with tissue-specific transcription programs responsible for distinct physiological functions of androgens in these tissues. The key finding in this work was that tissue-specific AR binding is directed by divergent pioneer factors, and that previously identified forkhead box protein A1 (FoxA1) is prostate-specific rather than general pioneer factor for AR. Two novel pioneer factors for AR were identified in this study hepatocyte nuclear factor 4 alpha (Hnf4α) in murine kidney and activating protein 2 alpha (AP-2α) in murine epididymis. ChIP-seq was also utilized to study in vivo role and characteristics of selective AREs cis-elements not bound by other steroid receptors. Transgenic SPARKI mice have the second zinc finger of the AR DNA-binding domain swapped with the respective part of glucocorticoid receptor, resulting in a chimeric AR unable to bind to selective AREs. A significant proportion of in vivo binding events of wild-type AR were not shared by SPARKI AR in prostate and epididymis, highlighting the importance of selective AREs in AR-specific functions in vivo. Differential receptor binding was also linked to differentially expressed genes in the epididymides of wild-type and SPARKI mice. De novo sequence analysis revealed that the selective AREs are characterized by decreased sequence conversation, indicating that, counter-intuitively, AR selectivity in vivo is achieved by relaxed rather than increased cis-element stringency. In conclusion, both the AREs and the collaborating proteins contribute to precise AR-regulated transcriptional outcome in the context of native chromatin and distinct tissues. Overall, the results clarified several molecular mechanisms employed by AR in vivo that can potentially contribute to the development of better treatments and diagnostic tools for hormone-dependent disorders in the future.
  • Thompson, James (Helsingin yliopisto, 2006)
    The androgen receptor (AR) mediates the effects of the male sex-steroid hormones (androgens), testosterone and 5?-dihydrotestosterone. Androgens are critical in the development and maintenance of male sexual characteristics. AR is a member of the steroid receptor ligand-inducible transcription factor family. The steroid receptor family is a subgroup of the nuclear receptor superfamily that also includes receptors for the active forms of vitamin A, vitamin D3, and thyroid hormones. Like all nuclear receptors, AR has a conserved modular structure consisting of a non-conserved amino-terminal domain (NTD), containing the intrinsic activation function 1, a highly conserved DNA-binding domain, and a conserved ligand-binding domain (LBD) that harbors the activation function 2. Each of these domains plays an important role in receptor function and signaling, either via intra- and inter-receptor interactions, interactions with specific DNA sequences, termed hormone response elements, or via functional interactions with domain-specific proteins, termed coregulators (coactivators and corepressors). Upon binding androgens, AR acquires a new conformational state, translocates to the nucleus, binds to androgen response elements, homodimerizes and recruits sequence-specific coregulatory factors and the basal transcription machinery. This set of events is required to activate gene transcription (expression). Gene transcription is a strictly modulated process that governs cell growth, cell homeostasis, cell function and cell death. Disruptions of AR transcriptional activity caused by receptor mutations and/or altered coregulator interactions are linked to a wide spectrum of androgen insensitivity syndromes, and to the pathogenesis of prostate cancer (CaP). The treatment of CaP usually involves androgen depletion therapy (ADT). ADT achieves significant clinical responses during the early stages of the disease. However, under the selective pressure of androgen withdrawal, androgen-dependent CaP can progress to an androgen-independent CaP. Androgen-independent CaP is invariably a more aggressive and untreatable form of the disease. Advancing our understanding of the molecular mechanisms behind the switch in androgen-dependency would improve our success of treating CaP and other AR related illnesses. This study evaluates how clinically identified AR mutations affect the receptor s transcriptional activity. We reveal that a potential molecular abnormality in androgen insensitivity syndrome and CaP patients is caused by disruptions of the important intra-receptor NTD/LBD interaction. We demonstrate that the same AR LBD mutations can also disrupt the recruitment of the p160 coactivator protein GRIP1. Our investigations reveal that 30% of patients with advanced, untreated local CaP have somatic mutations that may lead to increases in AR activity. We report that somatic mutations that activate AR may lead to early relapse in ADT. Our results demonstrate that the types of ADT a CaP patient receives may cause a clustering of mutations to a particular region of the receptor. Furthermore, the mutations that arise before and during ADT do not always result in a receptor that is more active, indicating that coregulator interactions play a pivotal role in the progression of androgen-independent CaP. To improve CaP therapy, it is necessary to identify critical coregulators of AR. We screened a HeLa cell cDNA library and identified small carboxyl-terminal domain phosphatase 2 (SCP2). SCP2 is a protein phosphatase that directly interacts with the AR NTD and represses AR activity. We demonstrated that reducing the endogenous cellular levels of SCP2 causes more AR to load on to the prostate specific antigen (PSA) gene promoter and enhancer regions. Additionally, under the same conditions, more RNA polymerase II was recruited to the PSA promoter region and overall there was an increase in androgen-dependent transcription of the PSA gene, revealing that SCP2 could play a role in the pathogenesis of CaP.
  • Sarin, Peter (2010)
    Double-stranded RNA (dsRNA) viruses encode only a single protein species that contains RNA-dependent RNA polymerase (RdRP) motifs. This protein is a central component in the life cycle of a dsRNA virus, carrying out both RNA transcription and replication. The architecture of viral RdRPs resembles that of a 'cupped right hand' with fingers, palm and thumb domains. Those applying de novo initiation have additional structural features, including a flexible C-terminal domain that constitutes the priming platform. Moreover, viral RdRPs must be able to interact with the incoming 3'-terminus of the template and position it so that a productive binary complex is formed. Bacteriophage phi6 of the Cystoviridae family is to date one of the best studied dsRNA viruses. The purified recombinant phi6 RdRP is highly active in vitro and possesses both RNA replication and transcription activities. The extensive biochemical observations and the atomic level crystal structure of the phi6 RdRP provides an excellent platform for in-depth studies of RNA replication in vitro. In this thesis, targeted structure-based mutagenesis, enzymatic assays and molecular mapping of phi6 RdRP and its RNA were used to elucidate the formation of productive RNA-polymerase binary complexes. The positively charged rim of the template tunnel was shown to have a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. This work demonstrated that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the phi6 RdRP can be greatly enhanced. Furthermore, proteolyzed phi6 RdRPs that possess a nick in the polypeptide chain at the hinge region, which is part of the extended loop, were better suited for catalysis at higher temperatures whilst favouring back-primed initiation. The clipped C-terminus remains associated with the main body of the polymerase and the hinge region, although structurally disordered, is involved in the control of C-terminal domain displacement. The accumulated knowhow on bacteriophage phi6 was utilized in the development of two technologies for the production of dsRNA: (i) an in vitro system that combines the T7 RNA polymerase and the phi6 RdRP to generate dsRNA molecules of practically unlimited length, and (ii) an in vivo RNA replication system based on restricted infection with phi6 polymerase complexes in bacterial cells to produce virtually unlimited amounts of dsRNA. The pools of small interfering RNAs derived from dsRNA produced by these systems were validated and shown to efficiently decrease the expression of both exogenous and endogenous targets.
  • Gylling, Annette (Helsingin yliopisto, 2008)
    Hereditary non-polyposis colorectal carcinoma (HNPCC; Lynch syndrome) is among the most common hereditary cancers in man and a model of cancers arising through deficient DNA mismatch repair (MMR). It is inherited in a dominant manner with predisposing germline mutations in the MMR genes, mainly MLH1, MSH2, MSH6 and PMS2. Both copies of the MMR gene need to be inactivated for cancer development. Since Lynch syndrome family members are born with one defective copy of one of the MMR genes in their germline, they only need to acquire a so called second hit to inactivate the MMR gene. Hence, they usually develop cancer at an early age. MMR gene inactivation leads to accumulation of mutations particularly in short repeat tracts, known as microsatellites, causing microsatellite instability (MSI). MSI is the hallmark of Lynch syndrome tumors, but is present in approximately 15% of sporadic tumors as well. There are several possible mechanisms of somatic inactivation (i.e. the second hit ) of MMR genes, for instance deletion of the wild-type copy, leading to loss of heterozygosity (LOH), methylation of promoter regions necessary for gene transcription, or mitotic recombination or gene conversion. In the Lynch syndrome tumors carrying germline mutations in the MMR gene, LOH was found to be the most frequent mechanism of somatic inactivation in the present study. We also studied MLH1/MSH2 deletion carriers and found that somatic mutations identical to the ones in the germline occurred frequently in colorectal cancers and were also present in extracolonic Lynch syndrome-associated tumors. Chromosome-specific marker analysis implied that gene conversion, rather than mitotic recombination or deletion of the respective gene locus accounted for wild-type inactivation. Lynch syndrome patients are predisposed to certain types of cancers, the most common ones being colorectal, endometrial and gastric cancer. Gastric cancer and uroepithelial tumors of bladder and ureter were observed to be true Lynch syndrome tumors with MMR deficiency as the driving force of tumorigenesis. Brain tumors and kidney carcinoma, on the other hand, were mostly MSS, implying the possibility of alternative routes of tumor development. These results present possible implications in clinical cancer surveillance. In about one-third of families suspected of Lynch syndrome, mutations in MMR genes are not found, and we therefore looked for alternative mechanisms of predisposition. According to our results, large genomic deletions, mainly in MSH2, and germline epimutations in MLH1, together explain a significant fraction of point mutation-negative families suspected of Lynch syndrome and are associated with characteristic clinical and family features. Our findings have important implications in the diagnosis and management of Lynch syndrome families.
  • Laurikkala, Johanna (Helsingin yliopisto, 2004)
  • Biala, Agnieszka (Helsingin yliopisto, 2011)
    Hypertension is a major risk factor for stroke, ischaemic heart disease, and the development of heart failure. Hypertension-induced heart failure is usually preceded by the development of left ventricular hypertrophy (LVH), which represents an adaptive and compensatory response to the increased cardiac workload. Biomechanical stress and neurohumoral activation are the most important triggers of pathologic hypertrophy and the transition of cardiac hypertrophy to heart failure. Non-clinical and clinical studies have also revealed derangements of energy metabolism in hypertensive heart failure. The goal of this study was to investigate in experimental models the molecular mechanisms and signalling pathways involved in hypertension-induced heart failure with special emphasis on local renin-angiotensin-aldosterone system (RAAS), cardiac metabolism, and calcium sensitizers, a novel class of inotropic agents used currently in the treatment of acute decompensated heart failure. Two different animal models of hypertensive heart failure were used in the present study, i.e. hypertensive and salt-sensitive Dahl/Rapp rats on a high salt diet (a salt-sensitive model of hypertensive heart failure) and double transgenic rats (dTGR) harboring human renin and human angiotensinogen genes (a transgenic model of hypertensive heart failure with increased local RAAS activity). The influence of angiotensin II (Ang II) on cardiac substrate utilization and cardiac metabolomic profile was investigated by using gas chromatography coupled to time-of-flight mass spectrometry to detect 247 intermediary metabolites. It was found that Ang II could alter cardiac metabolomics both in normotensive and hypertensive rats in an Ang II receptor type 1 (AT1)-dependent manner. A distinct substrate use from fatty acid oxidation towards glycolysis was found in dTGR. Altered cardiac substrate utilization in dTGR was associated with mitochondrial dysfunction. Cardiac expression of the redox-sensitive metabolic sensor sirtuin1 (SIRT1) was increased in dTGR. Resveratrol supplementation prevented cardiovascular mortality and ameliorated Ang II-induced cardiac remodeling in dTGR via blood pressure-dependent pathways and mechanisms linked to increased mitochondrial biogenesis. Resveratrol dose-dependently increased SIRT1 activity in vitro. Oral levosimendan treatment was also found to improve survival and systolic function in dTGR via blood pressure-independent mechanisms, and ameliorate Ang II-induced coronary and cardiomyocyte damage. Finally, using Dahl/Rapp rats it was demonstrated that oral levosimendan as well as the AT1 receptor antagonist valsartan improved survival and prevented cardiac remodeling. The beneficial effects of levosimendan were associated with improved diastolic function without significantly improved systolic changes. These positive effects were potentiated when the drug combination was administered. In conclusion, the present study points to an important role for local RAAS in the pathophysiology of hypertension-induced heart failure as well as its involvement as a regulator of cardiac substrate utilization and mitochondrial function. Our findings suggest a therapeutic role for natural polyphenol resveratrol and calcium sensitizer, levosimendan, and the novel drug combination of valsartan and levosimendan, in prevention of hypertension-induced heart failure. The present study also provides a better understanding of the pathophysiology of hypertension-induced heart failure, and may help identify potential targets for novel therapeutic interventions.
  • Lagus, Markus (Helsingin yliopisto, 2013)
    BACKGROUND Sleep disturbances and mood alterations are highly interrelated. The majority of patients suffering from depression report a reduced sleep quality. Inversely, people with sleep complaints are at elevated risk to develop depression. The complex regulation of these phenomena involves several brain areas and mechanisms. The susceptibility to change in this system is influenced by several factors, such as age and stressful lifestyle that are considered in this study. HYPOTHESIS The hypothesis of this study was that sleep and mood share common genetic/molecular regulatory networks and that both are also regulated by epigenetic mechanisms and neural plasticity. METHODS The studies were conducted both on humans and using an animal model for depression. In the animal model we measured the genome wide expression of genes in different brain areas of clomipramine-treated pups and adults. Using these data we conducted both individual area and inter-area network analyses of basal forebrain, frontal cortex, hypothalamus and hippocampus. We also measured the amount of BDNF, one of the plasticity-related factors, in sleep restriction and under aging. In the human study we conducted epigenetic analysis of the serotonin transporter gene and related the epigenetic changes to stress in a stressful working environment. RESULTS In the models investigated changes were observed on the system, protein, transcript and transcriptional regulatory levels. Inter-tissue pathways related to synaptic transmission, regulation of translation and ubiquitinylation were disrupted. The involved pathways are within the cellular components of the axons, growth cones, melanosomes and pigment granules. The disturbed networks are centred around serotonin, Mn(II) and Rhoa. In the basal forebrain the imbalance in gene expression is widely controlled by CREB1. Some of the changes seem to be epigenetically induced by sleep deprivation and stress. Individuals working in a high stress environment have significantly less methylation in the promoter area of serotonin transporter gene SLC6A4, as compared to individuals working in a low stress environment. We also found that the expression of cortical BDNF correlated with the recovery non-REM (NREM) slow wave activity (SWA) response, and that both the cortical BDNF and the SWA response to sleep deprivation were decreased in the aged animals, as were the changes in sleep latency. CONCLUSIONS The disturbances in the models investigated, arise, largely, but not solely, due to disruption in neurological systems previously related to the regulation of sleep and mood. Novelty value could be ascribed to findings that suggest involvement of inter-tissue networks, and more precisely, imbalance of melanosome related gene expression and gene networks connected to Mn(II). The stress induced demethylation of the SLC6A4 promoter suggests a mechanism for the body to cope with prolonged excessive stress. The downside of this coping mechanism is the possibility that this reprogramming increases the long-term risk for mood disorders. The findings in the sleep deprived aging rats support the hypothesis that the age related decrease in homeostatic NREM SWA is related to a reduced sleep need.
  • Wasik, Anita Agnieszka (Helsingin yliopisto, 2014)
    Diabetic nephropathy (DN) is a major microvascular complication of diabetes and a common cause of end-stage renal disease. Mechanisms leading to the development of DN are not fully understood, but podocyte injury is involved. Interestingly, in respect to glucose uptake podocytes are uniquely insulin sensitive cells. Insulin rapidly induces remodeling of the actin cytoskeleton and leads to glucose uptake via glucose transporters GLUT1 and GLUT4. Defects in the trafficking of the glucose transporters may affect the insulin sensitivity of podocytes. Thus, regulators of glucose transporter trafficking may provide suitable targets to enhance insulin sensitivity of podocytes and prevent the development and progression of DN. However, the precise mechanisms regulating glucose transporter trafficking and glucose uptake into podocytes are largely uncharacterized. To identify changes in the expression of glomerular proteins at an early stage of DN we performed quantitative proteomic profiling of glomeruli isolated from rats with streptozotocin-induced diabetes and controls. Ezrin was found to be downregulated in diabetic glomeruli. In cultured podocytes depletion of ezrin increased glucose uptake by increasing translocation of GLUT1 to the plasma membrane. Loss of ezrin also induced actin remodelling, which involved cofilin-1. Phosphorylated cofilin-1 was upregulated in diabetic glomeruli suggesting altered actin dynamics. Furthermore, reduced expression of ezrin was found in the podocytes of human patients with diabetes. We found that the filament-forming septin 7 forms a complex with CD2AP and nephrin, both of which are essential for glomerular ultrafiltration. We showed that septin 7 negatively regulates GLUT4 storage vesicle (GSV) trafficking by forming a physical barrier between the vesicles and the plasma membrane. The novel interaction partner of septin 7, nonmuscle myosin heavy chain IIA (NMHC-IIA), was found to positively regulate insulin-stimulated glucose uptake into podocytes. Loss of NMHC-IIA reduced formation of the SNARE complex involved in GSV exocytosis. Furthermore, we presented that insulin regulates the association of septin 7 and phosphorylated RLC (pp-RLC), a part of myosin hexameric complex, with a plasma membrane SNARE, SNAP23. pp-RLC is upregulated in diabetic glomeruli and cultured human podocytes exposed to macroalbuminuric sera from patients with Type 1 diabetes. Our findings indicate that ezrin, septin 7 and NMHC-IIA regulate glucose uptake into podocytes and may play a role in the development of the renal complication in diabetes by regulating glucose transport and organization of the actin cytoskeleton in podocytes.
  • Nuotio, Krista (Helsingin yliopisto, 2007)
    Carotid artery disease is the most prevalent etiologic precursor of ischemic stroke, which is a major health hazard and the second most common cause of death in the world. If a patient presents with a symptomatic high-grade (>70%) stenosis in the internal carotid artery, the treatment of choice is carotid endarterectomy. However, the natural course of radiologically equivalent carotid lesions may be clinically quite diverse, and the reason for that is unknown. It would be of utmost importance to develop molecular markers that predict the symptomatic phenotype of an atherosclerotic carotid plaque (CP) and help to differentiate vulnerable lesions from stable ones. The aim of this study was to investigate the morphologic and molecular factors that associate with stroke-prone CPs. In addition to immunohistochemistry, DNA microarrays were utilized to identify molecular markers that would differentiate between symptomatic and asymptomatic CPs. Endothelial adhesion molecule expression (ICAM-1, VCAM-1, P-selectin, and E-selectin) did not differ between symptomatic and asymptomatic patients. Denudation of endothelial cells was associated with symptom-generating carotid lesions, but in studies on the mechanism of decay of endothelial cells, markers of apoptosis (TUNEL, activated caspase 3) were found to be decreased in the endothelium of symptomatic lesions. Furthermore, markers of endothelial apoptosis were directly associated with those of cell proliferation (Ki-67) in all plaques. FasL expression was significantly increased on the endothelium of symptomatic CPs. DNA microarray analysis revealed prominent induction of specific genes in symptomatic CPs, including those subserving iron and heme metabolism, namely HO-1, and hemoglobin scavenger receptor CD163. HO-1 and CD163 proteins were also increased in symptomatic CPs and associated with intraplaque iron deposits, which, however, did not correlate with symptom status itself. ADRP, the gene for adipophilin, was also overexpressed in symptomatic CPs. Adipophilin expression was markedly increased in ulcerated CPs and colocalized with extravasated red blood cells and cholesterol crystals. Taken together, the phenotypic characteristics and the numerous possible molecular mediators of the destabilization of carotid plaques provide potential platforms for future research. The denudation of the endothelial lining observed in symptomatic CPs may lead to direct thromboembolism and maintain harmful oxidative and inflammatory processes, predispose to plaque microhemorrhages, and contribute to lipid accumulation into the plaque, thereby making it vulnerable to rupture.
  • Vainio, Anni (Helsingin yliopisto, 2012)
    Methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae are major health problems worldwide, both found in symptomless carriage but also causing even life-threatening infections. The aim of this thesis was to characterise MRSA and S. pneumoniae in detail by using several molecular typing methods for various epidemiological purposes: clonality analysis, epidemiological surveillance, outbreak investigation, and virulence factor analysis. The characteristics of MRSA isolates from the strain collection of the Finnish National Infectious Disease Register (NIDR) and pneumococcal isolates collected from military recruits and children with acute otitis media (AOM) were analysed using various typing techniques. Antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), spa typing, staphylococcal cassette chromosome mec (SCCmec) typing, and the detection of Panton-Valentine leukocidin (PVL) genes were performed for MRSA isolates. Pneumococcal isolates were analysed using antimicrobial susceptibility testing, serotyping, MLST, and by detecting pilus islet 1 (PI-1) and 2 (PI-2) genes. Several international community- and hospital-associated MRSA clones were recognised in Finland. The genetic diversity among MRSA FIN-4 isolates and among FIN-16 isolates was low. Overall, MRSA blood isolates from 1997 to 2006 were genetically diverse. spa typing was found to be a highly discriminatory, rapid and accurate typing method and it also qualifies as the primary typing method in countries with a long history of PFGE-based MRSA strain nomenclature. However, additional typing by another method, e.g. PFGE, is needed in certain situations to be able to provide adequate discrimination for epidemiological surveillance and outbreak investigation. An outbreak of pneumonia was associated with one pneumococcal strain among military recruits, previously healthy young men living in a crowded setting. The pneumococcal carriage rate after the outbreak was found to be exceptionally high. PI-1 genes were detected at a rather low prevalence among pneumococcal isolates from children with AOM. However, the study demonstrated that PI-1 has existed among pneumococcal isolates prior to pneumococcal conjugate vaccine and the increased antimicrobial resistance era. Moreover, PI-1 was found to associate with the serotype rather than the genotype. This study adds to our understanding of the molecular epidemiology of MRSA strains in Finland and the importance of an appropriate genotyping method to be able to perform high-level laboratory-based surveillance of MRSA. Epidemiological and molecular analyses of S. pneumoniae add to our knowledge of the characteristics of pneumococcal strains in Finland.