Browsing by discipline "Muu"

Anxiety disorders are a group of psychiatric disorders characterized by excessive fear and anxiety that disrupts normal life. They are very common, having an estimated life-time prevalence of 16.6 %, and they start often at an early age. Anxiety disorders have a clear genetic component, but the onset is also largely affected by the environment. Although several brain regions and neurotransmitter systems have been shown to take part in the regulation of anxiety, the exact pathophysiological mechanisms behind anxiety disorders are largely unknown. Mouse models are a good tool when studying the genetic component behind anxiety disorders. Different behavioral tests measuring the anxiety-like behavior of mice exist, and these tests can be used to model certain aspects of pathological anxiety. Individual gene effects on anxiety can be studied by using transgenic mouse models, which was done in this study, where a knockout mouse model lacking the functional version of glutathione reductase (Gsr) was used to study the effect of Gsr in anxiety. Because of the reduced viability of the homozygotic knockout mice, our study was done using heterozygotic knockout mice, which have 64 % of normal glutathione reductase activity. Glutathione is one of the most important endogenous antioxidants, which protects cells from oxidative stress by getting oxidized by reactive oxygen species. Glutathione reductase takes part in this process by transforming glutathione back to its functional reduced form. Gsr expression in amygdala and cingulate cortex has been previously shown to correlate with anxiety-like behavior in mice such that mice with upregulated Gsr expression were more anxious. This and other results obtained from mice and human studies suggest that oxidative stress may be involved in the pathogenesis of anxiety. In this study we wanted to investigate, whether the heterozygous Gsr knockout mice have lower anxiety-like behavior than the wild-type mice and whether there is more oxidative damage present in the brain of heterozygous Gsr knockout mice compared to the wild-type mice. To assess the first question, we set up and validated two behavioral tests (novelty-induced hypophagia and marble burying) and tested the Gsr mice using these tests. To assess the second question, we used nitrotyrosine as a marker for oxidative damage and measured the amount of this marker from brain tissue samples of Gsr mice by a nitrotyrosine immunoblotting assay. We discovered that both our novelty-induced hypophagia and marble burying test settings were able to detect some behavioral differences between inbred mouse strains, but were not optimal in distinguishing highly anxious strains from less anxious strains. When testing the Gsr mice, no behavioral difference between the heterozygous knockout and wild-type mice was observed, but a significant difference in the behavior between sexes was detected. We obtained preliminary results showing that the nitrotyrosine levels are slightly increased in the cortex of male heterozygous Gsr knockout mice. Whether this is the case also in other brain regions and in female mice still needs to be tested. These results and previous results obtained by our group suggest that heterozygous Gsr knockout mice do not have a striking anxiety phenotype under baseline conditions, but might have slightly reduced anxiety-like behavior and slightly increased brain oxidative stress status when compared to the wild-type mice.

Recently, our research group together with the Hematology Research Unit Helsinki, found that 31 of 77 patients (40%) with T-cell large granular lymphocytic leukemia have somatic point mutations in the Src Homology 2 (SH2) domain of the STAT3 gene. LGL leukemia is a rare and indolent disease characterized by the clonal expansion of large granular lymphocytes of unknown etiology. The aim of this master's thesis study was to elucidate whether the identified Y640F and D661V mutations can cause hyperactivity of the STAT3 protein and excessive proliferation of T-cells. STAT3 is a transcription factor that is known to have a key role in cell proliferation and apoptosis, and which is activated by the phosphorylation of receptor-associated kinases. The phosphorylation of tyrosine residue 705 in STAT3 induces dimerization and localization of the STAT3 dimer to the nucleus. In several cancers STAT3 protein has been reported to be constitutively active. Furthermore, previously published data strongly support our hypothesis that the mutations identified in LGL patients might cause STAT3 to be hyperactive resulting in inhibition of apoptotic pathways in cytotoxic T cells. In order to evaluate the function of the novel mutations, expression constructs of STAT3 containing the D661V and Y640F mutations were generated. In addition, lentiviral vectors were produced to establish a T-cell line (Jurkat) with stable expression of mutant STAT3. After the successful generation of STAT3 constructs and cell line models, several functional assays were performed. Transcriptional activity of STAT3 was measured by luciferase reporter assay and immunocytochemistry was used to determine whether the mutations promote nuclear localization of STAT3. STAT3 phosphorylation was examined by immunoblotting. In addition, quantitative RT-PCR was used to detect differential expression of five STAT3 target genes from patient samples. The results, particularly the luciferase reporter assay, indicated a significant difference between the mutant and wild type STAT3 providing strong evidence that the STAT3 mutants are transcriptionally more active. The localization assay, imaged by fluorescence microscopy, showed more STAT3 D661V and Y640F protein present in the nucleus when compared to wild type STAT3. However, the proliferation rate of mutant STAT3 expressing Jurkat cells was not increased. In addition, STAT3 target gene expression levels of two patient samples did not show large differences when compared to healthy LGL cells. As a result of these findings, it can be strongly hypothesized that aberrant STAT3 signaling underlies the cause of T-cell LGL leukemia. Understanding the molecular basis of LGL leukemia is important in order to develop diagnostic and therapeutic strategies for patients suffering from the disease. Since constitutively active STAT3 is common amongst many cancers and autoimmune disorders, activating mutations could possibly be found in these diseases. More careful sequencing studies of STAT3 upstream molecules are warranted as well, and will be performed in the future from leukemic LGL samples.

Influenza viruses are a group of pathogens in the family Orthomyoviridae, which are classified into 6 genera (A,B,C, Thogotovirus, Isavirus and new unnamed genus). Type A influenza viruses are categorized based on their surface glycoproteins: hemagglutinin (HA) and neuraminidase (NA). So far 17 HA and 9 NA subtypes have been identified. Influenza genome comprises of eight single-stranded negative-sense RNA segments that encode ten to twelve proteins (HA, NA, NP M1/M2, NS1/NS2, PA, PB1 and PB2). Influenza replication cycle depends on the surface proteins binding to host cell receptors, pH mediated fusion and cell-mediated transcription and replication of the viral genome. Virus particles leave the host cell via budding. Influenza viruses cause global epidemic infections each year, the peak is from December to March. These pathogens have also contributed to six global pandemics identified so far. The latest pandemic outbreak was announced by WHO in 2009 which caused over 5000 hospitalizations in Finland. Factors contributing to the severity of clinical outcome can be either genetic, environmental or caused by human host features. This study aims to identify the susceptibility factors for severe influenza A infections and describe the phylodynamics of the latest pandemic A(H1N1)pdm09 from Finnish patient nasopharyngeal aspirates collected between 2009-2013. One-step reverse-transcription PCR was used to amplify all of the 8 segments equally. Fast and precise next-generation sequencing with Illumina 2000 sequencer was used to generate the sequences. Results were bioinformatically analysed using Bayesian modelling with Markov Chain Monte Carlo algorithms of probability distributions. Models analysed showed highest mutation rate in hemagglutinin protein. Phylodynamic analysis revealed higher mutation rate of HA and NA compared to other proteins. Subgroup specific polymorphisms (either in severe or mild cases) were not identified. In total 4657 amino acid substitutions were located in 135 pandemic A(H1N1)pdm09 patient isolates and 238 in 10 seasonal patient samples. Viral HA, NA and PB2 were more frequently mutated than other proteins. Interestingly this study identified double-resistant markers (E119K and S31N) to two antiviral drugs (amantadine and oseltamivir) in one patient isolate (A/Helsinki/598/2013). Previously reported D222 polymorphism (without the signalling peptide)causing more severe clinical outcome was not identified in any of the patient isolates in this study.

Miten aktiivisuusmittarin tulos korreloi kuuden minuutin kävelytestin tulokseen keuhkoahtaumatautipotilailla? Miten keuhkoahtaumatautipotilaiden omatoimista liikunnallista kuntoutusta pystyttäisiin tukemaan entistä paremmin? Tämän tutkimuksen tavoitteena on luoda tutkittuun tietoon perustuva malli keuhkoahtaumatautipotilaiden omatoimisen kuntoutuksen ohjaamiseen erikoissairaanhoidon konsultaationa. Vaasan keskussairaalassa järjestetyssä keuhkoahtaumataudin omahoitopäivässä kerättiin kyselylomakkeilla tietoa potilaiden nykyisistä elämäntavoista ja uskomuksista, kuinka he voivat elämäntavoillaan vaikuttaa keuhkoahtaumataudin ennusteeseen. Omahoitopäivässä annetun tiedon ja sen jälkeisen motivoivan keskustelun jälkeen selvitettiin potilaiden elämäntapamuutosaikomuksia. Potilaat osoittivat halukkuutensa tupakoinnin lopettamiseen, liikunnan lisäämiseen ja ruokailutottumustensa muutokseen lisääntyneen intervention myötä. Keuhkoahtaumatautipotilaille tarjotaan mahdollisuutta osallistua ryhmämuotoiseen elämäntapamuutosvalmennukseen yksilöllisen liikunnallisen omahoidon ohjauksen rinnalla. Kehonkoostumusmittari-, aktiivisuusmittari ja kuuden minuutin kävelytesti on todettu hyödyllisiksi työkaluiksi potilaiden ohjauksessa. Jatkossa potilasohjauksessa on kiinnitettävä huomiota riittävään ja monipuoliseen ravintoon, lihaskuntoharjoitusten tekemiseen sekä pahenemisvaiheiden tehostettuun hoitoon. Aktiivisuus- ja kehonkoostumusmittareita tullaan käyttämään kuuden minuutin kävelytestin rinnalla keuhkoahtaumatautipotilaiden motivoimisessa omatoimiseen liikunnalliseen kuntoutukseen.

Sirtuin-1 (Sirt-1), an NAD+-dependent deacetylase, maintains energy homeostasis upon stress-induced decline in energy levels. Sirt-1 possesses many other functions ranging from regulating cellular proliferation and apoptosis to maintaining glucose and lipid metabolism. By deacetylating its target proteins, Sirt-1 can also increase the lifespan of lower organisms such as yeast, flies, and worms. However, lifespan-prolonging ability of Sirt-1 in rodent models and human subjects has been investigated, exhibiting only partially promising results. Contrary to its role in increasing lifespan in rodents and humans, Sirt-1 has exhibited positive results on prolonging the health-span of these model organisms by delaying or inhibiting the development of diseases and disorders that present themselves with increasing age. Drug mediated Sirt-1 activation via resveratrol and Sirt-1 activation through calorie restriction were shown to bring a healthy balance to glucose and lipid metabolism especially in rodents fed on high-fat diet. Currently, Sirt-1 is investigated as a target gene for the treatment of Type-2 Diabetes in multiple clinical trials. In this study, we investigated the role of the Sirtuin-1 in the maintenance of intestinal epithelium. In our study, we utilized intestinal organoid models grown from isolated crypts of mouse small intestine. We performed knockdown of Sirt-1 at mRNA level via lentiviral shRNA. Additionally, we targeted Sirt-1 activity by applying resveratrol to the culture media of mouse intestinal organoids. Our main aim was to address how manipulations of Sirt-1 expression or activity would affect the function of intestinal stem cells and Paneth cells located in the crypt tips of intestinal organoids. Furthermore, we were interested in how these changes would alter organoid viability and crypt regeneration. In this study, we demonstrate that Sirt-1 is a fundamental gene for mouse intestinal organoid viability as well as organoid crypt regeneration. We also show that Sirt-1 controls crypt regeneration by regulating Paneth cell differentiation and thus intestinal stem cell niche conditions in the crypt bottoms of mouse intestinal organoids. Activation of Sirt-1 by resveratrol decreases total Paneth cell number in a crypt bottom leading to the relative retardation of crypt regeneration as well as the reduction in crypt size. High concentrations of resveratrol resulted in death of intestinal organoids, probably due to complete loss of Paneth cells.