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  • Priyadarshini, Madhusmita (Helsingin yliopisto, 2013)
    Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease (AD). The quest for better therapies to modify the progression of PD is still ongoing. During the last two decades, the concept of the etiological basis of PD has changed, which has been driven by genetics, the recognition of familial forms, knowledge of the effects of the environment and toxins, and genome-wide association studies. Although most cases are sporadic, approximately 5-10% of PD cases are due to genetic mutations that give rise to the familial forms. Studies using neurotoxins and also genetic mutations that underlie familial PD have implicated mitochondrial dysfunction in the pathogenesis of PD. Among the different genes associated with familial PD, PTEN-induced putative kinase1 (Pink1), responsible for the autosomal recessive type, is strongly linked to the mitochondria. To investigate in depth the underlying mechanisms of Pink1, we inhibited the function of pink1 in zebrafish using morpholino oligonucleotides (MOs). The MO was first thoroughly characterized with all necessary control experiments to avoid unspecific effects. Since the dopaminergic system is affected in PD, a marker for dopamine, tyrosine hydroxylase (TH), was used to assess damage to the system. Due to a genome duplication event that occurred early in the evolution of teleosts after the divergence from the mammals, two TH non-allelic isoforms were identified in zebrafish: th1 and th2. In the pink1 morphants, both the TH gene isoforms were altered. With in situ hybridization, the loss of th1 was found in the ventral diencephalon (dopaminergic cell groups 5, 6, 11) and th2 was reduced in the caudal hypothalamus (cell group 10b). Similar results were obtained with the cell counting method for TH1 immunoreactive cells. TH-ir indicated the loss of cells in the pretectum (group 7) and the ventral diencephalic cluster represented by cell groups 5,6,11. These pink1 morphants were exposed to subeffective doses of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). This amalgamation of the toxin and genetic manipulation caused a locomotor deficit and also facilitated the loss of TH-ir in the same cell populations in the larval brains as was instigated by pink1 knockdown alone. To investigate the involvement of pink1 in cell damage, we used a two-color gene expression-based microarray and identified a number of genes that were potentially involved in the pathogenic mechanism of the disease. After successful data analysis, the changes in critical genes were successfully validated by quantitative real-time PCR. The gene expression changes in the morphants, identified by the microarray, were rescued by pink1 mRNA injections, suggesting the specific involvement of pink1 in the differentially expressed gene regulation. One of the significant findings was HIF signaling, an important pathway affected by pink1 knockdown. Individual factors and genes in the same pathway were validated by independent methods in the pink1 morphants to reveal whether pink1 affected hif1α or the cascade of events in the signaling pathway. Changes in the VEGF transcripts, erythropoiesis, and reactive oxygen species were observed, as were other antioxidant system genes, including cat and sod2. These pathways may provide new targets for drug development in PD. To study the mechanisms underlying the involvement of pink1 in oxidative stress-mediated PD pathology using zebrafish as a tool, we generated a transgenic line, Tg(pink1:EGFP). The Tol2 transgenic approach was used to generate Tg(pink1:EGFP) by using the zebrafish pink1 promoter. Expression of the pink1 transgene was detected in the telencephalon, midbrain, and rhombencephalon in the CNS, and in the muscle, heart, and liver among the peripheral organs. The transgenic fish line was used to study the effect of oxidative stress. When subjected to a low concentration of hydrogen peroxide (H2O2), which had no effect on the mortality or phenotype of the fish, the transgenic fish showed an increase in pink1 transgene activity in the brain of the larval zebrafish. Oxidative stress-mediated changes in TH expression are valuable for PD study. H2O2 administration did not affect the th1 transcript levels, but it significantly increased pink1 expression and reduced the th2 transcript levels. This transgenic model will be highly useful for drug development and the screening of new potential therapeutic approaches as an in vivo model.
  • Korostenskaja, Milena (Helsingin yliopisto, 2008)
    Cognitive impairments of attention, memory and executive functions are a fundamental feature of the pathophysiology of schizophrenia. The neurophysiological and neurochemical changes in the auditory cortex are shown to underlie cognitive impairmentsin schizophrenia patients. Functional state of the neural substrate of auditory information processing could be objectively and non-invasively probed with auditory event-related potentials (ERPs) and event- related fields (ERFs). In the current work, we explored the neurochemical effect on the neural origins of auditory information processing in relation to schizophrenia. By means of ERPs/ERFs we aimed to determine how neural substrates of auditory information processing are modulated by antipsychotic medication in schizophrenia spectrum patients (Studies I, II) and by neuropharmacological challenges in healthy human subjects (Studies III, IV). First, with auditory ERPs we investigated the effects of olanzapine (Study I) and risperidone (Study II) in a group of patients with schizophrenia spectrum disorders. After 2 and 4 weeks of treatment, olanzapine has no significant effects on mismatch negativity(MMN) and P300, which, as it has been suggested, respectively reflect preattentive and attention-dependent information processing. After 2 weeks of treatment, risperidone has no significant effect on P300, however risperidone reduces P200 amplitude. This latter effect of risperidone on neural resources responsible for P200 generation could be partly explained through the action of dopamine. Subsequently, we used simultaneous EEG/MEG to investigate the effects of memantine (Study III) and methylphenidate (Study IV) in healthy subjects. We found that memantine modulates MMN response without changing other ERP components. This could be interpreted as being due to the possible influence of memantine through the NMDA receptors on auditory change- detection mechanism, with processing of auditory stimuli remaining otherwise unchanged. Further, we found that methylphenidate does not modulate the MMN response. This finding could indicate no association between catecholaminergic activities and electrophysiological measures of preattentive auditory discrimination processes reflected in the MMN. However, methylphenidate decreases the P200 amplitudes. This could be interpreted as a modulation of auditory information processing reflected in P200 by dopaminergic and noradrenergic systems. Taken together, our set of studies indicates a complex pattern of neurochemical influences produced by the antipsychotic drugs in the neural substrate of auditory information processing in patients with schizophrenia spectrum disorders and by the pharmacological challenges in healthy subjects studied with ERPs and ERFs.
  • Riekki, Tapani (Helsingin yliopisto, 2014)
    The present thesis consists of six studies that investigate different cognitive factors that contribute to believing and unbelieving in paranormal, superstitious, magical, and supernatural (commonly referred to as paranormal beliefs). Earlier studies have found several factors reaching from personality factors to cognitive factors to cultural factors that contribute to believing. However, the research has neglected the important factor of what sets paranormal beliefs apart from other beliefs. In addition, although we know a lot about demographical and personality features that contribute to differences in paranormal beliefs, neuro-cognitive differences are still not well known or empirically tested. One explanation that takes into account the difference between paranormal beliefs and other beliefs is that paranormal beliefs stem from core knowledge confusions about the ontological properties of mental, physical, and biological phenomena. The first study of the thesis tried to gain insight into the neural basis of core knowledge confusions in an event-related electroencephalography study. The next two studies tested the possibility that cognitive inhibition, the ability to flexibly switch between thinking modes and if needed, to inhibit unwanted or irrelevant thoughts, could contribute to believing and unbelieving. In these two studies group differences between paranormal believers and skeptics were first compared by using tests of cognitive inhibition and secondly by using brain imaging. Brain imaging was done during a task that invoked paranormal interpretations. The last three studies of the thesis examined the role of the social information processing differences between paranormal believers and skeptics. Methods included brain imagining, behavioral experiments, and self-report measurements. In the first study, we tested how conceptions about the mind are related to beliefs and core knowledge confusions. In the second and third of these studies, we tested group differences between paranormal believers and skeptics by using brain imaging and a behavioral test. We investigated if the groups differ in attribution of intentions to randomly moving objects and in tendency for illusory face perception. The results suggest that core knowledge confusions are based on intuitive world knowledge and that this intuitive world knowledge is less categorized among paranormal believers than among skeptics. Cognitive inhibition was also found to contribute to paranormal beliefs: strong cognitive inhibition downplays paranormal beliefs. Social information processing was connected to paranormal beliefs in several ways. First, understanding mind and its properties in a Cartesian dualistic way was associated with paranormal beliefs and ontological confusions preceded them. Second, believers when compared to skeptics assigned more intentions to randomly moving objects. This was associated with activation of the mentalizing system at the brain level. Finally, we found out that paranormal believers were more prone to illusory face perception than skeptics were. The results underline that if one seeks to understand believing and especially unbelieving, which both are complex phenomena, individual differences in cognitive processing must be taken into account.
  • Leminen, Alina (Helsingin yliopisto, 2012)
    The representation of morphologically complex words in the mental lexicon and their neurocognitive processing has been a vigorously debated topic in psycholinguistics and the cognitive neuroscience of language. This thesis investigates the effect of stimulus modality on morphological processing, the spatiotemporal dynamics of the neural processing of inflected (e.g., work+ed ) and derived (e.g., work+er ) words and their interaction, using the Finnish language. Overall, the results suggest that the constituent morphemes of isolated written and spoken inflected words are accessed separately, whereas spoken derived words activate both their full form and the constituent morphemes. The processing of both spoken and written inflected words elicited larger N400 responses than monomorphemic words (Study I), whereas the responses to spoken derived words did not differ from those to monomorphemic words (Study IV). Spoken inflected words elicited a larger left-lateralized negativity and greater source strengths in the left temporal cortices than derived words (Study IV). Thus, the results suggest different cortical processing for derived and inflected words. Moreover, the neural mechanisms underlying inflection and derivation seem to be not only different, but also independent as indexed by the linear summation of the responses to derived and inflected stimuli in a combined (derivation+inflection) condition (Study III). Furthermore, the processing of meaningless, spoken derived pseudowords was more difficult than for existing derived words, indexed by a larger N400-type effect for the pseudowords. However, no differences were observed between meaningful derived pseudowords and existing derived words (Study II). The results of Study II suggest that semantic compatibility between morphemes seems to have a crucial role in a successful morphological analysis. As a methodological note, time-locking the auditory event-related potentials/fields (ERP/ERF) to the suffix onset revealed the processes related to morphological analysis more precisely (Studies II and IV), which also enables comparison of the neural processes in different modalities (Study I).
  • Sarajuuri, Anne (Unigrafia Oy, 2012)
    Hypoplastic left heart syndrome (HLHS) and other forms of functionally univentricular heart (UVH) are complex congenital heart defects (CHD) that during the past few decades have gained viability with staged palliative surgery. It results in the passive return of systemic venous blood to the lungs without passing through the heart. The aim of this study was to evaluate the neurodevelopmental outcome of children with HLHS and UVH in Finland with neurological, neuropsychological, and motor assessments and brain imaging, and to detect possible risk factors associated with an adverse outcome. Parental perceptions of child behavior and parenting stress were also assessed with parental question-naires. The first study cohort of patients born between 1995 and 1999, which included most of the first HLHS survivors in Finland, showed a significantly lower cognitive outcome compared to normative data among both patients with HLHS (n = 7) and with UVH (n = 19). Cerebral palsy was present in 14% of the patients with HLHS and in 10% of those with UVH, and milder difficulties in gross and fine motor functions were also commonly found. Patients with HLHS or UVH born between 2002 and 2005 were recruited in a prospective neurodevelopmental follow-up study as newborns. The neurodevelopmental evaluations at the ages of 12 months, 30 months, and 5 years revealed a significantly lower cognitive and motor outcome among the patients with HLHS (n = 23) compared to healthy age- and gender-matched controls (n = 40). The patients with UVH (n = 14) only demonstrated a statistically significant difference to controls in motor development during the earlier assessments, and at the age of 5 years also in cognitive development. A smaller sample size may have contributed to the lack of statistically significant difference in cognitive development in the earlier assessments. Major neurodevelopmental impairment was found in 26% of the patients with HLHS, and 23% of the patients with UVH at the age of 5 years. Brain magnetic resonance imaging revealed abnormalities in 82% of the patients with HLHS and 56% of those with UVH most commonly infarcts or ischemic findings of different degrees. Other than minimal ischemic findings were significantly associated with neurodevelopmental outcome. The level of parenting stress was significantly higher among the patients with HLHS compared to controls at the age of 18 months. The parents of the patients with UVH, however, did not report more stress than those of the controls. In conclusion, neurodevelopmental deficits remain a major concern among patients with UVH, and especially among those with HLHS. Neurodevelopmental follow-up is recommended for this seriously ill patient group and psychosocial support for their parents.
  • Anders, Eriksson-Palojärvi (Helsingin yliopisto, 2013)
    Myxomatous mitral valve disease causing mitral regurgitation (MR) is a common cause of heart failure in dogs. However, many aspects of pathophysiology affecting diagnostic measurements are poorly defined. The objective of this study was to add to the knowledge of different pathophysiological processes affecting measures used. Focus was put on plasma parameters, including N-terminal pro A-type natriuretic peptide (NT-proANP) and nitric oxide (NO), and first pass radionuclide angiocardiography to evaluate heart pump function and possible right sided heart enlargement associated with pulmonary hypertension. Echocardiography and thoracic radiographs were used as reference methods. Results. In normal dogs both NT-proANP (P=0.002) and NO (P=0.01) increased with age. Plasma NT-proANP concentrations for dogs under 5.9 years of age were lower than for dogs older than 5.9 years (inter quartile range 190 270 pmol/l vs. 307 530, respectively), with no overlap between groups. This discrimination was not seen for NO. In healthy dogs heart rate normalized blood pulmonary transit time (nPTT) was 4.4±0.6, dogs with asymptomatic MR 6.3±1.6, and dogs with CHF 11.8±3.4 (P < 0.001). The size of the right heart chambers increased only late in MR. Pulmonary blood volume (PBV) was associated with nPTT (R2=0.85, P < 0.0001) but not with forward stroke volume. Increase in PBV appeared late in the phase before CHF. The hazard ratio for NT-proANP was 1.21 (per 100 pmol/l; P < 0.0002). The median time to failure was 11 months for dogs with NT-proANP concentrations >1000 pmol/l and 54 months for dogs with concentrations ≤1000 pmol/l (P < 0.0001). Dogs that developed CHF had a lower mean plasma level of NO than dogs not reaching CHF (mean 23 vs. 28 µmol/l (as nitrite), P=0.016). Increased heart rate (>130 beats/min, P < 0.001) and heart murmur (3-6/6 vs. 1-2/6, P < 0.001) increased risk. Conclusions. Specific normal values for natriuretic peptides should be established for different age groups of dogs. Heart rate, murmur and NT-proANP can be used to predict risk of and time to heart failure in dogs with MR. Heart rate normalized PTT (nPTT) is a robust measure of heart pump function in MR. Both nPTT and pulmonary blood volume increase before onset of CHF. Apparent right-sided heart enlargement on radiographs is due to them being displaced by left heart chambers as they enlarge only in severe MR.
  • Pischik, Elena (Helsingin yliopisto, 2006)
    Acute intermittent porphyria (AIP, MIM #176000) is an inherited metabolic disease due to a partial deficiency of the third enzyme, hydroxymethylbilane synthase (HMBS, EC: 4.3.1.8), in the haem biosynthesis. Neurological symptoms during an acute attack, which is the major manifestation of AIP, are variable and relatively rare, but may endanger a patient's life. In the present study, 12 Russian and two Finnish AIP patients with severe neurological manifestations during an acute attack were studied prospectively from 1995 to 2006. Autonomic neuropathy manifested as abdominal pain (88%), tachycardia (94%), hypertension (75%) and constipation (88%). The most common neurological sign was acute motor peripheral neuropathy (PNP, 81%) often associated with neuropathic sensory loss (54%) and CNS involvement (85%). Despite heterogeneity of the neurological manifestations in our patients with acute porphyria, the major pattern of PNP associated with abdominal pain, dysautonomia, CNS involvement and mild hepatopathy could be demonstrated. If more strict inclusion criteria for biochemical abnormalities (>10-fold increase in excretion of urinary PBG) are applied, neurological manifestations in an acute attack are probably more homogeneous than described previously, which suggests that some of the neurological patients described previously may not have acute porphyria but rather secondary porphyrinuria. Screening for acute porphyria using urinary PBG is useful in a selected group of neurological patients with acute PNP or encephalopathy and seizures associated with pain and dysautonomia. Clinical manifestations and the outcome of acute attacks were used as a basis for developing a 30-score scale of the severity of an acute attack. This scale can easily be used in clinical practice and to standardise the outcome of an attack. Degree of muscle weakness scored by MRC, prolonged mechanical ventilation, bulbar paralysis, impairment of consciousness and hyponatraemia were important signs of a poor prognosis. Arrhythmia was less important and autonomic dysfunction, severity of pain and mental symptoms did not affect the outcome. The delay in the diagnosis and repeated administrations of precipitating factors were the main cause of proceeding of an acute attack into pareses and severe CNS involvement and a fatal outcome in two patients. Nerve conduction studies and needle EMG were performed in eleven AIP patients during an acute attack and/or in remission. Nine patients had severe PNP and two patients had an acute encephalopathy but no clinically evident PNP. In addition to axonopathy, features suggestive of demyelination could be demonstrated in patients with severe PNP during an acute attack. PNP with a moderate muscle weakness was mainly pure axonal. Sensory involvement was common in acute PNP and could be subclinical. Decreased conduction velocities with normal amplitudes of evoked potentials during acute attacks with no clinically evident PNP indicated subclinical polyneuropathy. Reversible symmetrical lesions comparable with posterior reversible encephalopathy syndrome (PRES) were revealed in two patients' brain CT or MRI during an acute attack. In other five patients brain MRI during or soon after the symptoms was normal. The frequency of reversible brain oedema in AIP is probably under-estimated since it may be short-lasting and often indistinguishable on CT or MRI. In the present study, nine different mutations were identified in the HMBS gene in 11 unrelated Russian AIP patients from North Western Russia and their 32 relatives. AIP was diagnosed in nine symptom-free relatives. The majority of the mutations were family-specific and confirmed allelic heterogeneity also among Russian AIP patients. Three mutations, c.825+5G>C, c.825+3_825+6del and c.770T>C, were novel. Six mutations, c.77G>A (p.R26H), c.517C>T (p.R173W), c.583C>T (p.R195C), c.673C>T (p.R225X), c.739T>C (p.C247R) and c.748G>C (p.E250A), have previously been identified in AIP patients from Western and other Eastern European populations. The effects of novel mutations were studied by amplification and sequencing of the reverse-transcribed total RNA obtained from the patients' lymphoblastoid or fibroblast cell lines. The mutations c.825+5G>C and c.770T>C resulted in varyable amounts of abnormal transcripts, r.822_825del (p.C275fsX2) and [r.770u>c, r.652_771del, r.613_771del (p.L257P, p.G218_L257del, p.I205_L257del)]. All mutations demonstrated low residual activities (0.1-1.3 %) when expressed in COS-1 cells confirming the causality of the mutations and the enzymatic defect of the disease. The clinical outcome, prognosis and correlation between the HMBS genotype and phenotype were studied in 143 Finnish and Russian AIP patients with ten mutations (c.33G>T, c.97delA, InsAlu333, p.R149X, p.R167W, p.R173W, p.R173Q, p.R225G, p.R225X, c.1073delA) and more than six patients in each group. The patients were selected from the pool of 287 Finnish AIP patients presented in a Finnish Porphyria Register (1966-2003) and 23 Russian AIP patients (diagnosed 1995-2003). Patients with the p.R167W and p.R225G mutations showed lower penetrance (19% and 11%) and the recurrence rate (33% and 0%) in comparison to the patients with other mutations (range 36 to 67% and 0 to 66%, respectively), as well as milder biochemical abnormalities [urinary porphobilinogen 47±10 vs. 163±21 mol/L, p<0.001; uroporphyrin 130±40 vs. 942±183 nmol/L, p<0.001] suggesting a milder form of AIP in these patients. Erythrocyte HMBS activity did not correlate with the porphobilinogen excretion in remission or the clinical of the disease. In all AIP severity patients, normal PBG excretion predicted freedom from acute attacks. Urinary PBG excretion together with gender, age at the time of diagnosis and mutation type could predict the likelihood of acute attacks in AIP patients.
  • Nevalainen, Päivi (Helsingin yliopisto, 2010)
    Until recently, objective investigation of the functional development of the human brain in vivo was challenged by the lack of noninvasive research methods. Consequently, fairly little is known about cortical processing of sensory information even in healthy infants and children. Furthermore, mechanisms by which early brain insults affect brain development and function are poorly understood. In this thesis, we used magnetoencephalography (MEG) to investigate development of cortical somatosensory functions in healthy infants, very premature infants at risk for neurological disorders, and adolescents with hemiplegic cerebral palsy (CP). In newborns, stimulation of the hand activated both the contralateral primary (SIc) and secondary somatosensory cortices (SIIc). The activation patterns differed from those of adults, however. Some of the earliest SIc responses, constantly present in adults, were completely lacking in newborns and the effect of sleep stage on SIIc responses differed. These discrepancies between newborns and adults reflect the still developmental stage of the newborns’ somatosensory system. Its further maturation was demonstrated by a systematic transformation of the SIc response pattern with age. The main early adult­like components were present by age two. In very preterm infants, at term age, the SIc and SIIc were activated at similar latencies as in healthy fullterm newborns, but the SIc activity was weaker in the preterm group. The SIIc response was absent in four out of the six infants with brain lesions of the underlying hemisphere. Determining the prognostic value of this finding remains a subject for future studies, however. In the CP adolescents with pure subcortical lesions, contrasting their unilateral symptoms, the SIc responses of both hemispheres differed from those of controls: For example the distance between SIc representation areas for digits II and V was shorter bilaterally. In four of the five CP patients with cortico­subcortical brain lesions, no normal early SIc responses were evoked by stimulation of the palsied hand. The varying differences in neuronal functions, underlying the common clinical symptoms, call for investigation of more precisely designed rehabilitation strategies resting on knowledge about individual functional alterations in the sensorimotor networks.
  • Havia, Mari (2013)
    Neuronaaliset kolinergiset nikotiinireseptorit (nAChR:t) vaikuttavat moniin elimistön fysiologisiin toimintoihin ja patofysiologisiin tiloihin. nAChR:t ovat ligandin sitovia ionikanavareseptoreita, jotka esiintyvät laajalti eri aivoalueilla usein presynaptisesti moduloiden näin muiden keskushermoston välittäjäaineiden vapautumista. nAChR:t koostuvat viidestä alayksiköstä, joita on tunnistettu toistaiseksi yhdeksän, muodostaen lukuisia eri nAChR-alatyyppejä. nAChR:t välittävät endogeenisen agonistin, asetyylikoliinin, lisäksi nikotiinin sekä useiden muiden ligandien, vaikutuksia elimistössä. Riippuvuutta aiheuttavat yhdisteet kuten nikotiini ja opioidit aiheuttavat adaptiivisia muutoksia keskushermostossa. NAChR:ssa on tunnistettu asetyylikoliinin sitoutumiskohdan lisäksi useita allosteerisia sitoutumiskohtia, joihin sitoutumalla ligandi voi moduloida agonistin vastetta. Pro gradun kokeellisen osuuden tarkoituksena oli selvittää kolmen eri opioidin: kodeiinin, oksikodonin ja tramadolin ja nikotiinin yhteisvaikutuksia in vitro SH-SY5Y-solulinjassa, joka ilmentää endogeenisesti α3* ja α7-nAChR:ta. Opioidien sitoutumista nAChR:hin tutkittiin reseptorisitoutumiskokein radioaktiivisen ligandin [3H]-epibatidiinin avulla. Nikotiinin ja opioidien toiminnallista yhteisvaikutusta nAChR:ssa tutkittiin 86Rb+- ulosvirtauskokeilla. SH-SY5Y-solulinjassa kodeiinilla, oksikodonilla ja tramadolilla todettiin olevan reseptoritason yhteisvaikutuksia nikotiinin kanssa, jotka välittyivät neuronaalisten nikotiinireseptorien kautta. Opioidiagonistit kodeiini, oksikodoni ja tramadoli estävät nikotiinin nACh-reseptoreita aktivoivaa vaikutusta sitoutumatta 3H-epibatidiinin sitoutumispaikkaan. Kodeiini, oksikodoni ja tramadoli ovat heikkoja ei-kilpailevia antagonisteja. Nämä tulokset antavat lisätietoa nikotiinin ja opioidien reseptoritason yhteisvaikutuksista. Nikotiinilla ja opioideilla on havaittu olevan yhteisvaikutuksia myös in vivo, jotka saattavat osittain selittyä havaituilla reseptoritason interaktioilla.
  • Lonka, Liina (Helsingin yliopisto, 2004)
  • Vanhanen, Jenni (Helsingin yliopisto, 2015)
    Neuronal histamine and its H3 receptor (H3R) regulate several physiological functions and are involved in the pathophysiology of various central nervous system disorders such as Parkinson s disease, Alzheimer s disease, Tourette syndrome and narcolepsy. Studies conducted in experimental animals have also suggested a role for histamine and especially H3R in the effects of drugs of abuse. In this thesis, the main aim was to study how histamine and H3R regulate alcohol-related behaviors. Furthermore, our goal was to investigate the underlying mechanisms in the observed behaviors. By using both wild type mice in different background strains and genetically modified mice, we studied whether histamine and H3R regulate the behavioral responses to alcohol. Three different H3R antagonists (ciproxifan, JNJ-10181457 and JNJ-39220675) were used and it was found that both pharmacological antagonism and genetic knockout of H3R (H3R KO) lead to diminished alcohol consumption and reward. By using histamine deficient histidine decarboxylase knockout (HDC KO) mice, we found that the lack of histamine does not alter alcohol consumption or reward but histamine is indeed required for the H3R-mediated alcohol reward inhibition. We also found that JNJ-39220675 inhibited the acute stimulation of amphetamine, but failed to inhibit the rewarding properties of amphetamine. This indicates that although H3R antagonists inhibit alcohol reward, they may not possess the same ability on psychostimulants, such as amphetamine. The findings obtained from the behavioral experiments led us to hypothesize that H3R interacts with the dopaminergic system. This was further studied on a molecular level using both radioactive in situ hybridization and semi-quantitative Western blotting. We found that compared with control mice, H3R KO mice displayed lower levels of dopamine D1 receptor messenger RNA in the striatum, which is an area important in the regulation of e.g. reward. In addition, we found that activation of dopamine D1 and D2 receptors resulted in abnormal striatal cell signaling in the absence of H3Rs. Taken together, these findings demonstrate that H3R is an important regulator of alcohol-related behaviors. The mechanism by which H3R regulates these phenomena might involve the interaction between the striatal H3R and dopamine receptors. In addition, these results provide preclinical evidence that H3R antagonists may serve as a novel approach to treat alcohol dependence.
  • Uvarov, Pavel (Helsingin yliopisto, 2010)
    K-Cl cotransporter 2 (KCC2) maintains a low intracellular Cl concentration required for fast hyperpolarizing responses of neurons to classical inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine. Decreased Cl extrusion observed in genetically modified KCC2-deficient mice leads to depolarizing GABA responses, impaired brain inhibition, and as a consequence to epileptic seizures. Identification of mechanisms regulating activity of the SLC12A5 gene, which encodes the KCC2 cotransporter, in normal and pathological conditions is, thus, of extreme importance. Multiple reports have previously elucidated in details a spatio-temporal pattern of KCC2 expression. Among the characteristic features are an exclusive neuronal specificity, a dramatic upregulation during embryonic and early postnatal development, and a significant downregulation by neuronal trauma. Numerous studies confirmed these expressional features, however transcriptional mechanisms predetermining the SLC12A5 gene behaviour are still unknown. The aim of the presented thesis is to recognize such transcriptional mechanisms and, on their basis, to create a transcriptional model that would explain the established SLC12A5 gene behaviour. Up to recently, only one KCC2 transcript has been thought to exist. A particular novelty of the presented work is the identification of two SLC12A5 gene promoters (SLC12A5-1a and SLC12A5-1b) that produce at least two KCC2 isoforms (KCC2a and KCC2b) differing by their N-terminal parts. Even though a functional 86Rb+ assay reveals no significant difference between transport activities of the isoforms, consensus sites for several protein kinases, found in KCC2a but not in KCC2b, imply a distinct kinetic regulation. As a logical continuation, the current work presents a detailed analysis of the KCC2a and KCC2b expression patterns. This analysis shows an exclusively neuron-specific pattern and similar expression levels for both isoforms during embryonic and neonatal development in rodents. During subsequent postnatal development, the KCC2b expression dramatically increases, while KCC2a expression, depending on central nervous system (CNS) area, either remains at the same level or moderately decreases. In an attempt to explain both the neuronal specificity and the distinct expressional kinetics of the KCC2a and KCC2b isoforms during postnatal development, the corresponding SLC12A5-1a and SLC12A5-1b promoters have been subjected to a comprehensive bioinformatical analysis. Binding sites of several transcription factors (TFs), conserved in the mammalian SLC12A5 gene orthologs, have been identified that might shed light on the observed behaviour of the SLC12A5 gene. Possible roles of these TFs in the regulating of the SLC12A5 gene expression have been elucidated in subsequent experiments and are discussed in the current thesis.
  • Palva, Satu (Helsingin yliopisto, 2007)
    The synchronization of neuronal activity, especially in the beta- (14-30 Hz) /gamma- (30 80 Hz) frequency bands, is thought to provide a means for the integration of anatomically distributed processing and for the formation of transient neuronal assemblies. Thus non-stimulus locked (i.e. induced) gamma-band oscillations are believed to underlie feature binding and the formation of neuronal object representations. On the other hand, the functional roles of neuronal oscillations in slower theta- (4 8 Hz) and alpha- (8 14 Hz) frequency bands remain controversial. In addition, early stimulus-locked activity has been largely ignored, as it is believed to reflect merely the physical properties of sensory stimuli. With human neuromagnetic recordings, both the functional roles of gamma- and alpha-band oscillations and the significance of early stimulus-locked activity in neuronal processing were examined in this thesis. Study I of this thesis shows that even the stimulus-locked (evoked) gamma oscillations were sensitive to high-level stimulus features for speech and non-speech sounds, suggesting that they may underlie the formation of early neuronal object representations for stimuli with a behavioural relevance. Study II shows that neuronal processing for consciously perceived and unperceived stimuli differed as early as 30 ms after stimulus onset. This study also showed that the alpha band oscillations selectively correlated with conscious perception. Study III, in turn, shows that prestimulus alpha-band oscillations influence the subsequent detection and processing of sensory stimuli. Further, in Study IV, we asked whether phase synchronization between distinct frequency bands is present in cortical circuits. This study revealed prominent task-sensitive phase synchrony between alpha and beta/gamma oscillations. Finally, the implications of Studies II, III, and IV to the broader scientific context are analysed in the last study of this thesis (V). I suggest, in this thesis that neuronal processing may be extremely fast and that the evoked response is important for cognitive processes. I also propose that alpha oscillations define the global neuronal workspace of perception, action, and consciousness and, further, that cross-frequency synchronization is required for the integration of neuronal object representations into global neuronal workspace.
  • Ning, Lin (Helsingin yliopisto, 2013)
    In the central nervous system, neurons are connected through local contacting structures called synapses. The initial contacts between axons and dendrites undergo changes in morphology and protein composition, and differentiate into fully functional synapses. Precise regulation on synapse formation is essential for normal brain functioning. Cell adhesion molecules are the key regulators during this process, by connecting the membranes of synapses and initiating signaling cascades that mediate synapse formation. A neuron-specific cell adhesion molecule ICAM-5 (telencephalin) controls immune responses by suppressing T-cell activation or by mediating neuron-microglia interaction. On the other hand, ICAM-5 is so far the only identified CAM that slows synaptic development. Deletion of ICAM-5 from mice leads to accelerated synapse formation, enhanced synaptic capacity, and improved memory and learning. Clinically, changes in ICAM-5 levels are associated with various diseases, such as acute encephalitis, epilepsy, and Alzheimer s disease. The major goal of my thesis is to address the molecular mechanisms by which ICAM-5 regulates synapse formation as well as maturation of dendritic spines, the post-synaptic components of excitatory synapses. In my study, ICAM-5 was observed as a substrate for MMP-2 and -9. Activation NMDA receptors in neurons elevated the level of MMP activity, and subsequently induced ICAM-5 ectodomain cleavage, which in turn promoted spine maturation. In addition, I identified the pre-synaptic β1-integrins as counter-receptors for ICAM-5. This trans-synaptic interaction inhibited the MMP-induced ICAM-5 cleavage, and thereby prevented spine maturation. At the intracellular side, α-actinin, an actin cross-linking protein, was found as a binding partner for ICAM-5. This binding linked ICAM-5 to the actin cytoskeleton and was important for the membrane distribution of ICAM-5 and neurite outgrowth. The GluN1 subunit of the NMDAR is also known to bind to α-actinin. We found here that GluN1 and ICAM-5 competed for the same binding region in α-actinin. Activation of NMDAR changed α-actinin binding property to ICAM-5, resulting in α-actinin accumulation and actin reorganization. In conclusion, my thesis defines a novel, ICAM-5-dependent mechanism, which regulates synapse formation, spine maturation and remodeling.
  • Carlsson, Emilia (Helsingin yliopisto, 2012)
    This thesis is based on the original observation of an allelic deletion of the recently described Neuron navigator 3 (NAV3) gene in patients with primary cutaneous T-cell lymphoma (CTCL) and CTCL-associated lung cancer. Thereafter mutations or copy number changes of NAV3 have been reported in melanoma, glioblastoma (GBM) and adrenal carcinoma. The aim of this study has been to shed light on the function and interactions of the NAV3 protein, as well as characterizing NAV3 copy number changes and their effect on patient survival in several forms of cancer. NAV3 is a novel cancer-associated gene at 12q21. The specific function of NAV3 is not known except that it carries actin-binding domains with ATPase activity, and is therefore likely to have an action on microtubules and cytoskeleton reorganization. The three Navigator genes, NAV1, NAV2 and NAV3 share homology among themselves and among different species. This suggests a central role for the encoded proteins in cell biology. In this study, NAV3 copy number changes have been studied by fluorescence in situ hybridisation (FISH) and array comparative genomic hybridisation (aCGH) in non-melanoma skin cancers, colorectal cancer and neural system tumours, and the relevant NAV3-regulated target genes have been identified. Furthermore, the expression levels of NAV3 and NAV3-regulated signalling molecules have been correlated to disease progression and patient outcome. In Basal cell carcinomas (BCC) and Squamous cell carcinomas (SCC) we found NAV3 copy number loss and corresponding absence of protein in 21% of the BCC and in 25% of the SCC tumours. In the nodular/superficial BCC subgroup, also low-level NAV3 amplification was found. NAV3 aberrations were independent of the known chromosome 6 amplification in BCC. Chromosome 12 polysomy, also independent of chromosome 6 polysomy, was found in 33% and 25% of the invasive type of BCC and in SCC, respectively. In colorectal carcinomas NAV3 deletion and chromosome 12 polysomy were detected in 30% and 70% of MSS carcinomas, and in 12.5% and 50% of MSI carcinomas, but also in 23% and 30% of adenomas, respectively. Low copy number amplification of NAV3 was found in 25% of MSS samples. 119 patient samples representing different central and peripheral nervous system tumours were studied for NAV3 copy number changes. Neuronally differentiated tumours (neuroblastomas and medulloblastomas) entailed more NAV3 aberrations than the glial tumours. In glial tumours, those with grade IV (gliobalstoma (GBM)) had significantly more NAV3 deletions than tumours with grades I, II or III. Log rank analysis also linked NAV3 deletion to a poor prognosis in gliomas. In contrast, glioma patients with NAV3 amplification showed better prognosis than those with normal NAV3 copy numbers. The FISH result was also supported by aCGH analysis, which showed results matching the FISH analysis for tumour samples with NAV3 amplification and deletion. To understand the in vivo functional consequences of NAV3 copy number changes, especially of NAV3 deletion, we silenced NAV3 in normal colon, GBM and primary keratinocyte cells, using a commercially available small inhibitory ribonucleic acid (siRNA) construct. Post transfection RNA samples from several time points were analyzed with Agilent 44K microarray. In GBM and colorectal cell lines we identified, among others, GnRHR and IL-23R as upregulated by NAV3 gene silencing. The upregulation of the selected genes were confirmed by quantitative PCR. In primary keratinocytes, NAV3 silencing led to consistent upregulation of 22 annotated genes, and several Wnt/HH pathway genes were slightly overexpressed too. Taken together the results of this thesis support the previously suggested role of NAV3 as a novel cancer associated gene, and we suggest that NAV3 affects the malignant potential of a given tumour through multiple pathways. This assumption is based on the fact that gene expression analysis of NAV3 silenced cells indicates that NAV3 affects genes with involvement in both inflammation and carcinogenesis.
  • den Hollander, Bjørnar (Helsingin yliopisto, 2015)
    In recent years there has been a large increase in the use of a new kind of amphetamine- type stimulants known as substituted cathinones. These compounds have a short history of human use, and little is known about their potential neurotoxicity. Two of the most popular substituted cathinones, 4-methylmethcathinone (4-MMC, mephedrone) and 3,4- methylenedioxymethcathinone (MDMC, methylone} are, aside from their β-ketone group, close structural analogues of potentially neurotoxic amphetamines such as methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy). This has led to concern about the potential neurotoxicity of these novel compounds, and warrants a closer investigation into their possible long-term neurotoxic effects. METHODS The long-term effects of METH and MDMA as well as 4-MMC and MDMC were assessed using a range of biochemical assays, including assessment of monoamine levels and their transporters. The effects on brain activity were investigated using manganese-enhanced magnetic resonance imaging. Furthermore, behavioral experiments assessing cognition and neuropsychiatric function were performed. Finally, in vitro experiments in a neuroblastoma cell line were performed to identify mechanisms responsible for the observed differences in toxicity between the amphetamines and cathinones. RESULTS Unlike METH and MDMA, which produced strong reductions in dopamine and serotonin levels or brain activation, 4-MMC produced few notable effects on monoamine levels and had only minor effects on brain activation, although MDMC produced a reduction in 5-HT levels similar to MDMA. No clear effects on behavioral tests of memory function were observed as both increases and decreases in test performance were seen following 4- MMC and MDMC. In vitro experiments revealed that cathinones differ from amphetamines in their redox properties, and 4-MMC produced different effects than METH on the mitochondrial electron transport chain. CONCLUSIONS The substituted cathinones 4-MMC and MDMC do not appear to be more neurotoxic than METH and MDMA. If anything, they show a more favorable safety profile. Therefore, these substances do not appear to present an imminent and severe threat to public health. From a harm reduction perspective, these compounds may be good alternatives toMETH and MDMA. However, future work is needed to assess with certainty the long- term effects of amphetamine-type stimulants in humans.
  • Takala, Anna (2012)
    Neurosteroidit ovat aivoissa ja keskushermostossa vaikuttavia steroideja, joilla on monia biologisia ja fysiologisia vaikutuksia elimistössä. Neurosteroidien pitoisuuksien muutoksilla arvellaan olevan vaikutuksia monissa taudeissa, esimerkiksi masennuksessa, skitsofreniassa ja epilepsiassa. Neurosteroidien pitoisuuksia mitataan, jotta pystytään paremmin ymmärtämään steroidien roolia aivojen toiminnassa ja ihmisen käyttäytymisessä. Työn tarkoituksena on kehittää edelleen menetelmää neurosteroidien analysoimiseksi virtsanäytteistä kaasukromatografia-ilmanpaine fotoionisaatio-tandem massaspektrometria –tekniikalla (GC-APPI-MS/MS). Tutkimuksessa on mukana 19 eri neurosteroidia tai niiden aineenvaihduntatuotetta. Neurosteroidit erittyvät virtsaan pääasiassa konjugaatteinaan, joten näytteet hydrolysoidaan ennen analyysiä. Hydrolysoinnin jälkeen näyte käsitellään neste-nesteuutolla ja derivatisoidaan haihtuvuuden parantamiseksi. Koska yleisesti käytetty Helix pomatiasta peräisin oleva β-glukuronidaasi/aryylisulfataasi-entsyymi hapettaa 3β-hydroksi-5-eeni ja 3β-hydroksi-5α-pelkistyneitä steroideja, valittiin vaihtoehtoisiksi hydrolyysimenetelmiksi Escherichia colin β-glukuronidaasientsyymi sekä sulfaattikonjugaattien hajottamiseen happohydrolyysi. Neurosteroidien kokonaismäärän määrityksen havaittiin olevan haasteellista valituilla menetelmillä, koska E. colin entsyymit eivät hydrolysoineet glukuronideja täydellisesti ja happohydrolyysi hajotti sulfaatti-konjugaattien lisäksi myös glukuronidikonjugaatteja. Lisäksi sisäisenä standardina käytetyn d4-allopregnanolonin havaittiin olevan epäpuhdasta ja hajoavan ainakin happohydro-lyysikäsittelyssä. Käytetyllä menetelmällä havaintoalarajat olivat suhteellisen alhaisia (2 pg/ml-1 ng/ml). Menetelmän toistettavuus oli melko hyvä kaikille yhdisteille (RSD alle 27 %) ja menetelmä oli hyvin toistettava retentioajan (RSD 0,06-0,11 %) suhteen. Alustavasti validoidulla menetelmällä analysoitiin kahden miehen ja kahden naisen virtsanäytteitä. Virtsasta pystyt-tiin määrittämään arvioidut pitoisuudet dehydroepiandrosteronille, dihydrotestosteronille, androsteenidionille, testosteronille, estronille, β-estradiolille, estriolille, 5α-tetrahydrodeok-sikortikosteronille, kortisonille, kortikosteronille sekä hydrokortisonille. Kehitetty mene-telmä ei täyttänyt kaikkia sille asetettuja tavoitteita. Menetelmä täytyy validoida perusteelli-semmin sekä tutkia tarkemmin hydrolyysien vaikutus kantayhdisteisiin. Myös sisäinen standardi on vaihdettava toiseen, mieluiten ei-deuteroituun yhdisteeseen.