Browsing by Subject "Physiology and Neuroscience"

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  • Hlushchenko, Iryna (Helsingin yliopisto, 2019)
    This thesis explores the role of several actin-binding proteins in the regulation of brain physiology with a focus on dendritic spines. Dendritic spines are considered the plausible physical substrate for learning and memory, as their morphology allows for modulating incoming signals. Disruptions in spine density and morphology are also often associated with neuropsychiatric disorders. The two cellular processes representing neuronal learning are long-term potentiation (LTP) and long-term depression (LTD). Here, I show that the actin-severing protein gelsolin transiently relocates to dendritic spines upon LTD induction, but not LTP induction or spontaneous neuronal activity. It is plausible that the modest – but relatively long-lasting – LTD-induced elevation of Ca2+ concentration increases the affinity of gelsolin to F-actin, thus inducing the relocalization of gelsolin to dendritic spines. Proper spine regulation is crucial for learning in live animals. MIM is an I-BAR containing membrane curving protein, shown to be involved in dendritic spine initiation and dendritic branching in Purkinje cells in the cerebellum. Behavioral analysis of MIM knock-out (KO) mice revealed defects in both learning and reverse-learning, alterations in anxiety levels and reduced dominant behavior, and confirmed the previously described deficiency in motor coordination and pre-pulse inhibition. Anatomically, we observed a decreased density of thin dendritic protrusions, enlarged brain ventricles and decreased cortical volume. Genetic studies have pointed out that genes often disturbed in neuropsychiatric disorders encode synaptic actin regulators. We selected five genes encoding different actin-regulating proteins and induced ASD-associated de novo missense mutations in these proteins. These mutations induced changes in the localization of α-actinin-4, which localized less to dendritic spines, and for SWAP-70 and SrGAP3, which localized more to dendritic spines. Among the wild-type proteins studied, only α-actinin-4 expression caused a significant change in dendritic spine morphology by increasing mushroom spine density and decreasing thin spine density. We hypothesized that mutations associated with ASD shift dendritic spine morphology from mushroom to thin spines. An M554V mutation in α-actinin-4 (ACTN4) resulted in the expected shift in dendritic spine morphology by increasing the density of thin spines. In addition, we observed a trend toward higher thin spine density with mutations in myosin IXb and SWAP-70. Myosin IIb and myosin IXb expression increased the proportion of inhibitory synapses in spines. The expression of mutated myosin IIb (Y265C), SrGAP3 (E469K), and SWAP-70 (L544F) induced variable changes in inhibitory synapses.
  • Yukin, Alexey (Helsingin yliopisto, 2016)
    This Thesis addresses fundamental mechanisms of brain disorders that occur in preterm infants or during early childhood. To this end, we have developed animal models to mimic disorder-specific pathological conditions in the brain. During critical periods of development of the mammalian brain, cell migration and synapse formation are crucially important to set up newly developed neuronal circuits that will support complex network activity. A particular set of cells, the subplate neurons, is the first to mature during the earliest stages of cortical development. These neurons act as transient relay and processing station for signals coming from subcortical structures to the cortex. Their high dependence on oxygen supply makes them vulnerable to injuries that take place during pregnancy. In the present work, conditions of this kind were mimicked by specific toxin-based ablation of subplate neurons. At a more advanced stage of brain development, a very common type of brain disorder is febrile seizures (FS). They represent convulsive events in humans that are promoted by respiratory alkalosis during febrile illness. The global incidence of FS is estimated from 2 to 14 % (depending on ethnicity) of all children in the age of 6 months to 5 years. Using an animal based on 13-14 day old rats exposed to hyperthermia, we show that a pro-excitatory action of GABA based on the neuron-specific carbonic anhydrase (CA) isoform VII is crucially involved in the generation of these seizures. Finally, while it is widely assumed that FS are limbic in origin, this Thesis provides new evidence that the brainstem can have an independent and prominent role in the generation of FS, and also of convulsions induced by kainic acid, which so far, have been considered prototypical in studies of limbic seizures. As a whole, this work demonstrates the high utility and heuristic value of custom-designed animal models in studies of basic mechanisms and consequences of disorders in the developing brain.
  • Andersson, Oliver (Helsingin yliopisto, 2020)
    This study focused on the spectral sensitivity of two Norwegian lake populations of opossum shrimp (Mysis relicta), with a common implantation history and a temporal separation of about 50 years. Previous findings have indicated a difference in the absorption maximum (λmax) between sea and lake populations of Nordic mysids that have been separated about 9 000 years. Between the population groups, the spectral sensitivity correlates to the Wavelength of Maximal Transmission (WLMT) in the habitats. This may be considered a form of adaptive tuning. It is not known if the species-specific mechanism is based on chromophore shift or opsin modification or a combination of both, neither is the timescale of the adaption well understood. The intent was to determine λmax of both populations, what chromophore(s) they use and possible variations of the opsin gene. By comparison to spectrometric data of the habitats, the study aimed to broaden the insight into the mentioned unknowns. The light conditions of the lakes were determined by spectrometry down to depths of three and five meters. As predicted a positive, lake-bound correlation between the attenuation coefficient (k) and WMLT was found. Single-rhabdom microspectrophotometry (MSP) was used to determine λmax of the visual pigment in situ. Absorbance spectra were analysed by manual fitting to mathematical pigment templates and by script-based automation. Neither the chromophore nor differences in λmax could be determined, due to a small sample size that limited the statistical power of the results. The opsin genes from both populations were sequenced. No differences expected to have an effect on spectral sensitivity were found. Spectral tuning could not be demonstrated to have occurred in the populations due to the limited sample size. Nor did the results give support for any new findings on the mechanism or the time scale of spectral tuning among mysids. To answer the proposed questions of the study, additional sampling of both populations is needed.
  • Pitkänen, Stina (Helsingin yliopisto, 2018)
    The arylhydrocarbon receptor (AHR) is known for its xenobiotic role. In the last decades we have realized it has an important role even in normal physiology. Earlier studies have shown different circadian behavior in mice and rats when AHR is activated with the environmental toxoid TCDD. Also, AHR knock-out (AHRKO) mice have shown to adapt quicker to new lighting conditions. The aim of this study was to chart AHRs role on the circadian behavior in rats, by comparing daily eating and drinking habits under normal lighting condition for 7 days and for 7 days after a 12-hour light shift. Tissue samples to be used in continuing studies were taken after the 14 days long follow up. These studies will chart how the circadian timekeeping genes are expressed in the central (suprachiasmatic nucleus) and periphery (liver) cells in AHRKO rats after an adaptation to phase shift compared to wild type rats. This way the study will provide information that will help us understand the role of AHR in different species regarding behavior and in continuing studies gene expression. In our study no differences in drinking and eating activity could be seen between AHRKO and wild type rats. Both groups adapted to new lighting conditions equally fast.
  • Fredrikson, Linda (Helsingin yliopisto, 2019)
    The consumption of omega (n-) 3 polyunsaturated fatty acids (PUFA) from fish has been associated with lower rates of cardiovascular diseases with one mechanism being lowering LDL cholesterol levels in blood. When incorporated into LDL particle n-3 PUFAs can modify the lipid composition and reduce atherogenicity of the particle, e.g. by influencing inflammatory processes. The effects of n-3 PUFA of plant origin are less studied. This study investigated the effects of Camelina sativa oil (CSO), a rich source of alpha-linolenic acid (ALA), on lipid species of human LDL including phosphatidylcholines (PC), lysophosphatidylcholines (LPC), sphingomyelin (SM), triacylglycerols (TAG) and cholesterol esters (CE). A total of 38 subjects with a history of impaired fasting glucose, were randomly divided into two groups; CSO (ALA 10 g/day) and the control group (limited fish and ALA intake) for 12 weeks. Blood samples were collected from the subjects at the beginning and at the end of the experiment after 12 weeks. LDL particles were isolated from blood and the lipids were analyzed by mass spectrometry. The CSO affected more the LDL core lipids (TAG and CE) than lipid species of the shell (PC, LPC, SM). CSO is high in ALA and linoleic acid (LA). Thus, the diet reduced mole fractions of lipid species containing saturated acyl chains while acyl chains in the core lipids with ALA, LA and EPA, that is formed in the body from ALA, were increased. Based on the results, having CSO in the diet changed the LDL particle lipid composition in a favorable direction for cardiovascular health.
  • Karvonen, Eira (Helsingin yliopisto, 2020)
    APECED (Autoimmune-Polyendocrinopathy-Candidiasis-Ectodermal-Dystrophy) is a severe, multiorgan autoimmune disease caused by mutations in the AIRE (autoimmune regulator) gene. APECED is a rare disease, however in Finland the frequency is significantly high (1:25 000) and APECED belongs to the ‘Finnish Disease Heritage’. The most common mutation worldwide is the so-called Finn-major mutation R257X that results in a truncation of the AIRE protein, which disrupts the indispensable functions of AIRE. Immune reactions towards body’s own components are typically prevented with various central and peripheral immune tolerance mechanisms. AIRE is essential for the proper development of central and peripheral tolerance and the absence of functional AIRE leads to a loss of immune tolerance and various autoimmune manifestations. Recent studies have suggested that AIRE also has functions in stem cells and actively contributes to the regulation network of pluripotency. Currently, the development of induced pluripotent stem cell (iPSC) technology has opened opportunities for precision medicine and for defining the cure for genetic diseases, such as APECED. The ultimate objective of our research group is to examine whether APECED could be cured via autologous, gene-corrected cell transplants with the use of induced pluripotent stem cells (iPSCs). As a requirement for such later therapeutic use and iPSC differentiation, the APECED patient-derived iPS cells needed to be characterized in detail. To assess, whether AIRE R257X mutation, present in APECED patients’ iPSCs, would cause defects in their stemness properties, the expression of AIRE and classical stem cell markers were examined with qPCR and immunocytochemistry and compared to healthy control iPSCs. The iPSC cells were also treated with spontaneous differentiation -inducing dimethyl sulfoxide (DMSO) to study, whether AIRE R257X mutation would affect the spontaneous differentiation of iPS cells. To further investigate the stemness and early developmental phase properties of APECED patient derived iPSCs, self-aggregated embryoid bodies (EBs) were generated and cultured. Immunocytochemistry was used to examine whether APECED EBs differ in stemness, proliferation or apoptosis from healthy individual’s EBs. The comparative Ct method (ΔΔCt) i.e. fold change revealed that APECED iPSC clones expressed all the classical stem cell markers similarly to healthy control iPSCs. DMSO treatment reduced the expression of stem cell markers in both healthy and APECED-derived iPSCs. The immunostaining results of iPSCs were consistent with the qPCR analysis. The overall growth properties as well as the immunocytochemical assays of stemness, proliferation and apoptosis markers did not show any significant difference between the APECED patient and healthy control derived EBs. Together the results indicate that the R257X mutation of the APECED patients does not affect stem cell properties such as stem cell marker expression and colony or the EB formation of the iPSCs. The results are contrary to previous studies in mice demonstrating the interspecific difference between mouse and human and denoting the importance of human samples completing the studies with animal models. As the APECED patient derived iPSCs did not exhibit any defects in their stemness properties, the later iPS differentiation and therapeutic use could be accomplished without hindrance. However, future work is still needed, as the small sample size in this preliminary test might introduce some biases to the results and hindered a relevant statistical analysis. Nevertheless, this thesis project was the first time APECED patient-derived iPSCs were characterized and has provided new information about the effect of AIRE mutation in APECED patient derived iPSCs.
  • Moliner, Rafael (Helsingin yliopisto, 2019)
    Classical and rapid-acting antidepressant drugs have been shown to reinstate juvenile-like plasticity in the adult brain, allowing mature neuronal networks to rewire in an environmentally-driven/activity-dependent process. Indeed, antidepressant drugs gradually increase expression of brain-derived neurotrophic factor (BDNF) and can rapidly activate signaling of its high-affinity receptor TRKB. However, the exact mechanism of action underlying drug-induced restoration of juvenile-like plasticity remains poorly understood. In this study we first characterized acute effects of classical and rapid-acting antidepressant drugs on the interaction between TRKB and postsynaptic density (PSD) proteins PSD-93 and PSD-95 in vitro. PSD proteins constitute the core of synaptic complexes by anchoring receptors, ion channels, adhesion proteins and various signaling molecules, and are also involved in protein transport and cell surface localization. PSD proteins have in common their role as key regulators of synaptic structure and function, although PSD-93 and PSD-95 are associated with different functions during development and have opposing effects on the state of plasticity in individual synapses and neurons. Secondly, we investigated changes in mobility of TRKB in dendritic structures in response to treatment with antidepressant drugs in vitro. We found that antidepressant drugs decrease anchoring of TRKB with PSD-93 and PSD-95, and can rapidly increase TRKB turnover in dendritic spines. Our results contribute to the mechanistic model explaining drug-induced restoration of juvenile-like neuronal plasticity, and may provide a common basis for the effects of antidepressant drugs.
  • Jenkins, Cherie (Helsingin yliopisto, 2020)
    Reptiles have long been studied in search of the mechanisms behind neuronal regeneration. This thesis delves into the regenerative areas of two emerging model species to the field of regenerative research: Pogona vitticeps (bearded dragon) and Pantherophis guttatus (corn snake). This fluorescent immunohistochemical study maps out and compares the constitutive proliferative zones in these two species to better define the focus of future comparative neurodegenerative experiments. A BrdU pulse chase experiment in conjunction with PCNA reveals proliferative zones in the lateral ventricular ependyma of both species. Stem cell niches were found in the ependymal lining adjacent to the medial cortex and dorsal ventricular ridge in both species, however, the nucleus sphericus ependyma was an active proliferative zone only in Pantherophis. Imaging of further markers in this study support the findings of the pulse chase experiment. High levels of the stem cell marker Sox2 was found in lateral ventricular ependymal cells in both species. The glial marker GFAP reveals a highly ordered array of radial glia in the cortical areas of Pogona, which is significantly reduced or absent in Pantherophis. And lastly the neuronal marker HU was found in the same cells that were BrdU positive and had migrated a short distance from the proliferative zones, which shows that the proliferative areas in the lateral ventricular lining do indeed produce neurons. The BrdU and PCNA marked cells were quantified in both species, and a brief comparison between the species showed that Pogona had a significantly higher number and concentration of proliferative cells in the proliferative zones than Pantherophis. Scattered BrdU positive cells that were neither neuronal nor positive for any other marker were also found scattered throughout the parenchyma of Pogona, and these cells remain uncharacterized. Differences between these two species are not surprising, as lizards are known to have better regenerative capabilities than snakes, however, more comparative research between these species is needed to gain further insight into the mechanisms behind their contrasting regenerative capabilities.
  • Sirola, Roosa (Helsingfors universitet, 2013)
    Visual working memory (VWM) maintains information for future usage. Several studies show that the cortical oscillations in the γ-frequency band (from 30 to 120 Hz) are modulated by the VWM performance. However, less is known about the cortical sources underlying the modulation of these oscillations in VWM. To address this question, we recorded human neuronal activity with magneto- and electroencephalography (M/EEG) during a delayed-matching-to-sample VWM task with three different task conditions, within which participants were instructed to focus on different object features in turn. In addition, anatomical data was acquired with magnetic resonance imaging for source modeling purposes. We then estimated the cortical amplitude dynamics across frequencies from three to 90 Hz during the VWM retention period for these three different conditions. We found that the amplitudes of the γ –frequency band oscillations were strengthened in the occipito-temporal cortical areas during the VWM for shapes but not for color or spatial locations. These data suggest that γ –band oscillations are fundamental in VWM, especially for visual stimuli requiring perceptual feature binding. Furthermore, cortical γ –band oscillations were found to be load dependently strengthened in the frontal cortex, where the central executive and attention associated processes are believed to take place. These data support the previous hypotheses stating that γ –band oscillations contribute to the maintenance of object representations in VWM.
  • Puskarjov, Martin (Helsingfors universitet, 2010)
    The Cl- and HCO3- electrochemical gradients across the plasma membrane dictate the electrical consequences of GABAA receptor (GABAAR) function and thereby play a significant role in neuronal GABA-mediated signalling. In adult pyramidal neurons, responses to GABA are maintained hyperpolarizing mainly by the action of K-Cl cotransporter isoform 2 (KCC2). KCC2 acts as a Cl- extrusion mechanism responsible for setting the intracellular Cl- concentration below the electrochemical equilibrium, a necessary condition for hyperpolarizing inhibition mediated by GABAARs. Recent evidence suggests that plasmalemmal KCC2 has a very high rate of turnover, pointing to a novel role for changes in KCC2 expression in diverse manifestations of neuronal plasticity. Some studies indicate that rapid down-regulation of KCC2 may be a general early response involved in various kinds of neuronal trauma. In this work, whole-cell patch-clamp was used to examine KCC2 function under a pharmacologically induced arrest of protein synthesis in living hippocampal brain slices from rat. The stability of KCC2 function was quantitatively assessed on the basis of the dendritic Cl- extrusion capacity in the presence of protein synthesis inhibitors cycloheximide and emetine. The parameter used for assessing extrusion capacity was a somato-dendritic Cl- gradient, which was imposed by a somatic Cl- load that resulted in a gradient of EGABA (ΔEGABA). The results of this study show that under general protein synthesis inhibitor-induced arrest of translation, KCC2 function persists unperturbed for at least 4 hours and hence that the cessation of mRNA translation cannot rapidly induce downregulation of KCC2-mediated Cl- extrusion. This finding precludes the use of protein synthesis inhibitors for rapid modulation of KCC2 function. Indirectly, the results presented here imply that the levels of KCC2 under pathophysiological conditions are primarily determined by the degradation rate and not by de novo synthesis.
  • Spoljaric, Inkeri (Helsingin yliopisto, 2019)
    Spontaneously arising network events are a characteristic feature of all developing neural networks. This activity is crucial for normal neuronal development and the establishment of appropriate synaptic connectivity. In the developing hippocampus, depolarizing GABAergic drive is essential in generation of early network events, known as giant depolarizing potentials (GDPs). Blockade of GABAergic signaling leads to hypersynchronization of the network and emergence of ictal-like events, pointing to dual, both excitatory and inhibitory roles for GABA, in regulation of these events. In Studies I-III of this thesis, we examined the role of GABAA receptor (GABAAR) -mediated neurotransmission with some parallel work on glycinergic signaling as well as neuronal Cl- regulation in modulation of GDPs in the developing rodent hippocampus. In Study I, we demonstrate that low levels of GABA and glycine suppress GDPs by activating extrasynaptic receptors. This implies that regardless of the depolarizing drive for Cl- currents at this developmental stage, a low conductance via Cl- -permeable GABAARs and glycine receptors (GlyRs) can cause efficient shunting and inhibition of the network events. In Study II, we discovered that sustained activation of a subset of hippocampal interneurons, caused by the neuropeptide arginine vasopressin (AVP), silences the network events in the perinatal hippocampus, regardless of the maturational level of the GABAergic system as compared across species. This is attributed to decreased synchronous interneuronal input that is essential for the GDP generation. In Study III, we demonstrate that transport-functional K-Cl cotransporter 2 (KCC2) is present in the CA3 pyramidal neurons already in the perinatal stages in mice and rats. Cl- extrusion by KCC2 counteracts the dominant Na-K-2Cl cotransporter 1 (NKCC1) -mediated Cl- uptake and restrains the depolarizing GABAergic drive onto the CA3 pyramidal cells. Thereby, function of KCC2 limits pyramidal neuron spiking and synchronization during GDPs and participates in the modulation of GDPs from their developmental onset. This work describes novel physiological GABAergic mechanisms that control GDPs in the perinatal rodents and establishes a role for KCC2 in regulation of pyramidal neuron excitability and synchronization during GDPs starting from their developmental onset.
  • Ollonen, Joni (Helsingin yliopisto, 2020)
    The skull represents the most highly diversified and evolutionarily adapted anatomical aspect of metazoans, and its development and evolution have been a major driving force in the expansion of vertebrates. The evolution of skull and lower jaw bones have led to the adaptive radiation of jawed vertebrates, and skull tissues have changed rapidly over time and were finely tuned to meet functional and ecological demands with tremendous precision. Because of the long-lasting interest in conventional animal models, there is no general genetic or developmental model of skull evolution and diversity in vertebrates. Squamate reptiles represent the best model to study those aspects because of their key basal phylogenetic position within amniotes (i.e., mammals, birds, reptiles) and their exceptionally high levels of morphological variation (including their kinetic skulls). In particular, their lower jaw bones display tremendous variation. In order to assess this variation and the ecological and developmental factors connected to it, several methods from different fields of biology have to be used. In this study, morphometric, embryology and developmental approaches are used to investigate the ecological and developmental factors associated with the diversification of lower jaw bones in snakes and lizards. The shape diversity of squamate lower jaw bones was approached in a systematic way, using geometric morphometrics. Embryological methods were used to compare the embryonic stage of available squamate model animals at oviposition and to assess the order of ossification of embryo with earliest developmental stage at oviposition (bearded dragon, Pogona vitticeps). In addition, expression of major conserved candidate genes at different stages of lower jaw development (pharyngeal arches, mesenchyme patterning, ossification) were assessed in this species. The results indicate that the lower jaw bones of snakes versus lizards but also of fossorial squamates versus other habitats are significantly different. Heterochrony was also detected at both early stages (pharyngeal arche development at oviposition) and at the onset of ossification in lizards and snakes. Coherent with that, alterations in the expression pattern of Dlx genes in pharyngeal arches were observed in bearded dragon in comparison to earlier studies with mice, while other conserved markers of skeletogenesis were rather conserved. This analysis of the genotype and phenotype map of the reptilian skull provides some new insights into the development, origin and divergence of vertebrate tissues. The results will establish a good basis for future studies involving comparative developmental biology of bearded dragon. Future studies will offer excellent new opportunities to link craniofacial morphology, genetics/genomics and development to both ecological adaptation and evolutionary biology.
  • Hirvonen, Jonni (Helsingfors universitet, 2013)
    Tässä pro gradu -tutkielmassa on tarkasteltu aivosähkö- ja aivomagneettikäyrien amplitudien vaihteluiden vastaavuussuhteita koehenkilön suoriutumiseen audiovisuaalisten ärsykkeiden tarkkaavaisuustehtävissä. Aikaisemmista tutkimuksista tiedetään, että koehenkilön osumatarkkuus ei pysy vakiona koko tehtävän ajan, vaan on monesti jaksottunut valppauden ja herpaantumisen jaksoihin. Lisäksi osumatarkkuus koko kokeen ajalta on alhaisempi kuin lyhyen kalibraatiojakson ajalta mitattuna. Tämän intuitiiviseltä tuntuvan keskittymiskyvyn järkkymisen taustalla on esitetty olevan henkilön introspektiiviset ja mielenvaelteluun liittyvät kognitiiviset toiminnot. Ennen tätä tutkimusta on jäänyt kuitenkin osoittamatta osumatarkkuuden ailahtelun yhteys aivokuoren hermostollisen aktiivisuuden pitkällä ajalla autokorreloiviin muutoksiin lähdemallintamisella. Tämän pro gradun tutkimustulokset osoittavat, että näiden kahden lajin välillä on olemassa merkittävä korrelaatioyhteys. Lisäksi lepovaiheen aivotoiminnasta modaliteettispesifeillä tarkkaavaisuus- ja oletustilan verkoston alueilla voidaan ennustaa psykofyysisen suoriutumisen vaihteluja jatkuvan audiovisuaalisen ärsykekynnyksen tarkkaavaisuustehtävän aikana. Keskittymiskyvyn vaihtelun muutoksia hermostollisella tasolla ja näitä mahdollisesti ilmentäviä käyttäytymisen ailahteluja psykofyysisinä parametreinä, kuten osumatarkkuutena ja reaktionopeutena, voidaan luonnehtia skaalauslakianalyysilla. Ilmiön skaalaton käyttäytyminen heijastelee monimutkaisen järjestelmän taipumusta luoda sisäisiä vastaavuussuhteita eli autokorrelaatioita, jotka heikkenevät hitaammin ja ulottuvat kauemmaksi ajassa ja/tai paikassa kuin mitä alla piilevistä mekanismeista voidaan suoraan ennustaa. On havaittu, että osumatarkkuuden jaksottuminen ja spontaani aivotoiminta noudattavat potenssilain skaalauskäyttäytymistä ajan suhteen. Psykofyysisen ja hermostollisen skaalauslain mukaisen käyttäytymisen kvantifioimiseksi tässä opinnäytetyössä on käytetty vaihtelun ikkunallista autokorrelaatioanalyysiä, DFA:ta. DFA paljastaa ilmiön sisällä olevien peräkkäisten tapahtumien autokorrelaatioiden kestävyyden tarkasteluvälin kasvaessa. Skaalausluvut eli DFA-eksponentit on johdettu tässä kokeessa jatkuvan audiovisuaalisen ärsykekynnyksen tarkkaavaisuustehtävän ja levon aikana rekisteröidyistä aivosähkö- ja aivomagneettikäyräsignaalien verhokäyrästä sekä psykofyysisen osuma/huti -binäärisekvenssistä rakennetusta keinotekoisesta satunnaiskulun kaltaisesta käyrästä. Jatkuvat ärsykekynnystehtävät soveltuvat hyvin tarkkaavaisuuden top-down mekanismien tutkimiseen, koska heikoista, vain juuri ja juuri havaintokyvyn säteellä olevista ärsykkeistä seuraa verraten heikko bottom-up hermostovaste. Näin keskittymiskykyyn vaikuttavat top-down säätelymekanismit kuten motivaatio, päämäärät tai mielenvaeltelu eli spontaanilta vaikuttava aivotoiminta edustuu selkeämmin aivosähkö- ja -magneettikäyrissä. Aivokuoren kokonaisvaltaisen skaalautumisen lisäksi ollaan kiinnostuneita psykofyysisten ja hermostollisten vastaavuussuhteiden jakaumamallista tietyille aivoalueille. Mitattujen hermostollisten signaalien paikantaminen tarkalleen tietyille aivokuoren alueille aiheuttaa käänteisen ongelman, joka on ratkaistu tässä MNE -lähdemallintamisella. Lähdemallintamisen algoritmit tuottavat todennäköisimmän mallin aivokuoren alueista, joiden aktiivisuudella voidaan selittää mitatut MEEG signaalit. Mallintaminen on työn kriittinen vaihe, koska sillä yhdistetään neuroanatominen tieto fysiologisen ja psykofyysisen tiedon kanssa. Yksilötason data on käsitelty lopuksi ryhmätasolla tilastollisin menetelmin korrelaatiotulosten merkittävyyksien arvioimiseksi.
  • Sundman, Elina (Helsingin yliopisto, 2020)
    Hypothyroidism affected 337 370 people in Finland in 2019. The hypothalamus-pituitary negative feedback loop is used in the diagnostics of hypothyroidism. TSH, a pituitary hormone, is the most used diagnostic tool with the free thyroxine (T4) in a supporting role. L-T4 has been the main treatment option, since the discovery of peripheral deiodination. Biochemical and clinical euthyroidism is the aim of L-T4 therapy. There are no nationwide official treatment guidelines for hypothyroidism in Finland. The Finnish Endocrine Society has published their recommended guidelines in 2019. Studies have shown that 5–15 % of levothyroxine treated patients continue to report symptoms when they are biochemically euthyroid. The symptoms consist of typical symptoms of hypothyroidism: fatigue, cognitive symptoms, depression, anxiety and weight gain. The molecular basis of the symptoms is not yet known. These symptoms have notbeen studied in Finnish population. The aim of this study was to find out what symptoms levothyroxine treated patients report on Finnish social media support groups and view the current treatment of hypothyroidism and guidelines of treatment. This study was conducted by surveying patient-reported information from social media hypothyroidism related support groups. The posts were divided into two groups: hypothyreotic and athyreotic. The biochemical data and symptoms were collected from 137 posts in the athyreotic group and 191 in the hypothyreotic group. Only posts with TSH under or within the refence range and free T4 within the reference range wereincluded in the study. The results show that patients reported symptoms in 74 % of the posts viewed. 81 % of patients reported symptoms in the hypothyroidism group and 64.2 % in the athyreotic group. The most reported symptom in both groups was fatigue. Symptoms were reported within the normal TSH range (0.5-4 mU/l) and below the normal range (<0.5 mU/l). In asymptomatic patients the median TSH was 0.38 mU/l in the athyreotic group and 0.54 mU/l in the hypothyreotic group. Free T4 seems to be a little higher in the asymptomatic patients in both groups. 16.6-25 % the patients reported that they had had Free T3 measured. Free T3 seem to be higher in relation to free T4 in the athyreotic group that reported having no symptoms. L-T4 is the recommended treatment modality for hypothyroidism. Other options are synthetic combination treatment with L-T4 + L-T3 and desiccated thyroid extract (DTE).This study supports the view that hypothyroidism patients can have symptoms on levothyroxine treatment
  • Kalha, Solja (Helsingin yliopisto, 2020)
    Cornea is the outermost surface of the eye that refracts light to the lens and protects the sensitive ocular machinery. The cornea is divided to three cellular compartments; epithelium, stroma and endothelium. Our work focuses on the corneal epithelium, which is located closest to the tear film and, together with the film, forms a physiological barrier to pathogens and small particles from the environment. We followed the maturation of the mouse corneal epithelium from birth to adulthood and discovered a novel marker, Krt19, in this process. Krt19 expression gradually restricted from the central cornea to the limbus, concomitantly with eyelid opening and epithelial stratification, which are the hallmarks of postnatal maturation of the murine cornea. Corneal epithelium is renewed continuously throughout life by stem cells. Previous studies demonstrated that the limbus, located in the periphery of the cornea, houses the corneal stem cells. Immediate progeny of the stem cells, the progenitor cells, localize to the limbus, peripheral, and central cornea. We identified the gene Bmi1 in the corneal, epithelial progenitor cells. By lineage tracing of the Bmi1+ cells, we followed renewal dynamics in the central cornea and estimated the turnover of the epithelium to be 2-8 weeks in adult mice. However, we noticed a decrease in renewal rate with older animals. This is in line with evidence from renewal studies of the limbal stem cells, suggesting a general decrease of corneal epithelial renewal upon aging. We optimized a method to perform in vivo epithelial abrasion injury on mouse cornea. The development of this assay was instrumental for the experiments that followed. Using the abrasion model, we showed that the Bmi1+, central, corneal progenitor cells do not contribute to wound healing. Instead, the wound closed by rearrangement and migration of the remaining epithelial cells. We extended our analysis of the corneal barrier to encompass an accessory organ of the eye, the lacrimal gland (LG, tear gland). LG produces and secretes the aqueous part of the tear film, which is the largest portion of the film. The tear film provides another layer of protection to the ocular surface, because it contains anti-inflammatory and antimicrobial components as well as assists eyelid movements. We studied the role of Ectodysplasin-A (Eda) gene in the LG. Eda is critical in the development of ectodermal appendages, however LG development was not affected by the loss-of-function mutation in Eda. Instead, lack of EDA resulted in modulation of LG secretion and the development of a dry eye disease (DED). Furthermore, we discovered that Eda signalling activity was inhibited in response to corneal injury and suggest that this is necessary for the production of reflex tears that are released in ocular insult. In this assay, we shed light on the cooperation between cornea and the LG in homeostasis and injury. Our work is part of the research that aims to understand maturation and homeostatic maintenance of the anterior segment of the eye, cornea and the LG. This work provides new information regarding the development of Eda-linked DED. This is of importance, because the DED affects a large part of the population. Furthermore, we call for further studies on the mechanisms of how these two tissues communicate, as they are intricately linked and dependent of each other.
  • Kalha, Solja (Helsingin yliopisto, 2020)
    Cornea is the outermost surface of the eye that refracts light to the lens and protects the sensitive ocular machinery. The cornea is divided to three cellular compartments; epithelium, stroma and endothelium. Our work focuses on the corneal epithelium, which is located closest to the tear film and, together with the film, forms a physiological barrier to pathogens and small particles from the environment. We followed the maturation of the mouse corneal epithelium from birth to adulthood and discovered a novel marker, Krt19, in this process. Krt19 expression gradually restricted from the central cornea to the limbus, concomitantly with eyelid opening and epithelial stratification, which are the hallmarks of postnatal maturation of the murine cornea. Corneal epithelium is renewed continuously throughout life by stem cells. Previous studies demonstrated that the limbus, located in the periphery of the cornea, houses the corneal stem cells. Immediate progeny of the stem cells, the progenitor cells, localize to the limbus, peripheral, and central cornea. We identified the gene Bmi1 in the corneal, epithelial progenitor cells. By lineage tracing of the Bmi1+ cells, we followed renewal dynamics in the central cornea and estimated the turnover of the epithelium to be 2-8 weeks in adult mice. However, we noticed a decrease in renewal rate with older animals. This is in line with evidence from renewal studies of the limbal stem cells, suggesting a general decrease of corneal epithelial renewal upon aging. We optimized a method to perform in vivo epithelial abrasion injury on mouse cornea. The development of this assay was instrumental for the experiments that followed. Using the abrasion model, we showed that the Bmi1+, central, corneal progenitor cells do not contribute to wound healing. Instead, the wound closed by rearrangement and migration of the remaining epithelial cells. We extended our analysis of the corneal barrier to encompass an accessory organ of the eye, the lacrimal gland (LG, tear gland). LG produces and secretes the aqueous part of the tear film, which is the largest portion of the film. The tear film provides another layer of protection to the ocular surface, because it contains anti-inflammatory and antimicrobial components as well as assists eyelid movements. We studied the role of Ectodysplasin-A (Eda) gene in the LG. Eda is critical in the development of ectodermal appendages, however LG development was not affected by the loss-of-function mutation in Eda. Instead, lack of EDA resulted in modulation of LG secretion and the development of a dry eye disease (DED). Furthermore, we discovered that Eda signalling activity was inhibited in response to corneal injury and suggest that this is necessary for the production of reflex tears that are released in ocular insult. In this assay, we shed light on the cooperation between cornea and the LG in homeostasis and injury. Our work is part of the research that aims to understand maturation and homeostatic maintenance of the anterior segment of the eye, cornea and the LG. This work provides new information regarding the development of Eda-linked DED. This is of importance, because the DED affects a large part of the population. Furthermore, we call for further studies on the mechanisms of how these two tissues communicate, as they are intricately linked and dependent of each other.
  • Harmoinen, Katri (Helsingin yliopisto, 2019)
    New Zealand is an isolated landmass laying in the Southwest Pacific waters, far away from any major islands or continents. It was the last major landmass to be colonized by people, discovered by the first Polynesian explorers around a thousand years ago. Historically, New Zealand lacked all native mammals (apart from three species of bats) and so has developed a plethora of bird species and other endemic wildlife. The absence of mammalian predators, combined with the continuous isolation for millions of years, has led the evolution of some very unique and charismatic species. One of these species is the iconic symbol of New Zealand – the kiwi (Apteryx spp). The biggest challenge to the New Zealand wildlife has been the introduction of mammalian species to the New Zealand ecosystem. There are 25 species of introduced mammals in New Zealand today that are regarded as pests. The devastation caused by these species is the main cause for the dramatic decline of the endemic New Zealand wildlife, including the iconic kiwi. Nationally, kiwi continue to decline by more than 2% annually and there are estimates of the species going extinct from the wild within 50 years. Since the first more permanent human settlement, more than 50% of the New Zealand breeding birds have gone extinct. In this thesis, the relation between kiwi and introduced mammalian species around the township of Whakatāne, New Zealand, was studied. During summer 2018-2019, three out of eight monitored kiwi chicks were predated by a suspected mustelid/mustelids and DNA swabs were obtained from the bite sites. Volunteer pest trappers were then asked to bring in all their catches in an attempt to catch the individual/individuals responsible for the predations. Molecular tools including microsatellites were used to create ID profiles in an attempt to match the profiles to those obtained from the kiwi chicks. In the second part of the study, the stoats’ stomachs were analysed as part of a diet study. A new, kiwi specific DNA probe was trialled and the remaining stomach contents were sequenced for other native wildlife species. Out of the three predated kiwi chicks, all of them were confirmed to be stoat predations. Unfortunately, none of the stoat ID profiles obtained matched the profile of the kiwi chick Ranui who was the only chick a good micro-satellite profile was obtained for. This confirmed that the stoat/stoats responsible for the predation of Ranui was not caught as part of this study. In the diet part of this thesis, we trialled the kiwi specific probe but could not identify any kiwi DNA in the stoat stomach contents. The DNA sequencing however revealed five other species: tomtit (lat. Petroica macrocephala, 100%), common chaffinch (lat. Frigilla coelebs, 100%), tui (lat. Prosthemadera novaseelandiae, 96%), European hare (lat. Lepus europaeus,100%) and copper skink (lat. Cyclodina aenea, 100%). These findings shed new light on the extent introduced mammalian species contribute to the species loss taking place in the New Zealand forests today. The use of molecular techniques and tools in conservation offers an often faster, cost-efficient and more reliable alternative to traditional monitoring methods of introduced species. The rapid development of these tools has seen New Zealand taking critical steps towards one day becoming predator free. The ambitious goal to rid New Zealand of target introduced species (mustelids, possums and rats) by year 2050 (Predator Free 2050), has been compared as the New Zealand equivalent of putting the man on the moon.
  • Lankinen, Tuuli (Helsingin yliopisto, 2020)
    Our hearing perception is based on the ability to discriminate mechanical sound waves and to amplify and transduce them into electrical stimuli.This function is based on the complex cellular organization of the cochlea, the hearing organ. The sensory epithelium in the organ of Corti spirals along the cochlear duct in a tonotopic arrangement: every sound frequency elicits the strongest response at allocation along this duct. Sound stimulus is detected by three rows of outer hair cells (OHCs) which amplify- and tone-discriminate the sound stimulus, and by one row of inner hair cells (IHCs), which transduce the mechanical stimulus into electric impulses. Basal regions of the cochlea detect high- frequency sounds and apical regions detect low- frequency sounds. The complexity and sensitivity of the cochlea is linked with its vulnerability to various traumas. Most kinds of damage to the mammalian hair cells is irreversible, because these cells are not capable of regeneration. Hearing impairment has many etiologies. Common to them is that damage is permanent and no pharmacotherapy is available. Hearing impairment is often a disabling condition and it has vast societal consequences. The number of hearing impaired people is constantly increasing and the WHO has estimated that 10% of the world`s population will suffer from disabling hearing loss in 2050. Mesencephalic astrocyte- derived neurotrophic factor (MANF) is an unconventional, ER-resident protein that promotes ER- homeostasis. It has been associated with cytoprotective functions in many neurodegenerative disease- models and shown to promote recovery after ischemic trauma. MANF expression has been previously found in many cell-types in the cochlea, including OHCs and IHCs. Its deficiency in a mouse model led to upregulation of ER-stress markers and a robust, tonotopic base –to apex gradient loss of outer hair cells and severe hearing loss. This study examines the role of MANF in noise-induced trauma in the hair cells of the cochlea. In a conditionally inactivated (Manf -/- cKO) mouse model in the C57BL/6J – background, where Manf has been inactivated from most of the cochlear cells, I studied, if Manf -deficiency sensitizes the cells to noise-induced cell death in two age-groups. I also examined the basic and noise- induced MANF expression, using two mouse- strains, C57BL/6J and CBA/Ca. I also examined OHC stereociliary bundle morphology to find out if noise induces morphological changes in Manf cKO-mice that differ from noise-exposed C57BL/6j wild type mice. This study found that OHCs have a low MANF- expression, whereas in IHCs the expression is strong. MANF is expressed in a base- to apex gradient in the OHCs of the two mouse-strains examined, in a uniform pattern, that correlates with vulnerability, implicating that low levels of MANF predispose basal OHCs to vulnerability. MANF expression in the IHCs was non-gradiental. Noise did not induce upregulation, as was expected, but instead noise induced downregulation of MANF in the basal region of the OHCs by an unknown mechanism in both mouse-strains.This suggests that noise-induced trauma induces ER dyshomeostasis, possibly independent of ER stress response pathways ,unfold protein response (UPR). This study also demonstrates that MANF deficiency sensitizes the OHCs to noise- induced trauma, resulting in more elevated OHC loss and hearing thresholds. This sensitization is mainly caused by a progressive degenerative changes seen in the OHC stereociliary bundles of Manf cKO-mice, and is associated with more severe noise-induced hearing loss. The results of my study suggest that MANF has an important, yet unknown, protective role in noise-induced trauma in OHCs. These results support the possible role of MANF as a therapeutic agent in a noise-induced trauma.
  • Tentke, Annika (Helsingfors universitet, 2014)
    This project was about the molecular mechanisms involved in the generation of eicosanoids in human mast cells with particular emphasis on lipid bodies as a source and/or site of lipid mediator biogenesis. The cells to be used are isolated from human peripheral blood provided by Finnish Red Cross Blood Transfusion Service and collected from healthy donors. Human mast cells are found in connective tissue. They contain granules filled with histamine, heparine and proteases. Human mast cells are potent effector cells in host-defense mechanisms of innate immunity, including inflammatory diseases such as atherosclerosis. Activation of mast cells by different stimuli triggers the release of a huge range of mediators, including de-novo synthesized eicosanoids, which are highly biologically active lipid mediators. The major eicosanoid released by activated mast cells is prostanoid prostaglandin D2 (PGD2). The aim of this project was to find out whether mast cell lipid bodies are the cellular compartments of PGD2 synthesis, what are the enzymes involved in AA liberation from TGs, and whether TG-derived AA is a source for PGD2 production. The enzymes of special interest were hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL). We were also interested about hematopoietic prostaglandin D synthase (HPGDS), the key enzyme in the production of D and J series of prostanoids. Methods used in this pro gradu work include siRNA transfections, RNA isolation, cDNA synthesis, qPCR, immunoblotting, ELISA and conventional fluorescence microscopy. Immediate increase in the amount of PGD2 released from mast cells sensitized with human IgE (1 µg/ml) and activated by polyclonal rabbit anti-human IgE (1 µg/ml) was observed. The increase was most prominent after one hour of activation, and slowly decreased to basal levels at 48 h post-activation. siRNA transfection affected the amount of enzyme DNA in mast cells and the amount of PGD2 released. HSL, ATGL and HSL+ATGL double knockdowns all reduced the amount of PGD2 released in acute (5 to 30 minutes) term activation compared to control cells. However, no significant changes were observed in the mRNA expression levels of ATGL, HSL, CGI-58, HPGDS or COX-1 under mast cell activation. The only significant changes in mRNA expression levels were observed with COX-2. However, the relative expression of HPGDS increased in IgE treated mast cells compared to control treated cells and the expression was even greater in mast cells treated with αIgE also. Both ATGL and HPGDS were recognized throughout the cytosolic area in the non-activated Ctrl cells. Although HPGDS located also in the circumference of mast cells, no clear localization of HPGDS was observed in the circumference of mast cell lipid droplets. The experiments carried out at the Wihuri Research Institute, including those presented here, have established that, in addition to phospholipids, the triglycerides present in mast cell lipid droplet core are also an important source of eicosanoids, and that also ATGL and HSL, not just cPLA, can release arachidonic acid for eicosanoid production. The ramifications of this study include the possibility that arachidonic acid release from triglycerides for the formation of eicosanoids could take an indirect or a direct route to supply precursors for cellular eicosanoid biosynthesis. The key is the pathway of AA release. In the direct pathway, AA is released from LD TGs by ATGL or HSL and this free AA is used for the generation of PGs by either COX-1 or COX-2, depending on the status of the cell. In the indirect pathway, AA is liberated from LD TGs by ATGL or HSL and then further re-esterified into phospholipids from where AA is then finally released by cPLA2 for the generation of eicosanoids.
  • Hannukainen, Riikka (Helsingfors universitet, 2013)
    Työssä tutkittiin rasvahappojen kerrostumista itämerennorpan (Phoca hispida botnica) traanissa ja tämän merkitystä rasvahappokoostumukseen perustuvassa ravintokohteiden arvioinnissa. Vertaamalla sisätraanin rasvahappokoostumusta plasman ja maksan rasvahappokoostumuksiin pyrittiin selvittämään siirtyvätkö jotkut tietyt ravinnon rasvahapot toisia tehokkaammin traanin sisäosiin, eli heijastelevatko jotkut sisätraanin rasvahapoista ravinnon rasvahappokoostumusta toisia paremmin. Itämerennorpan traanin eri kerrosten rasvahappokoostumusta verrattiin myös sen tärkeimmän ravintokalan, Itämeren silakan rasvahappokoostumukseen, jotta nähtäisiin minkä kerroksen koostumus muistuttaa eniten ravinnon rasvahappokoostumusta. Vertailun vuoksi työssä tutkittiin myös makeassa vedessä elävän saimaannorpan (Phoca hispida saimensis) traanin ja maksan rasvahappokoostumuksia. Lisäksi määritettiin plasma- ja maksanäytteiden kuljettaman tai lyhytaikaisesti varastoiman varastorasvan määrät. Tutkimuksessa käytettiin Perämerellä ammutuista itämerennorpista kerättyjä traani-, maksa- ja plasmanäytteitä, sekä kuolleina löydetyistä saimaannorpista kerättyjä traani- ja maksanäytteitä. Kudosnäytteiden rasvahappokoostumusten määritys tehtiin analysoimalla niistä valmistettuja rasvahappojen metyyliesteriseoksia kaasukromatografisesti (GC). Lipidiluokkakoostumukset puolestaan määritettiin korkean erotuskyvyn ohutlevykromatografialla (HPTLC). Analyysien tuloksia käsiteltiin tilastollisesti pääkomponenttianalyysin (PCA) ja sen tuloksia ohjatusti luokittelevan menetelmän (SIMCA) avulla, regressioanalyysillä, sekä laskemalla koostumusten euklidisia etäisyyksiä eri näytteiden välillä. Jokaisen yksilön traanille luotiin vertikaalinen rasvahappoprofiili toisiaan nahasta lihakseen seuraavien osanäytteiden rasvahappokoostumuksen perusteella. Itämerennorpan traanin kerrostuneisuutta tutkittiin nyt ensimmäistä kertaa ja sen havaittiin olevan rakenteeltaan kerrostunut, kuten on havaittu myös aiemmin tutkituilla kahdella norpan alalajilla. Aiemmista tutkimustuloksista poiketen keskitraani ei kuitenkaan eronnut rasvahappokoostumukseltaan merkitsevästi muista traanikerroksista. Sisä- ja ulkotraanin väliset rasvahappokoostumuksen erot olivat sen sijaan merkitseviä. Traanikerroksista sisätraani muistutti eniten itämerennorpan tärkeän saalislajin, silakan, rasvahappokoostumusta. Itämerennorpan kudosten rasvahappokoostumus erosi selvästi saimaannorpan kudosten rasvahappokoostumuksista. Näiden kahden alalajin ulkotraanit kuitenkin muistuttivat toisiaan rasvahappokoostumukseltaan sisätraaneja enemmän, mikä viittaa siihen, että niiden ulkotraanin koostumusta säätelevät samankaltaiset lämmönsäätelyyn liittyvät geneettisesti määräytyvät tekijät. Rasvahappokoostumusten alalajikohtaisista eroista huolimatta traanin rasvahappojen kerrostumistapa oli samanlainen molemmilla tutkituista alalajeista. Traanin vertikaaliset rasvahappoprofiilit olivat kuitenkin hyvin yksilöllisiä. Useiden rasvahappojen suhteelliset määrät plasmassa ja sisätraanissa korreloivat tilastollisesti merkitsevästi keskenään. Tämä tulos vahvistaa oletuksen, että viimeaikaisella ravinnolla on vaikutusta erityisesti sisätraanin rasvahappokoostumukseen ja, että sen perusteella voidaan saada tietoa eläimen ravinnosta. On kuitenkin huomattava, että tietyt sisätraanin rasvahapot ilmentävät ravinnon rasvahappokoostumusta toisia paremmin. Vaikka traanien vertikaaliset rasvahappoprofiilit antavat mitä ilmeisimmin yksilökohtaista tietoa eläinten ravinnosta ja aineenvaihdunnasta, niitä ei ole pystytty tulkitsemaan aiemmissa tutkimuksissa kovinkaan syvällisesti. Nämä tulokset voivatkin osaltaan auttaa tulkitsemaan traanin vertikaalisia rasvahappoprofiileja tulevaisuudessa. Saatuja tuloksia voidaan lisäksi hyödyntää myös hylkeiden ravintokohteiden arvioinnissa käytettävien näytteenottoprotokollien suunnitteluun ja kehittämiseen.