Browsing by Subject "Magisterprogrammet i neurovetenskap"

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  • Doutel Figueira, Joana Filipa (Helsingin yliopisto, 2022)
    The general question of this research is how beta oscillations are implicated in stopping an ongoing movement. Previous studies regarding movement cancellation have found a significant increase in beta activity in sensorimotor areas, especially in the form of transient increases in beta oscillations, called beta bursts. However, the functional role of beta band activity in stopping is still unclear, mainly because the behavioural tasks used cannot measure the exact timing when the subjects start the stopping process and therefore it is only possible to infer the stopping time. To resolve this, we used head-fixed rats running on a treadmill while performing a Go/NoGo task. In some NoGo trials, the rat starts to run, realizes the mistake and stops before crossing a fixed distance threshold. These are the events being analyzed, called near-mistake events (N=39,366). We found a single beta burst occurring prior to stopping in all five brain regions analyzed (from 44.2±20.1 ms to 55.8±16.0 ms) and positive correlations of beta burst number and power increase with movement speed before stopping. We also found a single alpha burst prior to and during stopping in all brain regions (from 45.9±20.1 ms to 57.1±19.3 ms), supporting previous studies’ findings of alpha band involvement in inhibitory motor actions. Our findings on beta bursts suggest a causality role in stopping an ongoing movement while our results of alpha bursts need to be further analyzed to understand its functional role.
  • Enberg, Emma (Helsingin yliopisto, 2021)
    Kiinnostus käyttää psykedeelejä, kuten lysergihapon dietyyliamidia (LSD) ja psilosybiiniä, erinäisten psykiatristen sairauksien hoidossa ei ole jättänyt huomiotta päihteiden väärinkäyttöä. Tutkimukset ovat osoittaneet alustavia positiivisia vaikutuksia LSD:n käyttämisessä erinäisten addiktioiden, kuten kokaiini-, nikotiini- ja alkoholiriippuvuuksien hoidossa. LSD:n on raportoitu auttaneen joitain alkoholismista kärsiviä pysymään raittiina jopa 6-12 kuukautta yksittäisen LSD annoksen jälkeen. Valitettavasti näitä tuloksia on hankala tulkita, ja vaikutusten taustalla olevat mekanismit tunnetaan huonosti. Tutkimme hiirimallimme avulla, kuinka yksittäinen LSD annos vaikuttaa ahmimiskäyttäytymiseen. Käytimme sukroosiliuosta ahmivaa eläinmallia palkkionottamiskäyttäytymisen mallintamiseen, mikä on yksi addiktioihin liittyvän käyttäytymisen tunnusmerkeistä. Tutkimuksemme tavoitteena oli selvittää vaikuttaako LSD palkkionottamiskäyttäytymiseen, ja siten mahdollisesti aivojen palkkiojärjestelmään. LSD -annostelu (0,05 ja 0,1 mg/kg, i.p.) vähensi akuutisti sukroosiliuoksen ahmimiskäyttäytymistä, mutta vaikutus loppui viikon kuluessa. Tästä havaitusta akuutista vaikutuksesta huolimatta erot ryhmien välillä eivät olleet tilastollisesti merkittäviä. Täten oletettiin, että nettovaikutukset aivojen palkkiojärjestelmään ovat epätodennäköisiä. Kuitenkin pelkän i.p. injektion (10 ml/kg) havaittiin vaikuttavan veden juomiseen. Havaitsimme merkittävän piikin veden juonnissa injektointipäivänä, mikä palautui normaalitasolle jo seuraavaan päivään mennessä. Nämä tulokset johtivat jatkotutkimukseemme, jossa osoitettiin injektion aiheuttavan piikin vedenjuontiin riippumatta siitä, injektoidaanko saliinia vai LSD:tä. Tätä vaikutusta ei enää havaittu, mikäli injektioita annettiin perättäisinä päivinä, mutta jopa yhden tai kahden päivän väli injektioiden välillä riitti palauttamaan injektion aiheuttaman piikin vedenjuontiin. Koska onnistuimme poistamaan vedenjuontiin aiheutuneen vaikutuksen toistetuilla saliini-injektioilla, eikä vaikutus palautunut injektoitaessa LSD:tä, voimme todeta, että vaikutus liittyi injektiotoimenpiteeseen. Keskeisin havaintomme tässä tutkimuksessa oli, ettei LSD:llä ole merkittävää akuuttia vaikutusta sukroosiliuoksen ahmimiskäyttäytymiseen tässä hiirimallissa.
  • Kallo, Henna (Helsingin yliopisto, 2021)
    During the brain development, GABAergic neurons, also referred as interneurons, migrate tangentially from the subpallium to the pallium. After intracortical dispersion, the interneurons start radial migration towards their final location in the cortex. Although the radial migration of interneurons is extensively studied, mechanisms guiding the migration remain relatively unknown. Here we studied how manipulation of cortical activity affects the radial migration and allocation of the cortical GABAergic neurons in the developing mouse brain. For this purpose, we utilized whisker trimming induced sensory deprivation in GAD67-GFP mice at postnatal days 2-5 (P2-P5) followed by cell counting in brain slices derived from P5 and P10-aged mice. In addition, we performed live-imaging of migrating neurons in organotypic cultures derived from P2 SST-TdTomato and 5HT3aR-GFP mice and cultured for 1 day in vitro. These two mouse lines roughly represent early- and late-born subpopulations of the GABAergic neurons. Live-imaging was accompanied by activity manipulations using different drugs and the Designer Receptors Exclusively Activated by Designer Drugs (DREADD) technology. Analysis of the interneurons’ allocation on the barrel cortex after the unilateral sensory deprivation revealed misallocation of GAD67+ neurons on deep cortical layers of the contralateral hemisphere of the ablation group at P5. Analysis of the tracks from the live-imaged migrating interneurons revealed altered saltatory movement behaviour of 5HT3aR+ interneurons when clozapine-N-oxide (CNO) was used to activate the electroporated GFP-GCaMP3-mCherry-hM3Dq neurons located on L2/3 of the cortex. Moreover, we observed reduced motility of migrating interneurons in the organotypic cultures treated with a KCC2 inhibitor that alters GABAA-receptor mediated transmission. Altogether, our results suggest that activity is important in promoting the radial migration of late-born interneurons during the first days of the postnatal development.
  • Paranko, Birgitta (Helsingin yliopisto, 2019)
    Introduction and aims. Multiple different neurobiological alterations have been hypothesized to underlie Major Depression Disorder (MDD), but no unifying theory exists to explain the mechanisms of the disorder. The aberrant brain dynamics in MDD can be seen in the alterations of long-range temporal correlations (LRTCs), which have been proposed to be an indication of criticality in healthy brain. Alterations in LRTCs have been suggested to reflect deficiencies in excitation-inhibition (E/I) balance, neuromodulation or connectivity patterns, which have also been proposed to be the underlying mechanisms of MDD. There has been controversy whether the pathology is related to attenuated or increased LRTCs, and the sources of altered brain dynamics have not yet been localized. The aim of this study was to find in which frequency bands and where in the brain the neuronal LRTCs are altered in MDD on source level. In addition to analyzing the correlations between neuronal LRTCs and depression severity in parcel level, we studied correlations in functional networks to get a better understanding of the system level alterations in MDD. We also studied whether behavioral LRTCs correlate with depression severity or with behavioral performance. Methods. We investigated the long-range temporal correlations in a cohort of 19 depressed subjects by using magnetoencephalography (MEG) for recording brain activity during resting state and response inhibition task and performed DFA analysis on the amplitude envelopes of cortical oscillations. The depression severity was measured with BDI-21 questionnaire. Results and conclusions. We found the LRTCs to be positively correlated with depression severity in the alpha frequency band (8–12Hz) predominantly in the limbic system that underlies emotional control. This result was supported by the parcel level analysis in which correlations between alpha band LRTCs and depression severity were observed in the orbitofrontal cortex and temporal pole, indicating that the hyper-activation of limbic system could explain the negative bias characteristic to depression. Positive correlations were also found in frontoparietal, ventral, and dorsal attentional networks that support cognitive control. Alpha band LRTCs correlated also with behavioral LRTCs during both resting state and task conditions. However, we observed more wide-spread correlations between alpha range LRTCs and depression severity than between neuronal LRTCs and behavioral LRTCs. Behavioral LRTCs correlated with depression severity, but not with behavioral performance. These results indicate that depression is characterized by vast alterations in the brain dynamics and imply that the wide range of different symptoms in MDD could be explained by alterations in the excitation/inhibition balance in the limbic system and cognitive networks.
  • Llach Pou, Maria (Helsingin yliopisto, 2019)
    Parvalbumin (PV) interneurons are GABAergic inhibitory neurons that shape neuronal network activity and plasticity. They are involved in both developmental and adult plasticity and have recently been divided into subpopulations that differ in birthdate, intrinsic properties and are involved in different types of learning; while late born PV neurons, expressing low levels of PV, are required for the acquisition of new information, early born PV neurons, expressing high levels of PV, are involved in the consolidation of the information. PV cells can be enwrapped with perineuronal nets (PNNs), an extracellular matrix structure that stabilizes synapses and indicates a mature state of the cell. The development of PNNs correlates with the closure of critical period of plasticity in development, and the enzymatic removal in adulthood can reopen those periods. Similarly, antidepressants like fluoxetine have been proven to reopen critical periods of plasticity in adulthood (iPlasticity) and decrease PNN structures in PV cells. However, whether the effect of fluoxetine is restricted to a subpopulation of PV interneurons is unknown. In addition, no previous studies have yet investigated the maturity state of the PV subpopulation by analyzing its PNN structures. In this thesis we aimed to elucidate differences in the maturity state of the subpopulations and the fluoxetine effect in those. To do that, we treated a cohort of adult mice with a chronic fluoxetine treatment previously reported to be capable of the reopening of critical periods. Following, we performed an immunohistochemistry analysis to detect PV and PNN levels in the CA3b hippocampal area. In addition, our mice line expressed TdTomato (TdT) in PV cells which allowed a more sensitive detection of PV neurons. After imaging the slices with a confocal microscope, we analyzed the PV and PNN intensity both by manual counting and with a semi-automatic macro script in ImageJ software that we developed and validated. The PV intensity of control mice was used to divide the cells in two groups; low PV and high PV expressing cells. PNNs in those subpopulations in both the control and fluoxetine treated group were analyzed and statistically compared. The low PV subpopulation showed a significantly low PNN intensity compared to the high PV subpopulation, indication a plastic or immature low PV subpopulation and a mature or consolidated high PV subpopulation. Interestingly, fluoxetine selectively decreased the PNN structures in the high PV subpopulation, by bringing the PNN intensity to comparable levels found in the low PV network. No effect of fluoxetine in the low PV network was detected. Fluoxetine induced a change towards a plastic state in the network believed to be involved in memory consolidation by decreasing its PNNs structures. This discovery gives new insights on the understanding of antidepressant plastic actions, suggesting that a chance for strong memories to change could be facilitated with the drug, and explain the antidepressant’s effects when combined with psychotherapy. However, supplementary experiments to compare and define PV subpopulations and a confirmation of the selective effect of fluoxetine are needed to confirm the preliminary hypothesis suggested by our data.
  • Srinivasan, Rakenduvadhana (Helsingin yliopisto, 2019)
    Caged photolysable compounds have served to be pivotal to neuroscientific investigations; allowing the cognizing of molecular kinetics and properties of neuronal micro-machinery such as neurotransmitter receptors. Precision in terms of temporal and spatial resolution of neurotransmitter release endowed by photolysis has multitudinal applicabilities in the realm of GABAA receptors (GABAARs), their neuronal niche and effects on neuronal and network activity. Caged compounds, in their caged form, may display certain unideal traits such as undesired interactions with the system and antagonistic activity on the target receptor. This study aims to reevaluate the GABAAR antagonistic actions of caged Rubi-GABA, which was found to antagonize these receptors at significantly lower concentrations than those reported in the literature. Furthermore, this study electrophysiologically characterizes the possible antagonistic properties of a novel quinoline-derived UV-photolysable caged GABA compound, 8 DMAQ GABA, whose activity, in its caged form appears to have a much more favorable antagonism profile compared to the widely used RuBi-GABA. To assess the antagonistic effects of these compounds on GABAAR-mediated miniature inhibitory postsynaptic currents (mIPSCs) patch-clamp recordings were carried out in the whole-cell voltage clamp configuration on cortical layer 2/3 cortical pyramidal neurons in acute neocortical slices prepared from 16-18 day-old rat rats. The results of this study indicate a revised antagonism profile for caged Rubi-GABA, with marked GABAAR toxicity in the low micromolar range. The study also scrutinizes the photo-kinetic properties of both caged GABA compounds and reveals that the rate of GABA release from 8-DMAQ is slower than from RuBi-GABA.
  • Voipio, Mikko Emil Olavi (Helsingin yliopisto, 2020)
    Nitric oxide (NO) is an important signalling molecule in the brain. NO regulates the function of many proteins by e.g. interacting with tyrosine and cysteine residues, thus inducing post-translational modifications. In animal models, inhibition of NO production triggers behavioural effects similarly to those induced by antidepressant drugs. Receptor tropomyosin-related kinase B (TRKB) has been identified as a key player mediating antidepressant drug (AD) induced effects, and it’s a potential target for NO since it displays multiple potential sites for nitration. Preliminary results from our group indicate that TRKB nitration impairs its signalling, and AD uncouple many proteins from TRKB and reorganizes TRKB protein complex. We examined the effect of selective nitric oxide synthase (NOS) inhibitor N⍵-propyl-L-arginine (NPA) in mice submitted to the contextual fear conditioning and found out that inhibiting NO production with NPA has an antidepressant-like effect on mice. We also found out that AD fluoxetine prevents nitration of TRKB receptors in vivo and antidepressant drugs fluoxetine, phenelzine and imipramine disrupt the interactions of TRKB, NOS1 and NOS1 adaptor protein (CAPON) in co-immunoprecipitation assay. To understand the nature of TRKB and NOS1 interaction, we thus examined the protein domains in NOS1 and TRKB using Uniprot database, and we were unable to identify sites that could interact directly. Literature search for NOS1 adapting proteins followed by Uniprot data mining indicated CAPON as a potential candidate to mediate NOS1: TRKB interaction. Our data shows for the first time that antidepressant drugs disrupt TRKB:CAPON:NOS1 interaction, thus protecting TRKB from NOS1-induced nitration. ADs might induce their behavioural effects by preventing NO-induced impair in TRKB signalling
  • Toissalo, Emilia (Helsingin yliopisto, 2022)
    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective loss of upper and lower motor neurons (MN), which causes progressive muscle weakness and paralysis. ALS leads to death typically from 2 to 4 years after diagnosis. It is important to find more effective treatment options for this devastating disease, as the current treatments can prolong the survival by only a few months. Mesencephalic astrocyte-derived neurotrophic factor (MANF) belongs to an evolutionary conserved neurotrophic factor (NTF) family, whose mode of action differs from classical NTFs. MANF is an endoplasmic reticulum (ER) resident protein and is secreted upon ER stress from the ER, and it can protect the cells from ER stress-induced cell death. MANF has shown to be neuroprotective and –restorative in Parkinson’s disease and stroke rodent models. Adeno-associated viral (AAV) vectors can be used to express therapeutic genes in the target tissues for several months, which lessens the need for repetitive dosing. In this master’s thesis project, we aimed to investigate the neuroprotective effects of intrathecally injected AAV1-MANF gene therapy in a SOD1-G93A mouse model of ALS. We used two different MANF genes; full-length MANF and MANF with deleted ER retention signal (MANF-d), to assess the differences between normal and only secreted MANF. Red fluorescent protein (RFP) was used as a control and to further evaluate the transduction and expression of the viral vectors. Intrathecal injections were performed once on 13 weeks old mice, just before the disease onset. Clinical symptom analyses together with a set of behavioral tests were conducted once a week. Mice were sacrificed at the endpoint of the study when they could no longer use their hind limbs for forwarding propulsion. Immunohistochemical staining was performed on spinal cord paraffin samples, where MN count, microglia activation, and RFP expression were evaluated. AAV1-MANF and AAV1-MANF-d treatments improved the motor behavior of the SOD1-G93A mice one-week post- injection. More specifically, a statistically significant difference was seen in the turning times in the static rods test on two different diameter rods compared to control, but there was no difference between MANF groups. In addition, there was a notable difference between AAV1-MANF and the control group on week 16 rotarod scores. There were no statistically significant differences in other tests, survival of the mice, MN counts, or microglia activation between the groups. RFP expression was detected mainly in the ventral areas of the spinal cord with immunohistochemistry. Our results indicate the potential of MANF gene therapy in the treatment of ALS. Furthermore, we showed that intrathecal AAV1-MANF injections were well tolerated.
  • Niemelä, Miska Aleksanteri (Helsingin yliopisto, 2022)
    Master's thesis project includes the backbone assignment of the human activity-regulated cytoskeleton-associated protein C-lobe (hArc, Uniprot ID: Q7LC44), 7-fluoroindole-based tryptophan-labeling method, and comparing that with the 100% double-labeled and 20%(13C) fractionally labeled samples. The project focuses on the effects of 7-fluoroindole-based fluorotryptophan-labeling. hArc C-lobe has only one tryptophan, which makes the analysis easier. Typically fluorotryptophan-labeling is a costly method – fluorotryptophan itself is very expensive and attaching the fluorine to the tryptophan while expressing is expensive and complicated. Fluoroindolebased labeling circles around the problem, as indole and serine are used in procaryotic systems for tryptophan biosynthesis – meaning that fluoroindole, which is cheap, could be used as an alternative for previous methods. Fluoro-labeled tryptophan is used in protein NMR; for example, in binding studies – fluorine-probes are sensitive, and binding of ligand or protein would move these peaks, indicating binding. This project aims to get an insight into the application of this labeling method. The goal is to see if one could utilize one sample with both (1H, 15N, 13C) labeling and 7-fluorotryptophan labeling for binding and structural studies. However, fluorine is very electronegative, affecting surrounding structures and possibly sequentially nearby amino acids. This possible effect will be observed and determined by comparing the 1H15N-chemical shifts between well-established labeling methods and fluoroindolebased labeling. To determine what amino acids in the protein are affected, if they are affected, will be determined by using the backbone assignment results and the results from the sample comparisons.
  • Garnier Artiñano, Tomás (Helsingin yliopisto, 2021)
    Effective population coding is dependent on connectivity, active and passive postsynaptic membrane parameters but how it relates to information transfer and information representation in the brain is still poorly understood. Recently, Brendel et al. (2020) showed how spiking neuronal networks can efficiently represent a noise input signal. This "D_Model” successfully showed that spiking neural networks can recreate input signal representations and how these networks can be resilient to the loss of neurons. However, this model has multiple unphysiological characteristics, such as instantaneous firing and the lack of units related to physical values. The aim of the present study is to build upon the D_Model to add biological accuracy to it and study how information transfer is affected by biophysical parameters. We first modified the D_Model in the MATLAB environment to allow for the simultaneous firing of the neurons. Using our CxSystem2 simulator in a Python environment (Andalibi et al. 2019), we built a network replicating the one used in the D_Model. We quantified the information transfer of Leaky Integrate-and-Fire units that had identical physiological values for both inhibitory and excitatory units (Comrade class) as well as more biologically accurate physiological values (Bacon class). We used various information transfer metrics such as granger causality, transfer entropy, and reconstruction error to quantify the information transfer of the network. We examined the behaviour of the network while altering the values of the capacitance, synaptic delay, equilibrium potential, leak conductance, reset potential, and voltage threshold. Broad parameter searches showed that no single set of biophysical parameters maximised all information transfer metrics, but some ranges fully blocked information transfer by either saturating or stopping neuronal firing. This suggests theoretical boundaries on the possible electrophysiological values neurons can have. From narrow searches within electrophysiological ranges, we conclude that there is no single optimal set of physiological values for information transfer. We hypothesise that different neuronal types may specialise in transferring different aspects of information such as accuracy, efficiency, or to act as frequency filters.
  • Jalanko, Petri (Helsingin yliopisto, 2021)
    Physical fitness has declined during the last decades in adolescents. Furthermore, several studies have found a positive association between physical fitness and brain volume in adolescents, which is noteworthy since the adolescent brain undergoes substantial changes during growth and maturation. However, despite the importance of the cerebellum on adolescents' cognition and coordination, there remains a paucity of evidence on the associations between physical fitness and cerebellum characteristics. Thus, a cross-sectional approach was used to explore the relationship of cardiorespiratory fitness (CRF), power, speed-agility, coordination and overall neuromuscular performance index (NPI) with total gray matter (GM) volume of the cerebellum as well as lobules VI & VIIb, and crus I volume in 40 (22 girls; 18 boys) adolescents. Peak oxygen uptake (V̇O2peak) was measured by the maximal ramp test on a cycle ergometer, lower limb power was determined with standing long jump (SLJ), speed-agility was assessed with the 10 x 5-m shuttle-run test, upper limb coordination was determined with the Box and Block Test (BBT) and NPI was calculated as the sum of SLJ, BBT and shuttle-run z-values. Lean mass (LM) and body fat percentage (BF%) were measured using a bioelectrical impedance analysis. Cerebellum GM volume, lobules VI & VIIb, and crus I volumes were measured by magnetic resonance imaging (MRI). Results demonstrated that V̇O2peak/LM was negatively associated (β = -.045 P= .014) with cerebellum GM volume. No statistically significant associations were found between SLJ, shuttle-run, BBT scores or NPI and cerebellum characteristics in all participants. However, a poorer shuttle-run time was associated (β = -.363 P = .024) with smaller crus I volume in girls and V̇O2peak/LM was negatively associated (β = -.501 P = .031) with lobule VIIb volume in boys. These findings suggest that, in general, CRF and speed agility are associated with cerebellum characteristics in adolescents and there may be sex differences. The results extend our knowledge of the associations between physical fitness and brain volume, but more studies should be conducted to understand the relationship further.
  • Emre, Dusunceli (Helsingin yliopisto, 2022)
    The degree of neurogenesis in the adult hippocampal dentate gyrus (DG) is the center of the discussion in the field of adult neurogenesis. Although there is an on-going controversy, accumulating evidence suggests that the neural stem cells (NSCs) in the adult human DG are very few. The question remains open as to why there are so few NSCs in the adult human DG when compared with the rodent DG. In order to address these questions, it seems necessary to understand the developmental process of the NSCs in the adult human DG. In this thesis, the neural stem and progenitor cells in the fetal human DG are characterized. In addition to these findings, a semi-automatic method for counting and categorizing cells in their expressions of immunochemistry markers is developed.
  • Rönkkö, Julius (Helsingin yliopisto, 2020)
    Charcot-Marie-Tooth disease (CMT) is a collective name for inherited neuropathies affecting the peripheral nerves. CMT affects 1:2500 children and adults worldwide. The disease is genetically highly heterogeneous, and the pathogenic mechanisms are largely unknown. Thus far, there is no cure known for the Charcot-Marie-Tooth disease. Therefore, the study of the genetic factors involved in the disease and the understanding of the underlying molecular mechanisms will benefit the development of strategies to prevent or treat these diseases. In this thesis, a new candidate gene for CMT was investigated in patient fibroblasts. The novel gene variant was originally found at University of Helsinki in a pair of Finnish brothers with CMT; and in later examinations, in their affected family members. The gene encodes an ER calcium channel receptor that is responsible for Ca2+ release from the endoplasmic reticulum (ER) and plays an important role in the regulation of various cellular processes. In this thesis, I studied the effect of the variant in patient fibroblasts by Western blotting, quantitative reverse transcriptase PCR (RT-qPCR) and calcium imaging. I also knocked down the gene using siRNA in healthy fibroblasts to investigate if the loss of the receptor has a similar effect on calcium signaling as the patient variant. My results showed that siRNA treatment significantly decreased the targeted protein levels and delayed the ATP-evoked Ca2+ release from ER without profound effect on the amplitude of the release. Similar effects of the studied mutation were observed in one patient cell line, but not in the other. Patient cell line, which did not have alterations in the levels of the protein and Ca2+ release, had elevated levels of mRNA of the affected gene. The results suggest that the gene variant does not impair the total volume of the ATP-evoked Ca2+ release from ER. The possible effect of the studied mutation may be related to the decreased levels of the mutated protein, which at the functional level may affect the timing of total Ca2+ release from ER. However, the functional effect of the variant could not be confirmed with the fibroblast cells; further experiments are needed to clearly confirm the variant’s effect on calcium signaling.
  • Aksentjeff, Katri (Helsingin yliopisto, 2020)
    The progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is a neurodegenerative disease caused by loss-of-function mutations in the cystatin B gene (CSTB) with juvenile onset, stimulus sensitive action-activated myoclonus, generalized tonic-clonic seizures and ataxia. The cystatin B (CSTB) protein inhibits cysteine proteases, such as cathepsin L, which has been reported to cleave histone H3 N-terminal tails in mouse embryonic stem cell differentiation. We have shown previously that histone H3 cleavage is an irreversible epigenetic chromatin modification, which occurs in cystatin B-deficient (Cstb-/-) mice derived neural progenitor cells during differentiation. In this study, first, we used the wild-type E13.5 mice brain derived neural cells in culture to determine the effect of extrinsic signaling factors to our earlier developed ex vivo neurosphere cell model. We also confirmed that the histone H3 cleavage positive progenitor cells are primarily neuronal cells. Then, we used phenotype rescue of Cstb-/- neural progenitor cells and showed that CSTB is a negative regulator of histone H3 cleavage. In wt mouse neurosphere cryosections, we showed that cathepsin B and L are not expressed in the nucleus of neural cells before differentiation.
  • Lackman, Madeleine Helena (Helsingin yliopisto, 2022)
    Diabetes mellitus is an incurable disease caused by dysfunctional insulin signaling. The brown adipose tissue (BAT) serves as a hotspot for both lipid and glucose consumption and is thus an attractive target for treating metabolic diseases. Newly surfacing evidence suggest that the endothelial cells (ECs) lining the inner layer of vessels might regulate the morphology and function of adipose tissues. Several studies, including our own, suggest that the vessel density is negatively affected by metabolic diseases. As the BAT is an important organ for systemic lipid and glucose metabolism, and as the effects of metabolic diseases on BAT vessels are not adequately explored, I wanted to investigate how the BAT vasculature changes upon early time points of type 1 (T1D) and 2 (T2D) diabetes in this thesis work. To this end, I used mouse models with chemically induced T1D and genetic T2D and characterized these models with immunohistochemical analyses and immunoassays. To explore the transcriptomic landscapes of ECs and adipose stem cells (ASCs), I analyzed scRNAseq data of BAT stromal vascular fractions (SVF), focusing on changes in gene expression and EC-ASC interactions at a transcriptomic level. Also, by using a publicly available single-cell RNA sequencing (scRNAseq) dataset, I compared BAT SVF gene expression to complement the data resulting from our experiments. The results from this work reveal differential angiogenic responses in the T1D and T2D mouse models and open new avenues of research into how these different pathways are activated and how we can take advantage of these differences to treat diseases. All in all, this work will support the efforts in developing better options for future diabetes prevention, diagnosis, and care.
  • Rappe, Anna (Helsingin yliopisto, 2021)
    Aging is the progressive accumulation of cellular dysfunction, stress and inflammation. The mitochondrial network plays a central role in the maintenance of cellular homeostasis, with a growing body of evidence assigning dysfunctional regulation of this network as cause or effect of age-related diseases including metabolic disorders, neuropathies, various forms of cancer and neurodegenerative diseases. Neuronal sensitivity to changes in energy supply and metabolic homeostasis make neurons especially susceptible to alterations in the mitochondrial network. Mitophagy, a specified form of autophagy, is the selective degradation and quality control mechanism of mitochondria by engulfment and fusion with acidic endolysosomal compartments of the cell. Mitophagy has been extensively characterised in cultured cells and short-lived model organisms. However, our understanding of physiological mitophagy during mammalian aging is unknown. This study utilizes mito-QC mitophagy reporter mice that enable in vivo detection and monitoring of mitochondrial turnover due to the distinct physicochemical properties of the tandem GFP-mCherry reporter. Using cohort groups of young and aged reporter mice, age-dependent alterations of mitophagy were quantified in the cerebellum and the outer nuclear layer (ONL) of the retina. Specific autophagy and mitophagy markers were used to assess the longitudinal alterations in the mitophagic landscape. Images of fixed brain tissue sections were attained by high-speed spinning disc confocal microscopy for the quantitative and histological analysis. This study characterises the longitudinal alterations of mitophagy in distinct regions of the central nervous system (CNS) of mitophagy reporter mice, demonstrating tissue-specific alterations in mitochondrial turnover throughout physiological time. Åldrande kan definieras som den successiva ackumuleringen av cellulär dysfunktion, stress och inflammation. I upprätthållandet av cellens funktioner och homeostas har det mitokondriella nätverket en central roll. Omfattande forskning visar att åldersrelaterade sjukdomar såsom neuropati, ämnesomsättningssjukdomar, olika cancerformer samt neurodegenerativa sjukdomar föranleds av mitokondriell dysfunktion. Neuroner är beroende av oavbruten energitillförsel och upprätthållen metabolisk homeostas, vilket gör dem speciellt mottagliga för förändringar i det mitokondriella nätverket. Mitofagi är en selektiv form av autofagi som degenererar och kvalitetskontrollerar mitokondrier genom att leverera dem till lysosomer där de bryts ned av hydrolytiska enzymer. Den aktuella kunskapen inom regleringen av och mekanismerna bakom mitofagi baserar sig på gedigen forskning av kortlivade organismer och cellkulturer. Däremot är vår kunskap inom åldrandets inverkan på mitofagi i däggdjur begränsad. I denna studie används musmodellen mito-QC vars rapportörgen består av ett binärt GFP-mCherry-komplex som besitter olika fysikaliska och kemikaliska egenskaper, vilket möjliggör upptäckt och analys av mitofagi in vivo. En kvantitativ jämförelse av mitofagi i unga och åldrande möss genomfördes i vävnadssnitt av cerebellum och av det yttre nukleära lagret av retinan. Specifika autofagi- och mitofagimarkörer användes för att utvärdera de longitudinella förändringarna i mitokondriell degenerering. Bilder för kvantitativ och histologisk analys erhölls med höghastighets spinning-disk-konfokalmikroskop. Denna forskning karaktäriserar de longitudinella förändringarna av mitofagi i definierade regioner av det centrala nervsystemet i musmodellen mito-QC och presenterar vävnadsspecifika förändringar i degenereringen av mitokondrier under åldrandets framskridande.
  • Ouabbou, Sophie (Helsingin yliopisto, 2019)
    Tiivistelmä – Referat – Abstract Mental disorders are among the leading causes of global disease burden and years lived with disability. Their pathogenesis is poorly understood and there are enormous challenges in the development of biomarkers to aid in diagnosis and more effective therapeutic options. It has been documented that the microbiota-gut-brain axis shows alterations in mental disorders such as anxiety, depression, autism spectrum disorders, bipolar disorder and schizophrenia. Here we study the gut microbiota of individuals with axis I mental disorders and their unaffected siblings by 16S RNA gene amplicon sequencing. In the Central Valley of Costa Rica, a total of 37 participants were recruited and diagnosed using a Best Estimate Diagnosis protocol. For each of the individuals diagnosed with a mental disorder a healthy sibling was selected after matching by age and gender. A total of 13 pairs of 26 siblings, affected and unaffected, was used for the analysis. In a subsequent analysis, individuals were also divided into the three categories of “unaffected” (UA), “affected without psychosis” (AA) and “affected with psychosis” (AP). They underwent clinical assessments about their habits and diet and about resilience (Connor-Davidson Resilience Scale), current status (SADS-C) and disability (WHODAS 2.0). Their fecal samples were collected freshly and stored at -80°C. DNA was extracted, libraries constructed by PCR and subjected for Illumina MiSeq 300 paired-end 16S RNA amplicon sequencing for analysis of the gut microbiota. The sequencing data were analyzed using the R packages mare and vegan for gut microbiota composition, diversity and richness, taking into account the identified confounders. All participants were of Hispanic ethnicity, residents of the San José Greater Metropolitan Area, adults and 69% of them were women. Affected individuals had major depression, bipolar affective disorder, psychosis non-otherwise specified or schizoaffective disorder. Based on beta-diversity analysis as a measure of the community-level microbiota variation, it was found that the use of levothyroxine (R2=0.08, p=0.005) and of irbesartan (R2=0.068 ,p=0.001) had a significant impact on the microbiota composition and hence the use of these drugs was included as confounder in further analyses. Several statistically significant differences in the relative abundance of intestinal bacteria were identified: Differences were found in the relative abundance of bacterial families Peptostreptococcaceae, Ruminococcaceae, Porphyromonadaceae, and in bacterial genera Pseudomonas, Barnesiella, Odoribacter, Paludibacter, Lactococcus, Clostridium, Acidaminococcus and Haemophilus. Our results indicate that affected individuals have more pro-inflammatory Proteobacteria (Pseudomonas) and less bacteria associated to healthy phenotype, such as Barnesiella and Ruminococcaceae, the former being dose-dependently depleted in AP and AA compared to UA. Furthermore, we documented decreased bacterial richness among affected participants while no significant differences were detected in alpha diversity. Our study identified significant differences in the microbiota of individuals affected by mental illness when comparing to their healthy siblings. The results may have important implications for the holistic understanding of mental health and its diagnosis and therapeutics. Larger studies to confirm these findings would be justified.
  • Tienhaara, Samu (Helsingin yliopisto, 2021)
    In visual detection, thresholds for light increments are higher than thresholds for light decrements. This asymmetry has been often ascribed to the differential processing of ON and OFF pathways in the retina, as ON and OFF retinal ganglion cells have been found to respond to increments and decrements, respectively. In this study, the performance of human participants in detecting spatially restricted (diameter 1.17 degrees of visual angle) and unrestricted increments and decrements was measured using a two-interval forced choice task. Background light intensities ranged from darkness through scotopic to low photopic levels. The detection threshold asymmetry found in earlier experiments was replicated with local stimuli. In contrast, however, the asymmetry between increment and decrement detection thresholds disappeared with fullfield stimuli. An ideal observer model was constructed to evaluate the role of two factors, Poisson variations and dark noise, in determining detection thresholds. Based on the model, these factors are insufficient to account for the increment-decrement asymmetry.
  • Blom, Sonja (Helsingin yliopisto, 2022)
    Pain is a subjective feeling often difficult to interpret or study and thus, pain of those unable to communicate their pain is difficult to recognize. According to the new definition of pain by IASP (Raja et al 2020), verbal description is only one of the many behaviours that can be used to express pain, and the inability to communicate pain does not negate the possibility of experiencing it. This addition to the definition points out that non-human animals, too, even if they cannot express it in words, are capable of both experiencing and communicating pain. Can we as humans interpret a state of pain in an animal in a trustworthy way – and in a manner that would be respectful and non-invasive to the animal? Infrared thermography (IRT) is a technology based on using infrared radiation instead of normal light to form images. These images can be used to quantify the surface temperature of an object with high resolution. The intensity of the radiation emitted by the object being imaged depends on the surface temperature and for this reason thermal imaging enables detecting and measuring changes of surface temperature. Pain and stress might manifest physiologically as activation of the autonomic nervous system, which in turn might result in changes in surface temperatures of the body. These changes might be detectable with a thermal camera. If we could establish a link between certain intricate temperature changes of the head area to certain type of activation of the sympathetic nervous system resulting from pain, thermal imaging could have the potential to detect this. In this study I investigated if there were detectable temperature changes in animal patients before and after a standard examination conducted to each patient admitted to the Wildlife Hospital of Helsinki Zoo, where my data was gathered. Another question was whether the patients that had pain differed in their temperature changes as compared to other patients. The question at the heart of my research was whether there would be a change in peripheral facial temperatures of patients before and after the examination. Another question was whether thermal patterns would be different for pain- and non-pain patients. I found that for some parameters, the temperature differences between pain- and non-pain patients were indeed different, for example the crown temperature of birds seemed to change with examination for patients without pain but not for patients with pain. A more prominent finding was that temperatures decrease across many parameters after an examination as compared to prior to it, across all or many patient groups. My research does not univocally show that thermal imaging could be used to detect pain; rather it affirms the thought that the measurement of changes in peripheral temperatures could be a potential window to non-invasively detect some changes of activation of the sympathetic nervous system in animals.
  • Tervi, Anniina (Helsingin yliopisto, 2020)
    The diversity of different neuronal types lays the foundation for different functions in the brain. The development of different subpopulations and special features of neurons in the central nervous system are still partly unknown. Finding answers to these developmental issues could help in the process of characterisation of cell types and mapping of neuronal networks between the brainstem nuclei in the brain. Previous studies have shown that a ventrolateral neuroepithelial domain in the anterior hindbrain, rV2, produces excitatory (glutamatergic) and inhibitory (GABAergic) neurons, which are related to monoaminergic nuclei in the brainstem (Lahti et al., 2016). In this master’s thesis project, the development of a subpopulation of neurons expressing Gsc2 transcription factor in the interpeduncular nucleus was studied. This project was based on single-cell RNA sequencing results conducted in E13.5 mice. Predicted by single-cell RNA sequencing results, Gsc2 expressing cells are GABAergic interneurons and originate from the rV2 domain of the rhombomere 1 region in the hindbrain. Co-expression pattern with another transcription factor Sall3 with Gsc2 during development was also addressed in the study. Furthermore, the role of Notch signalling in the binary cell fate decision between GABAergic and the glutamatergic fate of rV2 neurons was investigated. Validation of single-cell RNA sequencing results was performed using in situ hybridisation and immunohistochemistry methods with mice embryos at the age of E12.5 and E15.5. This study verified previously shown origin of Gsc2 expressing cells to the rhombomere 1 region and in addition, showed that Gsc2 expressing cells are GABAergic. Co-expression pattern of Gsc2 with Sall3 neither in the rV2 domain nor in the interpeduncular nucleus was seen in our results. In the rV2 domain, the depletion of Notch signalling decreased the expression of differentiating GABAergic neurons. This indicates that Notch has a role in GABAergic neurotransmitter identity during the development of brainstem neurons in mice. Based on our results, Gsc2 could be used as a lineage marker for GABAergic interneurons originating from the rhombomere 1 region and as a marker for a subpopulation of the interpeduncular nucleus. Furthermore, results from the role of Notch signalling could help in discovering the mechanisms related to the determination of neurotransmitter identity in rV2 neurons. Further investigations, in different developmental time points and with additional markers, are needed to verify these results.