Browsing by master's degree program "Magisterprogrammet i neurovetenskap"

Intersectins (ITSNs) are important scaffold and adaptor proteins that play an important role in various cellular processes such as endocytosis. Although we know a lot about their function, there is little information on the regulation of these proteins. On the other hand, microRNAs have been shown to have an extensive function in regulating numerous genes in animals and their dysfunction is credited for down regulation of many proteins. In this study, I demonstrate that microRNAs are potential regulators of ITSNs in HEK293 cells and human neuronal cell cultures. In this study, I cloned 3’UTRs of different isoforms of intersectins (ITSNs) and microRNAs to the expression vectors to express them in cells. I then transfected HEK293T or neuronal stem cell line (HEL47.2) with the constructed vectors and used various methods to analyse the effect of microRNAs on the expression of ITSNs. The main methods I used were dual-luciferase assay, reverse transcription quantitative PCR and western blotting, human neuronal stem cell culturing and lentiviral transduction. My results demonstrate that there were two microRNAs that stood out from other and had a significant downregulation of ITSNs mRNA levels in HEK293T cells. Those were miR-124 and miR-19. However, in the human neuronal cell line I did not observe a significant alteration of the ITSNs transcript level. Additionally, I suggest that the given microRNAs regulate protein levels by promoting the decay of the ITSN transcripts. However, more studies are needed to show a stronger causative effect of microRNAs on ITSNs. Subsequent studies should also look at how multiple microRNAs can influence gene expression cooperatively.

Kalvojännitteen syntyyn ja sen muutoksiin liittyvät prosessit mielletään usein haastaviksi aiheiksi oppia ja opettaa. Aiheen opetuksesta ja oppimisesta lukiossa ei juurikaan ole tehty tutkimusta, mutta aiemmat tutkimukset yliopisto-opiskelijoilla ovat osoittaneet, että haasteita esiintyy erityisesti lepokalvojännitteen muodostumisen ymmärtämisessä. Aihetta käsitellään suomalaisissa lukiossa pääasiassa ihmisen biologian kontekstissa vapaavalintaisella kurssilla 4 (LOPS 2015) tai 5 (LOPS 2019) ja aihe onkin yksi ihmisen biologian kurssin keskeisistä sisällöistä oppia ja ymmärtää. Kalvojännitteen synty ja sen muutokset on kuvattu suomenkielisissä lukion biologian oppikirjoissa usein yksinkertais-tetusti ja ajoittain virheellisesti. Tämä saattaa johtaa virhekäsitysten syntymiseen, jolloin opiskeltavaa aihetta ei opita riittävällä tasolla. Siksi tämän maisterintutkielman tavoitteena on selvittää, miten solukalvon sähköistä aktiivisuutta opetetaan suomenkielisissä lukioissa ja millaisia virhekäsityksiä opiskelijoilla aiheesta esiintyy. Näiden tulosten pohjal-ta luodaan kehittämistuotos eli opetusmateriaali biologian aineenopettajien käyttöön, jolla voidaan tukea solukalvon sähköisen aktiivisuuden opetusta ja oppimista. Tutkimusmenetelmänä tutkimuksessa käytettiin kehittämistutkimusta, jossa yhdisteltiin teoreettisen ja empiirisen ongelma-analyysin periaatteita. Teoreettisen ongelma-analyysin kautta pyrittiin kartoittamaan lukio-opetuksen kannalta solukalvon sähköiseen toimintaan liittyvät keskeisimmät seikat, joihin monet virhekäsitykset liittyvät, sekä tarkastelemaan niitä eri näkökulmista. Tämä toteutettiin aiemman tutkimuskirjallisuuden avulla. Empiirisessä ongelma-analyysissä tarkasteltiin sekä lukion biologian oppikirjoja (N=3) että kevään 2021 biologian ylioppilaskoevastauksia (N=400) hermosolun aktiopotentiaalin kulkuun liittyen. Molempia aineistoja analysoitiin laadullisen sisällönanalyysin periaatteiden mukaisesti. Teoreettisesta ja empiirisestä ongelma-analyysistä saatujen tulosten perusteella kehitettiin opetusmateriaalin ensimmäinen versio. Opetusmateriaali luetutettiin läpi maisterintutkielman ohjaajilla ja opetusmateriaalia kehitettiin heiltä saatujen kommenttien perusteella. Jatkokehittämisen tuloksena syntyi opetusmateriaalin toinen ja virallinen versio. Tutkimuksen tulokset osoittivat, että solukalvon sähköinen aktiivisuus on haastava aihe sekä opetuksen että oppimi-sen näkökulmasta. Lukion biologian oppikirjoissa esiintyi epätarkkoja kohtia solukalvon sähköiseen aktiivisuuteen liit-tyen ja nämä epätarkkuudet näkyivät opiskelijoiden ylioppilaskokeen vastauksissa yleisinä virhekäsityksinä. Erityisesti esiin nousi virheellinen käsitys natrium-kaliumpumpun ioneja ”palauttavasta” vaikutuksesta aktiopotentiaalin jälkeen. Toinen yleinen virhekäsitys oli, että uusi aktiopotentiaali on mahdollinen vasta kun kalvojännite on hyperpolarisaation jälkeen palautunut lepotilaan. Tulosten perusteella voidaan todeta, että ainakin osa opiskelijoiden virhekäsityksistä on lähtöisin oppikirjoista eikä näitä virhekäsityksiä ole pystytty korjaamaan opettajan toimesta opetustilanteessa. Ylioppi-laskokeessa tehtävään vastanneista opiskelijoista 11,3 % ei osannut vastata tehtävän ensimmäiseen kysymykseen ollenkaan. Vastauksista, joissa opiskelija oli vähintään välttävällä tasolla onnistunut vastaamaan kysymykseen (N=381), 92,4 % sisälsi yhden tai useamman virhekäsityksen. Virhekäsitysten karsimiseksi paras keino on estää niiden syntyminen. Siksi tämän tutkimuksen perusteella voidaan todeta, että lukion uuden opetussuunnitelman biologian oppikirjat tulisi päivittää sellaisiksi, että virheellistä käsitystä ei pääse syntymään. Tämä ei kuitenkaan pelkästään riitä, vaan myös lukion biologian opettajien tulisi luopua vanhoista opetusdioistaan ja varmistaa, että he opettavat aihetta nykytiedon valossa oikein. Tässä apuna toimii tämän tutkimuksen osana kehitetty opetusmateriaali solukalvon sähköisestä aktiivisuudesta hermosolun aktiopotentiaalin kontekstissa. Jotta opetusmateriaalin toimivuutta ja opiskelijoiden käsitteellistä muutosta voidaan arvioida, jatkotutkimukset ai-heesta ovat tarpeellisia.

Parkinsonin tauti on maailman yleisin hermorappeumaa aiheuttava liikehäiriösairaus. Taudin ilmaantuvuus- ja esiintyvyysluvut ovat jatkuvassa nousussa, mitä väestön ikääntyminen ei yksin selitä. Taudin patologisia löydöksiä ovat alfasynukleiinin kertyminen ja vääränlaisesta laskostumisesta johtuva aggregaatio, Lewy neuriittien ja kappaleiden kertyminen sekä dopaminergisten hermosolujen solukato mustatumakkeesta. Taudin pidemmälle edenneille vaiheille on tyypillistä vaikea toimintakyvyttömyys ja elinajanodotteen lasku. Nykyiset hoitomuodot niin Parkinsonin taudille, kuin muillekin hermorappeumasairauksille ovat ainoastaan oireita lievittäviä. Onnistuneeseen lääkekehitykseen vaaditaan parannusta eläinmallien validiteetin jokaisella alatasolla. Parkinsonin taudin käytössä olevissa prekliinisissä eläinmalleissa on huono ilmivaliditeetti monien potilailla tehtyjen patologisten löydösten puuttuessa. Tässä tutkielmassa esitän uudenlaisen SynFib rottamallin Parkinsonin tautiin. Eksogeenisesti valmistettuja ihmisen alfasynukleiinifibrillejä injisoitiin yhdessä alfasynukleiinia ekspressoivien virusvektoreiden kanssa mustatumakkeeseen. Injektio aiheutti intensiivisen ja etenevän alfasynukleiinista johtuvan patologian ja merkittävän dopaminergisen soluvaurion. Taudin etenemistä seurattiin pitkittäistutkimuksessa positroniemissiotomografialla ja toiminnallisia puutteita arvioitiin synapsitiheydessä, inflammaatiossa ja dopaminergisessa järjestelmässä 16 viikon ajan. Havaitsin aivokudoksen tulehduksen ja dopaminergisen ipsilateraalisen soluvaurion lisääntyneen merkittävästi. Kahden viikon kohdalla synapsitiheys oli merkittävästi vähentynyt ipsilateraalisesti ja taudin leviäminen kontralateraaliselle puolelle oli alkanut.

Neural Oscillations at large-scale local and global neural synchrony levels can be detected at the scalp using electroencephalography. This neural activity presents itself in a varied range of frequencies referred to as ‘Brain Waves’. These frequency bands have cognitive significance and have been implicated in several neural functions due to its important role in communicating with functionally-similar but spatially-distinct brain regions. Frontal Asymmetry is the difference in activity between the right and left hemispheres in frontal areas of the brain recorded via EEG and is seen to be a strong indicator of emotional states. Specifically, approach and withdrawal motivation which have been associated with positive and negative emotions respectively. Using a combination of behavioural and physiological methods in measuring preference and responses gives us an accurate representation of the participant responses. In this study, three tests were conducted during a continuous EEG recording. Test 1: The implication of inducing a positive mood before the onset of stimulus line-up and the extent of its effect on emotions and alpha asymmetry is not extensively studied. In this test, we employed the use of an instrumental soundscape for one experimental group before beginning the stimulus presentation to test this effect against a ‘silent’ control group. Test 2: This test aims to compare the participants’ physiological measures (EEG) and behavioural self-reports to audio advertisement stimuli consisting of different categories of music: ‘Brand music’ vs. ‘Campaign’ music or ‘No music’ Controls. Test 3: There is ambiguity in research regarding how frontal alpha asymmetry as measured by EEG and self-report preferences might change with changing the format of the advertisement to: only Audio, Audiovisual and Silent videos. There has been contradictory evidence regarding the impact of music on an individual’s emotions and consequent memory and decision-making. This thesis delves into these questions through the post-study behavioural test and simple binary choice paradigm that measure the above-mentioned in relation with the stimuli presented to participants. Our results did not show a significant difference in frontal asymmetry in the stimulus presentation across the three tests conducted during EEG recording. The behavioural data however indicated significant preference in behavioural self-report ratings for Brand Music- associated stimuli in Test 2 and for Audiovisual advertisement stimuli in Test 3. Results also revealed a significant correlation between ratings given to a stimulus and post-study memorability. The final binary choice paradigm test indicated higher preference to products related to stimulus presentation (‘advertised’ brand) vs similar products not related to the presented stimulus (‘non-advertised’ brand). We anticipate that these results will further help us understand and predict general preferences that can help companies, government policy-makers and the general public be more aware and better equipped to manage their valuable resources of money, time, attention and memory.

Meningeal lymphatic vessels (mLVs), a recent functionally characterized structure in the meninges, contribute to the clearance of macromolecules, immune cells and metabolic waste from the central nervous system to peripheral lymph nodes. Having been identified as a route of clearance, there is a focus on understanding their role in neurological disease pathology. Here we consider their function in the pathology of traumatic brain injury (TBI) particularly in blood solute clearance, lesion progression and neuroinflammatory response. We use a transgenic model of mLV developmental dysfunction, K14-VEGFR3-Ig, to analyze the progression and severity of a controlled cortical impact (CCI) injury. We show that in mice lacking mLVs there are a higher percentage of microglia cells in an activated state in the hippocampus whereas the progression of hematoma and lesion size does not differ from wild type. Our results suggest that at two months post injury, meningeal lymphatics could be functionally important in modulating microglia activation, which is associated with chronic inflammation.

Sleep is one of the most vital functions of newborns and infants, and it is essential for neuronal network development. Therefore, long-term sleep disturbances have been associated with growth delays and behavioral disorders. Commonly reported infant sleep disturbances, such as night awakenings and difficulties falling asleep, cause distress to parents. Yet, the development of infant sleep in the home environment has not been fully elucidated due to lack of objective measurement parameters. In the current study, we assessed the feasibility of a motion sensor, attached to wearable pants, and ECG textile electrodes to monitor sleep-related respiration and heart rate of newborns and infants. First, we compared signals recorded by the motion sensor’s measurement channels to the standard respiratory piezo effort belt’s signal during daytime EEG recordings. According to our results, the motion sensor’s gyroscope proved to measure respiratory rate most accurately, while the ECG signal transmitted by the sensor was reliable in interpretable sections. We then provided wearable garments and smartphones to families with infants to assess overnight home-use. Our results indicate that different sleep states could likely be identified based on respiration fluctuation visible in the gyroscope’s signals. Moreover, the wearable system was considered practical and easy to use by the parents. Future studies should focus on validating the sensor with clinically approved measures, in order to train the algorithms to automatically identify different sleep-wake states. By doing so, the wearable sensor could provide information on natural infant sleep structure development over long time periods. Additionally, clinical validation of the sensor may result in the development of a companion diagnostic tool for infant cardiorespiratory and movement disorders.

Efficient processing of auditory information begins to emerge early in human ontogeny and establishes foundations for learning language from speech exposure. Here we show that repeated exposure to spoken words causes in neonatal brain attenuated neural responses that are linked to language skills at the age of 24 months. In the study, 75 newborn infants were exposed to repeated presentation of two spoken disyllabic pseudowords. During the word exposure, event-related potentials to presented pseudowords were measured with electroencephalography. The study provides three kinds of findings regarding neonatal brain dynamics and repetitive word exposure. Firstly, the results show that continuous exposure to spoken pseudowords modulates neonatal brain activity and can lead to attenuation of neural responses. This neural suppression likely reflects neonates’ early capacity to recognize spoken words and form neural representations of the stimuli through repetition. Secondly, the attenuated neural responses were bound to the presentation of the first syllable and did not occur after presentation of the second syllable. Thirdly, occurrence of neonatal neural suppression was associated with better expressive language skills later, at the age of 2 years. Altogether, the results provide preliminary evidence that neonatal brain responses to word repetition can be utilized to indicate efficiency of learning language from speech exposure and later state of language development.

Alzheimer's disease (AD) is a degenerative brain disorder that exhibits deterioration as one gets older. Although much remains to be learned about the pathophysiology of AD, there is strong evidence links amyloid beta (Aβ) plaques, which are responsible for cognitive impairment, to GABAergic interneurons. Model systems are of prime importance for adequately studying the pathophysiology of this disorder; however, existing in vitro models have limitations in producing patient-specific cells. The development of induced pluripotent stem cell (iPSC) technology has provided a novel opportunity for the effective production of disease-relevant cell types while preserving the molecular traits of the patient. In this thesis, the differentiation protocol established by Nicholas et al. (2013) was used to promote the development of interneurons derived from iPSCs. To enhance the efficiency of differentiation, the protocol was modified with the use of small molecules combined in different ways. The end result of the differentiation was characterized using immunocytochemistry (ICC) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The combination of molecules that produced greater efficiency in differentiation was selected, and the optimized protocol was carried out with iPSCs derived from an AD patient harbouring the APP Swedish mutation. The differentiation of cortical interneurons, demonstrated by the expression of pan-neuronal and specific GABAergic neuronal markers, signifies the successful generation of differentiated interneurons in the context of AD. AD iPSCs upregulated several markers related to AD pathology, such as APP and BACE1. However, the cell lines tolerated the small molecules differently, and thus, the protocol needs more optimization in the future. In summary, iPSC-based differentiation protocols are capable of producing disease-specific cell types that would be helpful in developing accurate AD models for revealing the mechanisms of Aβ pathology.

The developing human hindbrain and its role in neuronal pathogenesis have been relatively difficult to study for ethical reasons. By using the dual-SMAD inhibition and WNT signalling induction, a new method to culture brain organoids to resemble the human hindbrain has recently been established. In this study the new method has been used to detect the developing hindbrain’s response to flaviviral infection. Model virus used in this study is the Zika virus (ZIKV) which is known to alter the development of central nervous system and cause microcephalia. Pathogenic activity of the virus is measured by detecting the morphology of the organoids during infection as well as screening the organoids activation against oxidative stress, in a form of KEAP1/Nrf2-ARE pathway activation. Three different clones of ZIKV, which differ from each other by one amino acid in their non-structural protein 1 (NS1) gene, were used in the infections as well as two different time points of development. Controversially to previous findings on ZIKV infections to brain organoids, our findings show that developing hindbrain-like organoids do not change in shape or size during ZIKV infection. There are no differences in the lack of morphological changes between one-month olds or two months old organoids or between the different ZIKV clone infected organoids. The activation of the KEAP1/Nrf2-ARE pathway was measured by screening the two final products of the pathway, Nqo1 and HO-1. By screening the mRNA levels of these two genes, it showed that different ZIKV clones affect the activation of the KEAP1/Nrf2-ARE pathway in different levels at different times of development. Also, the expression of the same gene can be altered by the age of the organoids. Additionally, the expression of the two genes were different from each other at given time points and in response to the different clones. These findings suggest that the different isoforms of NS1 of ZIKV may alter the developing hindbrain’s response to oxidative stress. Findings also show that the time of the infection can additionally play a critical role to the ZIKV infection. The altered response to oxidative stress may contribute to microcephaly: the oxidation homeostasis of the developing hindbrain is modified, and apoptotic cell death can take place.

Gliomas are the most common malignant brain tumours. The most aggressive and lethal type of glioma is glioblastoma. It has a dismal prognosis, and, despite aggressive treatment, the average patient survival is 1-2 years. Although glioblastoma has a heavy impact on individuals and their families, as well as on healthcare systems, our current lack of mechanistic knowledge hinders the development of improved treatments and diagnostics. Recent studies showed that glutaminolysis, a metabolic pathway utilizing glutamine to produce α-ketoglutarate, is promoted in tumour cells, suggesting a significant role of α-ketoglutarate concentration in tumour progression. Therefore, I hypothesise that reduction of α-ketoglutarate concentration in glioblastoma might suppress glioblastoma aggressiveness. To address this hypothesis, I focus on another metabolic pathway controlling α-ketoglutarate concentration, namely the GABA metabolism. Here, I show that the expression of ABAT and GAD1, which encode rate-limiting enzymes of the GABA metabolism, is associated with the lower-grade of glioma and a better prognosis for patients. Interestingly the expression of ABAT and GAD1 negatively correlates with the expression of CD109, a glioma stemness marker. Furthermore, suppression of glioblastoma stemness by CD109 silencing induces ABAT and GAD1 expression. Taken together these results suggest that the upregulation of the GABA metabolism reduces glioblastoma stemness and proliferation. In future, I am planning to examine the effect of ABAT and GAD1 overexpression and knockdown on glioblastoma stemness and proliferation, as well as the underlying molecular mechanisms to understand how the GABA metabolism suppresses the glioblastoma progression.