Browsing by discipline "Biochemistry"
Now showing items 21-40 of 41
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(2013)Epithelial tissue is characterized by close cell-cell and cell-extracellular matrix (ECM) contacts as well as by apico-basal polarization. Integrity of these two features is important for functionality of epithelium. Additionally, proteins regulating polarity and cell junctions have been linked to cell cycle and apoptosis control. Consequently, defects in many of the polarity proteins have been linked to oncogenic events and loss of polarity is a hallmark of advanced cancers but whether it is causal to tumorigenesis is yet unknown. However, large body of knowledge on apico-basal polarity regulation and its connection on homeostasis control is derived from studies in Drosophila. This is mainly due to fact that efficient high throughput organotypic three dimensional (3D) culture methods enabling apico-basal polarization have not been available until the last decade. Large screens for epithelial polarity regulators have not been carried out in mammalian cells. Moreover, as cancer is the leading cause of death in developed countries and most of the cancers originate from epithelial tissues, knowledge of polarity regulation can be medically relevant. Oncogene MYC is overexpressed or amplified in variety of human cancers. The tumorigenic function of MYC is mainly due to its ability to drive cell cycle. We have previously shown that intact epithelial architecture is protective from cell cycle deregulating activities of MYC in 3D MCF10A mammary epithelial cell model and in vivo. This resistance can be overcome by inactivating LKB1 which is the human homologue of the polarity protein PAR4 implying a tumour suppressive role for epithelial architecture in mammalian cells. To identify regulators of epithelial architecture in mammalian cells, we have established lentiviral shRNA library (human epithelial architecture library, hEAL) encompassing 219 constructs targeting 77 genes associated with polarity regulation in Drosophila. We have previously screened the shRNA constructs for downregulation and quantified their effects on acinar morphology in the MCF10A 3D model. In this Master's Thesis I have validated the downregulation and phenotypes observed in a subset of the shRNAs during primary screening of the constructs of the library. Additionally, the possible co-operation with downregulation of the polarity regulators and conditional activation of MYC was determined. Most dramatically, downregulation of Wnt pathway gene DVL3 was shown to cause formation of enlarged multiacinar structures, which have increased proliferation. Additionally, downregulation of another Wnt pathway gene, GSK3β, resulted in acini with increased size and filled lumens. Thus these results propose a role for these genes in epithelial architecture regulation and tumour suppression in the used model even though apico-basal polarization of the acini was intact and no synergy with MYC was observed. Interestingly, no role in epithelial architecture regulation for Hippo pathway related genes FAT4 and MOBKL1A was found. Importantly, this study was able to validate primary screen showing relevance of the pipeline. Lastly, the study characterized the synthetic lethality phenotype found in the primary screen caused by downregulation of GTPase RHOA and chronic MYC activation. The shRHOA acini exhibited perturbed α6-integrin localization. When combined with MYC activation, the percentage of apoptotic acini was significantly increased. Importantly, the results suggest the observed synthetic lethality to be specific for the 3D context and to be associated with MEK/ERK and ROCK pathways. Taken together, in this study I have validated the role of novel epithelial architecture regulators and candidate tumour suppressors in MCF10A cells which may have medical relevance by helping to characterize tumorigenic processes. Furthermore, I characterized a novel 3D specific RHOA-MYC synthetic lethal interaction, which may prove to have therapeutic significance in MYC-driven cancers in future.
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(2015)The International Biology Olympiad is a yearly science competition; hundreds of high school students from over 60 countries take part in it. In Finland, the students are chosen by the national biology competition and the national Olympiad training camp. The research subject of this study is Finnish training for International Biology Olympiad (IBO). About ten high school students who are interested in biology take part at each training camp. Science competitions, such as IBO and training camps, are classified as non-formal or out-of-school science education. Since there is very little scientific knowledge about non-formal biology education, especially about science competitions, the theoretical framework of this study consists of non-formal science education, the relevance of science education and the development of interest. In this study, design research methodology with three research cycles was applied to develop the course. In the study, both theoretical and empirical problem analyses were used. There were two main research questions: 1) What are the needs for the development of the training camp? 2) What kind of training is relevant for the participants? The first research question was elaborated by examining a) what kind of topics of interest the participants have, b) how the participants expect the training to be relevant for them, c) what kind of relevance the previous participants experienced, and d) what kind of effects the previous training camps had on the participants interest in biology and career choice. The features of relevant biology Olympiad training were searched in the second research question. The data was collected from pre-camp and post-camp questionnaires, post-camp interviews and a questionnaire sent for the previous camp participants. The qualitative data was analyzed by content analysis and quantitative data from the questionnaires was analyzed by statistical methods. The main results were following: 1) The biggest needs for development were diversifying the contents and balancing the workload of the camp. The participants of the training camps were interested mostly about medicine and human biology -related topics and cell and molecular biology. In addition, it was found out that the previous participants considered biology education to be individually, vocationally and societally relevant for them. According to them, the training camps were especially individually relevant and had some effects on the career choice. 2) The new training camp for biology Olympiad is especially individually relevant for the participants but it has also some vocational relevance. In the science Olympiad training, special attention should be directed towards developing vocational and societal relevance. A new model for Biology Olympiad training camps was developed based on the collected research data. The individual dimension can be improved by i) diversifying the contents of the camps, ii) including inquiry-based learning modules and by iii) taking a student-centered approach to the development process. The vocational and societal dimensions of relevance can be targeted by iv) allowing the teachers of the participants to take part at camps. The vocational dimension of relevance can be enhanced v) by organizing visits to research laboratories and companies and vi) by enabling the participants to meet university students. The results of this study can be applied to not only to Science Olympiad training but also to non-formal biology education. This research provides models for developing out-of-school biology education and its relevance. This is the first design research study about Biology Olympiad training and it opens up discussion about the relevance of science competitions and Science Olympiad training.
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(University of HelsinkiHelsingin yliopistoHelsingfors universitet, 1998)Kilpailunjälkeinen maitohapon jakaantuminen plasmaan ja punasoluihin hevosella - toistettavuustutkimus - tutkimuksen tarkoituksena oli selvittää, kuinka ja millä tavalla maitohappo jakaantuu veritilassa ravihevosella heti kilpailun jälkeen. Toinen tutkittava asia oli, onko maitohapon jakautuminen plasmaan ja punasoluihin toistettavissa kilpailusta toiseen samalla hevosella. Tämän työn lähtökohtana oli havainto, että hevosella tietyn monokarboksylaattikuljettajaproteiinin (MCT) aktiivisuudessa on suuria yksilöllisiä eroja. Tämä proteiini vastaa suurelta osin maitohapon kuljetuksesta plasmasta punasoluihin (n. 70 %). Punasolut tarjoavat maitohapolle lisävaraston veressä työskenneltäessä korkeilla plasman maitohappotasoilla. Maitohapon mahdollisimman tehokas kuljetus pois lihaksista mahdollistaa korkeamman suorituskyvyn. Tutkimuksessa oli mukana 7 lämminveristä ravihevosta, joista kustakin otettiin heti kilpailun jälkeen verinäyte, josta mitattiin maitohappopitoisuudet kokoveressä, plasmassa ja punasoluissa. Lisäksi hevosilta mitaKiin lepoverinäytteestä monokarboksylaattikuljettaja -aktiivisuudet kahdessa eri pH:ssa ja maitohappokonsentraatiossa. Tutkimuksessa kokoveren maitohappotasot vaihtelivat kilpailutilanteessa paljon hevosten ja saman hevosenkin välillä. Positiivinen korrelaatio havaittiin kokoveren maitohappopitoisuuden ja punasolujen maitohappopitoisuuden välillä. Kahdella hevosista havaittiin positiivinen korrelaatio kokoveren maitohappopitoisuuden ja maitohapon punasoluosuuden välillä. MCT - aktiivisuuden ja pH:n korrelaatio havaittiin laktaattikonsentraatiossa 30 mM. MCT - aktiivisuudessa ja punasolujen laktaattipitoisuuksissa ei havaittu korrelaatiota. Maitohapon aineenvaihduntaan ja maitohapon tuottoon kilpailutilanteessa vaikuttavat lukuisat tekijät, joiden keskinäinen roolijako vaihtelee kilpailusta toiseen.
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(2014)Mobile zinc is involved in the pathogenesis of several fatal neurodegenerative diseases, such as amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease. Design of novel Zn(II) chelators is a promising research field in the development of new medical treatments for these diseases. However, depletion of zinc using a high affinity chelator can lead to cell death. The Zn(II) chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) can reverse some zinc related pathologies, but its high zinc affinity makes it unsuitable for use as a medical treatment. In this thesis, calculations at the density functional theory (DFT) level have been performed on TPEN and TPEN derivatives. The aim was to suggest modifications in the molecular structure which lower the zinc affinity of the chelator to a less toxic level, and which therefore could potentially lead to new medical therapies for neurodegenerative diseases. A further aim was to develop a computational protocol that is suitable for studies and in silico design of Zn(II) chelators. The results show that DFT methods, which include a correction for dispersion forces and which treat the solvent implicitly, can yield free energies for ligand exchange reactions which agree well with experimental data. The employed computational methodology is also suitable for similar studies involving other metals. The zinc affinity of TPEN can be lowered by substituting hydrogens on its pyridyl rings with electron-attracting groups. Substitution with weakly electron-donating groups can also lower the zinc affinity, provided that it results in a conformational change which stabilises the free chelator. Substitution of carbon atoms with nitrogens on the pyridyl rings also lowers the zinc affinity. The computational methodology needs improvement if one wishes to address more complicated problems, such as studies of complexation energies for chelators with varying denticities, in which solvent molecules may play a more significant role as one of the ligands.
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(University of HelsinkiHelsingin yliopistoHelsingfors universitet, 1994)Tämä tutkimus kuuluu osana laajempaan hevosen rasitusfysiologiaa käsittelevään tutkimukseen, jota suoritetaan EKK:n biokemian laitoksella ja Ypäjän hevostutkimusasemalla. Tämän työn tarkoituksena oli tutkia kovatehoisen, lyhytkestoisen sekä matalatehoisen, pitkäkestoisen liikuntarasituksen vaikutusta hevosen plasman E-vitamiinipitoisuuteen. Koemateriaalina käytettiin Ypäjän Hevostutkimusaseman ravihevosia sekä yksityisomistuksessa olevia, kilpailevia matkaratsastushevosia. Näytteitä kerättiin kontrolloidusta rasituskokeesta, jossa hevoset juoksivat 3 hiittiä kahtena päivänä sekä matkaratsastuskilpailusta (toukokuussa -94) yhteensä kymmeneltä hevoselta. Näytteistä määritettiin vapaa alfa-tokoferoli, Ekk:n biokemian laitoksella käytössä olevalla fluorometrisellä menetelmällä. Osasta näytteitä tutkittiin myös vapaiden rasvahappojen ja eri glyseridien pitoisuus. Tulokset käsiteltiin tilastollisesti toistuvien mittausten varianssianalyysillä. Kontrolloidussa liikuntarasituskokeessa (kovatehoinen, lyhytkestoinen liikuntarasitus) todettiin tilastollisesti merkitsevä ero plasman E-vitamiinipitoisuuksissa ensimmäisen ja toisen rasituspäivän välillä. Kun E-vitamiinipitoisuus määritettiin suhteessa plasman triglyserideihin, rasituspäivien välille ei saatu tilastollisesti merkitsevää eroa. Matkaratsastuskilpailusta (matalatehoinen, pitkäkestoinen liikuntarasitus) kerätyissä näytteissä ei todettu tilastollisesti merkitsevää eroa.
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(University of HelsinkiHelsingin yliopistoHelsingfors universitet, 1998)Tässä tutkimuksessa selvitettiin porovasojen (Rangifer tarandus tarandus L.) lihassyykoostumuksen muutoksia ja lihaksen proteiinien hajoamista niukkaravinteisen talven aikana. Tutkimuksessa käytettiin kahta eläinryhmää, joista toista ruokittiin mahdollisimman luonnonmukaisella talviravinnolla ja toista lisäksi jäkälällä ja kaupallisella rehulla. Lihasbiopsioista valmistettiin ATPaasi-värjäysleikkeet sekä määritettiin proteiineja hajottavien entsyymin, katepsiini B:n aktiivisuudet. Kokeen taustalla oli Huippuvuorten peuroilla (Rangifer tarandus platyrhynchus) tehty tutkimus, jossa tyypin IIB lihassyiden osuudet ja pinta-alat pienenivät huomattavasti talvella, jolloin eläimet nälkiintyvät voimakkaasti ankarissa luonnonoloissa. On esitetty, että Huippuvuorten peura käyttäisi tyypin IIB lihassyitä energiantuottoon luonnonravinnon ehtyessä. Meidän tutkimuksemme tarkoituksena oli selvittää, havaitaanko vastaavia muutoksia poron keskimmäisessä pakaralihaksessa (M. gluteus medius). Lisäruokittujen eläinten paino lisääntyi kokeen aikana n. 6 kg, kun taas nälkiintyneet eläimet laihtuivat keskimäärin 7 kg. Lisäruokittujen porojen ryhmässä tyypin I ja IIA lihassyiden yhteenlaskettu pinta-ala sekä tyypin IIA lihassyiden prosentuaalinen osuus kasvoivat talven aikana. Nälkiintyneiden eläinten lihassyykoostumuksessa ei havaittu vastaavia muutoksia talven aikana. Tyypin IIB lihassyiden osuudessa ja pinta-aloissa ei tässä tutkimuksessa havaittu muutoksia. Lihaksen katepsiini B -aktiivisuudet laskivat kummassakin ryhmässä talven aikana, mutta ryhmien välillä ei ollut eroja missään vaiheessa kokeen aikana. Koska kokeessa mitattujen katepsiini B -aktiivisuksien perusteella lihasproteiinien hajoamisessa ei ollut ryhmien välillä eroa, ryhmien välisen eron voidaan olettaa olevan proteiinisynteesissä; lisäruokittujen porojen lihasmassa kasvoi talven aikana, kun taas nälkiintyneiden eläinten lihasmassa pysyi muuttumattomana koko talven ajan.
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(University of HelsinkiHelsingin yliopistoHelsingfors universitet, 2001)Maitohapon kertyminen lihaksistoon liikuntasuorituksen aikana on yksi suorituskykyä heikentävistä tekijöistä ja sen pitoisuuden määrittämistä verestä on jo pitkään käytetty liikuntarasituksen intensiteetin mittarina. Maitohappo on myös erittäin käyttökelpoinen energianlähde sekä rasituksen että palautumisvaiheen aikana, joten sen jakautuminen elimistössä ja siihen vaikuttavat tekijät ovat nousseet tärkeäksi tutkimuskohteeksi. Kirjallisuuskatsauksessa käsitellään lihasten koostumusta ja energiantuotantoa sekä maitohapon muodostumista, jakautumista, kuljetusta ja merkitystä liikuntasuorituksen kannalta. Fysiologisessa pH:ssa maitohappo on pääosin dissosioituneessa muodossa: laktaattianionina ja vetyionina. Koska dissosioitunut muoto ei kykene siirtymään solukalvojen läpi diffuusiolla, tarvitaan kuljettajamekanismi siirtämään dissosioitunutta laktaattia. Kuljettajamekanismeja ovat monokarboksylaattikuljettaja (MCT) ja anioninvaihtajamekanismi. Lihas- ja punasoluissa anioninvaihtajamekanismilla on vähäinenen merkitys, MCT:n vastatessa pääosin laktaatin kuljetuksesta. MCT perheeseen kuluu nisäkkäillä ainakin 7 eri muotoa eli isoformia (MCT1-MCT7), joilla on kudosspesifisiä ominaisuuksia. Hevosen punasoluissa laktaatin kuljetuksesta solukalvon läpi vastaa MCT2. Lämminverisillä MCT2:n laktaatin kuljetusaktiivisuudessa on todettu suuria yksilöllisiä vaihteluja. Muilla tutkituilla eläinlajeilla vastaavaa ei ole todettu Tämän tutkimuksen tarkoituksena oli selvittää onko suomenhevosella samankaltaista suurta yksilöllistä laktaatinkuljettajan aktiivisuuden vaihtelua kuin lämminverihevosilla. Verinäytteet kerättiin 50:ltä suomenhevoselta ja punasolujen laktaatinkuljetusaktiivisuus määritettiin radioaktiivisen laktaatin avulla. Tulokset vastasivat lämminverihevosilla tehtyjen tutkimusten tuloksia. Yksilölliset erot MCT2:n aktiivisuudessa kuljettaa laktaattia punasoluihin olivat huomattavia, aktiivisuuden vaihdellessa 1,0-10,5 nmol/(mg x minuutti). Vaihtelua oli havaittavissa riippumatta hevosen iästä tai sukupuolesta. Tutkitut hevoset olivat jaettavissa kahteen ryhmään punasolujen MCT:n laktaatin kuljetusaktiivisuuden mukaan. Hevosten, joilla oli matala kuljetusaktiivisuus, osuus oli 36 %. Vastaavasti hevosten, joilla oli korkea kuljetusaktiivisuus, osuus oli 64 %. Jako matalan tai korkean kuljettaja-aktiivisuuden omaaviin ryhmiin ei kuitenkaan ollut yhtä selkeä kuin lämminverisillä. Aikuisilla, yli kolmevuotiailla, tammoilla havaittiin laktaatin kuljetusaktiivisuuden olevan merkittävästi (p < 0,01) korkeampi kuin aikuisilla oreilla. Hevosen suorituskykyä kuvaamaan käytettiin Suomen Hippoksen laskemia yksilöindeksejä. Yksilöindeksejä verrattiin laktaatin kuljetusaktiivisuuteen, mutta tilastollisesti merkittävää korrelaatiota ei havaittu.
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(University of HelsinkiHelsingin yliopistoHelsingfors universitet, 1990)Tässä tutkimuksessa tutkittiin maksimaalisen rasituksen vaikutusta plasman alaniini- ja kortisolikonsetraatioihin ja mahdollisia eroja hyvin suoriutuvien ja huonojen hevosten välillä. Sekä alaniinin että kortisolin on todettu aiemmin nousevan rasituksessa. Alaniini on merkityksellinen glukoosi-alaniinisyklissä, jossa se mahdollisesti kuljettaa rasituksessa syntyvää ammoniakkia sekä pyruvaattiaa maksaan käsiteltäväksi. On esitetty, että alaniinille voisi olla merkitystä suorituskykyä parantavana tekijänä. Kortisolin eritys nousee intensiteetiltään tarpeeksi voimakkaassa rasituksessa. Paitsi rasituksen kestosta on nousun päätelty riippuvan ennen kaikkea psyykkisistä tekijöistä, jotka varsinkin hevosella usein ratkaisevat. Tässä tutkimuksessa ei todettu alaniinikonsentraatioiden nousussa eroja hyvien ja huonojen välillä. Sen sijaan havaittiin, että keskiryhmässä alaniiniarvot alkoivat laskea 60 min jälkeen, kun taas hyvillä ja huonoilla alaniinikonsentraatiot 60 min jälkeen yhä nousivat. Koko otoksessa alaniinikonsentraatiot nousivat tilastollisesti merkittävästi lepoarvoihin nähden. Näytteistä mitattiin vielä 60 min kilpailun jälkeen merkittävästi korkeampi kortisoliarvo hyvillä kuin keskiryhmän hevosilla ja myös lepoarvot olivat matalammat hyvillä. Sekä kortisoli että alaniiniarvoissa havaittiin lievää sukupuoleensidonnaisuutta. Tämä havainto vaatisi kuitenkin varmistusta suuremmalla materiaalilla. Yhteenvetona voidaan sanoa että kortisoli ja varsinkin alaniinikonsentraatioiden tutkimuksella on mielenkiintoa hevosen rasitusfysiologiassa. Uusia ulottuvuuksia voidaan saada lihashiopsioiden perusteella ja laajempialaisesta tutkimuksesta, jossa mitataan alaniinin lisäksi esim. glutamaatin vaihteluita. Tässä tutkimuksessa havaitut kortisolikonsentraatioiden erot hyvä- ja huonokuntoisilla todennäköisesti kuvastavat hyväkuntoisten parempaa "stressikapasiteettia".
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(2020)Lipoproteins are biochemical carriers of the insoluble lipids. They are complexes combining lipids and proteins for the transport of lipids. Amongst the type of lipoproteins are low-density lipoproteins (LDL) which are prevalent in various diseases such as obesity, diabetes, atherosclerosis, and other cardiovascular diseases (CVD). Omega-3 fatty acids are polyunsaturated fatty acids (PUFA) that are essential components of lipid metabolism and play a significant role in the human diet. Omega-3 PUFAs such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are derived from fish and are necessary for proper cardiovascular functioning. Because the human body is unable to produce enough quantities of some omega-3, diet is an important source for its availability. When a diet is rich in saturated fats, the above-mentioned diseases transpire. This study investigated how consumption of two fish diets, Lean fish and Fatty fish, influence the lipid species of human LDL particles. The lipid species analysed in this study are phospholipids such as phosphatidylcholine (PC), sphingomyelin (SM), and lysophosphatidylcholine (LPC), and cholesteryl esters (CE), and triacylglycerols (TAG). A total of 42 volunteers with a history of impaired fasting glucose had randomly been divided into two groups: fatty fish (4 fish meals/week) and lean fish (4 fish meals/week) for 12 weeks. Blood samples had been collected from the volunteers before and after consumption of the fish meals and LDL particles had been isolated from the blood samples by ultracentrifugation. In this study, the lipids were extracted by Folch method, and the extracted lipids were analysed using Triple quadrupole mass spectrometry. The lipid class profile did not change due to the two fish type diets. However, the consumption of fatty fish diet increased the levels of lipid species of PC, LPC, and CE containing EPA and DHA acyl chains, while decreasing levels of several TAG species. Lean fish induced minor changes in the lipid composition of LDL particles. Based on these results, fatty fish diet alters the plasma LDL lipidome profile with changes induced to both the surface and the core composition of the LDL particles in a positive way regarding cardiovascular health.
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Novel inhibitors of lipid phosphatase SHIP2 and their effects on the phosphorylation of Akt kinase (2020)Type 2 diabetes mellitus (T2DM) has been shown to be associated with hyperglycemia, insulin resistance, hyperinsulinemia and impaired insulin secretion from pancreatic β-cells leading to micro- and macro-vascular complications including multiorgan failures. At the cellular level, the mechanism of insulin resistance is associated with complex PI3K-Akt mediated insulin signaling pathway. Moreover, lipid phosphatase SHIP2 (Src homology 2 domain containing inositol 5-phosphatase 2) plays a vital role as a negative regulator of the insulin signaling pathway downstream of PI3K by hydrolyzing phosphatidylinositol- 3,4,5-trisphosphate (PIP3) into phosphatidylinositol- 3,4- biphosphate (PIP2). Scientific reports have shown that inhibition of SHIP2 activity might improve Akt phosphorylation and thus PI3K-Akt mediated insulin signaling pathway. Considering this, I am interested in the SHIP2 inhibitors with drug like properties such as improved solubility, pharmacokinetic and bioavailability properties with little to no contraindications. In the present thesis, I have attempted to detect indirectly the capacity of 8 novel small molecule SHIP2 inhibitors, #160, #161, #162, #163, #167B, #170A, #171, #172 for their ability to phosphorylate Akt kinase in L6 myotubes using immunoblotting as a tool and compared data using graphical representation to pick up the best candidate. Two inhibitors, #163 and #170A were further chosen for alamarBlue® cytotoxicity assay. Treatment with #163 did not display direct cytotoxic effects on the myotubes. The viability of myotubes was not affected at low concentrations of #170A, but it started to reduce at concentrations >200 µM. In my study, I came up with #163 and #170A as the best lead candidates for further analysis. In future, more trials need to be performed with these inhibitors. Moreover, there are several other novel small molecule SHIP2 inhibitors identified from chemical library that need to be tested. Briefly, in this thesis, I have first time reported 8 novel small molecule SHIP2 inhibitors which could be a significant step in the discovery of new T2DM drugs for more efficient, cost effective and safe treatment of the disease with least contraindications.
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(2014)Modic-muutokset (MC) liittyvät alaselkäkipuun (LBP). Tulehdusta on pidetty erityisesti 'tyypin I' -MC:n avaintekijänä, mutta tähän mennessä potentiaalisista tulehduksellisista välittäjäaineista vain TNFα on yhdistetty MC:n. Tutkimuksen tavoitteena oli analysoida tiettyjä tulehduksen välittäjäaineita ihmisen kirurgisesti poistetuista välilevyistä, ja määrittää niiden yhteys MC:n kanssa viereisissä selkänikamissa. Tutkimuspopulaatio koostui 55 välilevynäytteestä; 20 'Ei MC' -välileyä, 20 'tyypin I' -välilevyä ja 15 'tyypin II' -välilevyä. 47 välittäjäaineen mRNA -ekspressio määritettiin eristetystä RNA:sta. Märitetyistä välittäjäaineista tilastollisesti merkittävät assosiaatiot löydettiin RANKL:sta (p=0.020), M-CSF1:stä (p=0.019), NFATc1:stä (p=0.021) ja RUNX1:stä (p=0.023), jotka olivat lisääntyneet 'tyypin II' MC -ryhmässä, kun taas OSCAR (p=0.023) oli lisääntynyt 'tyypin I' –ryhmässä verrattuna 'ei MC' -ryhmään. Kun nämä välittäjäaineet ovat yhteydessä osteoklastien erilaistumiseen ja lisääntymiseen, hypoteesimme mukaan välilevyjen erittämien aineiden aiheuttama nikamien osteoklastien stimulaatio on osallisena MC:n patofysiologiassa.
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(2015)Autophagy is a eukaryotic cellular process where intracellular material is recycled by transporting it in newly formed vesicles to lysosomes for degradation. In normal conditions autophagy supports cellular homeostasis. Different stress conditions can induce autophagy and then it helps the cell to avoid an unnecessary or uncontrolled cell death. RAB proteins are small GTPases that regulate vesicle traffic and fusion events in endocytic and exocytic pathways. RAB24 has recently been shown to participate in autophagy, but there is very little information about how it works at the molecular level. GOSR1 is a Golgi SNARE protein that regulates membrane fusion events, and it has been observed to interact indirectly with RAB24. The participation of GOSR1 in autophagy has not been studied yet. The aim of the study was to find out if RAB24 and GOSR1 colocalize into the same vesicle structures and if they interact within each other. HeLa cells were used as a model organism, and to induce autophagy amino acid starvation was used. For GOSR1 detection a DNA construct was created where GOSR1 was tagged with green fluorescent protein GFP-sequence. Localization was studied with immunofluorescence staining where in addition to RAB24 and GOSR1 also the autophagosomal marker protein LC3 was labeled. The labeled cells were photographed with a confocal microscope. The pictures were analyzed with ImagePro software. Interaction between the proteins was studied using immunoprecipitation. GOSR1 and RAB24 were not observed to colocalize into same structures in significant amount. Instead it was found that GOSR1 colocalized into LC3-positive autophagic vesicles. In immunoprecipitation studies no interaction between RAB24 and GOSR1 could be shown. In order to ensure the results more immunofluorescence stainings should be done using several time points and GFP-tagged GOSR1. Also GOSR1 silencing with siRNA should be used in order to find out if GOSR1 is necessary for autophagy. The immunoprecipitation protocol should be optimized, and the possible interaction could be studied by using other methods, for example yeast-two hybride technology.
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(2020)Extracellular vesicles (EVs) are lipid bilayer-enclosed nano-sized particles that are found in all body fluids. EVs are part of normal cell functions and they can carry for example proteins, RNA and lipids between cells. This makes them potential candidates as drug delivery vehicles. When nanoparticles are introduced to blood plasma, a plasma protein structure is formed on their surface, called the protein corona. The formation of a protein corona is a dynamic process, and the proteins are binding to the corona depending on their affinity to the nanoparticle surface. When administrating nanoparticles to cells, protein corona has a big impact on the half-life and cellular uptake of the particles. The aim of this work was to study the plasma protein corona of PC-3 derived EVs, and to investigate methods for protein corona isolation. First, EVs and lipoprotein particles were removed from fresh frozen plasma by membrane filtration. Using this filtrated plasma, we then tested plasma-EV incubation and removing of free plasma proteins by simple ultrafiltration. Removing of free plasma proteins was also examined by size-exclusion chromatography (SEC). The resulting EV-protein corona complex was visualized with SDS-PAGE. EVs were characterized with nanoparticle tracking analysis and western blotting. Of these two methods used, SEC appeared to be more convenient and efficient way to remove the free plasma proteins from the EV-protein corona complex. The method still requires further development and testing in order to perform optimally.
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(2018)In this study 39 methicillin-resistant Staphylococcus aureus CC398 isolates from Finnish pigs and pork were sequenced by whole-genome sequencing. Also, phenotypic antimicrobial resistance of the strains was examined. The strains originated from the culture collection of Finnish Food Safety Authority (Evira) and they were isolated between the years of 2008 and 2017. MRSA strains were isolated from both asymptomatic and infected pigs and pork. The strains represented the four most common spa types in Finnish pigs (t034, t2741, t011 and t108). DNA extraction method for S. aureus was optimized by comparing automatic DNA extraction robot and manual DNA extraction method. Extracted DNA samples were sent to Germany to be sequenced with Illumina sequencing. Paired-end raw read sequences were assembled using three different algorithms: Spades, Idba and Velvet. The assembled sequences were analysed by bioinformatics tools. Possible antimicrobial resistance genes, virulence genes, plasmids and SCCmec cassettes were identified. The strains were also typed by spa typing and sequence typing. spa typing was already performed in Evira using PCR and the new spa typing results were compared to the PCR results. Using the assembled sequences, a phylogenetic tree was also constructed. Phenotypic antimicrobial susceptibility testing was performed using broth microdilution panel. The manual extraction method was slightly better since the extracted DNA was purer. The best assembly algorithm was Spades and because of that all sequences were assembled with that algorithm for further analyses. The strains carried only a few virulence genes and many genes that are important for human infection were missing. All the strains carried the same SCCmec cassette type and sequence type ST398 both of which are known to be typical for pig related MRSA. Illumina sequencing and spa typing were not working perfectly since the used program was not able to distinguish spa types t034 and t011 from each other. The problem was probably due to very similar short tandem repeats that these two spa types share. Also, Illumina sequencing is known to produce short reads that could be problematic for sequence assembly. All the strains were resistant to beta-lactams and tetracycline which is typical for MRSA CC398 strains. Many strains were also resistant to clindamycin and trimethoprim. Antimicrobial resistance seemed to be carried by plasmids in some cases because the strains carried several plasmids that are known to contain antimicrobial resistance genes. The antimicrobial resistance profiles were different between different spa types since some spa types were more resistant to certain antimicrobials than others. According to the phylogenetic tree, the strains seemed to be a very heterogenic group. The strains that belonged in the same spa type were most related to each other as expected. There were no differences between strains originating from pigs and strains originating from pork which strongly indicates that bacteria could have spread from pigs to pork. Asymptomatic pigs also carried similar strains as infected pigs. The strains that were causing infections in pigs did not contain any more virulence factors compared to the other strains. The study gave plenty of new information concerning MRSA CC398 in Finnish pigs and pork. The antimicrobial resistance and virulence patterns in addition to other qualities of the strains were very similar that was described in the literature before. The strains seemed not to be very virulent for humans, but the situation must be observed in case of increasing resistance and virulence. The best way to prevent spreading of MRSA is to decrease the amount of MRSA in pig farms since MRSA bacteria are most likely to spread from pigs to the environment and to the people who work in pig farms and from there to the rest of the population.
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(2018)The motivation of this study was to find new treatment options for the rare cancer pseudomyxoma peritonei (PMP). PMP is a slowly progressing mucinous adenocarcinoma that originates from the appendix and disseminates into the peritoneum where the cancer cells secrete large amounts of MUC2, the main component of intestinal mucus, into the peritoneum. The disulfide isomerase AGR2 helps MUC2 with forming the correct intramolecular disulfide bonds prior secretion, and is essential to MUC2 protein production. The mucus build-up into the peritoneum causes stress on vital organs, and eventually death. Therefore, inhibiting MUC2 production in PMP cancer cells might slow down the disease progression significantly. MAPK/ERK and cAMP/PKA signaling pathways stimulate MUC2 production, and activating mutations in KRAS and GNAS of these pathways are common in PMP. The aim of this study was to elucidate how MUC2 and AGR2 affect each other’s expression levels, and how the MAPK/ERK pathway and the cAMP/PKA pathway targeting substances caffeine, theophylline, cromolyn, fudosteine, octreotide, and lanreotide, affect MUC2 expression in vitro. This study was conducted on human colorectal adenocarcinoma cell lines LS174T, LoVo, and HT29 that all produce large amounts of MUC2. In addition, LS174T and LoVo cell lines carry activating heterozygous mutations in their KRAS genes. MUC2 and AGR2 expression levels were measured on mRNA level with real-time quantitative reverse transcriptase polymerase chain reaction. The effects of MUC2 on AGR2 expression, and vice versa, were tested by silencing each at a time with the appropriate siRNA. MUC2 siRNA suppressed both MUC2 and AGR2 mRNA levels down to 50 % in LS174T cells and down to 40 % in LoVo cells in respect to their control groups. AGR2 siRNA suppressed AGR2 mRNA levels down to 50 % in LS174T cells and even down to 30 % in LoVo cells in respect to their control groups, while there were no statistically significant changes in MUC2 mRNA levels. Caffeine and theophylline inhibit phosphodiesterase of the cAMP/PKA signaling pathway, and in consequence, the hydrolysis of the secondary messenger cAMP, prolonging the activation of the pathway. Caffeine stimulated MUC2 production in LS174T and LoVo cells. In addition, AGR2 mRNA levels increased in LoVo cells, in which the fold change in MUC2 mRNA levels was much greater. Theophylline, a compound found in tea, and used for the treatment of asthma, did not affect MUC2 production. PMP cancer cells express protein S100P. Cromolyn is a pharmaceutical substance used for the treatment of asthma, and it inhibits S100P, and thereby S100P/RAGE signaling -dependent activation of MAPK/ERK pathway. Fudosteine, on the other hand, is a mucoactive pharmaceutical substance that lowers the production of MUC5AC, the main component of lung mucus. Since activating GNAS mutation stimulates the production of both MUC2 and MUC5AC in HT29 cells, the expression of both must be at least partially controlled by same mechanisms. In addition, Somatostatin analogues octreotide and lanreotide inhibit ERK1/2 of the MAPK/ERK pathway, as well as protein kinase A of the cAMP/PKA pathway, and hence cAMP production. However, none of these four tested pharmaceutical substances were able to inhibit MUC2 production in the cell lines used this study in vitro.
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(University of HelsinkiHelsingin yliopistoHelsingfors universitet, 1997)Yhdeksän ratsuhevosta suoritti viisi kertaa juoksumatolla kilpailurasitustestin kuumassa ja kosteassa kahden viikonvälein. Kilpailurasitustestin jälkeen hevosille annettiin nenä-nieluletkun kautta glukoosi-elektrolyyttiliuosta javiimeisellä kerralla pelkkää vettä. Vuorokauden kuluttua kilpailurasitustestin alusta hevoset suorittivat vakioidun rasitustestin, jossa seurattiin hevosten palautumista kilpailurasitustestistä. Rasituksessa syntyi paljon ylimääräistä lämpöä, joka poistetaan elimistöstä pääasiassa hikoilemalla. Hien mukana hevoset menettävät runsaasti nesteitä ja elektrolyyttejä. Nesteiden menetyksen arvioimiseksi hevosten paino mitattiinennen ja jälkeen rasitustestien. Niiden rektaalilämpö mitattiin ja hevosilta otettiin verinäytteitä rasitustestin aikana ja sen jälkeen. Plasmasta määritettiin plasmanmäärä, kokonaisproteiinit ja osmolaalisuus. Lisäksi siitä mitattiin natriumin, kloridin, kaliumin, kalsiumin, magnesiumin, aldosteronin ja argiini-vasopressiinin pitoisuudet. Hevosten painon palautuminen oli nopeampaa kokeen edetessä. Lisäksi hevosten plasmamäärä lisääntyi, plasmanosmolaalisuus laski, natriumin ja kloridin menetysrasitustestin aikana väheni ja kloridipitoisuus palautui nopeammin rasituskertojen lisääntyessä. Muutokset johtuvat hevosten sopeutumisesta kuumaan ja kosteaan ilmastoon. Glukoosi-elektrolyyttiliuoksen antaminen nopeutti hevosten palautumista rasituksen aiheuttamasta neste- ja elektrolyyttihukasta paremmin kuin pelkän veden antaminen. Viidennen kilpailurasitustestin jälkeen, jolloin hevosille annettiin vettä nenä-nieluletkun kautta, painon palautuminen oli huomattavasti hitaampaa, kun sitä verrataan muihin testikertoihin.
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(2014)Alzheimerin tauti (AT) on yleisin dementian syy ja perintötekijöiden vaikutus tautiriskiin on suuri. APOE-geenialue on vahvimmin AT:iin liittyvä geenialue. APOE:sta on kolme yleistä alleelia, joiden koodamassa proteiinissa on 1-2 aminohapon eroja: E2, E3 ja E4, joista APOE4 on voimakas AT-riskitekijä. Suomalaistutkimuksissa on osoitettu, että eräs yleinen APOE3-haplotyyppi lisää ja toinen APOE3-haplotyyppi pienentää AT-riskiä. Etiologinen variantti APOE3-haplotyypissä on tuntematon. Tässä tutkimuksessa selvitettiin kolmen pistepolymorfismin avulla APOE:n viereisen TOMM40-geenialueen vaikutusta tautiriskiin, erityisesti APOE3-haplotyypin osana. Aikaisemmin kerätystä vantaalaisesta ikäkohortista monistettiin tutkittavat alueet polymeraasiketjureaktiolla (PCR), käsiteltiin PCR-tuotteet restriktioenstyymeillä ja tutkittiin restriktiotuotteet geelielektroforeesilla. Saatuja tuloksia vertailtiin aikaisemmin aineistosta kerättyyn kliiniseen, neuropatologiseen ja geneettiseen tutkimustietoon. Tulokset osoittivat TOMM40-geenialueen olevan kytkentäepätasapainossa APOEpromoottorialueeseen, mutta ei APOE-proteiinia määrittävään eksoni-4:ään. TOMM40-polymorfismien avulla voitiin erottaa myös APOE-tyypistä riippumaton heikko AT-riskivaikutus. TOMM40- ja APOE-polymorfismien analyysi viittasi siihen, että primaari geneettinen riskitekijä APOE3-haplotyypeissä on APOE-geenin ei-koodavalla alueella, mahdollisesti promoottori/enhancer-alueella. Jatkotutkimuksissa selvitetään, vaikuttaako tämä riskitekjä APOE:n, TOMM40:n tai muiden lähigeenien ekspressioon.
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(2012)Core-fucosylation of N-glycoproteins is associated with different cancers and other pathologies. Identification of glycoproteins and determination of their glycan structure manually by mass spectrometry (MS) is time-consuming and laborious. In this Pro gradu thesis, the use of the mass spectrum-analyzing software Glycopeptide ID for identification of core-fucosylation from a known standard, immunoglobulin G, was studied. Also, a plasma sample with unknown glycoproteins was analyzed. For the MS analysis, the proteins were digested with trypsin, and the resulting glycopeptides were enriched using lectin affinity chromatography. From IgG and plasma, also samples treated with α-Lfucosidase were prepared in order to cleave the core fucose. The presence of glycopeptides was determined by high-performanve liquid chromatographymass spectrometry (HPLC-MS) analysis, and they were fragmented using collision-induced dissociation (CID) in a tandem-MS (MS/MS) analysis. The MS/MS spectra were analyzed with the Glycopeptide ID software. The software was found to identify core-fucosylation reliably from high-quality spectra, but identification of proteins were often incomplete from spectra with poor quality. From the plasma sample with unknown proteins, a probable corefucosylation was found from IgG2, fetuin A, serotransferrin, hemopexin and ceruloplasmin. As a conclusion, the software Glycopeptide ID can be considered as an appropriate tool for identification of core-fucosylation in N-glycopeptides.
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(2014)Nykyiset DNA:n eristämiseen ja puhdistamiseen käytetyt myrkyttömät proteiinien ulossuolausmenetelmät (protein-salting-out) ovat vielä melko aikaavieviä ja eräissä tilanteissa jopa epäluotettavia. Tämän työn tarkoituksena on kehittää luotettava ja entisiä menetelmiä nopeampi proteiinien ulossuolausmenetelmä kromosomaalisen DNA:n eristämiseksi viljellyistä ihmisen soluista (Jurkat, K562 ja U937). Tämän uuden proteiinien ulossuolausmenetelmän avulla voidaan 60 minuutissa eristää puhdasta ja suurimolekyylimassaista DNA:ta erilaisiin vaativiin sovelluksiin, kuten genomisten DNA kirjastojen valmistukseen ja niiden analyysiin. Tässä työssä kehitetty DNA:n eristysmenetelmä validoitiin tekemällä puhdistetusta DNA:sta geenikirjasto, josta vektoretti-PCR:ään perustuvalla kromosomikävelyllä eristettiin ihmisen kasvurajoitegeenin, tp53-geenin, promoottorialue, joka analysoitiin restriktioentsyymikartoituksella ja sekvennoinilla.
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(2014)Microvesicles (MVs) are lipid bilayered membranous vesicles containing functional lipids, proteins, RNA and DNA that are produced by most cells. The physiological significance of MVs has become evident, and increased MV counts and the contents of MVs are nowadays also associated with different pathophysiological phenomena. The goal of the field is to use MVs as diagnostic and therapeutic tools. To achieve this, the understanding of the mechanisms of the functions of MVs should be understood better and additionally, reliable methods for the quantification and characterization of MVs should be developed and standardized. The aim of the study was to determine differences in platelet-derived MVs produced by different activation mechanisms. The second aim was to set up and optimize a protocol based on the reaction of sulphur, phosphate and vanillin (SPV) for measuring lipid content of MVs. The third aim was to study the effect of thrombin and proteinase inhibitor PPACK to the vesiculation of platelets. Platelets were isolated from the whole blood of healthy volunteers and vesicles were produced by platelet agonists mediating thrombogenic activation (thrombin and collagen, TC), pathophysiological activation (lipopolysaccharide, LPS) and Ca-ionophore (A23187) as positive control for vesiculation. Quantification and size determination of produced MVs was done using Nanoparticle Tracking Analysis (NTA). MVs were characterized by protein content using bicinchonic acid assay (BCA) and by lipid content using SPV-reaction. MVs had great activation-dependent differences in the lipid and the protein content. Activation with Ca-ionophore produced the most MVs, but the lipid and protein content was only a fraction from (patho)physiologically induced MVs. Only TC increased vesiculation. Vesicle subpopulations had significant difference in lipid content. Thrombin and proteinase inhibitor PPACK mediated inhibition of platelet formation in all of the activations, but the effect was not statistically significant. The mechanism of inhibition was likely to be proteinase inhibitor mediated. The isolation of vesicle populations using differential centrifugation proved to isolate studied populations only partially and the quantification method with NTA was susceptible to concentrated samples. SPV protocol reacted with different intensity to different lipids. In the future, quantification and isolation methods for MVs and the subpopulations of MVs should be improved. Additionally, to understand the physiologically relevant mechanisms of platelet-derived vesicle formation, the inhibitor experiments with PPACK should be continued, because the number of replicates was too low to see significant effects due to a large donor-dependent deviation. Since MVs are heterogenous cellular multitools affecting varying (patho)physiological phenomena, optimization and standardization of methods should be continued in order to study MVs properly.
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