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Tutkimuksen tavoitteena oli selvittää mahdollisuutta hyödyntää metsätalousalueita ja metsätaloudessa käytettyjä vesiensuojelumenetelmiä maatalouden vesiensuojelussa. Kolmelta Keski-Suomessa sijaitsevalta esimerkkivaluma-alueelta etsittiin sellaisia teoreettisia kosteikoiksi tai pintavalutuskentiksi soveltuvia kohteita, joihin olisi mahdollista johtaa peltojen valumavesiä. Tutkimuksessa hyödynnettiin RiverLifeGIS-paikkatietotyökalua. Lisäksi selvitettiin kirjallisuuden avulla kosteikkojen ja pintavalutuskenttien toimivuuteen ja käyttökelpoisuuteen vaikuttavia tekijöitä sekä verrattiin maatalouden ravinnekuormitusta metsätalouden kuormitukseen. Esimerkkiarvio maatalouden todellisesta ravinnekuormituk-sesta saatiin käyttämällä Hovin valuma-alueen yhden vuoden seurantajakson jatkuvatoimiseen mittaukseen perustuvia kuormituslukuja. Kyseisiä kuormituslukuja käytettiin arvioitaessa tutkittujen esimerkkivaluma-alueiden laskennallista kuormitusta ja teoreettisten kosteik-kokohteiden hyödyntämisen vaikuttavuutta. Hovin valuma-alueen seurantajaksolla mitattu kokonaistypen kuormitus oli 5,9 kg peltohehtaarilta vuodessa ja kokonaisfosforin kuormitus 2,6 kg peltohehtaarilta vuodessa. Kaikilta tutkituilta esimerkkivaluma-alueilta löydettiin teoreettisia kosteikkokohteita. Valuma-alueiden pelloista 19–49 % oli sellaisia, joiden valumavedet voitaisiin johtaa teoreettisiin kosteikkokohteisiin. Laskennallisen arvion mukaan maatalouden kokonaistypen kuormitusta voitaisiin tutkituilla valuma-alueilla vähentää 10–23 % ja kokonaisfosforin kuormitusta 9-21 %, mikäli teoreettisia kosteikkokohteita hyödynnettäisiin tehokkaasti. Jo 10–15 %:n kuormitusvähenemää voidaan pitää merkittävänä valuma-aluetasolla. Kokonaisuutena maatalouden ravinnekuormitus ja sen hallinta on kuitenkin erittäin vaikeasti hallittavissa oleva kokonaisuus. Sekä ravinteiden huuhtoutumiseen että niiden pidättymiseen vaikuttavat useat tekijät, minkä vuoksi yksittäisten tulosten yleistämiseen on suhtauduttava varauksella.
Tämä maisterin kirjallinen opinnäyte käsittelee herkkyyden ja näyttelijäntekniikan suhdetta. Opinnäytteessä esitellään näyttelijän näkökulmasta käsin erilaisia kokemuksia herkkyydestä ja sen muodoista näyttämöllä ja kameratyöskentelyssä. Tulokulmat herkkyyteen vaihtelevat ilmaisun, esitystilanteen, prosessin ja esiintyjän oman henkilökohtaisen tason hyödyntämisessä osana näyttelemistä. Lopuksi opinnäytteessä pohditaan itkuvirsiharjoittelun kautta sitä, miten herkkyys voi toimia myös teoksen sisällöllisenä lähtökohtana.
Metsäpalojen sammuttaminen on vaikeakulkuisen maaston ja pitkäkestoisuuden vuoksi hyvin kuormittavaa, ja tämän vuoksi sammutukseen on kehitetty sammutussäiliö, jonka avulla voitaisiin helpottaa sammuttajan kuormitusta ja tehostaa myös itse sammutustyötä. Metsäkoneen ja sammutussäiliön yhdistelmällä saadaan kuljetettua sammutusvesi ja -kalusto hyvinkin vaativaan maastoon ja näin ollen voidaan säästyä myös pitkiltä selvitysmatkoilta. Opinnäytetyön tavoitteena oli testata sammutussäiliön toimintoja, jolloin sammutussäiliöstä saataisiin metsäkoneeseen yhdistettynä tehokas työkalu metsäpalojen sammutukseen. Tutkimus toteutettiin testaamalla sammutussäiliön ominaisuuksia sekä ennalta sovituilla testipaikoilla että hälytystehtävillä. Tutkimustulokset osoittivat, että sammutussäiliön käyttö metsäpalojen sammutuksessa antoi toimivan vaihtoehdon perinteiselle sammutustekniikalle. Metsäkoneen kyky kulkea maastossa ja sammutusveden vieminen säiliöllä kohteeseen ilman pitkiä selvitysmatkoja helpotti sammuttajan kuormitusta. Säiliössä olevan kaluston avulla pystytään kohteessa toimimaan hyvinkin itsenäisesti, jos lähistöllä on käytettävissä luonnonvesilähde. Metsäkoneen ja sammutussäiliön tehokkaassa käytössä on oleellista pelastustyön johtajan, sammuttajan ja metsäkoneen kuljettajan onnistunut yhteistyö. Sammutusominaisuuksien ohella nousi esille myös metsäkoneen ja sammutussäiliön käyttö apuna kaluston viemiseen ja letkujen selvittämiseen kohteessa. Käyttökokemusten myötä saadaan varmasti lisää tietoa ja ajatuksia sammutussäiliön toimintojen kehittämiseen.
Tutkimuksessa selvitettiin tulvavesien tilapäisen varastoinnin vaikutusta kunnostusojitusalueen puuston kasvuun. Viitasaarelta pohjoisesta Keski-Suomesta valittiin 10 mäntyvaltaista metsikköä, joissa putkipato oli ollut käytössä vähintään 5 kasvukautta inventointihetkeen mennessä. Kuhunkin metsikköön perustettiin padon läheinen toimenpidekoeala sekä vastaava vertailukoeala, jotka inventoitiin syksyllä 2009. Keskimääräiset tilavuuskasvut mitattiin kairatuista lustonäytteistä lustomikroskoopilla. Puuston kokonaistilavuudet puolestaan laskettiin mitattujen puustotunnusten perusteella. Koealoilta kerätyistä turvenäytteistä määritettiin ylimmän turvekerroksen pääravinteiden pitoisuudet. Toimenpidekoealoilla seurattiin lisäksi pohjavesipinnan syvyyttä. Putkipatojen ja koealakeskipisteiden korkeusasemat määritettiin vaaitsemalla, jotta voitiin arvioida padotuksen vaikutusalueen ulottuvuutta. Putkipadot eivät aiheuttaneet keskimääräisen pohjavesipinnan haitallista kohoamista loppukesällä, jolloin puiden vedenkestävyys on heikoimmillaan. Tulvavesi ei myöskään ollut huuhtonut pääravinteita juuristokerroksesta. Puuston keskimääräiset tilavuuskasvut eivät poikenneet toisistaan tilastollisesti merkitsevästi toimenpide- ja vertailukoealoilla. Tulosten perusteella putkipatojen rakentamista voidaan suositella. Padotuksen aiheuttama virtausnopeuden hidastuminen vähentää eroosiota ja tehostaa liikkeelle lähteneen kiintoaineen laskeutumista ojan pohjalle. Putkipatojen toimivuudesta kunnostusojitusalueiden vesiensuojelussa on saatu lupaavia tuloksia.
The objective of the bachelor's thesis was to develop a responsive web application that would use a database via REST API and be available without a network connection. Furthermore, the web application should have a possibility to use a QR code scanner. The thesis introduced technologies used and described the full stack development. In addition to the description of the development, it examined what was required for offline use of the developed web application. The web application was created by using ASP.NET Core with React. ASP.NET Core was used as backend and the database was created on SQL Server. Entity Framework Core was chosen for the interaction with the database. Frontend was created with React and its component library called Mate-rial-UI. An open source application opening the camera of the device and identifying the contents of the QR code was chosen as a QR code scanner of the developed application. The offline functionality of the web application was carried out with the technologies of progressive web application. The purpose of the web application was to help inspect different targets, such as industrial equipment. The view of the inspection target would open by scanning the QR code. The main results of this thesis were the actual web application with offline functionality and description of its development. Service Worker code enabled the web application to run offline using cache API. The caching strategies of the web application were Cache first and Network first. The web application uses the local IndexedDB database for storing data offline and it fetches data from the local database to the server when the network is available. Based on the results it seemed that the PWA technologies would in general need more development for storing data offline. The thesis and the web application achieved their objectives. The web application would need further development due to the thesis framework and features which were identified during development. The technologies used for development were suitable and operated well together.
Abstract Objective Computational models of calcium (Ca2+) signaling have been constructed for several cell types. There are, however, no such models for retinal pigment epithelium (RPE). Our aim was to construct a Ca2+ signaling model for RPE based on our experimental data of mechanically induced Ca2+ wave in the in vitro model of RPE, the ARPE-19 monolayer. Methods We combined six essential Ca2+ signaling components into a model: stretch-sensitive Ca2+ channels (SSCCs), P2Y2 receptors, IP3 receptors, ryanodine receptors, Ca2+ pumps, and gap junctions. The cells in our epithelial model are connected to each other to enable transport of signaling molecules. Parameterization was done by tuning the above model components so that the simulated Ca2+ waves reproduced our control experimental data and data where gap junctions were blocked. Results Our model was able to explain Ca2+ signaling in ARPE-19 cells, and the basic mechanism was found to be as follows: 1) Cells near the stimulus site are likely to conduct Ca2+ through plasma membrane SSCCs and gap junctions conduct the Ca2+ and IP3 between cells further away. 2) Most likely the stimulated cell secretes ligand to the extracellular space where the ligand diffusion mediates the Ca2+ signal so that the ligand concentration decreases with distance. 3) The phosphorylation of the IP3 receptor defines the cell’s sensitivity to the extracellular ligand attenuating the Ca2+ signal in the distance. Conclusions The developed model was able to simulate an array of experimental data including drug effects. Furthermore, our simulations predict that suramin may interfere ligand binding on P2Y2 receptors or accelerate P2Y2 receptor phosphorylation, which may partially be the reason for Ca2+ wave attenuation by suramin. Being the first RPE Ca2+ signaling model created based on experimental data on ARPE-19 cell line, the model offers a platform for further modeling of native RPE functions.
AIMS: A fast, non-invasive and observer-independent method to analyze the homogeneity and maturity of human pluripotent stem cell (hPSC) derived retinal pigment epithelial (RPE) cells is warranted to assess the suitability of hPSC-RPE cells for implantation or in vitro use. The aim of this work was to develop and validate methods to create ensembles of state-of-the-art texture descriptors and to provide a robust classification tool to separate three different maturation stages of RPE cells by using phase contrast microscopy images. The same methods were also validated on a wide variety of biological image classification problems, such as histological or virus image classification. METHODS: For image classification we used different texture descriptors, descriptor ensembles and preprocessing techniques. Also, three new methods were tested. The first approach was an ensemble of preprocessing methods, to create an additional set of images. The second was the region-based approach, where saliency detection and wavelet decomposition divide each image in two different regions, from which features were extracted through different descriptors. The third method was an ensemble of Binarized Statistical Image Features, based on different sizes and thresholds. A Support Vector Machine (SVM) was trained for each descriptor histogram and the set of SVMs combined by sum rule. The accuracy of the computer vision tool was verified in classifying the hPSC-RPE cell maturation level. DATASET AND RESULTS: The RPE dataset contains 1862 subwindows from 195 phase contrast images. The final descriptor ensemble outperformed the most recent stand-alone texture descriptors, obtaining, for the RPE dataset, an area under ROC curve (AUC) of 86.49% with the 10-fold cross validation and 91.98% with the leave-one-image-out protocol. The generality of the three proposed approaches was ascertained with 10 more biological image datasets, obtaining an average AUC greater than 97%. CONCLUSIONS: Here we showed that the developed ensembles of texture descriptors are able to classify the RPE cell maturation stage. Moreover, we proved that preprocessing and region-based decomposition improves many descriptors' accuracy in biological dataset classification. Finally, we built the first public dataset of stem cell-derived RPE cells, which is publicly available to the scientific community for classification studies. The proposed tool is available at https://www.dei.unipd.it/node/2357 and the RPE dataset at http://www.biomeditech.fi/data/RPE_dataset/. Both are available at https://figshare.com/s/d6fb591f1beb4f8efa6f.
Aims A fast, non-invasive and observer-independent method to analyze the homogeneity and maturity of human pluripotent stem cell (hPSC) derived retinal pigment epithelial (RPE) cells is warranted to assess the suitability of hPSC-RPE cells for implantation or in vitro use. The aim of this work was to develop and validate methods to create ensembles of state-ofthe- art texture descriptors and to provide a robust classification tool to separate three different maturation stages of RPE cells by using phase contrast microscopy images. The same methods were also validated on a wide variety of biological image classification problems, such as histological or virus image classification. Methods For image classification we used different texture descriptors, descriptor ensembles and preprocessing techniques. Also, three new methods were tested. The first approach was an ensemble of preprocessing methods, to create an additional set of images. The second was the region-based approach, where saliency detection and wavelet decomposition divide each image in two different regions, from which features were extracted through different descriptors. The third method was an ensemble of Binarized Statistical Image Features, based on different sizes and thresholds. A Support Vector Machine (SVM) was trained for each descriptor histogram and the set of SVMs combined by sum rule. The accuracy of the computer vision tool was verified in classifying the hPSC-RPE cell maturation level. Dataset and Results The RPE dataset contains 1862 subwindows from 195 phase contrast images. The final descriptor ensemble outperformed the most recent stand-alone texture descriptors, obtaining, for the RPE dataset, an area under ROC curve (AUC) of 86.49% with the 10-fold cross validation and 91.98% with the leave-one-image-out protocol. The generality of the three proposed approaches was ascertained with 10 more biological image datasets, obtaining an average AUC greater than 97%. Conclusions Here we showed that the developed ensembles of texture descriptors are able to classify the RPE cell maturation stage. Moreover, we proved that preprocessing and region-based decomposition improves many descriptors' accuracy in biological dataset classification. Finally, we built the first public dataset of stem cell-derived RPE cells, which is publicly available to the scientific community for classification studies.
Retinal pigment epithelium (RPE) performs important functions for the maintenance of photoreceptors and vision. Malfunctions within the RPE are implicated in several retinal diseases for which transplantations of stem cell‐derived RPE are promising treatment options. Their success, however, is largely dependent on the functionality of the transplanted cells. This requires correct cellular physiology, which is highly influenced by the various ion channels of RPE, including voltage‐gated Ca2+ (CaV) channels. This study investigated the localization and functionality of CaV channels in human embryonic stem cell (hESC)‐derived RPE. Whole‐cell patch‐clamp recordings from these cells revealed slowly inactivating L‐type currents comparable to freshly isolated mouse RPE. Some hESC‐RPE cells also carried fast transient T‐type resembling currents. These findings were confirmed by immunostainings from both hESC‐ and mouse RPE that showed the presence of the L‐type Ca2+ channels CaV1.2 and CaV1.3 as well as the T‐type Ca2+ channels CaV3.1 and CaV3.2. The localization of the major subtype, CaV1.3, changed during hESC‐RPE maturation co‐localizing with pericentrin to the base of the primary cilium before reaching more homogeneous membrane localization comparable to mouse RPE. Based on functional assessment, the L‐type Ca2+ channels participated in the regulation of vascular endothelial growth factor secretion as well as in the phagocytosis of photoreceptor outer segments in hESC‐RPE. Overall, this study demonstrates that a functional machinery of voltage‐gated Ca2+ channels is present in mature hESC‐RPE, which is promising for the success of transplantation therapies.
Background: Wound healing of retinal pigment epithelium (RPE) is a complex process that may take place in common age-related macular degeneration eye disease. The purpose of this study was to evaluate whether wounding and wound healing has an effect on Ca2+ dynamics in human embryonic stem cell (hESC)-RPEs cultured different periods of time. Methods: The 9-day-cultured or 28-day-cultured hESC-RPEs from two different cell lines were wounded and the dynamics of spontaneous and mechanically induced intracellular Ca2+ activity was measured with live-cell Ca2+ imaging either immediately or 7days after wounding. The healing time and speed were analyzed with time-lapse bright field microscopy. The Ca2+ activity and healing speed were analysed with image analysis. In addition the extracellular matrix deposition was assessed with confocal microscopy. Results: The Ca2+ dynamics in hESC-RPE monolayers differed depending on the culture time: 9-day-cultured cells had higher number of cells with spontaneous Ca2+ activity close to freshly wounded edge compared to control areas, whereas in 28-day-cultured cells there was no difference in wounded and control areas. The 28-day-cultured, wounded and 7-day-healed hESC-RPEs produced wide-spreading intercellular Ca2+ waves upon mechanical stimulation, while in controls propagation was restricted. Most importantly, both wave spreading and spontaneous Ca2+ activity of cells within the healed area, as well as the cell morphology of 28-day-cultured, wounded and thereafter 7-day-healed areas resembled the 9-day-cultured hESC-RPEs. Conclusions: This acquired knowledge about Ca2+ dynamics of wounded hESC-RPE monolayers is important for understanding the dynamics of RPE wound healing, and could offer a reliable functionality test for RPE cells. The data presented in here suggests that assessment of Ca2+ dynamics analysed with image analysis could be used as a reliable non-invasive functionality test for RPE cells.
Age-related macular degeneration (AMD) is a multi-factorial disease that is the leading cause of irreversible and severe vision loss in the developed countries. It has been suggested that the pathogenesis of dry AMD involves impaired protein degradation in retinal pigment epithelial cells (RPE). RPE cells are constantly exposed to oxidative stress that may lead to the accumulation of damaged cellular proteins, DNA and lipids and evoke tissue deterioration during the aging process. The ubiquitin-proteasome pathway and the lysosomal/autophagosomal pathway are the two major proteolytic systems in eukaryotic cells. NRF-2 (nuclear factor-erythroid 2-related factor-2) and PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1 alpha) are master transcription factors in the regulation of cellular detoxification. We investigated the role of NRF-2 and PGC-1α in the regulation of RPE cell structure and function by using global double knockout (dKO) mice. The NRF-2/PGC-1α dKO mice exhibited significant age-dependent RPE degeneration, accumulation of the oxidative stress marker, 4-HNE (4-hydroxynonenal), the endoplasmic reticulum stress markers GRP78 (glucose-regulated protein 78) and ATF4 (activating transcription factor 4), and damaged mitochondria. Moreover, levels of protein ubiquitination and autophagy markers p62/SQSTM1 (sequestosome 1), Beclin-1 and LC3B (microtubule associated protein 1 light chain 3 beta) were significantly increased together with the Iba-1 (ionized calcium binding adaptor molecule 1) mononuclear phagocyte marker and an enlargement of RPE size. These histopathological changes of RPE were accompanied by photoreceptor dysmorphology and vision loss as revealed by electroretinography. Consequently, these novel findings suggest that the NRF-2/PGC-1α dKO mouse is a valuable model for investigating the role of proteasomal and autophagy clearance in the RPE and in the development of dry AMD.
This thesis studies a new possible method for testing the functionality of human embryonic stem cells (hESC) that are differentiated into retinal pigment epithelium (RPE) cells by using electroretinography (ERG). RPE is a cell layer that is situated behind the neurosensory retina at the back of the eye. The main function of the RPE is to support the light sensitive photoreceptor cells of the retina. ERG is a commonly used method for evaluating the functionality of the retina electrophysiologically. In ERG, a light stimulus is given to the retina and an electrical response to the stimulus is recorded. RPE functionality can be seen in the ERG signal in different ways: 1) the presence of the cwave, which is generated in the RPE, 2) increased amplitude of the b-wave when recovering from light adaptation and 3) ability to record light responses longer than without the RPE, because a functional RPE helps the retina to retain its viability. A literature review is done to evaluate the theoretical possibilities for developing a functionality test for hESC-derived RPE cells that is based on ERG. The idea is to bring a mouse retina in contact with a layer of hESC-derived RPE cells and record the effect of the RPE with ERG. Based on the review, no such test has been done before. However, it has been studied that RPE cell layer forms contacts with the retina even after the two have been detached and then reattached, and this can be registered with ERG measurement. This indicates that the hESC-derived RPE cells could behave similarly. A measurement setup is developed for measuring light responses first from isolated mouse retinas alone and later from mouse retinas together with hESC-derived RPE cell layers. The ERG measurements are done with a microelectrode array (MEA) system (Multi Channel Systems, MCS GmbH, Germany). The development of the system included designing a light stimulator, finding a suitable way for performing the tissue preparation and the measurements in darkness as much as possible and constructing a functional method for a short term culture for retina-RPE complexes. The functionality of the measurement setup developed is evaluated by the recorded responses. The recorded light responses of the isolated mouse retinas were good even though an undesired artefact caused by the light stimulator is present in the signals during stimulation. In the measurements from retina-RPE complexes the light stimulator was replaced with a monochromator so that the artefact is not present in the responses. Even though responses to light could be recorded, the stimulus intensity appeared to be too small to gain good responses. The measurement setup developed was found to be functional. Based on these measurements the functionality of the hESC-derived RPE cells could not yet be evaluated, and further development of the setup especially with the light stimulation is still needed. However, the results were promising and this kind of an approach for testing the functionality of the hESC-derived RPE cells might work also in practice. /Kir10