Lääkeaineiden solunsisäinen jakautuminen (kirjallisuuskatsaus) : Solunosien fraktionaatioon perustuva menetelmä plasma membraanin ja sytoplasman eristykseen MDCKII -soluista (erikoistyö)

Intracellular drug sequestration is useful to understand when designing new drugs with intracellular targets. The knowledge of the intracellular distribution can also help to understand the side effects and pharmacokinetics of a drug, as well as the lack of response in e.g. some multidrug resistant cancer cells. Intracellular concentrations are also important to know when predicting the role of active transport in the overall transport process when binding site of the transporter is intracellular. The literature review describes the mechanisms causing intracellular drug sequestration along with the consequences of intracellular drug sequestration and methods that are used to study it. Alterations of intracellular distribution of anticancer drugs in multidrug resistant cancer cells are also described as an example of the many factors affecting the distribution pattern of the drugs inside cells. Understanding these mechanisms is valuable when designing strategies to overcome the multidrug resistance. The most commonly applied methods for studying intracellular concentrations of drugs are based on fluorescence microscopy. In experimental work, subcellular fractionation protocol is introduced and applied to determine the concentration of CDCF, clotrimazole and celiprolol in vitro in the plasma membrane and cytoplasm of MDCKII cells. CDFC and celiprolol are substrates of the MDR1 transporter and clotrimazole is an inhibitor. Concentrations in the fractions were measured in wild type cells and in MDR1-transfected cells with and without MDR1 inhibitor verapamil to see if the transporter had an effect on the concentrations. Also the effect of lipophilicity of the drug on partition between plasma membrane and cytoplasm was reviewed. Celiprolol showed a typical behaviour of the MDR1 substrate whereas CDCF and clotrimazole did not. Clotrimazole as a lipophilic compound was accumulated more to the plasma membrane than less lipophilic CDCF and celiprolol. Lipophilicity affected also to the ratio of Km (or Ki)(determined from the concentration in extracellular fluid) and Km (or Ki)(membrane) (determined from the plasma membrane concentration) values, with clotrimazole Ki(membrane) value being larger than respective Ki value, and CDCF and celiprolol Km(membrane) values being smaller than their respective Km values.Lääkeaineiden solunsisäinen jakautuminen on hyödyllistä ymmärtää kehitettäessä uusia lääkkeitä, joiden vaikutuskohteet ovat solunsisäisiä. Tieto solunsisäisestä jakautumisesta voi auttaa myös ymmärtämään lääkkeiden haittavaikutuksia ja farmakokinetiikkaa, sekä tehon puutetta esim. lääkeaineille vastustuskykyisissä syöpäsoluissa. Solunsisäiset pitoisuudet ovat myös tärkeitä ennustettaessa aktiivisen kuljetuksen osuutta koko lääkeaineen kulkeutumisprosessissa, silloin kun kuljettajaproteiinin sitoutumiskohta on solun sisällä. Kirjallisuuskatsauksessa tarkastellaan lääkeaineen solunsisäisen jakautumisen mekanismeja, seurauksia joita jakautumisella on ja tapoja joita käytetään sen tutkimiseen. Osassa lääkkeille vastustuskykyisistä syöpäsoluista lääkeaineen solunsisäinen jakautuminen on muuttunut ja kirjallisuuskatsauksen lopussa tarkastellaan näitä tapauksia esimerkkeinä havainnollistamaan monia jakautumiseen vaikuttavia tekijöitä. Näiden tekijöiden ja mekanismien ymmärtäminen on tärkeää suunniteltaessa tapoja vastustuskykyisten syöpäsolujen tuhoamiseen. Yleisimmin käytetyt tavat tutkia lääkkeiden solunsisäisiä pitoisuuksia perustuvat fluoresenssimikroskopiaan. Erikoistyössä esitellään menetelmä, jossa eri sentrifugaationopeuksia käyttäen erotellaan solunosia toisistaan. Menetelmää hyväksikäyttäen määritettiin CDCF:n, klotrimatsolin ja seliprololin pitoisuudet in vitro MDCKII -solujen plasma membraanissa ja sytoplasmassa. CDCF ja seliprololi ovat MDR1-proteiinin substraatteja ja klotrimatsoli on sen inhibiittori. Osioiden pitoisuudet mitattiin villityypin soluissa ja MDR1-proteiinia ilmentävissä soluissa MDR1-inhibiittori verapamiilin kanssa ja ilman sitä, jotta nähtiin onko kuljettajaproteiinilla vaikutusta pitoisuuksiin. Myös rasvaliukoisuuden vaikutusta lääkeaineiden jakautumiseen plasma membraanin ja sytoplasman välillä tutkittiin. Seliprololi käyttäytyi kokeissa kuten MDR1-substraatin oletetaan käyttätyvän, mutta CDCF ja klotrimatsoli eivät. Rasvaliukoisena yhdisteenä klotrimatsoli kertyi enemmän plasma membraaniin kuin vähemmän rasvaliukoiset CDCF ja seliprololi. Rasvaliukoisuus vaikutti myös Km (tai Ki)(määritetty solunulkoisen nesteen pitoisuuksista) ja Km (tai Ki)(membraani) (määritetty pitoisuuksista plasma membraanissa) suhteeseen, siten että rasvaliukoisella klotrimatsolilla Ki(membraani) oli suurempi kuin vastaava Ki ja vähemmän rasvaliukoisilla CDCF:llä ja seliprololilla Km(membraani) oli pienempi kuin vastaava Km

Effects of a tree row on greenhouse gas fluxes, growing conditions and soil microbial communities on an oat field in Southern Finland

Agricultural ecosystems are facing critical loss of biodiversity, soil nutrients, and cultural values. Intensive crop production has caused landscape homogenisation, with trees and hedges increasingly disappearing from agricultural land. Changes in farming practices are essential to increase biodiversity and improve soil biogeochemical processes, such as nutrient cycling, soil carbon uptake, and sequestration, as well as to improve the resilience and fertility of farming systems. Agroforestry is an important practice for implementing and improving natural and cultural value of landscapes, but in northern countries, agroforestry methods remain rarely utilised. Our study was conducted in Southern Finland on an agricultural field where a row of willow and alder was planted 6 years prior to our study. We concentrated on the effects of the tree row on crop growing conditions and how far from the trees possible impacts can be observed. We studied soil properties, carbon dioxide (CO2), nitrous oxide (N2O), and methane (CH4) exchange, and soil microbial communities. The impact of trees on crop growing conditions, biomass production, and greenhouse gas fluxes was modest and did not extend further than few meters from the tree row in the warm and dry growing season of 2019. N2O and CH4 fluxes were negligible and the tree row did not increase greenhouse gas emissions from soil. Soil microbial diversity was clearly improved by the presence of trees due to more diverse habitats. The tree row also slightly decreased the estimated annual net emissions of carbon into the atmosphere. Due to positive indications of the effects of agroforestry on biodiversity and carbon uptake, we highly recommend further studies within various agroforestry practices in Nordic countries

Assessing the relation between geodiversity and species richness in mountain heaths and tundra landscapes

Context Recent studies show that geodiversity—the diversity of Earth's landforms, materials, and processes—has a positive relationship with biodiversity at a landscape scale. However, there is a substantial lack of evidence from finer scales, although this knowledge could improve the understanding of biodiversity patterns. Objectives We investigate whether plot-scale geodiversity and plant species richness (vascular plants, bryophytes, lichens, and total richness) are positively linked in different tundra landscapes. Methods We collected geodiversity (presence of different geofeatures) and plant species richness data from 165 sites in three distinct regions: isolated low-lying mountain heaths, and in sporadic and continuous mountain heaths and tundra. We used non-metric multidimensional scaling (NMDS) ordination to explore the correlations between the composition of geofeatures and species richness, followed by univariate and multivariate generalized linear models (GLM), to assess whether georichness is important for species richness. Results Geofeature composition was linked to species richness in all regions, as indicated by NMDS ordination. Both univariate and multivariate GLM models showed statistically significant relationship between species richness and georichness in all studied species richness groups in continuous Arctic-alpine tundra. Additionally, there was a positive link between georichness and lichen richness in isolated boreal mountain tops. Main conclusions We showed that plot-scale geodiversity has a positive relationship with species richness, yet the effect varies regionally and between species groups. Our study provides strong empirical evidence that geodiversity supports species richness in continuous Arctic-alpine tundra. This information can be used in species richness models but also be applied in biodiversity management and conservation.publishedVersio

Too much diversity—Multiple definitions of geodiversity hinder its potential in biodiversity research

Geodiversity—the diversity of abiotic features and pro-cesses of the Earth's surface and subsurface—is an increasingly used concept in ecological research. A growing body of scientific literature has provided evidence of positive links between geodiversity and biodiversity. These studies highlight the potential of geodiversity to improve our understanding of biodiversity patterns and to complement current biodiversity conservation practices and strategies. However, definitions of geodiversity in eco-logical research vary widely. This can hinder the progress of geodiversity–biodiversity research and make it difficult to synthesize findings across studies. We therefore call for greater awareness of how geodiversity is currently defined and for more consistent use of the term ‘geodi-versity’ in biodiversity research

Carbon guide : An outlook on carbon in soil and on carbon cultivation

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Pohjaveden suojausmenetelmät alikulkukäytävän kohdalla

Pohjavedensuojauksen rakentaminen vaatii hyvät esitiedot ja suunnittelun, jotta rakentaminen on ympäristölle turvallinen ja kestävä. Oikein toimiva pohjavesisuojaus mahdollistaa pohjaveden käyttämisen yhdyskunnan käyttövetenä myös onnettomuustilanteen sattuessa. Opinnäytetyön tavoitteena oli laatia Elinkeino-, liikenne- ja ympäristökeskukselle (ELY-keskus) suunnittelutyökalu, jonka avulla voidaan helposti valita sopiva pohjaveden suojausmenetelmä alikulkukäytävän kohdalle. Suunnittelutyökalun tarkoitus oli linjata, miten suojaustapa valitaan ympäristöolosuhteiden mukaan. Opinnäytetyössä perehdyttiin kloridisuojauksen sekä onnettomuussuojauksen toimivuuteen alikulkukäytävillä ja -silloilla ympäristölle hyvästä näkökulmasta. Työssä selvitettiin, mitkä geotekniset asiat voivat vaikuttaa pohjavesisuojauksen toimivuuteen. Lisäksi selvitettiin, mitkä yhteiset tekijät vaikuttavat suojauksen rakennustavan valintaan. Opinnäytetyössä laadittiin suunnittelutyön tueksi apuohje, jota suunnittelijat tai aikataulusta vastaavat henkilöt voivat käyttää. Työssä laadittua suunnittelutyökalua voivat käyttää myös henkilöt, joilla on vähäistä kokemusta pohjavesirakentamisesta alikulkukäytävän kohdalla. Opinnäytetyö tehtiin yhteistyössä Plaana Oy:n kanssa.This thesis is written to Centre for Economic Development, Transport and the Environment. The purpose was to make a tool for designing work. The main point is to help in selection of groundwater protection method. It was important to make clear what protection method is proper in certain conditions. There is being compared which groundwater protection method is more efficient environmentally or financially in this thesis. Someone who is not experienced with constructing on groundwater area can use this tool. This thesis is made in cooperation with Plaana Ltd. Plaana is a urban planning company. Most of the materials are from Plaana and Finnish Transport Infrastructure Agency. In this thesis groundwater protection is regarded from theoretical and practical angles. The practical parts are examples from real building projects

Dynamics of root-associated fungal communities in relation to disturbance in boreal and subarctic forests

Abstract Disturbance may shift microbial communities from one state to another. However, species differ in their ecological characteristics and their abilities to withstand disturbance. No single species or individuals of a species exist alone, but they are parts of complex interaction networks including species above- and belowground. In boreal and subarctic forests, almost all plants and a high number of fungi form mycorrhizas at the plant roots. In mycorrhiza, the fungal partner harvests nutrients for the host plant and, in return, gains carbon from the plant. In general, these common associations benefit both partners, but as heterotrophs, fungi are dependent on carbon photosynthesized by plants, whereas plants can survive alone as autotrophs. In addition to mycorrhizal fungi, also other fungi, such as endophytes, saprotrophs and pathogens, live in and on plant roots. This thesis concerns the impacts of disturbance on fungi living in plant roots and in soil near the roots. I hypothesized that i) root-associated fungal (RAF) and soil fungal communities and colonization types change after disturbance, that ii) the observed shifts relate to disturbance intensity and that iii) they co-occur with changes in soil conditions and vegetation. Changes in RAF were studied as changes in root fungal colonization, or in fungal community composition. The latter were detected with next-generation sequencing methods. The responses of RAF to disturbance seemed to be context dependent and related to sources of fungal communities (e.g. soil, RAF networks), environmental conditions (e.g. soil pH and nutrients) and host performance. It seems that abundances of those RAF species, which are present in the roots first (priority effect), may be increased by disturbance. Research produced new information related to ecological roles of the genera Phialocephala and Meliniomyces. Altogether, the results indicate connections between both abiotic and biotic environments and RAF, and host species viability and RAF.Tiivistelmä Häiriöt voivat siirtää eliöyhteisön tilasta toiseen. Lajien ominaisuudet ja häiriönsietokyvyt eroavat toisistaan. Mikään laji tai yksilö ei elä yksin, vaan lajit ovat osa maan ylä- ja alapuolelle ulottuvia monimutkaisia vuorovaikutusverkostoja. Boreaalisissa ja subarktisissa metsissä lähes kaikki kasvit ja useat sienet muodostavat sienijuuren eli mykorritsan. Mykorritsassa sieniosakas hankkii isäntäkasville ravinteita ja saa vastavuoroisesti kasvilta hiiltä. Tavallisesti nämä vuorovaikutussuhteet hyödyttävät molempia sienijuuren osakkaita, mutta toisenvaraisina (heterotrofeina) sienet ovat riippuvaisia kasvien yhteyttämästä hiilestä, kun taas tuottajina (autotrofeina) kasvit voivat elää itsenäisesti. Mykorritsasienten lisäksi kasvien juurissa elää yleisesti myös muita sieniä kuten endofyyttejä, saprotrofeja ja patogeeneja. Tämä väitöskirja käsittelee häiriön vaikutuksia sieniin, jotka elävät kasvien juurissa ja juuria ympäröivässä maassa. Hypoteesieni mukaan i) juurissa ja maassa elävien sienten yhteisöt ja kolonisaatiotyypit muuttuvat häiriön jälkeen, ii) muutokset liittyvät häiriön voimakkuuteen ja iii) muutokset tapahtuvat samanaikaisesti maan olosuhteiden ja kasvillisuuden muutoksien kanssa. Juurissa elävien sienten esiintymisen muutokset tutkittiin sienten kolonisaation tai yhteisörakenteen muutoksina. Sieniyhteisöt selvitettiin NGS-menetelmien avulla. Juurissa elävien sienten vasteet häiriöön vaikuttavat olevan tilannesidonnaisia ja liittyvän sienilajien lähteisiin (esim. maa, juurisieniverkostot), ympäristömuuttujiin (esim. maan pH, ravinteet) ja isäntäkasvin menestymiseen. Häiriö voi vahvistaa juurissa ensimmäisenä läsnä olevien sienilajien menestymistä (prioriteettivaikutus). Uutta tietoa tuotettiin Phialocephala ja Meliniomyces –sienisukujen ekologiasta, jota tunnetaan huonosti. Kaiken kaikkiaan, tulokset osoittavat yhteydet sekä elottoman ja elollisen ympäristön ja juurten sieniyhteisön että isäntäkasvin elinkyvyn ja juurten sieniyhteisön välillä

Effects of a tree row on greenhouse gas fluxes, growing conditions and soil microbial communities on an oat field in Southern Finland

Abstract Agricultural ecosystems are facing critical loss of biodiversity, soil nutrients, and cultural values. Intensive crop production has caused landscape homogenisation, with trees and hedges increasingly disappearing from agricultural land. Changes in farming practices are essential to increase biodiversity and improve soil biogeochemical processes, such as nutrient cycling, soil carbon uptake, and sequestration, as well as to improve the resilience and fertility of farming systems. Agroforestry is an important practice for implementing and improving natural and cultural value of landscapes, but in northern countries, agroforestry methods remain rarely utilised. Our study was conducted in Southern Finland on an agricultural field where a row of willow and alder was planted 6 years prior to our study. We concentrated on the effects of the tree row on crop growing conditions and how far from the trees possible impacts can be observed. We studied soil properties, carbon dioxide (CO₂), nitrous oxide (N₂O), and methane (CH₄) exchange, and soil microbial communities. The impact of trees on crop growing conditions, biomass production, and greenhouse gas fluxes was modest and did not extend further than few meters from the tree row in the warm and dry growing season of 2019. N₂O and CH₄ fluxes were negligible and the tree row did not increase greenhouse gas emissions from soil. Soil microbial diversity was clearly improved by the presence of trees due to more diverse habitats. The tree row also slightly decreased the estimated annual net emissions of carbon into the atmosphere. Due to positive indications of the effects of agroforestry on biodiversity and carbon uptake, we highly recommend further studies within various agroforestry practices in Nordic countries

Temporal changes in boreal vegetation under 70 years of conservation

Abstract Biodiversity conservation through protected areas (PAs) is often based on the idea that biodiversity is relatively static. This assumption is increasingly being challenged as species and communities shift their distributions in response to changing environmental conditions. Empirical evidence on the performance of PAs over decades is still sparse or lacking from several environments, although it is needed to understand species dynamics, support modelling of PA performance, assist PA management and ultimately, to achieve global biodiversity conservation goals. In 2021, we resurveyed vegetation of five boreal habitat types (heath forests, paludified forests, sun-exposed sites, mires and eulittoral sites) in Rokua National Park in Finland, where one of the conservation targets is to preserve the flora characteristic of the area. The study sites were originally surveyed in 1945‐49, just before the National Park was established. Study sites have also remained free from the disturbances (forest fires and reindeer grazing) typical of boreal regions. We show that the compositional similarity of plant communities between habitat types has increased over time and is associated with the increase of forest species in several habitat types and the loss of many habitat-specific species. Drivers of change were most often linked to ongoing succession (understory closure) and changes in moisture conditions. Our results suggest that without natural disturbance or appropriate management efforts, the original conservation targets may be compromised over the decades. Our study demonstrates that resurvey of historical vegetation data can be effectively used to estimate long-term PA performance, helping to fill in missing temporal evidence