Finnlines Oyj:n liikuntaluotsitoiminnan kehittäminen

Tämän opinnäytetyön tavoitteena oli kehittää Finnlines Oyj:n liikuntavastaavien eli -luotsien toimintaa ja antaa heille konkreettisia työkaluja sekä neuvoja heidän toimintaansa laivaympä-ristössä. Projektin tarkoituksena oli, että luotsit pystyvät toiminnallaan jatkossa tavoittamaan laajemmin merihenkilöstöä sekä auttamaan henkilöstöä motivoitumaan liikkumiseen laivoilla. Tässä projektissa tutkittiin ensiksi kyselyllä merihenkilöstön motiiveja liikkumiseen laivatyö-jaksoilla sekä heidän kokemuksiaan liikuntaluotsien toiminnasta tähän asti. Kyselyyn vastasi yhteensä 83 henkilöä. Tulosten pohjalta suunniteltiin liikuntaluotseille kahden päivän koulutus, johon osallistui kahdeksan henkilöä Finnlines Oyj:n henkilöstöstä. Koulutuksen tavoitteena oli ohjatun oivaltamisen ja ongelmanratkaisujen avulla ohjata luotseja kehittämään yhdessä toimintatapoja merihenkilöstön liikuntamotivaation ja liikunta-aktiivisuuden edistämiseksi. Merihenkilöstöstä 54 % oli laivatyöjaksoilla liikunnallisesti passiivisia ja he motivoituivat liik-kumaan eniten sisäisistä motivaatiotekijöistä, kuten liikunnasta saadusta hyvän olon tunteesta. Vastaajista 43 % ei ollut kuullut liikuntaluotseista ja 83 % eivät olleet ollut luotsien kanssa tekemisissä. Koulutuksen tuloksena liikuntaluotsit suunnittelivat yhdessä laivoille toteutettaviksi konkreettiset liikuntakampanjat, jotka motivoivat laajalti merihenkilöstöä liikkumaan. Lisäksi he kehittivät tapoja tuoda omaa toimintaansa enemmän esille, kuten ilmoitustauluilla luotsien esittely, ja luotsien välisen yhteydenpitokanavan. Liikuntaluotsit ovat innokkaita ja liikunta-asioista kiinnostuneita vapaaehtoisia. He ovat oivaltaneet, että heidän aktiivisuudellaan on mahdollista saada paljon aikaan. Jatkossa luotsien ja heidän yhteyshenkilöidensä välinen yhteydenpito sekä suunniteltujen toimintojen toteuttaminen ovat merkittävässä asemassa luotsien toiminnan sekä motivaation ylläpitämiseksi

Localization of substituted amino acids in the SDH subunits of <i>M. graminicola</i> UV mutants and degree of conservation across species.

<p>Asterix indicate substituted residues, conserved residues are shaded in black, dark grey and light grey corresponding to 100%, 80% and 60% conservation respectively. Sequences are from <i>M. graminicola</i> (Mg), <i>Alternaria alternata</i> (Aa), <i>Alternaria oryzae</i> (Ao), <i>B. cinerea</i> (Bc), <i>Magnaporthe grisea</i> (Mag), <i>S. cerevisiae</i> (Sc), <i>Ustilago maydis</i> (Um), <i>G. gallus</i> (Gg), <i>S. scrofa</i> (Ss) and <i>E. coli</i> (Ec).</p

Absence of a major oxidative stress related fitness penalty in <i>M. graminicola</i> homologous recombinant strains carrying Qp site mutations.

<p>A: Respiratory growth of wild type and mutants in the presence of oxidative stresses. Upper panel, AE agar plate growth, middle panel: AE agar supplemented with 10 mM Paraquat, lower panel: AE agar plates were placed in a constant 100% atmosphere. All plates were incubated in same room at room temperature for 6 days. B: Sensitivity towards hydrogen peroxide in liquid AE media. Values presented are the averages from replicates within 3 individual experiments. C: Evaluation of mitochondrial ROS produced <i>in vivo</i> as determined with MitoSOX™ Red fluorescent indicator. Evaluations based on values from over 3 replicates within 3 individual experiments. +PQ corresponds to 1 mM Paraquat supplementation during the incubation period, +H₂O₂ correspond to 10 mM H₂O₂ supplementation during the incubation period. WT (IPO323), and homologous recombinant strains HRAV: SDHC_A84V, HRDG: SDHD_D129G, HRHL: SDHB_H267L, HRHR: SDHC_H152R, HRHY: SDHB_H267Y, HRIV: SDHB_I269V, HRNK: SDHC_N86K, HRRP: SDHB_R265P, HRSG: SDHC_S83G.</p

Scatter plot presenting <i>in vivo</i> LogIC₅₀ (nM) versus <i>in vitro</i> LogIC₅₀ (nM) adjusted (B) or not (A) for enzyme efficiency.

<p>All data extracted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035429#pone-0035429-t003" target="_blank">table 3</a> and adjusted for amount of enzyme used in the sensitivity test in panel B. Adjustment was performed using enzyme efficiency data extracted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035429#pone-0035429-t004" target="_blank">table 4</a> following a simple equation: if percent efficiency is denoted by E then the efficiency adjusted IC₅₀ = observed IC₅₀×E/100. Following this adjustment the correlation was improved for all compounds.</p

Comparison of the resistance phenotypes displayed by transformants of the SDHB or SDHC subunits.

<p>Two types of transformants were created, (i) Tr strains where the genes encoding either SDHB or SDHC subunits were ectopically inserted under the control of a GPDA promoter, (ii) HR strains where the WT gene was replaced by a mutated version in its original genomic context. A: SDH inhibition displayed by mitochondrial extracts as measured with the succinate: Q₀/DCPIP reduction test in the presence of varying concentrations of Boscalid. Fitted curve is monophasic with the ectopic transformant containing the WT SDHB expression cassette (black dots) and with the homologous recombinant strain carrying the SDHB_H267L mutation (HR B_H267L, black triangles). Inhibition is biphasic with the ectopic transformant containing the SDHB_H267L expression cassette (Tr B_H267L, black squares). B: Southern blot of genomic DNA extracted from (left to right), the WT (IPO323), one ectopic transformant carrying the WT_SDHB expression cassette, one ectopic transformant carrying the SDHB_H267L expression cassette and signal obtained with one SDHB_H267L homologous recombinant. Boscalid (C) and Fluopyram (D) resistance phenotypes displayed by ectopic and homologous recombinants transformants. In both cases when additional WT SDHB (Tr_WT_SDHB) and SDHC (Tr_WT_SDHC) were inserted ectopically, no significant increase in resistance was observed. When an additional mutated copy of SDHB (Tr_SDHB_H267L) or of SDHC (Tr_SDHC_A84V) was inserted ectopically, significant resistance to the compounds was observed, clearly indicating a dominant effect of the mutated alleles. However, in the homologous recombinant strains where only the mutated subunit SDHB (HR_SDHB_H267L) or SDHC (HR_SDHC_A84_V) was present a further increase in resistance is observed. Whiskers represent minimum and maximum RFs obtained, boxes represent 95% confidence interval and bars value of the median. The data presented correspond to average values of duplicated tests with four independent events of each kind.</p

Tridimensional model of the <i>M. graminicola</i> SDH with Qp site docked carboxamides and interactions to substituted residues.

<p>A: Superposition of all complex II crystal structures with a resolution higher than 3 Å. Only amino acids that are in a 5 Å radius to the bound ligands in the quinone binding site are shown. Amino acids and the heme are represented in lines, the ligands in sticks and water molecules as non bonded spheres. The color code is presented according to atom types. Amino acid and heme carbons are colored in green, ligands carbons in salmon. B: putative binding mode of carboxin in a tridimensional model of <i>M.graminicola</i> SDH. The heme carbons are represented in cyan sticks, the carboxin carbon atoms in salmon. Amino acids that are involved in resistance after mutation are colored in dark blue, amino acids that make key interactions are shown in green and amino acids that are in close proximity to the ligand but which were not found substituted in this study are colored in grey. Hydrogen bonds are shown as yellow dotted lines. C: Putative binding mode of Boscalid in <i>M.graminicola</i> SDH. D: Putative binding mode of Fluopyram in <i>M. graminicola</i> SDH. E: Putative binding mode of Isopyrazam in <i>M. graminicola</i> SDH. F: Model of <i>M. graminicola</i> SDH where SDHB histidine 267 is mutated into a tyrosine. The putative binding mode of the mutated enzyme with Carboxin is shown.</p

Mutagenesis experiments overview.

<p>Mutagenesis experiments overview.</p

Comparison of <i>in planta</i> necrosis symptoms displayed by individual homologous recombinant strains carrying mutations conferring resistance to carboxamides.

<p>Data presented correspond to the individual means (average of 12 leaves each) of 3 individual experiments ± S.D. Numbering corresponds to individual homologous recombinants strains carrying: HRHL1, 4 and 8: SDHB_H267L substitution, HRSG1 and 2: SDHC_S83G substitution, HRHR1 and 4: SDHC_H152R substitution, HRNK1: SDHC_N86K substitution, HRAV1, 2 and 3: SDHC_A84V substitution.</p

Selected colonies and detailed quantitative and qualitative overview of the SDH substitutions selected with different carboxamides concentrations.

<p>Numbers correspond to the total number of colonies belonging to each sub-group (true/false positives and with/without mutation in the SDH encoding genes) for each selection condition (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035429#s4" target="_blank">material and methods</a>). The second part details the number of colonies for the 27 SDH substitution type selected on each carboxamide concentration.</p

<i>In vivo</i> and <i>in vitro</i> IC50s and resistance factors overview.

<p>Upper panel: <i>In vivo</i> and <i>in vitro</i> IC50 values obtained for the WT (IPO323) ±S.E. (triplicate). Lower panel: resistance factors (RFs) based on IC50 assessment for a selected subset of representative strains. Presented values are based on the ratio of the means of three individual determinations for the <i>in vivo</i> values and based on the ratio of a single determination for the <i>in vitro</i> values. Presented <i>in vitro</i> values were obtained by calibrating mitochondrial dilutions to obtain similar initial velocity (see<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035429#s4" target="_blank"> material and methods</a>). Cbx strains were originally obtained on Carboxin selection media, Flu on Fluopyram, Bos on Boscalid, Izm on Isopyrazam, Ol on pyrrole compound A. nd*: IC₅₀ could not be determined because fitted curves were not tending to 100% inhibition at infinite AI concentration. FR** (full resistance), no sufficient inhibition detected at highest inhibitor concentration tested.</p