Tuotantolaitoksen logiikoiden kartoitus ja modernisointi

Opinnäytetyön tarkoituksena oli suorittaa ensin yrityksen toimitiloissa sijaitsevien laitteiden logiikoiden kartoitus, jonka pohjalta saatiin selville erinäisten logiikkamerkkien ja -mallien määrät ja näiden pohjalta voitiin tarkistaa varaosien saatavuuksia. Lisäksi opinnäytetyössä oli tarkoitus suorittaa modernisointi yhdelle logiikkayksikölle sekä suunnitteluasteella että myös käytännössä. Kartoituksessa käytiin läpi kaikki logiikoita sisältäneet laitteet ja niiden sähkökaapit ja kirjattiin ylös tarvittavat komponentit. Lopuksi komponentit kirjattiin vielä Excel-taulukkoon ja järjesteltiin osastoittain. Modernisointiin siirryttäessä oli saatu kartoitusten pohjalta hyvin selville erilaisia tarpeellisia kohteita, jotka olisivat soveltuneet operaatioon parhaiten ja olisivat samalla myös palvelleet yrityksen tarpeita parhaiten. Modernisoitavan kohteen valitsemisen jälkeen siirryttiin suunnitteluvaiheeseen, jossa selvitettiin kyseisen laitteen tietoja perusteellisesti ja pohdittiin modernisoinnin eri vaiheita. Modernisointi onnistui kokonaisuudessaan melko hyvin. Logiikan komponenttien valinta ja tilaukset onnistuivat melko vaivattomasti ja itse asennustyöt sujuivat myös hyvin. Testiajojen perusteella laite ja kaikki sen ominaisuudet toimivat kuten pitikin. Lisäksi laitteen logiikoihin liittyvien sähkökuvien piirtäminen onnistui muutamia ongelmia lukuun ottamatta hyvin.The purpose of this thesis is to survey the programmable logic controllers of the devices on the company premises. On the basis of this information an inspection for the availability of the spare parts can be made. Another purpose is to perform a modernization for one PLC unit. The final project began by collecting information, during which all electrical devices with PLC units became familiar. Next, the modernization target was picked and the necessary designing and selection of the right kind of components for the new assembly were made. The components were also tested when they arrived and turned out to be in order. The installation of the new components managed quite easily. The wires of the old Siemens S5 -PLC were shifted to the new S7-assembly one by one and some wires were renewed. The electrical drawings were also updated. The device operated excellently and all the features were as they should be

Probiotic intervention in infancy is not associated with development of beta cell autoimmunity and type 1 diabetes

Non peer reviewe

Characteristics of MuA transposase-catalyzed processing of model transposon end DNA hairpin substrates

Bacteriophage Mu uses non-replicative transposition for integration into the host's chromosome and replicative transposition for phage propagation. Biochemical and structural comparisons together with evolutionary considerations suggest that the Mu transposition machinery might share functional similarities with machineries of the systems that are known to employ a hairpin intermediate during the catalytic steps of transposition. Model transposon end DNA hairpin substrates were used in a minimal-component in vitro system to study their proficiency to promote Mu transpososome assembly and subsequent MuA-catalyzed chemical reactions leading to the strand transfer product. MuA indeed was able to assemble hairpin substrates into a catalytically competent transpososome, open the hairpin ends and accurately join the opened ends to the target DNA. The hairpin opening and transposon end cleavage reactions had identical metal ion preferences, indicating similar conformations within the catalytic center for these reactions. Hairpin length influenced transpososome assembly as well as catalysis: longer loops were more efficient in these respects. In general, MuA's proficiency to utilize different types of hairpin substrates indicates a certain degree of flexibility within the transposition machinery core. Overall, the results suggest that non-replicative and replicative transposition systems may structurally and evolutionarily be more closely linked than anticipated previously

A gene truncation strategy generating N- and C-terminal deletion variants of proteins for functional studies: mapping of the Sec1p binding domain in yeast Mso1p by a Mu in vitro transposition-based approach

Bacteriophage Mu in vitro transposition constitutes a versatile tool in molecular biology, with applications ranging from engineering of single genes or proteins to modification of genome segments or entire genomes. A new strategy was devised on the basis of Mu transposition that via a few manipulation steps simultaneously generates a nested set of gene constructions encoding deletion variants of proteins. C-terminal deletions are produced using a mini-Mu transposon that carries translation stop signals close to each transposon end. Similarly, N-terminal deletions are generated using a transposon with appropriate restriction sites, which allows deletion of the 5′-distal part of the gene. As a proof of principle, we produced a set of plasmid constructions encoding both C- and N-terminally truncated variants of yeast Mso1p and mapped its Sec1p-interacting region. The most important amino acids for the interaction in Mso1p are located between residues T46 and N78, with some weaker interactions possibly within the region E79–N105. This general-purpose gene truncation strategy is highly efficient and produces, in a single reaction series, a comprehensive repertoire of gene constructions encoding protein deletion variants, valuable in many types of functional studies. Importantly, the methodology is applicable to any protein-encoding gene cloned in an appropriate vector

SNP discovery by mismatch-targeting of Mu transposition

Single nucleotide polymorphisms (SNPs) represent a valuable resource for the mapping of human disease genes and induced mutations in model organisms. SNPs may become the markers of choice also for population ecology and evolutionary studies, but their isolation for non-model organisms with unsequenced genomes is often difficult. Here, we describe a rapid and cost-effective strategy to isolate SNPs that exploits the property of the bacteriophage Mu transposition machinery to target mismatched DNA sites and thereby to effectively detect polymorphic loci. To demonstrate the methodology, we isolated 164 SNPs from the unsequenced genome of the Glanville fritillary butterfly (Melitaea cinxia), a much-studied species in population biology, and we validated 24 of them. The strategy involves standard molecular biology techniques as well as undemanding MuA transposase-catalyzed in vitro transposition reactions, and it is applicable to any organism

Allergic symptoms and sensitisation in adolescents with cows' milk allergy and atopic eczema in infancy

Background The association between atopic sensitisation, atopic eczema (AE) and asthma is known, but distinct roles of allergies on long-term health are unestablished. Objective Evaluation of allergic symptoms and sensitisation in adolescents who in infancy had AE and verified cows' milk allergy (CMA) or AE and a negative CMA challenge, and controls. Methods Children with AE, with and without CMA, from a randomised controlled study in 1999-2001 examining the effect of probiotics on AE severity at older than 12 months of age, attended a follow-up visit at age 16 to 18, with age-matched controls. Data came from a questionnaire (ISAAC questionnaire), analysis of serum antigen-specific immunoglobulin Es (IgEs), and clinical evaluation. Group comparisons were carried out (chi(2)tests and logistic regression). Results Fifty-two patients with AE and CMA (AE/CMA+ group), 52 with AE and suspicion of CMA (AE/CMA- group), and 57 controls attended a study visit. IgE-mediated sensitisation was significantly more prevalent in the AE/CMA+ group vs the controls, for horse, cat, dog, egg white and wheat (P <.024 for all). For birch, timothy and mugwort (P <.008 for all), sensitisation was more prevalent in both the AE/CMA+ group and the AE/CMA- group vs controls. On the basis of questionnaire data the AE/CMA + group reported a significantly higher lifetime prevalence of wheezing (64% vs 35% and 32%;P = .001), noninfectious rhinitis (85% vs 62% and 56%;P = .004), and hay fever (77% vs 52% and 33%;P <.001) vs the AE/CMA- group and the control group, respectively. Conclusion and Clinical Relevance Patients with AE and CMA in infancy, as opposed to patients with AE only, or controls, report more allergic symptoms and exhibit more allergic sensitisation in adolescence. This indicates that CMA in infancy is an independent risk factor of allergic disease in adolescence.Peer reviewe

Transposon insertion mutagenesis for archaeal gene discovery

Peer reviewe

Perinatal Probiotic Mixture and Development of Allergic Sensitization up to 13 Years of Age

Background: Probiotics have shown promising results in primary prevention of allergies in early years, but the long-term effects on allergic sensitization need more evaluation. Objectives: We conducted a randomized double-blind placebo-controlled study to determine whether the use of a mixture of pre- and probiotics perinatally affects the prevalence of immunoglobulin E (IgE) sensitization up to 13 years in high-risk children. Methods: One thousand two hundred twenty-three pregnant women were randomized to receiving probiotics or placebo from 36 gestational weeks until delivery, and their infants received pre- and probiotics or placebo from birth until 6 months. At 2, 5, and 13 years, blood samples were taken to determine specific IgE levels against common foods, pollen, and animal antigens. Results: The prevalence of IgE sensitization to any allergen was high and increased with age. No significant difference in the prevalence of IgE sensitization to any particular one of the tested allergens was found between the groups. At 2, 5, and 13 years these prevalence rates of IgE sensitization to any allergen were 31.1 and 34.1%, 50.1 and 45.6%, and 61.4 and 56.8% in the probiotic and placebo groups, respectively. At 13 years, IgE sensitization to cat/dog dander was more frequent in the probiotic group compared to the placebo group (40.2 vs. 31.0%, p = 0.03). Conclusions: In high-risk children, perinatal use of a mixture of probiotics did not affect the prevalence of sensitization to any one of the tested allergens, but it was associated with more frequent IgE sensitization to cat/dog dander at 13 years.Peer reviewe

Lehmänmaitoallergia ja toleranssin kehittyminen

Aims: To gain insight on the immunological processes behind cow’s milk allergy (CMA) and the development of oral tolerance. To furthermore investigate the associations of HLA II and filaggrin genotypes with humoral responses to early oral antigens. Methods: The study population was from a cohort of 6209 healthy, full-term infants who in a double-blind randomized trial received supplementary feeding at maternity hospitals (mean duration 4 days): cow’s milk (CM) formula, extensively hydrolyzed whey formula or donor breast milk. Infants who developed CM associated symptoms that subsided during elimination diet (n=223) underwent an open oral CM challenge (at mean age 7 months). The challenge was negative in 112, and in 111 it confirmed CMA, which was IgE-mediated in 83. Patients with CMA were followed until recovery, and 94 of them participated in a follow-up study at age 8-9 years. We investigated serum samples at diagnosis (mean age 7 months, n=111), one year later (19 months, n=101) and at follow-up (8.6 years, n=85). At follow-up, also 76 children randomly selected from the original cohort and without CM associated symptoms were included. We measured CM specific IgE levels with UniCAP (Phadia, Uppsala, Sweden), and β-lactoglobulin, α-casein and ovalbumin specific IgA, IgG1, IgG4 and IgG levels with enzyme-linked immunosorbent assay in sera. We applied a microarray based immunoassay to measure the binding of IgE, IgG4 and IgA serum antibodies to sequential epitopes derived from five major CM proteins at the three time points in 11 patients with active IgE-mediated CMA at age 8-9 years and in 12 patients who had recovered from IgE-mediated CMA by age 3 years. We used bioinformatic methods to analyze the microarray data. We studied T cell expression profile in peripheral blood mononuclear cell (PBMC) samples from 57 children aged 5-12 years (median 8.3): 16 with active CMA, 20 who had recovered from CMA by age 3 years, 21 non-atopic control subjects. Following in vitro β-lactoglobulin stimulation, we measured the mRNA expression in PBMCs of 12 T-cell markers (T-bet, GATA-3, IFN-γ, CTLA4, IL-10, IL-16, TGF-β, FOXP3, Nfat-C2, TIM3, TIM4, STIM-1) with quantitative real time polymerase chain reaction, and the protein expression of CD4, CD25, CD127, FoxP3 with flow cytometry. To optimally distinguish the three study groups, we performed artificial neural networks with exhaustive search for all marker combinations. For genetic associations with specific humoral responses, we analyzed 14 HLA class II haplotypes, the PTPN22 1858 SNP (R620W allele) and 5 known filaggrin null mutations from blood samples of 87 patients with CMA and 76 control subjects (age 8.0-9.3 years). Results: High IgG and IgG4 levels to β-lactoglobulin and α-casein were associated with the HLA (DR15)-DQB1*0602 haplotype in patients with CMA, but not in control subjects. Conversely, (DR1/10)-DQB1*0501 was associated with lower IgG and IgG4 levels to these CM antigens, and to ovalbumin, most significantly among control subjects. Infants with IgE-mediated CMA had lower β -lactoglobulin and α-casein specific IgG1, IgG4 and IgG levels (p<0.05) at diagnosis than infants with non-IgE-mediated CMA or control subjects. When CMA persisted beyond age 8 years, CM specific IgE levels were higher at all three time points investigated and IgE epitope binding pattern remained stable (p<0.001) compared with recovery from CMA by age 3 years. Patients with persisting CMA at 8-9 years had lower serum IgA levels to β-lactoglobulin at diagnosis (p=0.01), and lower IgG4 levels to β-lactoglobulin (p=0.04) and α-casein (p=0.05) at follow-up compared with patients who recovered by age 3 years. In early recovery, signal of IgG4 epitope binding increased while that of IgE decreased over time, and binding patterns of IgE and IgG4 overlapped. In T cell expression profile in response to β –lactoglobulin, the combination of markers FoxP3, Nfat-C2, IL-16, GATA-3 distinguished patients with persisting CMA most accurately from patients who had become tolerant and from non-atopic subjects. FoxP3 expression at both RNA and protein level was higher in children with CMA compared with non-atopic children. Conclusions: Genetic factors (the HLA II genotype) are associated with humoral responses to early food allergens. High CM specific IgE levels predict persistence of CMA. Development of tolerance is associated with higher specific IgA and IgG4 levels and lower specific IgE levels, with decreased CM epitope binding by IgE and concurrent increase in corresponding epitope binding by IgG4. Both Th2 and Treg pathways are activated upon CM antigen stimulation in patients with CMA. In the clinical management of CMA, HLA II or filaggrin genotyping are not applicable, whereas the measurement of CM specific antibodies may assist in estimating the prognosis.Lehmänmaitoallergiaa esiintyy 2-3 %:lla imeväisistä. Valtaosa (80-90%) paranee eli alkaa sietää lehmänmaitoa 3-4 vuoden ikään mennessä. Väitöskirjatutkimuksessa selvitettiin lehmänmaitoallergiaan ja sen paranemiseen liittyviä immunologisia ilmiöitä. Tutkittiin myös, onko tietyillä immunologiaan liittyvillä perinnöllisyystekijöillä yhteyttä ruokavasta-aineiden muodostumiseen. Lehmänmaitoallergian hoitolinjojen valinnassa olisi olennaista kyetä ennustamaan, miten nopeasti potilas todennäköisesti paranee. Tähän ei toistaiseksi ole menetelmiä käytettävissä. Tutkimusaineistona oli 6207 tervettä, täysiaikaista vastasyntynyttä, joita seurattiin lehmänmaitoallergian kehittymisen suhteen. Lapsia, joilla (111:lla) todettiin lehmänmaitoallergia (keskimäärin 7 kk:n ikäisenä), seurattiin paranemiseen asti. Heistä 94 osallistui 8-9 vuoden iässä seurantatutkimukseen. Tuolloin 13 lasta oli vielä lehmänmaitoallergisia. Lisäksi mukaan otettiin 76 tervettä lasta alkuperäisestä väestöstä. Tutkimme seeruminäytteitä kolmesta aikapisteestä: diagnoosihetkellä (keskimäärin 7 kk:n ikäisenä), vuotta myöhemmin ja seurantatutkimuksen hetkellä (keskimäärin 8,6 v:n iässä). Mittasimme lehmänmaidon ja kananmunan valkuaisaineita vastaan kohdistuvien vasta-aineiden (IgE-, IgA-, IgG1, IgG4) seerumipitoisuuksia. Tutkimme myös, mihin lehmänmaidon valkuaisaineiden osiin (peptideihin) IgE- ja IgG4-vasta-aineet sitoutuivat. Valkosolunäytteistä tutkimme T-auttajasoluihin liittyvien tekijöiden ilmentymistä vasteena lehmänmaitoproteiinistimulaatiolle. Vertasimme tuloksia lapsien, joilla oli lehmänmaitoallergia vielä kouluiässä, ja niiden, jotka parantuivat aikaisemmin, välillä. Käytimme bioinformatiikan menetelmiä tulosten analysoimiseen. Perinnöllisistä tekijöistä selvitimme HLA II-alatyyppejä, sekä filaggriini- ja PTPN22-geenien muunnelmia 87 lehmänmaitoallergiapotilaalla ja 76 verrokilla. Havaitsimme, että perintötekijä HLA (DR15)-DQB1*0602 oli yhteydessä korkeisiin IgG ja IgG-vasteisiin lehmänmaitoproteiineille allergiapotilailla, mutta ei verrokeilla. Sen sijaan (DR1/10)-DQB1*0501 oli yhteydessä mataliin IgG ja IgG-vasteisiin lehmänmaidon ja kananmunan proteiineille, ennen kaikkea verrokeilla. Lapsilla, joilla oli lehmänmaitoallergia vielä 8-9-vuotiaina, oli korkeimmat lehmänmaitospesifiset IgE-tasot kaikissa kolmessa mittausaikapisteessä. Pitkittyneeseen lehmänmaitoallergiaan liittyi myös matalat lehmänmaitospesifiset IgA-tasot diagnoosihetkellä ja matalat spesifiset IgG4-tasot 8-9-vuotiaana. Varhaiseen paranemiseen liittyi nousevat lehmänmaitospesifiset IgG4-tasot yhdistettynä laskeviin vastaaviin IgE-tasoihin. Neljän T-auttajasoluihin liittyvän tekijän ilmentymisen yhdistelmä erotteli neuroverkkoanalyysin perusteella diagnoosihetkellä lapset, jotka paranivat leikki-ikäisinä, niistä, joiden lehmänmaitoallergia kesti pidempään. Säätelijä T-soluihin liittyvä tekijä (FoxP3) ilmentyi vahvemmin lehmänmaitoallergiapotilailla kuin verrokeilla. Lehmänmaitoallergian kliinisessä arvioinnissa HLA II- tai filaggriiniperinnöllisyystekijöiden selvittämisestä ei ole hyötyä. Sen sijaan lehmänmaitospesifisten vasta-aineiden määrittäminen voi auttaa sen ennustamisessa, kuinka nopeasti potilas lehmänmaitoallergiasta paranee. T2-auttajasolujen ja säätelijä-T-solujen toiminnan tasapaino vaikuttaa olennaiselta lehmänmaitoallergian paranemisessa