Onko perunaruven hillitsijästä hyötyä sokerijuurikkaalle?

201

Potato virus A as a heterologous protein expression tool in plants

Viruksien käyttö tuotekehityksen ja tutkimuksen vaatimien proteiinien tuottamiseen, syötävien rokotteiden kehittämiseen ja geeniterapiaan edustavat kasvavia biotekniikan sovellusalueita. Perunan A-virus (PVA) kuuluu potyviruksiin, joiden proteiinit tuotetaan aluksi yhtenä suurena molekyylinä, joka pilkotaan yksittäisiksi proteiineiksi viruksen itsensä tuottamilla entsyymeillä. Siten virusgenomiin lisätty vieras geeni käännetään proteiiniksi virusproteiinien mukana. Lopputuloksena kaikkia proteiineja tuotetaan kasvisoluissa samansuuruinen määrä. Lisäksi, viruksen proteiinikuoren koontimekanismi sallii perintöaineksen merkittävän lisäyksen ilman että viruksen tartutuskyky merkittävästi heikkenee. Koska virus monistuu ja leviää koko kasviin, jo melko pieni määrä kasveja riittää huomattavan proteiinimäärän tuottamiseen esimerkiksi säännösten mukaisessa kasvihuoneessa. Tämän työn tarkoituksena oli muuntaa PVA:n genomia siten, että virus soveltuisi yhden vieraan proteiinin tai useiden erilaisten proteiinien samanaikaiseen tuottamiseen kasveissa. Aluksi kokeiltiin viruksen replikaasia ja kuoriproteiinia koodaavien genomialueiden välistä kohtaa ja ihmisestä peräisi olevaa geeniä, joka tuotti S-COMT-entsyymiä (katekoli-O-metyylitransferaasi). Sen aktiivisuuden rajoittaminen auttaa Parkinsonintaudin hoidossa. Kasvissa tuotettua S-COMT:ia voitaisiin käyttää lääkekehityksessä estolääkkeiden testaukseen. Kahden viikon kuluttua tartutuksesta tupakan lehdissä oli entsymaattisesti aktiivista S-COMT:ia n. 1 % lehden liukoisista proteiineista. PVA:n P1-proteiinia koodaavalta alueelta oli paikannettu kohta, johon ehkä voitaisiin siirtää vieras geeni. Asia varmistettiin siirtämällä tähän kohtaan meduusan geeni, joka tuottaa UV-valossa vihreänä fluoresoivaa proteiinia (GFP). GFP-geeniä kantava PVA levisi kasvissa ja lisääntyi n. 30-50 %:iin viruksen normaalista pitoisuudesta. Koko kasvi fluoresoi vihreänä UV-valossa. Vieras geeni voidaan sijoittaa myös potyviruksen P1- ja HCpro-proteiineja koodaavien alueiden väliin. Samaan PVA-genomiin siirrettiin kolme geeniä, yksi kuhunkin kolmesta kloonauskohdasta: GFP-geeni P1:n sisälle, merivuokon lusiferaasigeeni P1/HCpro-kohtaan ja bakteerin beta-glukuronidaasigeeni (GUS) replikaasi/kuoriproteiini-kohtaan. Virusgenomin ja itse viruksen pituudet kasvoivat 38 %, mutta virus säilytti tartutuskykynsä. Se levisi kasveissa saavuttaen n. 15 % viruksen normaalista pitoisuudesta. Kaikki kolme vierasta proteiinia esiintyivät lehdissä aktiivisina.An infectious clone of Potato virus A (PVA) (genus Potyvirus, family Potyviridae) was engineered to be used as an expression vector for production of heterologous proteins. The PVA genome (9565 nt) is translated to a large polyprotein that is subsequently processed to up to ten mature proteins. Hence, a foreign sequence inserted into an infectious cDNA clone of PVA will also be translated as part of the viral polyprotein in infected plants. Three sites in the genome of PVA were used for expression of heterologous protein encoding sequences in plants. Proteolytic cleavage sites for the viral proteinases were engineered and added for separation of the heterologous protein from the viral proteins. A novel genomic location for foreign encoding sequence expression was tested by inserting the Aequorea victoria gfp sequence encoding the green fluorescent protein (GFP) into the P1 encoding region. The vector-PVA expressing GFP accumulated to about 30-50 % of the levels reached with the wild-type PVA in Nicotiana benthamiana cells at 14 days post-inoculation. The vector-PVA continued to produce intact GFP in the systemically infected plants for 3 weeks post-inoculation. The cloning site between the polymerase (NIb) and CP encoding regions of PVA was tested for expression of human proteins. Soluble catechol-O-methyltransferase (S-COMT) that is involved in development of Parkinson’s disease, was produced from the vector PVA and constituted ca. 1% of the total soluble proteins in systemically infected N. benthamiana leaves. The third cloning site that can be used in PVA is between the P1 and HC-Pro encoding regions. Subsequently, all the three cloning sites were combined in the same vector-PVA clone to simultaneously produce three heterologous proteins: GFP from the P1 site, luciferase from the P1/HC-Pro site, and ß-glucuronidase from the NIb/CP site. Vector-virus amounts in the systemically infected leaves of N. benthamiana were 15 % of those of the wild-type virus at 14 days post-inoculation. All three heterologous proteins were detected in the leaf sap in an active form. In conclusion, PVA can be used for simultaneous production of at least three proteins (together consisting of over 1000 amino acid residues) in plants, which possibly will be useful for some research purposes and for heterologous protein production

Social Devices Client for Arduino

In the last few decades we have been witnessing great technology advancements regarding smart devices. At the same time we have accepted few social web services, such as Facebook or Twitter, to become a part of our everyday lives. Social Devices takes a step further regarding social services and proposes a new approach where people and devices would form together a new socio-digital system. In this system devices could participate proactively in social situations by enriching them somehow. According to the concept, the devices could, for instance, participate in a conversation or enable certain services that would encourage socialising. This thesis' research question is to find out if a modest embedded system, such as Arduino, is capable of functioning as a Social Device. For a device to function as a part Social Devices, it needs to have a client software that is tried to be implemented in this thesis. In addition, the Social Devices concept is introduced in the literature review part. In the literature review part we discussed about requirements and characteristics of Social Devices. Additionally, we presented two implementations, the Social Devices Platform and OrchestratorJS, and introduced their architecture and components. Furthermore, relating communication protocols and the target platform were presented. In the implementation part, we designed a client software that is compatible with the two implementations of the Social Devices. Additionally, general design paradigms and matters that influenced the design were introduced. Furthermore, the architecture and the components that the client consists of were presented as well. Finally, an example of Social Devices application that demonstrates the client was shown. We concluded that the implemented client functions as a proof of concept for Social Devices. The client fulfils the requirements as it is capable of registering itself to the server and communicating with it. In addition, the client is able to update its state values and informing RSSI values of nearby devices to the server. However, we acknowledged that there are specific circumstances where the client can not respond reliably. Performance-wise we stated that the client is fast enough for certain applications but can not meet the requirements of an application that needs real-time responsiveness

Moderni massasekvensointiin perustuva maaperämikrobitutkimus

201

Changes in soil bacteria community associated with an introduction of an antagonist of potato disease

201

Soil bacterial community in potato tuberosphere following repeated applications of a common scab suppressive antagonist

Disease suppressive soils are important for managing soil-borne diseases that cannot be controlled with chemicals. One such disease is the potato common scab caused by Streptomyces species. Suppressiveness against common scab can develop spontaneously in fields where potato is grown for years without interruption, and this has been attributed to non-pathogenic Streptomyces strains. Streptomyces spp. have been used as inoculants in biological control, but their long-term effects have gained less attention. In our previous studies, a nonpathogenic Streptomyces strain (Str272) isolated from a potato common scab lesion suppressed common scab in field trials lasting over 5 years. In this study, bacterial communities in the tuberosphere i.e. in the soil adjacent to potato tubers, were analysed by next generation sequencing (NGS). The aim was to compare bacterial communities in untreated control plots to those in which seed tubers were treated with Str272 in one or several growing seasons. Str272 applications increased soil bacterial diversity and affected the bacterial composition in the potato tuberosphere. The most pronounced differences were observed between the untreated control and the treatments in which the antagonist had been applied in three or four consecutive years. The differences remained similar until the following growing season. Bacterial composition after repeated antagonist applications was associated with lower common scab severity. The antagonist applications had no or only slight effect on the number or abundance of OTUs belonging to Actinobacteria or Streptomyces, and no differences in quantities of pathogenic Streptomyces populations were detected by qPCR. This indicates that suppression of common scab by Str272 may not be based on direct effect on the common scab pathogens but is more likely to be associated with the alterations of the soil bacterial community. The most abundant bacteria phyla in the potato tuberosphere were Actinobacteria, Proteobacteria and Acidobacteria. However, the OTUs responding greatest to the antagonist treatments belonged to Bacterioidetes and Gemmatimonadetes. Results indicate that repeated applications of Str272 can change the bacterial community in the potato tuberosphere and lead to development of soil that is suppressive against potato common scab for several growing seasons after the last application.Peer reviewe

Tuotekehityksessä ja tutkimuksessa tarvittavien proteiinien tuottaminen kasveissa perunan A-viruksen avulla

Viruksien käyttö tuotekehityksen ja tutkimuksen vaatimien proteiinien tuottamiseen, syötävien rokotteiden kehittämiseen ja geeniterapiaan edustavat kasvavia biotekniikan sovellusalueita. Perunan A-virus (PVA) kuuluu potyviruksiin, joiden proteiinit tuotetaan aluksi yhtenä suurena molekyylinä. Se pilkotaan yksittäisiksi proteiineiksi viruksen itsensä tuottamilla entsyymeillä. Siten virusgenomiin lisätty vieras geeni käännetään proteiiniksi virusproteiinien mukana. Lopputuloksena sekä viruksenproteiineja että vierasta proteiinia tuotetaan kasvisoluissa samansuuruinen määrä. PVA:n proteiini-kuoren koontimekanismi sallii perintöaineksen merkittävän lisäyksen ilman, että viruksen tartutuskykymerkittävästi heikkenee. Koska virus monistuu ja leviää koko kasviin, jo melko pieni määrä kasveja riittää huomattavan proteiinimäärän tuottamiseen esimerkiksi säännösten mukaisessa kasvihuoneessa.Tämän työn tarkoituksena oli hyödyntää PVA:n genomia yhden vieraan proteiinin tai useiden erilaisten proteiinien samanaikaiseen tuottamiseen kasveissa. Aluksi kokeiltiin vieraan geenin kloonaamista viruksen replikaasia ja kuoriproteiinia koodaavien genomialueiden väliin. Työhön valittiin kaksi ihmisestä peräisi olevaa geeniä. Toinen geeneistä tuotti sorsiiniproteiinia, joka toimii sydänlihaksen supistuksen säätelijänä. Toinen puolestaan tuotti S-COMT-entsyymiä (katekoli-O-metyylitransferaasi), jonka aktiivisuuden rajoittaminen auttaa Parkinsonin taudin hoidossa. Kasvissa tuotettua S-COMT:ia voitaisiin käyttää lääkekehityksessä estolääkkeiden testaukseen. Kahden viikon kuluttua tartutuksesta tupakan lehdissä oli entsymaattisesti aktiivista S-COMT:ia n. 1 % lehden liukoisista proteiineista. Sorsiini sen sijaan oli pysymätön kasvisoluissa, sillä sitä ei havaittu mitattavia määriä. Tulos osoitti, että kaikki ihmisen proteiinit eivät sellaisenaan sovellu kasvissa tuotettaviksi.Transposonimutaatioon perustuneella tutkimuksella oli paikannettu PVA:n P1-proteiinia koodaavalta alueelta kohta, johon voitaisiin siirtää vieras geeni. Asia varmistettiin siirtämällä tähän kohtaan meduusan geeni, joka tuottaa UV-valossa vihreänä fluoresoivaa proteiinia (GFP). GFP-geeniä kantava PVA levisi kasvissa ja lisääntyi n. 30-50 %:iin viruksen normaalista pitoisuudesta. Koko kasvi fluoresoi vihreänä UV-valossa. Sama voitiin havaita myös tuottamalla GFP jo edellä mainitusta, ihmisen proteiinien tuottamiseen käytetystä PVA-genomin kohdasta.Vieras geeni voidaan sijoittaa myös potyviruksen P1- ja HCpro-proteiineja koodaavien alueiden väliin. Tämä on mahdollista myös PVA:ssa, mikä osoitettiin tässä työssä. Siten samaan PVA-genomiin voitiin siirtää kolme geeniä, yksi kuhunkin kolmesta kloonauskohdasta. Lopputuloksena oli PVA-genomi, jossa GFP-geeni sijaitsi P1:n sisällä, merivuokon lusiferaasigeeni P1/HCpro-kohdassa ja bakteerin beta-glukuronidaasigeeni (GUS) replikaasi/kuoriproteiinikohdassa. Virusgenomin ja itse viruksen pituudet kasvoivat 38 %, mutta virus säilytti tartutuskykynsä. Se levisi kasveissa saavuttaen 10-15 % viruksen normaalista pitoisuudesta. Kaikki kolme vierasta proteiinia tuotettiin huomattavina pitoisuuksina ja aktiivisina kasvien lehdissä

TSC-22 up-regulates collagen 3a1 gene expression in the rat heart

Background: The transforming growth factor (TGF)-beta is one of the key mediators in cardiac remodelling occurring after myocardial infarction (MI) and in hypertensive heart disease. The TGF-beta-stimulated clone 22 (TSC-22) is a leucine zipper protein expressed in many tissues and possessing various transcription-modulating activities. However, its function in the heart remains unknown. Methods: The aim of the present study was to characterize cardiac TSC-22 expression in vivo in cardiac remodelling and in myocytes in vitro. In addition, we used TSC-22 gene transfer in order to examine the effects of TSC-22 on cardiac gene expression and function. Results: We found that TSC-22 is rapidly up-regulated by multiple hypertrophic stimuli, and in post-MI remodelling both TSC-22 mRNA and protein levels were up-regulated (4.1-fold, P <0.001 and 3.0-fold, P <0.05, respectively) already on day 1. We observed that both losartan and metoprolol treatments reduced left ventricular TSC-22 gene expression. Finally, TSC-22 overexpression by local intramyocardial adenovirus-mediated gene delivery showed that TSC-22 appears to have a role in regulating collagen type III alpha 1 gene expression in the heart. Conclusions: These results demonstrate that TSC-22 expression is induced in response to cardiac overload. Moreover, our data suggests that, by regulating collagen expression in the heart in vivo, TSC-22 could be a potential target for fibrosis-preventing therapies.Peer reviewe

Social Devices Client for Arduino

In the last few decades we have been witnessing great technology advancements regarding smart devices. At the same time we have accepted few social web services, such as Facebook or Twitter, to become a part of our everyday lives. Social Devices takes a step further regarding social services and proposes a new approach where people and devices would form together a new socio-digital system. In this system devices could participate proactively in social situations by enriching them somehow. According to the concept, the devices could, for instance, participate in a conversation or enable certain services that would encourage socialising. This thesis' research question is to find out if a modest embedded system, such as Arduino, is capable of functioning as a Social Device. For a device to function as a part Social Devices, it needs to have a client software that is tried to be implemented in this thesis. In addition, the Social Devices concept is introduced in the literature review part. In the literature review part we discussed about requirements and characteristics of Social Devices. Additionally, we presented two implementations, the Social Devices Platform and OrchestratorJS, and introduced their architecture and components. Furthermore, relating communication protocols and the target platform were presented. In the implementation part, we designed a client software that is compatible with the two implementations of the Social Devices. Additionally, general design paradigms and matters that influenced the design were introduced. Furthermore, the architecture and the components that the client consists of were presented as well. Finally, an example of Social Devices application that demonstrates the client was shown. We concluded that the implemented client functions as a proof of concept for Social Devices. The client fulfils the requirements as it is capable of registering itself to the server and communicating with it. In addition, the client is able to update its state values and informing RSSI values of nearby devices to the server. However, we acknowledged that there are specific circumstances where the client can not respond reliably. Performance-wise we stated that the client is fast enough for certain applications but can not meet the requirements of an application that needs real-time responsiveness