Perunan Y-viruksen testaaminen korkeassa happi-hiilidioksidipitoisuudessa idätetyistä perunoista

Perunalla (Solanum tuberosum L.) tällä hetkellä maailmanlaajuisesti eniten sato- ja laatutappioita aiheuttaa perunan Y-virus (PVY). Vaikka pelkän Y-viruksen aiheuttamaa satotappiota on vaikea mitata, on sen arvioitu olevan 20-80 %. Viruksen tärkein leviämistapa on viroottinen siemenperuna. Korkealaatuinen siemenperuna on edellytys ruoka-, ruokateollisuus- ja tärkkelysperunan tuotannolle. Kasvuston silmämääräinen tarkastelu aliarvioi yleensä Y-viruksen esiintyvyyttä. Laboratoriotestauksen avulla saadaan tarkempi tieto pellolta korjatun sadon saastunta-asteesta. Ongelmana Y-viruksen testaamisessa on, että sitä ei havaita dormanssissa olevista perunoista otetuista näytteistä yhtä luotettavasti kuin jo dormanssin ohittaneista perunoista testattaessa. Erilaisia menetelmiä kemikaaleista (Rindite, bromietaani) kasvihormoneihin (mm. gibberelliinihappo) ja varastointiolosuhteiden muutoksiin (kylmä- ja lämpökäsittely) on kokeiltu perunan dormanssin purkamiseen, mutta tulokset ovat olleet vaihtelevia. Tässä tutkielmassa perunan dormanssin purkamiseen käytettiin happi-hiilidioksidikäsittelyä (O2 40 % ja CO2 20 %) eripituisina käsittelyaikoina. Tarkoituksena oli selvittää, vaikuttaako käsittely perunan itämiseen ja dormanssin luontaista aikaisempaan purkautumiseen tai Y-viruksen havaitsemiseen. Lisäksi haluttiin selvittää, voiko Y-viruksen määrittämisen ELISA-testillä (Enzyme Linked Immunosorbent Assay) tehdä yhtä luotettavasti myös muista kasvinosista (mukula, itu), kuin tällä hetkellä yleisesti käytetystä perunan lehdestä. Idätyskäsittelyn vaikutuksista dormanssin purkautumiseen saatiin vaihtelevia, eikä kovinkaan yleistettäviä tuloksia. Käsittelyn ei myöskään havaittu vaikuttavan PYY-viroottisuuden havaitsemiseen eri näytemateriaaleilla testattaessa. Kun eri kasvinosien toimivuutta testissä vertailtiin, mukulamateriaalin todettiin aliarvioivan PVY-viroottisuutta kaikissa kokeissa. Myös itumateriaali aliarvioi pääsääntöisesti PVY-viroottisuutta ELISA:lla tehdyissä määrityksissä. Luotettavin testimateriaali oli perunan lehti.

Molekyylibiologisten menetelmien kehittäminen ihmisen suoliston sulfaattia pelkistävien bakteerien määrittämiseksi

Ihmisen ruuansulatuskanavan bakteeriston kehitys alkaa syntymästä, jolloin ensimmäiset bakteerit kansoittavat steriilin ruuansulatuskanavan. Bakteeristo kehittyy perimän, ympäristön ja varhaisen ruokavalion vaikutuksesta kohti monimuotoisempaa bakteeripopulaatiota. Aikuisen ruuansulatuskanavan normaalibakteeristo on varsin muuttumaton, mutta siihen vaikuttavat monet tekijät, kuten ikä, terveydentila, ruokavalio ja antibioottien käyttö. Bakteeriston koostumus vaihtelee ruuansulatuskanavan eri osissa ja bakteerimäärä kasvaa kohti paksusuolta, ollen paksusuolessa ja ulosteessa peräti 1010-1012 pmy/ml. Suurin osa ruuansulatuskanavan bakteereista on anaerobeja. Ruuansulatuskanavan bakteeristo vaikuttaa muun muassa suoliston kehittymiseen ja hiilihydraattien ja proteiinien hajotukseen sekä toimii osana immuunipuolustusta. Sulfaattia pelkistävät bakteerit (SRB) ovat monimuotoinen ryhmä pääosin anaerobisia bakteereita, jotka käyttävät aineenvaihdunnassaan elektronin vastaanottajana sulfaattia muuttaen sen lopulta sulfidiksi. SRB:t ovat sopeutuneet useisiin erilaisiin ympäristöihin. Niitä tavataan mm. vesistöjen sedimenteissä sekä ihmisen ruuansulatuskanavassa. Ihmisen ruuansulatuskanavassa on SRB:ta n. 105-108 pmy/g, ja niitä on löydetty erityisesti anaerobisista osista kuten suun ientaskuista ja paksusuolesta. SRB:t voivat olla haitaksi ruuansulatuskanavalle tuottamansa sulfidin vuoksi, joka esiintyy vesiliuoksessa vetysulfidina. Tämän on havaittu olevan toksista suoliston epiteelisoluille. Viimeaikoina on kiinnostuttu sulfaatinpelkistäjien yhteydestä suoliston sairaustiloihin, kuten tulehduksellisiin suolistosairauksiin (IBD). Pro gradu -tutkimukseni tavoitteena oli kehittää PCR-DGGE- ja qPCR-menetelmät ulosteen sulfaattia pelkistävien bakteerien määritykseen. Kohdegeeninä menetelmänkehityksessä käytettiin dsrAB-geeniä, joka koodaa dissimilatorista sulfiitinpelkistysentsyymiä. dsrAB-geeni on sulfaatinpelkistäjille ominainen konservoitunut geenialue, johon perustuvia tutkimuksia ei vielä ole paljon ihmispuolelta. qPCR-menetelmä saatiin optimoitua herkäksi ja spesifiseksi käyttäen dsrA-geenispesifisiä alukkeita, mutta PCR-DGGE-menetelmää ei saatu optimoitua käytössä olleilla alukkeilla, jotka monistivat PCR-DGGE:ssa myös negatiivikontrollikantoja. Tutkittaessa qPCR:lla IBD:tä (Crohn ja ulseratiivinen koliitti) sairastavien lasten ja terveiden kontrollihenkilöiden ulostenäytteistä eristettyä DNA:ta, merkittävää eroa SRB-määrissä ei havaittu eri ryhmien välillä. Crohnin tautia sairastavien aktiivisen vaiheen ja oireettoman vaiheen näytteiden välillä oli kuitenkin tilastollisesti merkitsevä ero (SRB-määrät; oireeton vaihe>oireellinen vaihe) (P <0,05).

Phytophthora in Finnish nurseries

Human-mediated movement of plants and plant products is now generally accepted to be the primary mode of introduction of plant pathogens. Species of the genus Phytophthora are commonly spread in this way and have caused severe epidemics in silviculture, horticulture as well as natural systems all over the world. The aims of the study were to gather information on the occurrence of Phytophthora spp. in Finnish nurseries, to produce information for risk assessments for these Phytophthora spp. by determining their host ranges and tolerance of cold temperatures, and to establish molecular means for their detection. Phytophthora cactorum was found to persist in natural waterbodies and results suggest that irrigation water might be a source of inoculum in nurseries. In addition to P. cactorum, isolates from ornamental nursery Rhododendron yielded three species new to Finland: P. ramorum, P. plurivora and P. pini. The only species with quarantine status, P. ramorum, was most adapted to growth in cold temperatures and able to persist in the nursery in spite of an annual sanitation protocol. Phytophthora plurivora and the closely related P. pini had more hosts among Nordic tree and plant species than P. ramorum and P. cactorum, and also had higher infectivity rates. All four species survived two weeks in -5 °C , and thus soil survival of these Phytophthoras in Finland is likely under current climatic conditions. The most common tree species in Finnish nurseries, Picea abies, was highly susceptible to P. plurivora and P. pini in pathogenicity trials. In a histological examination of P. plurivora in P. abies shoot tissues, fast necrotrophic growth was observed in nearly all tissues. The production of propagules in P. abies shoot tissue was only weakly indicated. In this study, a PCR DGGE technique was developed for simultaneous detection and identification of Phytophthora spp. It reliably detected Phytophthora in plant tissues and could discriminate most test species as well as indicate instances of multiple-species infections. It proved to be a useful detection and identification tool either applied alone or in concert with traditional isolation culture techniques. All of the introduced species of Phytophthora had properties that promote a high risk of establishment and spread in Finland. It is probable that more pathogens of this genus will be introduced and become established in Finland and other Nordic countries unless efficient phytosanitary control becomes standard practice in the international plant trade.

Basic and translational studies on species B adenoviruses

Human adenoviruses (Ads) have been classified into six species (A to F) currently containing 55 serotypes. For almost 2 decades vectors derived from group C serotype Ad5 have been extensively used for gene transfer studies. These Ad5 based vectors are able to efficiently infect many mammalian cell types (including both mitotic and post-mitotic cells) through interaction with a primary attachment receptor, the coxsackie and adenovirus receptor (CAR). Despite the many advantages of Ad5 based vectors a number of limitations have affected their therapeutic application to many diseases. Although they can transduce many tissue types, Ad5 based vectors are unable to efficiently transduce several potential disease target cell types, including hematopoietic stem cells and malignant tumor cells. Therefore, newer vectors have been developed based on Ad serotypes other than Ad5. This thesis focuses on species B Ads. Species B Ads are comprised of three groups based on their receptor usage. Group 1 of species B Ads (Ad16, 21, 35, 50) nearly exclusively utilize CD46 as a receptor; Group 2 (Ad3, Ad7, 14) share a common, unidentified receptor/s, which is not CD46 and which was tentatively named receptor X; Group 3 (Ad11) preferentially interacts with CD46, but also utilizes receptor X if CD46 is blocked. Species B group Ads are important human pathogens. Species B group 2 serotypes are isolated from patients with respiratory tract infections, whereas the Group 1 viruses are described as causing kidney and urinary tract infections. B-group Ad infections often occur in immunocompromised patients, including AIDS patients, recipients of bone marrow transplants, or chemotherapy patients. Recent studies performed in U.S. military training facilities indicate an emergence of diverse species B serotypes at the majority of sites. This included the group 1 serotype 21 and the group 2 serotypes 3, 7, and 14. CD46-targeting vectors derived from Ad35 and Ad11 are important tools for in vitro gene transfer into human stem cells, including hematopoietic stem cells and induced pluripotent stem cells. Ad35 and Ad11 have been used as tools for cancer therapy, because CD46 appears to be uniformely overexpressed on many cancers. Furthermore, receptor X-targeting vectors, i.e vectors derived from Ad3 or vectors containing Ad3 fibers have shown superior in the transduction of tumor cells both in vitro and in vivo and are currently being used clinically in cancer patients. While extensive basic virology studies have been done on Ad5, the information of species B group 1 interaction with CD46 is limited. Furthermore, the receptor for a major subgroup of species B Ads (receptor X) is unknown. The goal of this thesis was it therefore to better understand virological and translational aspects of species B Ads. The specific findings described in this thesis include i) the identification of CD46 binding sites within the Ad35 fiber knob, ii) the study of the in vitro and in vivo properties of Ad vectors with increased affinity to CD46. iii) the study of the receptor usage of a newly emergent Ad14a, iv) the identification of desmoglein 2 as the receptor for Ad3, Ad7, Ad11, and Ad14, v) the delineation of structural details of Ad3 virus interaction with DSG2, and vi) the analysis of functional consequences of Ad3-DSG2 interaction. As a result of these basic virology studies two Ad-derived recombinant proteins have been generated that can be used to enhance cancer therapy by monoclonal antibodies.

Moonlighting Proteins of Lactobacillus crispatus : Extracellular Localization, Cell Wall Anchoring and Interactions with the Host

Moonlighting functions have been described for several proteins previously thought to localize exclusively in the cytoplasm of bacterial or eukaryotic cells. Moonlighting proteins usually perform conserved functions, e. g. in glycolysis or as chaperonins, and their traditional and moonlighting function(s) usually localize to different cell compartments. The most characterized moonlighting proteins in Grampositive bacteria are the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which function in bacteria-host interactions, e. g. as adhesins or plasminogen receptors. Research on bacterial moonlighting proteins has focused on Gram-positive bacterial pathogens, where many of their functions have been associated with bacterial virulence. In this thesis work I show that also species of the genus Lactobacillus have moonlighting proteins that carry out functions earlier associated with bacterial virulence only. I identified enolase, GAPDH, glutamine synthetase (GS), and glucose-6-phosphate isomerase (GPI) as moonlighting proteins of Lactobacillus crispatus strain ST1 and demonstrated that they are associated with cell surface and easily released from the cell surface into incubation buffer. I also showed that these lactobacillar proteins moonlight either as adhesins with affinity for basement membrane and extracellular matrix proteins or as plasminogen receptors. The mechanisms of surface translocation and anchoring of bacterial moonlighting proteins have remained enigmatic. In this work, the surface localization of enolase, GAPDH, GS and GPI was shown to depend on environmental factors. The members of the genus Lactobacillus are fermentative organisms that lower the ambient pH by producing lactic acid. At acidic pH enolase, GAPDH, GS and GPI were associated with the cell surface, whereas at neutral pH they were released into the buffer. The release did not involve de novo protein synthesis. I showed that purified recombinant His6-enolase, His6-GAPDH, His6-GS and His6-GPI reassociate with cell wall and bind in vitro to lipoteichoic acids at acidic pH. The in-vitro binding of these proteins localizes to cell division septa and cell poles. I also show that the release of moonlighting proteins is enhanced in the presence of cathelicidin LL- 37, which is an antimicrobial peptide and a central part of the innate immunity defence. I found that the LL-37-induced detachment of moonlighting proteins from cell surface is associated with cell wall permeabilization by LL-37. The results in this thesis work are compatible with the hypothesis that the moonlighting proteins of L. crispatus associate to the cell wall via electrostatic or ionic interactions and that they are released into surroundings in stress conditions. Their surface translocation is, at least in part, a result from their release from dead or permeabilized cells and subsequent reassociation onto the cell wall. The results of this thesis show that lactobacillar cells rapidly change their surface architecture in response to environmental factors and that these changes influence bacterial interactions with the host.