Desorptioilmanpainefotoionisaatio- ja desorptiosähkösumutusionisaatio-massaspektrometria lipidien analytiikassa

Lipidit ovat rasvaliukoisia kudoksesta peräisin olevia yhdisteitä, joilla on monia eri fysiologisia tehtäviä. Lipidien analyysimenetelmien kehittämien on tärkeää, sillä niiden esiintymistä elimistössä voidaan käyttää biomarkkerina sairauksien diagnostiikassa ja apuna sairauksien kehittymismekanismien tutkimisessa. Lipideihin kuuluu polaarisuudeltaan ja rakenteeltaan hyvin erilaisia yhdisteitä. Niiden massaspektrometria-analytiikassa on aikaisemmin käytetty useita erilaisia ionisaatiomenetelmiä, jotka vaativat näytteen esikäsittelyn ennen analyysia. Desorptiosähkösumutusionisaatio-massaspektrometria (DESI-MS) ja desorptio-ilmanpainefotoionisaatio-massaspektrometria (DAPPI-MS) ovat uusia ionisaatio-menetelmiä, jotka mahdollistavat yhdisteiden analysoinnin suoraan eri matriiseista, kuten kudosnäytteistä, usein ilman esikäsittelyä. DESI-MS soveltuu parhaiten suhteellisen polaaristen yhdisteiden analytiikkaan, kun taas DAPPI:lla voidaan ionisoida myös poolittomia yhdisteitä. DESI-MS:lla on jo aikaisemmin analysoitu erilaisia lipidejä, kun taas DAPPI-MS:lla on aikaisemmin analysoitu vain steroideja. DAPPI- ja DESI-MS:lla tutkittiin erilaisten lipidien (fosfolipidit, triglyseridit, rasvahapot, rasvaliukoiset vitamiinit ja steroidit) ionisoitumista. Molemmilla menetelmillä optimoitiin standardiyhdisteille mittausolosuhteet. Lipidejä analysoitiin myös suoraan farmaseuttisista valmisteista. DAPPI:n ja DESI:n soveltuvuudessa erilaisten lipidien ionisoimiseen oli jonkin verran eroja. DAPPI toimi hyvin varsinkin poolittomampien lipidien, eli triglyseridien, steroidien, vitamiinien ja rasvahappojen ionisaatiossa, mutta huonosti hieman polaarisempien ja herkästi hajoavien fosfolipidien ionisaatiossa. Fosfolipidit fragmentoituivat DAPPI-ionisaatiossa, eikä moolimassatiedon sisältävää ionia saatu näkyviin. DESI puolestaan toimii hyvin fosfolipidien ionisoimisessa ja melko hyvin myös muiden tutkittavien lipidien ionisoimisessa, lukuunottamatta kaikkein poolittomimpia lipidejä. Uutta tietoa tutkimuksessa saatiin varsinkin DAPPI:n soveltuvuudesta erilaisten lipidien analytiikkaan. Tulosten perusteella voidaan sanoa, että DAPPI toimii yhtä hyvin tai jopa DESI:a paremmin useiden eri lipidien analytiikkassa. Menetelmää tulisi kuitenkin kehittää edelleen, jotta fosfolipidien, jotka ovat elimistön tärkeä lipidiryhmä, analysointi onnistuisi DAPPI:lla. Työssä ei analysoitu lipidejä suoraan kudosnäytteestä, joten DAPPI:n soveltuvuudesta lipidien analysointiin suoraan kudosnäytteistä ei voida tehdä johtopäätöksiä tämän työn perusteella.

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.

The Reception of the Maccabean Martyrs : Their Historiographical and Paradigmatic Functions in Antique and Late-antique Jewish and Christian Sources

The study attempts a reception-historical analysis of the Maccabean martyrs. The concept of reception has fundamentally to do with the re-use and interpretation of a text within new texts. In a religious tradition, certain elements become re-circulated and thus their reception may reflect the development of that particular tradition. The Maccabean martyrs first appear in 2 Maccabees. In my study, it is the Maccabean martyr figures who count as the received text; the focus is shifted from the interrelations between texts onto how the figures have been exploited in early Christian and Rabbinic sources. I have divided my sources into two categories and my analysis is in two parts. First, I analyze the reception of the Maccabean martyrs within Jewish and Christian historiographical sources, focusing on the role given to them in the depictions of the Maccabean Revolt (Chapter 3). I conclude that, within Jewish historiography, the martyrs are given roles, which vary between ultimate efficacy and marginal position with regard to making a historical difference. In Christian historiographical sources, the martyrs role grows in importance by time: however, it is not before a Christian cult of the Maccabean martyrs has been established, that the Christian historiographies consider them historically effective. After the first part, I move on to analyze the reception in sources, which make use of the Maccabean martyrs as paradigmatic figures (Chapter 4). I have suggested that the martyrs are paradigmatic in the context of martyrdom, persecution and destruction, on one hand, and in a homiletic context, inspiring religious celebration, on the other. I conclude that, as the figures are considered pre-Christian and biblical martyrs, they function well in terms of Christian martyrdom and have contributed to the development of its ideals. Furthermore, the presentation of the martyr figures in Rabbinic sources demonstrates how the notion of Jewish martyrdom arises from experiences of destruction and despair, not so much from heroic confession of faith in the face of persecution. Before the emergence of a Christian cult of the Maccabean martyrs, their identity is derived namely from their biblical position. Later on, in the homiletic context, their Jewish identity is debated and sometimes reconstructed as fundamentally Christian , despite of their Jewish origins. Similar debate about their identity is not found in the Rabbinic versions of their martyrdom and nothing there indicates a mutual debate between early Christians and Jews. A thematic comparison shows that the Rabbinic and Christian cases of reception are non-reliant on each other but also that they link to one another. Especially the scriptural connections, often made to the Maccabean mother, reveal the similarities. The results of the analyses confirm that the early history of Christianity and Rabbinic Judaism share, at least partly, the same religious environment and intertwining traditions, not only during the first century or two but until Late Antiquity and beyond. More likely, the reception of the Maccabean martyrs demonstrates that these religious traditions never ceased to influence one another.