Developing Bioimage Informatics – from Microscopy to Software Solutions – with alpha2beta1 Integrin as a Case Study

Biokuvainformatiikan kehittäminen – mikroskopiasta ohjelmistoratkaisuihin – sovellusesimerkkinä α2β1-integriini Kun ihmisen genomi saatiin sekvensoitua vuonna 2003, biotieteiden päätehtäväksi tuli selvittää eri geenien tehtävät, ja erilaisista biokuvantamistekniikoista tuli keskeisiä tutkimusmenetelmiä. Teknologiset kehitysaskeleet johtivat erityisesti fluoresenssipohjaisten valomikroskopiatekniikoiden suosion räjähdysmäiseen kasvuun, mutta mikroskopian tuli muuntua kvalitatiivisesta tieteestä kvantitatiiviseksi. Tämä muutos synnytti uuden tieteenalan, biokuvainformatiikan, jonka on sanottu mahdollisesti mullistavan biotieteet. Tämä väitöskirja esittelee laajan, poikkitieteellisen työkokonaisuuden biokuvainformatiikan alalta. Väitöskirjan ensimmäinen tavoite oli kehittää protokollia elävien solujen neliulotteiseen konfokaalimikroskopiaan, joka oli yksi nopeimmin kasvavista biokuvantamismenetelmistä. Ihmisen kollageenireseptori α2β1-integriini, joka on tärkeä molekyyli monissa fysiologisissa ja patologisissa prosesseissa, oli sovellusesimerkkinä. Työssä saavutettiin selkeitä visualisointeja integriinien liikkeistä, yhteenkeräytymisestä ja solun sisään siirtymisestä, mutta työkaluja kuvainformaation kvantitatiiviseen analysointiin ei ollut. Väitöskirjan toiseksi tavoitteeksi tulikin tällaiseen analysointiin soveltuvan tietokoneohjelmiston kehittäminen. Samaan aikaan syntyi biokuvainformatiikka, ja kipeimmin uudella alalla kaivattiin erikoistuneita tietokoneohjelmistoja. Tämän väitöskirjatyön tärkeimmäksi tulokseksi muodostui näin ollen BioImageXD, uudenlainen avoimen lähdekoodin ohjelmisto moniulotteisten biokuvien visualisointiin, prosessointiin ja analysointiin. BioImageXD kasvoi yhdeksi alansa suurimmista ja monipuolisimmista. Se julkaistiin Nature Methods -lehden biokuvainformatiikkaa käsittelevässä erikoisnumerossa, ja siitä tuli tunnettu ja laajalti käytetty. Väitöskirjan kolmas tavoite oli soveltaa kehitettyjä menetelmiä johonkin käytännönläheisempään. Tehtiin keinotekoisia piidioksidinanopartikkeleita, joissa oli "osoitelappuina" α2β1-integriinin tunnistavia vasta-aineita. BioImageXD:n avulla osoitettiin, että nanopartikkeleilla on potentiaalia lääkkeiden täsmäohjaussovelluksissa. Tämän väitöskirjatyön yksi perimmäinen tavoite oli edistää uutta ja tuntematonta biokuvainformatiikan tieteenalaa, ja tämä tavoite saavutettiin erityisesti BioImageXD:n ja sen lukuisten julkaistujen sovellusten kautta. Väitöskirjatyöllä on merkittävää potentiaalia tulevaisuudessa, mutta biokuvainformatiikalla on vakavia haasteita. Ala on liian monimutkainen keskimääräisen biolääketieteen tutkijan hallittavaksi, ja alan keskeisin elementti, avoimen lähdekoodin ohjelmistokehitystyö, on aliarvostettu. Näihin seikkoihin tarvitaan useita parannuksia,

Bioimage informatics in STED super-resolution microscopy

Optical microscopy is living its renaissance. The diffraction limit, although still physically true, plays a minor role in the achievable resolution in far-field fluorescence microscopy. Super-resolution techniques enable fluorescence microscopy at nearly molecular resolution. Modern (super-resolution) microscopy methods rely strongly on software. Software tools are needed all the way from data acquisition, data storage, image reconstruction, restoration and alignment, to quantitative image analysis and image visualization. These tools play a key role in all aspects of microscopy today – and their importance in the coming years is certainly going to increase, when microscopy little-by-little transitions from single cells into more complex and even living model systems. In this thesis, a series of bioimage informatics software tools are introduced for STED super-resolution microscopy. Tomographic reconstruction software, coupled with a novel image acquisition method STED< is shown to enable axial (3D) super-resolution imaging in a standard 2D-STED microscope. Software tools are introduced for STED super-resolution correlative imaging with transmission electron microscopes or atomic force microscopes. A novel method for automatically ranking image quality within microscope image datasets is introduced, and it is utilized to for example select the best images in a STED microscope image dataset.

Fluorescence in situ hybridization of potato somatohaploids and their somatic hybrid donors using two Solanum brevidens specific sequences

Selostus: Perunan somaattisten hybridien ja niiden somatohaploidien fluoresenssi in situ -hybridisaatio Solanum brevidens -lajin spesifisten sekvenssien avulla

Development of Raman microscopy methods for polymer coatings of polyfilms

Työn tavoitteena oli tutkia Raman-spektrometrin soveltuvuutta muovipäällystettyjen kartonkien syvyyssuuntaisiin mittauksiin. Lisäksi pyrittiin selvittämään voidaanko kiteisyyttä nähdä Raman-laitteistolla. Työn kirjallisessa osassa on selvitetty Raman-laitteiston teknisiä ominaisuuksia. Kokeellinen osa suoritettiin Lappeenrannan teknillisessä yliopistossa Membraanitekniikan ja teknillisen polymeerikemian laboratoriossa. Työssä käytettiin Horiban Jobin Yvon¿in valmistamaa konfokaalista Raman-spektrometri-laitteistoa (LabRam). Syvyyssuuntaisissa mittauksissa käytettiin apuna motorisoitua x-, y- ja z-suuntaan liikkuvaa tasoa. Mittaukset suoritettiin pistemäisesti tietyllä askelvälillä fokusoimalla näytteen pinnasta sisällepäin. Syvyysprofilointimittaukset aloitettiinmäärittelemällä laitteiston syvyysresoluutio eri konfokaalireikäkoolla. Lisäksityössä tehtiin syvyysprofilointimittauksia sekä läpinäkyvillä monikerrosmuoveilla että muovipäällystetyillä kartongeilla. Työssä mitatut muovipäällysteet sisälsivät pääasiassa polyeteeniä. Tulokset osoittivat, että Raman laitteistolla voidaan havainnoida Raman-aktiiviset ryhmät näytteen eri kerroksista. Lisäksi polyeteenin kiteisyysaste voidaan havaita tietyillä aallonpituuksilla.

Lanthanide Chelates as Donors in Fluorescence Resonance Energy Transfer: Exciting Prospects for Bioaffinity Assay Detection

Fluorescence resonance energy transfer (FRET) is a non-radiative energy transfer from a fluorescent donor molecule to an appropriate acceptor molecule and a commonly used technique to develop homogeneous assays. If the emission spectrum of the donor overlaps with the excitation spectrum of the acceptor, FRET might occur. As a consequence, the emission of the donor is decreased and the emission of the acceptor (if fluorescent) increased. Furthermore, the distance between the donor and the acceptor needs to be short enough, commonly 10-100 Å. Typically, the close proximity between the donor and the acceptor is achieved via bioaffinity interactions e.g. antibody binding antigen. Large variety of donors and acceptors exist. The selection of the donor/acceptor pair should be done not only based on the requirements of FRET but also the performance expectancies and the objectives of the application should be considered. In this study, the exceptional fluorescence properties of the lanthanide chelates were employed to develop two novel homogeneous immunoassays: a non-competitive hapten (estradiol) assay based on a single binder and a dual-parametric total and free PSA assay. In addition, the quenching efficiencies and energy transfer properties of various donor/acceptor pairs were studied. The applied donors were either europium(III) or terbium(III) chelates; whereas several organic dyes (both fluorescent and quenchers) acted as acceptors. First, it was shown that if the interaction between the donor/acceptor complexes is of high quality (e.g. biotin-streptavidin) the fluorescence of the europium(III) chelate could be quenched rather efficiently. Furthermore, the quenching based homogeneous non-competitive assay for estradiol had significantly better sensitivity (~67 times) than a corresponding homogeneous competitive assay using the same assay components. Second, if the acceptors were chosen to emit at the emission minima of the terbium(III) chelate, several acceptor emissions could be measured simultaneously without significant cross-talk from other acceptors. Based on these results, the appropriate acceptors were chosen for the dual-parameter assay. The developed homogeneous dual-parameter assay was able to measure both total and free PSA simultaneously using a simple mix and measure protocol. Correlation of this assay to a heterogeneous single parameter assay was excellent (above 0.99 for both) when spiked human plasma samples were used. However, due to the interference of the sample material, the obtained concentrations were slightly lower with the homogeneous than the heterogeneous assay, especially for the free PSA. To conclude, in this work two novel immunoassay principles were developed, which both are adaptable to other analytes. However, the hapten assay requires a rather good antibody with low dissociation rate and high affinity; whereas the dual-parameter assay principle is applicable whenever two immunometric complexes can form simultaneously, provided that the requirements of FRET are fulfilled.

Design of Installation for Transport Measurements

In the present work the aim was to prepare an automatic installation for studies of galvanomagnetic effects in solids and to test it by calibration measurements. As a result required automatic installation was created in this work and test measurements were performed. Created setup automatically provides measurements of the magnetoresistance of the Hall effect with an accuracy of ± 2 µV in the temperature range 2 – 300 K and steady magnetic fields up to 6 T. The test measurements of the glassy carbon samples showed that the setup is reliable, has high sensitivity and is easy to use. The results obtained in the research process are pioneer and will be separately analyzed.

Combining Near-Field and Far-Field Microscopy: A new method for nanoscale super-resolution imaging

Lähikenttä- ja kaukokenttämikroskopian yhdistäminen: Uusi korkearesoluutioinen menetelmä nanokuvantamiseen. Osteoporoosi on sairaus, jossa luun uudistumisprosessi ei ole enää tasapainossa. Uuden luun muodostuminen on hitaampaa johtuen osteoblastien laskeneesta aktiivisuudesta. Yksi keino estää osteoporoosin syntyä on estää osteoklastien sitoutuminen luun pinnalle, jolloin ne eivät aloita luun syömisprosessia. Tämän Pro gradu -tutkielman tarkoituksena on luoda uusi työkalu osteoklastien sitoutumisen tutkimiseen samanaikaisesti fluoresenssi- ja atomivoimamikroskoopilla. Tätä tarkoitusta varten yhdistettiin atomivoimamikroskooppi sekä STED mikroskooppi. Kirjallisuuskatsauksessa käydään läpi yksityiskohtaisesti molempien mikroskooppitekniikoiden teoriat. Kokeellisessa osiossa esitetään käytetyt metodit ja alustavat tulokset uudella systeemillä. Lisäksi keskustellaan lyhyesti kuvan analysoinnista ImageJohjelmalla. Konfokaalisen fluoresenssimikroskoopin ja atomivoimamikroskoopin yhdistelmä on keksitty jo aikaisemmin, mutta tavallisen konfokaalimikroskoopin erottelukyvyn raja on noin 200 nanometriä johtuen valon diffraktioluonteesta. Yksityiskohdat eivät erotu, jos ne ovat pienempiä kuin puolet käytettävästä aallonpituudesta. STED mikroskooppi mahdollistaa fluoresenssikuvien taltioimisen solunsisäisistä prosesseista 50 nanometrin lateraalisella erotuskyvyllä ja atomivoimamikroskooppi antaa topografista tietoa näytteestä nanometrien erotuskyvyllä. Biologisia näytteitä kuvannettaessa atomivoimamikroskoopin erotuskyky kuitenkin huononee ja yleensä saavutetaan 30-50 nanometrin erotuskyky. Kuvien kerrostaminen vaatii vertauspisteitä ja tätä varten käytettiin atomivoimamikroskoopin kärjen tunnistamista ja referenssipartikkeleita. Kuva-analysointi suoritettiin ImageJ-kuvankäsittelyohjelmalla. Tuloksista nähdään, että referenssipartikkelit ovat hyviä, mutta niiden sijoittaminen tarkasti tietylle kohdealueelle on hankalaa nanoskaalassa. Tästä johtuen kärjen havaitseminen fluoresenssikuvassa on parempi metodi. Atomivoimamikroskoopin kärki voidaan päällystää fluoresoivalla aineella, mutta tämä lisää kärjen aiheuttamaa konvoluutiota mittausdataan. Myös valon takaisinsirontaa kärjestä voidaan tutkia, jolloin konvoluutio ei lisäänny. Ensimmäisten kuvien kerrostamisessa käytettiin hyväksi fluoresoivalla aineella päällystettyä kärkeä ja lopputuloksessa oli vain 50 nanometrin yhteensopimattomuus fluoresenssi- ja topografiakuvan kanssa. STED mikroskoopin avulla nähdään leimattujen proteiinien tarkat sijainnit tiettynä ajankohtana elävän solun sisällä. Samaan aikaan pystytään kuvantamaan solun fyysisiä muotoja tai mitata adheesiovoimia atomivoimamikroskoopilla. Lisäksi voidaan käyttää funktinalisoitua kärkeä, jolla voidaan laukaista signalointitapahtumia solun ja soluväliaineen välillä. Sitoutuminen soluväliaineeseen voidaan rekisteröidä samoin kuin adheesiomediaattorien sijainnit sitoutumisalueella. Nämä dynaamiset havainnot tuottavat uutta informaatiota solun signaloinnista, kun osteoklasti kiinnittyy luun pintaan. Tämä teknologia tarjoaa uuden näkökulman monimutkaisiin signalointiprosesseihin nanoskaalassa ja tulee ratkaisemaan lukemattoman määrän biologisia ongelmia.

Luminescence-based assay methods in drug discovery

The drug discovery process is facing new challenges in the evaluation process of the lead compounds as the number of new compounds synthesized is increasing. The potentiality of test compounds is most frequently assayed through the binding of the test compound to the target molecule or receptor, or measuring functional secondary effects caused by the test compound in the target model cells, tissues or organism. Modern homogeneous high-throughput-screening (HTS) assays for purified estrogen receptors (ER) utilize various luminescence based detection methods. Fluorescence polarization (FP) is a standard method for ER ligand binding assay. It was used to demonstrate the performance of two-photon excitation of fluorescence (TPFE) vs. the conventional one-photon excitation method. As result, the TPFE method showed improved dynamics and was found to be comparable with the conventional method. It also held potential for efficient miniaturization. Other luminescence based ER assays utilize energy transfer from a long-lifetime luminescent label e.g. lanthanide chelates (Eu, Tb) to a prompt luminescent label, the signal being read in a time-resolved mode. As an alternative to this method, a new single-label (Eu) time-resolved detection method was developed, based on the quenching of the label by a soluble quencher molecule when displaced from the receptor to the solution phase by an unlabeled competing ligand. The new method was paralleled with the standard FP method. It was shown to yield comparable results with the FP method and found to hold a significantly higher signal-tobackground ratio than FP. Cell-based functional assays for determining the extent of cell surface adhesion molecule (CAM) expression combined with microscopy analysis of the target molecules would provide improved information content, compared to an expression level assay alone. In this work, immune response was simulated by exposing endothelial cells to cytokine stimulation and the resulting increase in the level of adhesion molecule expression was analyzed on fixed cells by means of immunocytochemistry utilizing specific long-lifetime luminophore labeled antibodies against chosen adhesion molecules. Results showed that the method was capable of use in amulti-parametric assay for protein expression levels of several CAMs simultaneously, combined with analysis of the cellular localization of the chosen adhesion molecules through time-resolved luminescence microscopy inspection.

Investigation of the two-photon induced fluorescence in Rb vapour excited by Ti:Sapphire femtosecond laser pulses

In this work we report the observation of the blue visible fluorescence at 420 nm in rubidium vapour as a result of two-photon absorption excited by femtosecond laser pulses 790 nm. After experimental investigation of the spa-tial and spectral characteristics of the obtained emission we can claim that mechanism of this coherent fluorescence at 420 nm was not caused by ampli-fied spontaneous emission, but represents the nondegenerate four-wave mixing. As a probable outcome of this investigation an opportunity of creation an ultrafast all-optical switcher might appear.

Two-Photon Excited Fluorescence Detection Technology in Screening for Methicillin-Resistant Staphylococcus aureus

Control of the world-wide spread of methicillin-resistant Staphylococcus aureus (MRSA) has been unsuccessful in most developed countries. A few countries have been able to maintain a low MRSA prevalence, plausibly due to their strict MRSA control policies. Such policies require wide-scale screening of patients with suspected MRSA colonization, in order to nurse the MRSA-positive patients in contact isolation. The aim of this study was to develop and introduce a 2-photon excited fluorescence detection (TPX) technique for screening of MRSA directly from clinical samples. The assay principle involves specific online immunometric monitoring of S. aureus growth under selective antibiotic pressure. After the novel TPX approach had been set up, its applicability for the detection of MRSA was evaluated using a large MRSA collection including practically all epidemic MRSA strains identified in Finland between 1991 and 2009. The TPX assay was found both sensitive (97.9%) and specific (94.1%) in this epidemiological setting, illustrating that the method is tolerant to wide biological variation as well as to environments with rapidly emerging MRSA strains. When MRSA was screened directly from colonization samples, all patients positive for MRSA by conventional methods were positive also by the TPX assay. The assay capacity was 48 samples per a test run, and the median time required for confirmation of a true-positive screening test result was 3 h 26 min. Collectively, the findings presented in this thesis suggest that the TPX MRSA screening assay could be applicable for direct screening of MRSA colonization samples without any prior steps of isolation. This can potentially mean that contact isolation of suspected carriers testing negative could be discontinued earlier, thereby reducing the costs and burden associated with the containment of MRSA. In case of infection, a positive test result would ensure an early onset of effective therapy.

JNK, a versatile regulator of kinesin-1 transport and cytoskeleton dynamics

C-Jun N-terminal kinase (JNK) is traditionally recognized as a crucial factor in stress response and inducer of apoptosis upon various stimulations. Three isoforms build the JNK subfamily of MAPK; generally expressed JNK1 and JNK2 and brain specific JNK3. Degenerative potency placed JNK in the spotlight as potential pharmacological option for intervention. Unfortunately, adverse effects of potential drugs and observation that expression of only JNK2 and JNK3 are induced upon stress, restrained initial enthusiasm. Notably, JNK1 demonstrated atypical high constitutive activity in neurons that is not responsive to cellular stresses and indicated existence of physiological activity. This thesis aimed at revealing the physiological functions of JNK1 in actin homeostasis through novel effector MARCKS-Like 1 (MARCKSL1) protein, neuronal trafficking mediated by major kinesin-1 motor protein and microtubule (MT) dynamics via STMN2/SCG10. The screen for novel physiological JNK substrates revealed specific phosphorylation of C-terminal end of MARCKSL1 at S120, T148 and T183 both ex vivo and in vitro. By utilizing site-specific mutagenesis, various actin dynamics and migrations assays we were able to demonstrate that JNK1 phosphorylation specifically facilitates F-actin bundling and thus filament stabilisation. Consecutively, this molecular mechanism was proved to enhance formation of filopodia; cell surface projections that allow cell sensing surrounding environment and migrate efficiently. Our results visualize JNK dependent and MARCKSL1 executed induction of filopodia in neurons and fibroblast indicating general mechanism. Subsequently, inactivation of JNK action on MARCKSL1 shifts cellular actin machinery into lamellipodial dynamic arrangement. Tuning of actin cytoskeleton inevitably melds with cell migration. We observed that both active JNK and JNK pseudo-phosphorylated form of MARCKSL1 reduce actin turnover in intact cells leading to overall diminished cell motility. We demonstrate that tumour transformed cells from breast, prostate, lung and muscle-derived cancers upregulate MARCKSL1. We showed on the example of prostate cancer PC-3 cell line that JNK phosphorylation negatively controls MARCKSL1 ability to induce migration, which precedes cancer cell metastasis. The second round of identification of JNK physiological substrates resulted in detection of predominant motor protein kinesin-1 (Kif5). Mass spectrometry detailed analysis showed evident endogenous phosphorylation of kinesin-1 on S176 within motor domain that interacts with MT. In vitro phosphorylation of bacterially expressed kinesin heavy chain by JNK isoforms displayed higher specificity of JNK1 when compared to JNK3. Since, JNK1 is constitutively active in neurons it signified physiological aspect of kinesin-1 regulation. Subsequent biochemical examination revealed that kinesin-1, when not phosphorylated on JNK site, exhibits much higher affinity toward MTs. Expression of the JNK non-phosphorable kinesin-1 mutant in intact cells as well as in vitro single molecule imaging using total internal reflection fluorescence microscopy indicated that the mutant loses normal speed and is not able to move processively into proper cellular compartments. We identify novel kinesin-1 cargo protein STMN2/SCG10, which along with known kinesin-1 cargo BDNF is showing impaired trafficking when JNK activity is inhibited. Our data postulates that constitutive JNK activity in neurons is crucial for unperturbed physiologically relevant transport of kinesin-1 dependant cargo. Additionally, my work helps to validate another novel physiological JNK1 effector STMN2/SCG10 as determinant of axodendritic neurites dynamics in the developing brain through regulation of MT turnover. We show successively that this increased MT dynamics is crucial during developmental radial migration when brain layering occurs. Successively, we are able to show that introduction of JNK phosphorylation mimicking STMN2/SCG10 S62/73D mutant rescues completely JNK1 genetic deletion migration phenotype. We prove that STMN2/SCG10 is predominant JNK effector responsible for MT depolymerising activity and neurite length during brain development. Summarizing, this work describes identification of three novel JNK substrates MARCKSL1, kinesin-1 and STMN2/SCG10 and investigation of their roles in cytoskeleton dynamics and cargo transport. This data is of high importance to understand physiological meaning of JNK activity, which might have an adverse effect during pharmaceutical intervention aiming at blocking pathological JNK action.

Kelvin Probe Force Microscopy (KPFM) characterization of lanthanum lutetium oxide high-k dielectric thin films

Lanthanum lutetium oxide (LaLuO3) thin films were investigated considering their perspective application for industrial microelectronics. Scanning probe microscopy (SPM) techniques permitted to visualize the surface topography and study the electric properties. This work compared both the material properties (charge behavior for samples of 6 nm and 25 nm width) and the applied SPM modes. Particularly, Kelvin probe force microscopy (KPFM) was applied to characterize local potential difference with high lateral resolution. Measurements showed the difference in morphology, chargeability and charge dissipation time for both samples. The polarity effect was detected for this material for the first time. Lateral spreading of the charged spots indicate the diffusive mechanism to be predominant in charge dissipation. This allowed to estimate the diffusion coefficient and mobility. Using simple electrostatic model it was found that charge is partly leaking into the interface oxide layer.

Switchable lanthanide fluorescence probes in homogenous molecular diagnostics

The number of molecular diagnostic assays has increased tremendously in recent years.Nucleic acid diagnostic assays have been developed, especially for the detection of human pathogenic microbes and genetic markers predisposing to certain diseases. Closed-tube methods are preferred because they are usually faster and easier to perform than heterogenous methods and in addition, target nucleic acids are commonly amplified leading to risk of contamination of the following reactions by the amplification product if the reactions are opened. The present study introduces a new closed-tube switchable complementation probes based PCR assay concept where two non-fluorescent probes form a fluorescent lanthanide chelate complex in the presence of the target DNA. In this dual-probe PCR assay method one oligonucleotide probe carries a non-fluorescent lanthanide chelate and another probe a light absorbing antenna ligand. The fluorescent lanthanide chelate complex is formed only when the non-fluorescent probes are hybridized to adjacent positions into the target DNA bringing the reporter moieties in close proximity. The complex is formed by self-assembled lanthanide chelate complementation where the antenna ligand is coordinated to the lanthanide ion captured in the chelate. The complementation probes based assays with time-resolved fluorescence measurement showed low background signal level and hence, relatively high nucleic acid detection sensitivity (low picomolar target concentration). Different lanthanide chelate structures were explored and a new cyclic seven dentate lanthanide chelate was found suitable for complementation probe method. It was also found to resist relatively high PCR reaction temperatures, which was essential for the PCR assay applications. A seven-dentate chelate with two unoccupied coordination sites must be used instead of a more stable eight- or nine-dentate chelate because the antenna ligand needs to be coordinated to the free coordination sites of the lanthanide ion. The previously used linear seven-dentate lanthanide chelate was found to be unstable in PCR conditions and hence, the new cyclic chelate was needed. The complementation probe PCR assay method showed high signal-to-background ratio up to 300 due to a low background fluorescence level and the results (threshold cycles) in real-time PCR were reached approximately 6 amplification cycles earlier compared to the commonly used FRET-based closed-tube PCR method. The suitability of the complementation probe method for different nucleic acid assay applications was studied. 1) A duplex complementation probe C. trachomatis PCR assay with a simple 10-minute urine sample preparation was developed to study suitability of the method for clinical diagnostics. The performance of the C. trachomatis assay was equal to the commercial C. trachomatis nucleic acid amplification assay containing more complex sample preparation based on DNA extraction. 2) A PCR assay for the detection of HLA-DQA1*05 allele, that is used to predict the risk of type 1 diabetes, was developed to study the performance of the method in genotyping. A simple blood sample preparation was used where the nucleic acids were released from dried blood sample punches using high temperature and alkaline reaction conditions. The complementation probe HLA-DQA1*05 PCR assay showed good genotyping performance correlating 100% with the routinely used heterogenous reference assay. 3) To study the suitability of the complementation probe method for direct measurement of the target organism, e.g., in the culture media, the complementation probes were applied to amplificationfree closed-tube bacteriophage quantification by measuring M13 bacteriophage ssDNA. A low picomolar bacteriophage concentration was detected in a rapid 20- minute assay. The assay provides a quick and reliable alternative to the commonly used and relatively unreliable UV-photometry and time-consuming culture based bacteriophage detection methods and indicates that the method could also be used for direct measurement of other micro-organisms. The complementation probe PCR method has a low background signal level leading to a high signal-to-background ratio and relatively sensitive nucleic acid detection. The method is compatible with simple sample preparation and it was shown to tolerate residues of urine, blood, bacteria and bacterial culture media. The common trend in nucleic acid diagnostics is to create easy-to-use assays suitable for rapid near patient analysis. The complementation probe PCR assays with a brief sample preparation should be relatively easy to automate and hence, would allow the development of highperformance nucleic acid amplification assays with a short overall assay time.

Fluorescence-based imaging of cellular defect in lysinuric protein intolerance (LPI)

Fluoresenssiperusteiset kuvantamismenetelmät lysinurisen proteiini-intoleranssin (LPI) soluhäiriön tutkimuksessa Lysinurinen proteiini-intoleranssi on suomalaiseen tautiperintöön kuuluva autosomaalisesti peit¬tyvästi periytyvä sairaus, jonka aiheuttaa kationisten aminohappojen kuljetushäiriö munuaisten ja ohutsuolen epiteelisolujen basolateraalikalvolla. Aminohappojen kuljetushäiriö johtaa moniin oirei¬siin, kuten kasvuhäiriöön, osteoporoosiin, immuunijärjestelmän häiriöihin, oksenteluun ja runsaspro¬teiinisen ravinnon nauttimisen jälkeiseen hyperammonemiaan. LPI-geeni SLC7A7 (solute carrier family 7 member 7) koodaa y+LAT1 proteiinia, joka on basolateraali¬nen kationisten ja neutraalien aminohappojen kuljettimen kevyt ketju, joka muodostaa heterodimee¬rin raskaan alayksikön 4F2hc:n kanssa. Tällä hetkellä SLC7A7-geenistä tunnetaan yli 50 LPI:n aiheut¬tavaa mutaatiota. Tässä tutkimuksessa erityyppisiä y+LAT1:n LPI-mutaatiota sekä yhdeksän C-terminaalista polypep¬tidiä lyhentävää deleetiota kuvannettiin nisäkässoluissa y+LAT1:n GFP (green fluorescent protein) -fuusioproteiineina. Tulokset vahvistivat muissa soluissa tehdyt havainnot siitä, että 4F2hc on edel¬lytyksenä y+LAT1:n solukalvokuljetukselle, G54V-pistemutantti sijaitsee solukalvolla samoin kuin vil¬lityyppinen proteiini, mutta lukukehystä muuttavia ja proteiinia lyhentäviä mutantteja ei kuljeteta solukalvoon. Lisäksi havaittiin, että poikkeuksena tästä säännöstä ovat y+LAT1-deleetioproteiinit, joista puuttui korkeintaan 50 C-terminaalista aminohappoa. Nämä lyhentyneet kuljettimet sijaitsevat solukalvolla kuten villityyppiset ja LPI-pistemutanttiproteiinit. Dimerisaation osuutta kuljetushäiriön synnyssä tutkittiin käyttämällä fluorescence resonance energy transfer (FRET) menetelmää. Heterodimeerin alayksiköistä kloonattiin ECFP (cyan) ja EYFP (yellow) fuusioproteiinit, joita ilmennettiin nisäkässoluissa, ja FRET mitattiin virtaussytometri-FRET -menetel¬mällä (FACS-FRET). Tutkimuksissa kaikkien mutanttien havaittiin dimerisoituvan yhtä tehokkaasti. Kul¬jetushäiriön syynä ei siten ole alayksiköiden dimerisaation estyminen mutaation seurauksena. Tutkimuksessa havaittiin, että kaikki mutantti-y+LAT1-transfektiot tuottavat vähemmän transfektoi¬tuneita soluja kuin villityyppisen y+LAT1:n transfektiot. Solupopulaatioissa, joihin oli tranfektoitu lu¬kukehystä muuttava tai stop-kodonin tuottava mutaatio havaittiin suurempi kuolleisuus kuin saman näytteen transfektoitumattomissa soluissa, kun taas villityyppistä tai G54V-pistemutanttia tuottavas¬sa solupopulaatiossa oli pienempi kuolleisuus kuin saman näytteen fuusioproteiinia ilmentämättö¬missä soluissa. Tulos osoittaa mutanttiproteiinien erilaiset vaikutukset niitä ilmentäviin soluihin, joko suoraan y+LAT1:n tai 4F2hc:n kautta aiheutuneina. LPIFin SLC7A7 lähetti-RNA:n määrä ei merkittävästi poikennut villityyppisen määrästä fibroblasteissa ja lymfoblasteissa. SLC7A7:n promoottorianalyysissä oli osoitettavissa säätelyalueita geenin 5’ ei-koo¬daavalla alueella sekä ensimmäisten kahden intronin alueella. LPI-taudin tautimekanismin kannalta keskeisin tekijä on kuitenkin aminohappokuljetuksen häiriö, jonka vaikutuksesta näistä aminohapoista riippuvaiset prosessit elimistössä eivät toimi normaalisti. Havaittu virheellinen y+LAT1/4F2hc kuljetuskompleksin sijainti edellyttää lisätutkimuksia sen mahdol¬lisen kliinisen merkityksen selvittämiseksi.

Surface studies of limestones and dolostones : characterisation using various techniques and batch dissolution experiments with hydrochloric acid solutions

In this thesis, stepwise titration with hydrochloric acid was used to obtain chemical reactivities and dissolution rates of ground limestones and dolostones of varying geological backgrounds (sedimentary, metamorphic or magmatic). Two different ways of conducting the calculations were used: 1) a first order mathematical model was used to calculate extrapolated initial reactivities (and dissolution rates) at pH 4, and 2) a second order mathematical model was used to acquire integrated mean specific chemical reaction constants (and dissolution rates) at pH 5. The calculations of the reactivities and dissolution rates were based on rate of change of pH and particle size distributions of the sample powders obtained by laser diffraction. The initial dissolution rates at pH 4 were repeatedly higher than previously reported literature values, whereas the dissolution rates at pH 5 were consistent with former observations. Reactivities and dissolution rates varied substantially for dolostones, whereas for limestones and calcareous rocks, the variation can be primarily explained by relatively large sample standard deviations. A list of the dolostone samples in a decreasing order of initial reactivity at pH 4 is: 1) metamorphic dolostones with calcite/dolomite ratio higher than about 6% 2) sedimentary dolostones without calcite 3) metamorphic dolostones with calcite/dolomite ratio lower than about 6% The reactivities and dissolution rates were accompanied by a wide range of experimental techniques to characterise the samples, to reveal how different rocks changed during the dissolution process, and to find out which factors had an influence on their chemical reactivities. An emphasis was put on chemical and morphological changes taking place at the surfaces of the particles via X-ray Photoelectron Spectroscopy (XPS) and Scanning Electron Microscopy (SEM). Supporting chemical information was obtained with X-Ray Fluorescence (XRF) measurements of the samples, and Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) and Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) measurements of the solutions used in the reactivity experiments. Information on mineral (modal) compositions and their occurrence was provided by X-Ray Diffraction (XRD), Energy Dispersive X-ray analysis (EDX) and studying thin sections with a petrographic microscope. BET (Brunauer, Emmet, Teller) surface areas were determined from nitrogen physisorption data. Factors increasing chemical reactivity of dolostones and calcareous rocks were found to be sedimentary origin, higher calcite concentration and smaller quartz concentration. Also, it is assumed that finer grain size and larger BET surface areas increase the reactivity although no certain correlation was found in this thesis. Atomic concentrations did not correlate with the reactivities. Sedimentary dolostones, unlike metamorphic ones, were found to have porous surface structures after dissolution. In addition, conventional (XPS) and synchrotron based (HRXPS) X-ray Photoelectron Spectroscopy were used to study bonding environments on calcite and dolomite surfaces. Both samples are insulators, which is why neutralisation measures such as electron flood gun and a conductive mask were used. Surface core level shifts of 0.7 ± 0.1 eV for Ca 2p spectrum of calcite and 0.75 ± 0.05 eV for Mg 2p and Ca 3s spectra of dolomite were obtained. Some satellite features of Ca 2p, C 1s and O 1s spectra have been suggested to be bulk plasmons. The origin of carbide bonds was suggested to be beam assisted interaction with hydrocarbons found on the surface. The results presented in this thesis are of particular importance for choosing raw materials for wet Flue Gas Desulphurisation (FGD) and construction industry. Wet FGD benefits from high reactivity, whereas construction industry can take advantage of slow reactivity of carbonate rocks often used in the facades of fine buildings. Information on chemical bonding environments may help to create more accurate models for water-rock interactions of carbonates.