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

Deep-hole drilling process, monitoring system and sensors modifications

 In the drilling processes and especially deep-hole drilling process, the monitoring system and having control on mechanical parameters (e.g. Force, Torque,Vibration and Acoustic emission) are essential. The main focus of this thesis work is to study the characteristics of deep-hole drilling process, and optimize the monitoring system for controlling the process. The vibration is considered as a major defect area of the deep-hole drilling process which often leads to breakage of the drill, therefore by vibration analysis and optimizing the workpiecefixture, this area is studied by finite element method and the suggestions are explained. By study on a present monitoring system, and searching on the new sensor products, the modifications and recommendations are suggested for optimize the present monitoring system for excellent performance in deep-hole drilling process research and measurements.

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.

Comparison and Evaluation of Eddy Current Sensors

In the work eddy current sensors are described and evaluated. Theoretical part includes physical basics of the eddy currents, overview of available commercial products and technologies. Industrial sensors operation was assessed based on several working modes. Apart from this, the model was created in Matlab Simulink with Xilinx Blockset and then translated into a Xilinx ISE Design Suite compatible project. The performance of the resulting implementation was compared to the existing implementation in the Xilinx Spartan 3 FPGA board with the custom made sensor. Additionally, an introduction to FPGAs and VHDL is presented.

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.

Modelling of potentiometric ion sensors

Potentiometric ion sensors are a very important subgroup of electrochemical sensors, very attractive for practical applications due to their small size, portability, low-energy consumption, relatively low cost and not changing the sample composition. They are investigated by the researchers from many fields of science. The continuous development of this field creates the necessity for a detailed description of sensor response and the electrochemical processes important in the practical applications of ion sensors. The aim of this thesis is to present the existing models available for the description of potentiometric ion sensors as well as their applicability and limitations. This includes the description of the diffusion potential occurring at the reference electrodes. The wide range of existing models, from most idealised phase boundary models to most general models, including migration, is discussed. This work concentrates on the advanced modelling of ion sensors, namely the Nernst-Planck-Poisson (NPP) model, which is the most general of the presented models, therefore the most widely applicable. It allows the modelling of the transport processes occurring in ion sensors and generating the potentiometric response. Details of the solution of the NPP model (including the numerical methods used) are shown. The comparisons between NPP and the more idealized models are presented. The applicability of the model to describe the formation of diffusion potential in reference electrode, the lower detection limit of both ion-exchanger and neutral carrier electrodes and the effect of the complexation in the membrane are discussed. The model was applied for the description of both types of electrodes, i.e. with the inner filling solution and solidcontact electrodes. The NPP model allows the electrochemical methods other than potentiometry to be described. Application of this model in Electrochemical Impedance Spectroscopy is discussed and a possible use in chrono-potentiometry is indicated. By combining the NPP model with evolutionary algorithms, namely Hierarchical Genetic Strategy (HGS), a novel method allowing the facilitation of the design of ion sensors was created. It is described in detail in this thesis and its possible applications in the field of ion sensors are indicated. Finally, some interesting effects occurring in the ion sensors (i.e. overshot response and influence of anionic sites) as well as the possible applications of NPP in biochemistry are described.

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.

Food packaging indicators and sensors

Various categories of food packaging indicators namely; VTT, Ageless Eye, Mocon, Åbo Akademi and Impak were selected and incorporated into food trays manufactured at LUT packaging laboratory. Each of these food packaging indicators was used to investigate (visually and qualitatively) the transmission of oxygen through the seal, and tray material, as well as to detect microbial activity within the content of the package. Applications of different methods of gas flushing, content variation and introduction of two distinct levels of oxygen scavengers were employed as treatments to evaluate the packaging performance of the food packaging indicators. Ease of handling of each food packaging indicator was also taken into considerations. Findings showed that for packages, which contained chicken product, the amount of oxygen in the package, measured immediately after the sealing operation on the first day gradually decreased to zero percent by the third day of the storage period. The oxygen level remained at this point throughout the duration of storage for the chicken packages. Besides, level of oxygen in the packages without product continued to increase with the storage time, at moderate rate of 0.1% for 100%N2 and 0.3% for 30%CO2/70%N2 empty packages. More carbon dioxide gas was recorded for packages flushed with 30%CO2/70%N2. Results also revealed that visual analysis of one of the color indicators for example Ageless Eye, conformed to the data derived from the luminescence food-packaging indicator. This shows that packaging operation of the packaging line was considerably stable, and efficient with negligible exception. However, it was found that most of the food packaging indicators investigated in this research study exhibited considerable packaging challenges, such as, reaction with the content of the package (Impak); over sensitivity (Åbo Akademi and Impak); ease of handling problem (Åbo Akademi); and ease of activation problem (VTT indicators). In this study, the strengths and limitations of different indicators were analyzed. This study demonstrates the applicability of various indicators in MAP using chicken package application.

Map matching by using inertial sensors: literature review

This literature review aims to clarify what is known about map matching by using inertial sensors and what are the requirements for map matching, inertial sensors, placement and possible complementary position technology. The target is to develop a wearable location system that can position itself within a complex construction environment automatically with the aid of an accurate building model. The wearable location system should work on a tablet computer which is running an augmented reality (AR) solution and is capable of track and visualize 3D-CAD models in real environment. The wearable location system is needed to support the system in initialization of the accurate camera pose calculation and automatically finding the right location in the 3D-CAD model. One type of sensor which does seem applicable to people tracking is inertial measurement unit (IMU). The IMU sensors in aerospace applications, based on laser based gyroscopes, are big but provide a very accurate position estimation with a limited drift. Small and light units such as those based on Micro-Electro-Mechanical (MEMS) sensors are becoming very popular, but they have a significant bias and therefore suffer from large drifts and require method for calibration like map matching. The system requires very little fixed infrastructure, the monetary cost is proportional to the number of users, rather than to the coverage area as is the case for traditional absolute indoor location systems.

Depth sensors in augmented reality solutions. Literature review

The emergence of depth sensors has made it possible to track – not only monocular cues – but also the actual depth values of the environment. This is especially useful in augmented reality solutions, where the position and orientation (pose) of the observer need to be accurately determined. This allows virtual objects to be installed to the view of the user through, for example, a screen of a tablet or augmented reality glasses (e.g. Google glass, etc.). Although the early 3D sensors have been physically quite large, the size of these sensors is decreasing, and possibly – eventually – a 3D sensor could be embedded – for example – to augmented reality glasses. The wider subject area considered in this review is 3D SLAM methods, which take advantage of the 3D information available by modern RGB-D sensors, such as Microsoft Kinect. Thus the review for SLAM (Simultaneous Localization and Mapping) and 3D tracking in augmented reality is a timely subject. We also try to find out the limitations and possibilities of different tracking methods, and how they should be improved, in order to allow efficient integration of the methods to the augmented reality solutions of the future.

Optical and photoelectric properties of dry bacteriorhodopsin sensors

In this thesis, bacteriorhodopsin (BR) photosensor’s optical and electrical properties were studied. The BR sensor consisted of a dry film with BR in polyvinyl alcohol and covered with transparent conductors. In the experiments the BR photocycle was started with two lasers. The characteristics of the BR sensor were measured in two ways. The first approach was theoretical and it required knowing the laser parameters. The second way required assembling a measurement setup for the optical response measurements. However, no measurable results were obtained due to low laser power. The photoelectric response was measured in the experiments with two laser systems and the amplifier was tested. In the experiment with a Cavitar laser, the photoelectric response was obtained. In the experiment with FemtoFiber Pro laser, the photoelectric response was not measured. The expected amplitude of the response was obtained. The experimental data was analyzed and possible solutions for reducing the interference were given.