Photoinhibition of Photosystem II. Kinetics, Photoprotection and Mechanism

Photosystem II (PSII) is susceptible to light-induced damage defined as photoinhibition. In natural conditions, plants are capable of repairing the photoinhibited PSII by on-going degradation and re-synthesis of the D1 reaction centre protein of PSII. Photoinhibition is induced by both visible and ultraviolet light and photoinhibition occurs under all light intensities with the same efficiency per photon. In my thesis work, I studied the reaction kinetics and mechanism of photoinhibition of PSII, as well as photoprotection in leaves of higher plants. Action spectroscopy was used to identify photoreceptors of photoinhibition. I found that the action spectrum of photoinhibition in vivo shows resemblance to the absorption spectra of manganese model compounds of the oxygen evolving complex (OEC) suggesting a role for manganese as a photoreceptor of photoinhibition under UV and visible light. In order to study the protective effect of non-photochemical quenching, the action spectrum was measured from leaves of wild type Arabidopsis thaliana and two mutants impaired in nonphotochemical quenching of chlorophyll a excitations. The findings of action spectroscopy and simulations of chlorophyll-based photoinhibition mechanisms suggested that quenching of antenna excitations protects less efficiently than would be expected if antenna chlorophylls were the only photoreceptors of photoinhibition. The reaction kinetics of prolonged photoinhibition was studied in leaves of Cucurbita maxima and Capsicum annuum. The results indicated that photoinhibitory decrease in both the oxygen evolution activity and ratio of variable to maximum fluorescence follows firstorder kinetics in vivo. The persistence of first-order kinetics suggests that already photoinhibited reaction centres do not protect against photoinhibition and that the mechanism of photoinhibition does not have a reversible intermediate. When Cucurbita maxima leaves were photoinhibited with saturating single-turnover flashes and continuous light, the light response curve of photoinhibition was found to be essentially a straight line with both types of illumination, suggesting that similar photoinhibition mechanisms might function during illumination with continuous light and during illumination with short flashes.

Dielectric spectroscopy and thermally stimulated discharge current in PEEK film

The complex permittivity of films of polyether ether ketone (PEEK) has been investigated over a wide range of frequency. There is no relaxation peak in the range of 1Hz to 10(5) Hz but in the low-frequency side (10-4 Hz) there is an evidence of a peak that also can be observed by thermally stimulated discharge current measurements. That peak is related with the glass transition temperature (Tg) of the polymer. The activation energy of the relaxation was found to be 0.44 eV, similar to that of several synthetic polymers. Space charges are important in the conduction mechanism as shown by discharging transient.

Lychnophoric acid from Lychnophora pinaster: a complete and unequivocal assignment by NMR spectroscopy

The investigation of the hexane extract from aerial parts of Lychnophora pinaster provided, besides others substances, the E-isomer of lychnophoric acid, a sesquiterpene derivative previously isolated from L. affinis.

Determination of hydrochlorothiazide in pharmaceutical formulations by diffuse reflectance spectroscopy

This paper describes a method for quantitative spot test analysis of hydrochlorothiazide using diffuse reflectance spectroscopy. The reflectance measurements were performed analyzing the colored compound (l = 585 nm) produced from the reaction between hydrochlorothiazide and p-dimethylaminocinnamaldehyde (PDAC) in acid medium. This reaction occurred on filter paper after heating to 80ºC for 8 minutes. Factorial designs allowed varying multiple reaction factors simultaneously in order to obtain the best reaction conditions. These factors included heating temperature, heating time, acid volume and PDAC volume. The linearity was studied in the range of 3.36x10-2 to 1.01x10-1 mol L-1 with a correlation coefficient of 0.998. The limit of detection was estimated to be 1.32x10² mol L-1. Commercial samples were analyzed using the proposed method and the results were favorably compared with those of the United States Pharmacopeia method, showing that quantitative spot test analysis by diffuse reflectance could be successfully used to determine hydrochlorothiazide in medicines.

A fluorescence probe study of gemini surfactants in aqueous solution: a comparison between n-2-n and n-6-n series of the alkanediyl-a,w-bis (dimethylalkylammonium bromides)

Two series of alkanediyl-a,w-bis (dimethylalkylammonium bromide (n-2-n and n-6-n; n=8, 10,12, and 16) have been synthesized and their micelles properties studied in aqueous solution using pyrene, pyrenecarboxaldehyde (PCA) and 1,8 anilinonaphtalene sulfonic acid sodium salt (ANS) as fluorescent probes. The micelles from these surfactants have been characterized on the basis of the information provided by micelle-solubilized fluorescent probes. The obtained results indicated that the surfactant concentration at which a marked decrease in l max parameter of pyrenecarboxaldehyde (PCA) occurs corresponds to the CMC determined by conductimetric measurements. Changes in the emission spectra of ANS and PCA observed in the submicellar range for both surfactants series (n-2-n and n-6-n) were interpreted as formation of pre-aggregates. It was found that the dimeric surfactants with long spacer (s= 6) form more hydrated aggregates when compared with those formed by the n-2-n and CnTAB surfactants series. This was attributed to a more difficult packing of n-6-n surfactant molecules to form micelles.

Determination of atenolol in pharmaceutical formulations by diffuse reflectance spectroscopy

A simple analytical method for quantification of atenolol in pharmaceutical formulations by diffuse reflectance spectroscopy is described. The method is based on the reaction, on the filter paper surface, between the drug and p-chloranil producing a colored compound. The best reaction conditions were obtained with 20 µL of atenolol solution and 20 µL of p-chloranil. All reflectance measurements were carried out at 550 nm and the linear range was from 1.13x10-2 to 7.88x10-2 mol L-1 (r = 0.9992). The limit of detection was 2.80 x 10-3 mol L-1. The proposed method was successfully applied to analysis of different commercial brands of pharmaceutical formulations and the results obtained by the proposed method were in good agreement with those obtained using the British Pharmacopoeia method.

Chlorophyll fluorescence of tropical tree species in a semi-deciduous forest gap

The characterization of different ecological groups in a forest formation/succession is unclear. To better define the different successional classes, we have to consider ecophysiological aspects, such as the capacity to use or dissipate the light energy available. The main objective of this work was to assess the chlorophyll fluorescence emission of tropical tree species growing in a gap of a semi-deciduous forest. Three species of different ecological groups were selected: Croton floribundus Spreng. (pioneer, P), Astronium graveolens Jacq. (early secondary, Si), and Esenbeckia febrifuga A. Juss. (late secondary, St). The potential (Fv/Fm) and effective (deltaF/Fm') quantum efficiency of photosystem II, apparent electron transport rate (ETR), non-photochemical (qN) and photochemical (qP) quenching of fluorescence were evaluated, using a modulated fluorometer, between 7:30 and 11:00 h. Values of Fv/Fm remained constant in St, decreasing in P and Si after 9:30 h, indicating the occurrence of photoinhibition. Concerning the measurements taken under light conditions (deltaF/Fm', ETR, qP and qN), P and Si showed better photochemical performance, i.e., values of deltaF/Fm', ETR and qP were higher than St when light intensity was increased. Values of qN indicated that P and Si had an increasing tendency of dissipating the excess of energy absorbed by the leaf, whereas the opposite was found for St. The principal component analysis (PCA), considering all evaluated parameters, showed a clear distinction between St, P and Si, with P and Si being closer. The PCA results suggest that chlorophyll fluorescence may be a potential tool to differentiate tree species from distinct successional groups.

Chloroplastid pigment contents and chlorophyll a fluorescence in Amazonian tropical three species

Plants react to changes in light and hydrological conditions in terms of quantity and composition of chloroplastidic pigments, which affects the photosynthetic properties and consequently the accumulation of plant biomass. Thus, the chloroplastidic pigment concentration and chlorophyll a fluorescence of three Amazonian species (Bertholletia excelsa, Carapa guianensis e Dipteryx odorata) were investigated in sun and shade leaves form the tree crown collected during two distinct periods of precipitation (dry and rainy seasons). Pigment contents were determined by spectrophotometry and fluorescence variables were determined using a portable fluorometer. The results demonstrated that the species showed high concentrations of Chl a, Chl b e Chl total during the wet season in relation to the dry season, especially in shade leaves. A higher concentration of carotenoids was found in B. excelsa, when compared with leaves of C. guianensis and D. odorata. In leaves of B. excelsa and D. odorata no significant difference was found in relation to the photochemistry of photosystem II (Fv/Fm) between the wet and dry seasons. In conclusion, the three species react differently to variations in the light and precipitation conditions regarding light capture, aspects that might be considered in the management of forest plantations.

White mold detection in common beans through leaf reflectance spectroscopy

ABSTRACT This study aimed to identify wavelengths based on leaf reflectance (400-1050 nm) to estimate white mold severity in common beans at different seasons. Two experiments were carried out, one during fall and another in winter. Partial Least Squares (PLS) regression was used to establish a set of wavelengths that better estimates the disease severity at a specific date. Therefore, observations were previously divided in two sub-groups. The first one (calibration) was used for model building and the second subgroup for model testing. Error measurements and correlation between measured and predicted values of disease severity index were employed to provide the best wavelengths in both seasons. The average indexes of each experiment were of 5.8% and 7.4%, which is considered low. Spectral bands ranged between blue and green, green and red, and red and infrared, being most sensitive for disease estimation. Beyond the transition ranges, other spectral regions also presented wavelengths with potential to determine the disease severity, such as red, green, and near infrared.

Reaaliaikaisen esikäsittelyn kehittäminen hemiselluloosa-analytiikalle

Tämän työn tavoitteena oli kehittää esikäsittelymenetelmä, jolla voidaan reaaliaikaisesti vähentää hydrolysaatin ligniinipitoisuutta ja näin vähentää fluoresenssia hemiselluloosan analysoimisessa. Työn kirjallisuusosassa käsitellään hemiselluloosien rakennetta sekä niiden erottamista puusta, sekä käydään läpi hemiselluloosien käyttömahdollisuuksia ja niiden reaaliaikaiseen analysointiin soveltuvia tekniikoita. Kokeellisessa osassa tutkittiin ultrasuodatusta sekä adsorptiohartsikäsittelyä hydrolysaatin esikäsittelynä ennen Raman-analyysiä. Esikäsittelyn tavoitteena oli vähentää Raman-analyysia häiritsevää ligniinistä johtuvaa fluoresenssia. Suodatukset tehtiin Amicon-suodatuslaitteistolla käyttäen viittä eri suodatuskalvoa. Hartsikäsittelyissä käytettiin Amberliten XAD16 ja XAD7HP adsorbtiohartseja. Hartsisuhteina käytettiin 1/80, 1/40, 1/19 ja 1/13. Käytetyt ultrauodatuskalvot osoittautuivat suodatusnäytteistä tehtyjen HPLC-analyysien perusteella cut-off-luvultaan liian pieniksi, sillä hemiselluloosien ja ligniinin erotus ei onnistunut, vaan molemmat väkevöityivät konsentraattiin. Hartsikäsittelyillä saatiin ligniiniä poistettua aiheuttamatta hemiselluloosahäviöitä. Parhaimmillaan ligniinin poistumista kuvaava UV-absorbanssi pieneni hartsilla XAD16 37 % ja hartsilla XAD7 25 %. Vaikka ligniinipitoisuus aleni, näytteet fluoresoivat edelleen voimakkaasti Raman-mittauksessa. Tulosten perusteella näyttäisi siltä, että hartsikäsittelyä optimoimalla analyysiä häiritsevää fluoresenssia olisi edelleen mahdollista vähentää.

Positron measurements in Mg-doped GaN and development of digital Doppler broadening spectroscopy

In this work parameters of Mg-doped GaN samples were studied using positron annihilation spectroscopy and analyzed. It is shown that gallium vacancies exist in an unintentionally doped sample. Next, the sample with higher concentration of Mg and low growth temperature contains vacancy clusters. In case of low concentration of Mg the growth temperature does not affect the formation of defects. Analog electronics can be replaced by a modern digital device. While promising a high quantity of benefits, the performance of these digitizers requires thorough adjustment. A 14-bit two channel digitizer has been tested in order to achieve better performance than the one of a traditional analog setup, and the adjustment process is described. It has been shown that the digital device is unable to achieve better energy resolution, but it is quite close to the corresponding attribute of the available analog system, which had been used for measurements in Mg-doped GaN.

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.

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.