Amperometric biosensor for ascorbic acid

A L-ascorbic acid biosensor based on ascorbate oxidase has been developed. The enzyme was extracted from the mesocarp of cucumber (Cucumis sativus) by using 0.05 mol L-1 phosphate buffer, pH 5.8 containing 0.5 mol L-1 NaCl. After the dialysis versus phosphate buffer 0.05 mol L-1 pH 5.8, the enzyme was immobilized onto nylon net through glutaraldehyde covalent bond. The membrane was coupled to an O2 electrode and the yielding reaction monitored by oxygen depletion at -600 mV using flow injection analysis optimized to 0.1 mol L-1 phosphate buffer pH 5.8, as the carrier solution and flow-rate of 0.5 mL min-1. The ascorbic acid calibration curve was linear from 1.2x10-4 to 1.0x10-3 mol L-1. The evaluation of biosensor lifetime leads to 500 injections. Commercial pharmaceutical samples were analyzed with the proposed method and the results were compared with those obtained by high-performance liquid chromatography (HPLC).

Chiral separations of mandelic acid by HPLC using molecularly imprinted polymers

Styrene is used in a variety of chemical industries. Environmental and occupational exposures to styrene occur predominantly through inhalation. The major metabolite of styrene is present in two enantiomeric forms, chiral R- and S- hydroxy-1-phenyl-acetic acid (R-and S-mandelic acid, MA). Thus, the concentration of MA, particularly of its enantiomers, has been used in urine tests to determine whether workers have been exposed to styrene. This study describes a method of analyzing mandelic acid using molecular imprinting techniques and HPLC detection to perform the separation of diastereoisomers of mandelic acid. The molecularly imprinted polymer (MIP) was prepared by non-covalent molecular imprinting using (+) MA, (-) MA or (+) phenylalanine, (-) phenylalanine as templates. Methacrylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA) were copolymerized in the presence of the template molecules. The bulk polymerization was carried out at 4ºC under UV radiation. The resulting MIP was grounded into 25~44¼m particles, which were slurry packed into analytical columns. After the template molecules were removed, the MIP-packed columns were found to be effective for the chromatographic resolution of (±)-mandelic acid. This method is simpler and more convenient than other chromatographic methods.

Functional characterization of proteins required for mitotic progression and the spindle assembly checkpoint

During mitotic cell division, the genetic material packed into chromosomes is divided equally between two daughter cells. Before the separation of the two copies of a chromosome (sister chromatids), each chromosome has to be properly connected with microtubules of the mitotic spindle apparatus and aligned to the centre of the cell. The spindle assembly checkpoint (SAC) monitors connections between microtubules and chromosomes as well as tension applied across the centromere. Microtubules connect to a chromosome via kinetochores, which are proteinaceous organelles assembled onto the centromeric region of the sister chromatids. Improper kinetochore-microtubule attachments activate the SAC and block chromosome segregation until errors are corrected and all chromosomes are connected to the mitotic spindle in a bipolar manner. The purpose of this surveillance mechanism is to prevent loss or gain of chromosomes in daughter cells that according to current understanding contributes to cancer formation. Numerous proteins participate in the regulation of mitotic progression. In this thesis, the mitotic tasks of three kinetochore proteins, Shugoshin 1 (Sgo1), INCENP, and p38 MAP kinase (p38 MAPK), were investigated. Sgo1 is a protector of centromeric cohesion. It is also described in the tension-sensing mechanism of the SAC and in the regulation of kinetochore-microtubule connections. Our results revealed a central role for Sgo1 in a novel branch of kinetochore assembly. INCENP constitutes part of the chromosomal passenger complex (CPC). The other members of the core complex are the Aurora B kinase, Survivin and Borealin. CPC is an important regulatory element of cell division having several roles at various stages of mitosis. Our results indicated that INCENP and Aurora B are highly dynamic proteins at the mitotic centromeres and suggested a new role for CPC in regulation of chromosome movements and spindle structure during late mitosis. The p38 MAPK has been implicated in G1 and G2 checkpoints during the cell cycle. However, its role in mitotic progression and control of SAC signaling has been controversial. In this thesis, we discovered a novel function for p38γ MAPK in chromosome orientation and spindle structure as well as in promotion of viability of mitotic cells.

Purified lignin for demanding applications

Diplomityön tarkoituksena oli puhdistaa kraft ligniiniä. Raaka-aineena käytettiin pääasiassa Lignoboost -menetelmän kaltaisella menetelmällä havupuu- mustalipeästä saostettua ligniiniä. Diplomityön ensimmäisenä tavoitteena oli löytää menetelmä, jolla voidaan poistaa kraft ligniinistä tuhkaa. Työssä raaka-aineena käytetyn saostetun ligniinin tuhkapitoisuus oli noin 3 %. Tarkoituksena oli saada laskettua tuhkapitoisuutta uudelleenlieton, suodatuksen ja pesun avulla. Työn toisena tavoitteena oli hiilihydraattien poisto kraft ligniinistä. Hiilihydraatit, pääosin hemiselluloosaa, ovat kiinnittyneet ligniiniin vahvoin kovalenttisin sidoksin. Aiempien kokemusten perusteella hemiselluloosat eivät irtoa ligniinistä vesipesun yhteydessä, vaan niiden irrottaminen vaatii onnistuakseen happo-, entsyymi- tai mikrobikäsittelyn, mikäli halutaan säilyttää ligniinin rakenne muuttumattomana. Tässä työssä käytetyt kraft ligniinin puhdistusmenetelmät olivat lietto, happohydrolyysi ja entsymaattinen hydrolyysi, joista kumpikin sisälsi ligniinin uudelleenlieton, suodatuksen ja muodostuneen kakun pesun vedellä.

The spindle assembly checkpoint as a drug target - Novel small-molecule inhibitors of Aurora kinases

Cell division (mitosis) is a fundamental process in the life cycle of a cell. Equal distribution of chromosomes between the daughter cells is essential for the viability and well-being of an organism: loss of fidelity of cell division is a contributing factor in human cancer and also gives rise to miscarriages and genetic birth defects. For maintaining the proper chromosome number, a cell must carefully monitor cell division in order to detect and correct mistakes before they are translated into chromosomal imbalance. For this purpose an evolutionarily conserved mechanism termed the spindle assembly checkpoint (SAC) has evolved. The SAC comprises a complex network of proteins that relay and amplify mitosis-regulating signals created by assemblages called kinetochores (KTs). Importantly, minor defects in SAC signaling can cause loss or gain of individual chromosomes (aneuploidy) which promotes tumorigenesis while complete failure of SAC results in cell death. The latter event has raised interest in discovery of low molecular weight (LMW) compounds targeting the SAC that could be developed into new anti-cancer therapeutics. In this study, we performed a cell-based, phenotypic high-throughput screen (HTS) to identify novel LMW compounds that inhibit SAC function and result in loss of cancer cell viability. Altogether, we screened 65 000 compounds and identified eight that forced the cells prematurely out of mitosis. The flavonoids fisetin and eupatorin, as well as the synthetic compounds termed SACi2 and SACi4, were characterized in more detail utilizing versatile cell-based and biochemical assays. To identify the molecular targets of these SAC-suppressing compounds, we investigated the conditions in which SAC activity became abrogated. Eupatorin, SACi2 and SACi4 preferentially abolished the tensionsensitive arm of the SAC, whereas fisetin lowered also the SAC activity evoked by lack of attachments between microtubules (MTs) and KTs. Consistent with the abrogation of SAC in response to low tension, our data indicate that all four compounds inhibited the activity of Aurora B kinase. This essential mitotic protein is required for correction of erratic MT-KT attachments, normal SAC signaling and execution of cytokinesis. Furthermore, eupatorin, SACi2 and SACi4 also inhibited Aurora A kinase that controls the centrosome maturation and separation and formation of the mitotic spindle apparatus. In line with the established profound mitotic roles of Aurora kinases, these small compounds perturbed SAC function, caused spindle abnormalities, such as multi- and monopolarity and fragmentation of centrosomes, and resulted in polyploidy due to defects in cytokinesis. Moreover, the compounds dramatically reduced viability of cancer cells. Taken together, using a cell-based HTS we were able to identify new LMW compounds targeting the SAC. We demonstrated for the first time a novel function for flavonoids as cellular inhibitors of Aurora kinases. Collectively, our data support the concept that loss of mitotic fidelity due to a non-functional SAC can reduce the viability of cancer cells, a phenomenon that may possess therapeutic value and fuel development of new anti-cancer drugs.

Yrityssaneeraukseen liittyvän erityistarkastuksen funktio ja sisältö

Suomalaisessa oikeusjärjestelmässä yrityssaneerauksen organisaatio koostuu pääosin kohdeyrityksestä, selvittäjästä ja velkojista. Selvittäjällä on lain mukaan mahdollisuus teettää tilintarkastajalla kohdeyrityksen toimintaan ja hallintoon liittyvä erityistarkastus saneerausmenettelyä edeltävältä ajalta. Saneeraukseen liittyvää erityistarkastusta kutsutaan myös nimellä saneeraustarkastus. Tutkielma käsittelee yrityssaneerausmenettelyn yhteydessä tehtävän saneeraustarkastuksen funktiota ja tarkastuksen lopputuloksena syntyvän tarkastuskertomuksen sisältöä. Tutkielman tarkoitus on selvittää mitä tehtävää saneeraustarkastus palvelee, mitä lisäarvoa saneeraustarkastus tuottaa selvittäjälle ja velkojille, sekä minkälainen tulisi olla tarkastuskertomuksen sisältö. Tutkielma pohjautuu kvalitatiiviseen kyselytutkimukseen, jossa vastaajina ovat toimineet 21 selvittäjää ja 28 velkojaa. Tutkimuksen perusteella velkojilla ja selvittäjillä on pitkälti samanlainen näkemys yrityssaneeraukseen liittyvän erityistarkastuksen funktiosta ja sisällöstä. Molemmat intressiryhmät katsoivat, että kohdeyhtiön vastuuhenkilöiden toimien selvittäminen, etenkin jos kohdeyhtiö ei nauti intressitahojen täyttä luottamusta, tuottaa paljon lisäarvoa. Luottamus kohdeyhtiöön on ratkaisevassa asemassa myös saneeraustarkastuksen tarvetta harkittaessa. Selkeimmäksi eroksi selvittäjien ja velkojien vastausten välil-lä muodostui ainoastaan syy jättää saneeraustarkastus teettämättä. Selvittäjien mielestä todennäköisin syy jättää saneeraustarkastus teettämättä on velallisen varojen puute, kun taas velkojien mielestä todennäköisin syy jättää saneeraustarkastus teettämättä on luottamus velalliseen. Tutkimuksen mukaan saneeraustarkastuskertomukset ovat sisältönsä puolesta korkealaatuisia. Kertomuksia tulisi kuitenkin kehittää tiiviimpään muotoon ilman kattavaa raporttiosuutta, sekä vain merkittäväksi arvioidut havainnot tulisi dokumentoida liiteaineistoon. Vastanneiden mielestä kolme tärkeintä saneeraustarkastuksen laadun mittaria ovat tärkeysjärjestyksessä hinta-laatu-suhde, laadukas raportointi ja nopea toimitusvarmuus. Kaksi tärkeintä laadun kriteeriä vaikuttavat myös eniten saneeraustarkastuksen tekijän valintaan.

Selective inhibition of human tankyrases

Tankyrases belong to the Diphtheria toxin-like ADP-ribosyltransferase (ARTD) enzyme superfamily, also known as poly(ADP-ribose) polymerases (PARPs). They catalyze a covalent post-translational modification reaction where they transfer ADP-ribose units from NAD+ to target proteins. Tankyrases are involved in many cellular processes and their roles in telomere homeostasis, Wnt signaling and in several diseases including cancers have made them interesting drug targets. In this thesis project, selective inhibition of human tankyrases was studied. A homogeneous fluorescence-based assay was developed to screen the compound libraries. The assay is inexpensive, operationally easy, and performs well according to the statistical analysis. Assay suitability was confirmed by screening a natural product library. Flavone was identified as the most potent inhibitor in the library and this motivated us to screen a larger flavonoid library. Results showed that flavones were indeed the best inhibitor of tankyrases among flavonoids. To further study the structure-activity relationship, a small library of flavones containing single substitution was screened and potency measurements allowed us to generate structure-activity relationship. Compounds containing substitutions at 4´-position were more potent in comparison to other substitutions, and importantly, hydrophobic groups improved isoenzyme selectivity as well as the potency. A flavone derivative containing a hydrophobic isopropyl group (compound 22), displayed 6 nM potency against TNKS1, excellent isoenzyme selectivity and Wnt signaling inhibition. Protein interactions with compounds were studied by solving complex crystal structures of the compounds with TNKS2 catalytic domain. A novel tankyrase inhibitor (IWR-1) was also crystallized in complex with TNKS2 catalytic domain. The crystal structure of TNKS2 in complex with IWR-1 showed that the compound binds to adenosine site and it was the first known ARTD inhibitor of this kind. To date, there is no structural information available about the substrate binding with any of the ARTD family members; therefore NAD+ was soaked with TNKS2 catalytic domain crystals. However, analysis of crystal structure showed that NAD+ was hydrolyzed to nicotinamide. Also, a co-crystal structure of NAD+ mimic compound, EB-47, was solved which was used to deduce some insights about the substrate interactions with the enzyme. Like EB-47, other ARTD1 inhibitors were also shown to inhibit tankyrases. It indicated that selectivity of the ARTD1 inhibitors should be considered as some of the effects in cells could come from tankyrase inhibition. In conclusion, the study provides novel information on tankyrase inhibition and presents new insight into the selectivity and potency of compounds.

Negotiating home and belonging. Young Kurds in Finland

This study looks at negotiation of belonging and understandings of home among a generation of young Kurdish adults who were born in Iraq, Iran, and Turkey and who reached adulthood in Finland. The young Kurds taking part in the study belong to the generation of migrants who moved to Finland in their childhood and early teenage years from the region of Kurdistan and elsewhere in the Middle East, then grew to adulthood in Finland. In theoretical terms, the study draws broadly from three approaches: transnationalism, intersectionality, and narrativity. Transnationalism refers to individuals’ cross-border ties and interaction extending beyond nationstates’ borders. Young people of migrant background, it has been suggested, are raised in a transnational space that entails cross-border contacts, ties, and visits to the societies of departure. How identities and feelings of belonging become formed in relation to the transnational space is approached with an intersectional frame, for examination of individuals’ positionings in terms of their intersecting attributes of gender, age/generation, and ethnicity, among others. Focus on the narrative approach allows untangling how individuals make sense of their place in the social world and how they narrate their belonging in terms of various mechanisms of inclusion and exclusion, including institutional arrangements and discursive categorisation schemes. The empirical data for this qualitative study come from 25 semi-structured thematic interviews that were conducted with 23 young Kurdish adults living in Turku and Helsinki between 2009 and 2011. The interviewees were aged between 19 and 28 years at the time of interviewing. Interview themes involved topics such as school and working life, family relations and language-learning, political activism and citizenship, transnational ties and attachments, belonging and identification, and plans for the future and aspirations. Furthermore, data were collected from observations during political demonstrations and meetings, along with cultural get-togethers. The data were analysed via thematic analysis. The findings from the study suggest that young Kurds express a strong sense of ‘Kurdishness’ that is based partially on knowing the Kurdish language and is informed by a sense of cultural continuity in the diaspora setting. Collective Kurdish identity narratives, particularly related to the consciousness of being a marginalised ‘other’ in the context of the Middle East, are resonant in young interviewees’ narrations of ‘Kurdishness’. Thus, a sense of ‘Kurdishness’ is drawn from lived experiences indexed to a particular politico-historical context of the Kurdish diaspora movements but also from the current situation of Kurdish minorities in the Middle East. On the other hand, young Kurds construct a sense of belonging in terms of the discursive constructions of ‘Finnishness’ and ‘otherness’ in the Finnish context. The racialised boundaries of ‘Finnishness’ are echoed in young Kurds’ narrations and position them as the ‘other’ – namely, the ‘immigrant’, ‘refugee’, or ‘foreigner’ – on the basis of embodied signifiers (specifically, their darker complexions). This study also indicates that young Kurds navigate between gendered expectations and norms at home and outside the home environment. They negotiate their positionings through linguistic repertoires – for instance, through mastery of the Finnish language – and by adjusting their behaviour in light of the context. This suggests that young Kurds adopt various forms of agency to display and enact their belonging in a transnational diaspora space. Young Kurds’ narrations display both territorially-bounded and non-territorially-bounded elements with regard to the relationship between identity and locality. ‘Home’ is located in Finland, and the future and aspirations are planned in relation to it. In contrast, the region of Kurdistan is viewed as ‘homeland’ and as the place of origins and roots, where temporary stays and visits are a possibility. The emotional attachments are forged in relation to the country (Finland) and not so much relative to ‘Finnishness’, which the interviewees considered an exclusionary identity category. Furthermore, identification with one’s immediate place of residence (city) or, in some cases, with a religious identity as ‘Muslim’ provides a more flexible venue for identification than does identifying oneself with the (Finnish) nation.

From Molecular Blocks To Bioanalytical Assays: Combining Lanthanide Luminescence and Bioaffinity Binders for Protein Detection

Measuring protein biomarkers from sample matrix, such as plasma, is one of the basic tasks in clinical diagnostics. Bioanalytical assays used for the measuring should be able to measure proteins with high sensitivity and specificity. Furthermore, multiplexing capability would also be advantageous. To ensure the utility of the diagnostic test in point-of-care setting, additional requirements such as short turn-around times, ease-ofuse and low costs need to be met. On the other hand, enhancement of assay sensitivity could enable exploiting novel biomarkers, which are present in very low concentrations and which the current immunoassays are unable to measure. Furthermore, highly sensitive assays could enable the use of minimally invasive sampling. In the development of high-sensitivity assays the label technology and affinity binders are in pivotal role. Additionally, innovative assay designs contribute to the obtained sensitivity and other characteristics of the assay as well as its applicability. The aim of this thesis was to study the impact of assay components on the performance of both homogeneous and heterogeneous assays. Applicability of two different lanthanide-based label technologies, upconverting nanoparticles and switchable lanthanide luminescence, to protein detection was explored. Moreover, the potential of recombinant antibodies and aptamers as alternative affinity binders were evaluated. Additionally, alternative conjugation chemistries for production of the labeled binders were studied. Different assay concepts were also evaluated with respect to their applicability to point-of-care testing, which requires simple yet sensitive methods. The applicability of upconverting nanoparticles to the simultaneous quantitative measurement of multiple analytes using imaging-based detection was demonstrated. Additionally, the required instrumentation was relatively simple and inexpensive compared to other luminescent lanthanide-based labels requiring time-resolved measurement. The developed homogeneous assays exploiting switchable lanthanide luminescence were rapid and simple to perform and thus applicable even to point-ofcare testing. The sensitivities of the homogeneous assays were in the picomolar range, which are still inadequate for some analytes, such as cardiac troponins, requiring ultralow limits of detection. For most analytes, however, the obtained limits of detection were sufficient. The use of recombinant antibody fragments and aptamers as binders allowed site-specific and controlled covalent conjugation to construct labeled binders reproducibly either by using chemical modification or recombinant technology. Luminescent lanthanide labels were shown to be widely applicable for protein detection in various assay setups and to contribute assay sensitivity.

Retinol-induced changes in the phosphorylation levels of histones and high mobility group proteins from Sertoli cells

Chromatin proteins play a role in the organization and functions of DNA. Covalent modifications of nuclear proteins modulate their interactions with DNA sequences and are probably one of the multiple factors involved in the process of switch on/off transcriptionally active regions of DNA. Histones and high mobility group proteins (HMG) are subject to many covalent modifications that may modulate their capacity to bind to DNA. We investigated the changes induced in the phosphorylation pattern of cultured Wistar rat Sertoli cell histones and high mobility group protein subfamilies exposed to 7 µM retinol for up to 48 h. In each experiment, 6 h before the end of the retinol treatment each culture flask received 370 KBq/ml [32P]-phosphate. The histone and HMGs were isolated as previously described [Moreira et al. Medical Science Research (1994) 22: 783-784]. The total protein obtained by either method was quantified and electrophoresed as described by Spiker [Analytical Biochemistry (1980) 108: 263-265]. The gels were stained with Coomassie brilliant blue R-250 and the stained bands were cut and dissolved in 0.5 ml 30% H2O2 at 60oC for 12 h. The vials were chilled and 5.0 ml scintillation liquid was added. The radioactivity in each vial was determined with a liquid scintillation counter. Retinol treatment significantly changed the pattern of each subfamily of histone and high mobility group proteins.

Absence of hepatitis B virus DNA in patients with hepatitis C and non-A-E hepatitis in the State of São Paulo, Brazil

Occult hepatitis B virus (HBV) infection has been reported as cases in which HBV DNA was detected despite the absence of any HBV serological markers or in cases in which anti-HBc antibody was the sole marker. The aim of the present study was to determine, using the polymerase chain reaction (PCR), whether HBV infection occurs in hepatitis C and non-A-E hepatitis patients without serological evidence of hepatitis B infection in São Paulo State. Two different populations were analyzed: 1) non-A-E hepatitis patients, including 12 patients with acute and 50 patients with chronic hepatic disorders without serological evidence of infection with known hepatitis viruses; 2) 43 patients previously diagnosed as hepatitis C with positive results for anti-HCV and HCV RNA. Among hepatitis C patients, anti-HBc was detected in 18.6% of the subjects. Three different sets of primers were employed for HBV DNA detection by nested PCR, covering different HBV genes: C, S and X. HBV-DNA was not detected in any sample, whereas the positive controls did produce signals. The lack of HBV DNA detection with these pairs of primers could be due to a very low viral load or to the presence of mutations in their annealing sites. The latter is unlikely as these primers were screened against an extensive dataset of HBV sequences. The development of more sensitive methods, such as real time PCR, to detect circular covalent closed DNA is necessary in order to evaluate this question since previous studies have shown that cryptic hepatitis B might occur.

Magnetic properties of defective graphene

In this thesis, the influence of the functionalization of graphene and graphite on their magnetic properties was investigated. The functionalization was performed by covalent attaching of a phenyl groups with three different radicals (4-bromoaniline, 4-chloroaniline and 4-nitroaniline). Magnetic properties were measured by SQUID magnetometer. Both pristine graphite and graphene showed strong diamagnetic behavior. For good quality graphite, diamagnetism was found to be temperature-dependent. All samples demonstrated noticeable paramagnetic contribution below 50 K. According to fitting experimental results with Brillouin function and Curie law, it was shown that paramagnetism is provided by small clusters of spins (superparamagnetic behavior). Moreover, the clusters size and spin concentrations were calculated. For the samples functionalized with nitroaniline the antiferromagnetic transition around 120 K was observed. To explain this behavior, a simple model was proposed. Additional analysis of the graphene quality, structure and composition of the samples was carried out by HRTEM, EDS mapping, Raman spectroscopy and X-ray diffraction techniques.

Epidermal growth factor receptor (EGFR) mutations in lung cancer: preclinical and clinical data

Lung cancer leads cancer-related mortality worldwide. Non-small-cell lung cancer (NSCLC), the most prevalent subtype of this recalcitrant cancer, is usually diagnosed at advanced stages, and available systemic therapies are mostly palliative. The probing of the NSCLC kinome has identified numerous nonoverlapping driver genomic events, including epidermal growth factor receptor (EGFR) gene mutations. This review provides a synopsis of preclinical and clinical data on EGFR mutated NSCLC and EGFR tyrosine kinase inhibitors (TKIs). Classic somatic EGFR kinase domain mutations (such as L858R and exon 19 deletions) make tumors addicted to their signaling cascades and generate a therapeutic window for the use of ATP-mimetic EGFR TKIs. The latter inhibit these kinases and their downstream effectors, and induce apoptosis in preclinical models. The aforementioned EGFR mutations are stout predictors of response and augmentation of progression-free survival when gefitinib, erlotinib, and afatinib are used for patients with advanced NSCLC. The benefits associated with these EGFR TKIs are limited by the mechanisms of tumor resistance, such as the gatekeeper EGFR-T790M mutation, and bypass activation of signaling cascades. Ongoing preclinical efforts for treating resistance have started to translate into patient care (including clinical trials of the covalent EGFR-T790M TKIs AZD9291 and CO-1686) and hold promise to further boost the median survival of patients with EGFR mutated NSCLC.

Phylogenetic Analysis of Avidins and Expression of Novel Avidins from Lactrodectus hesperus and Hoeflea phototrophica

Avidins (Avds) are homotetrameric or homodimeric glycoproteins with typically less than 130 amino acid residues per monomer. They form a highly stable, non-covalent complex with biotin (vitamin H) with Kd = 10-15 M (for chicken Avd). The best-studied Avds are the chicken Avd from Gallus gallus and streptavidin from Streptomyces avidinii, although other Avd studies have also included Avds from various origins, e.g., from frogs, fishes, mushrooms and from many different bacteria. Several engineered Avds have been reported as well, e.g., dual-chain Avds (dcAvds) and single-chain Avds (scAvds), circular permutants with up to four simultaneously modifiable ligand-binding sites. These engineered Avds along with the many native Avds have potential to be used in various nanobiotechnological applications. In this study, we made a structure-based alignment representing all currently available sequences of Avds and studied the evolutionary relationship of Avds using phylogenetic analysis. First, we created an initial multiple sequence alignment of Avds using 42 closely related sequences, guided by the known Avd crystal structures. Next, we searched for non-redundant Avd sequences from various online databases, including National Centre for Biotechnology Information and the Universal Protein Resource; the identified sequences were added to the initial alignment to expand it to a final alignment of 242 Avd sequences. The MEGA software package was used to create distance matrices and a phylogenetic tree. Bootstrap reproducibility of the tree was poor at multiple nodes and may reflect on several possible issues with the data: the sequence length compared is relatively short and, whereas some positions are highly conserved and functional, others can vary without impinging on the structure or the function, so there are few informative sites; it may be that periods of rapid duplication have led to paralogs and that the differences among them are within the error limit of the data; and there may be other yet unknown reasons. Principle component analysis applied to alternative distance data did segregate the major groups, and success is likely due to the multivariate consideration of all the information. Furthermore, based on our extensive alignment and phylogenetic analysis, we expressed two novel Avds, lacavidin from Lactrodectus Hesperus, a western black widow spider, and hoefavidin from Hoeflea phototrophica, an aerobic marine bacterium, the ultimate aim being to determine their X-ray structures. These Avds were selected because of their unique sequences: lacavidin has an N-terminal Avd-like domain but a long C-terminal overhang, whereas hoefavidin was thought to be a dimeric Avd. Both these Avds could be used as novel scaffolds in biotechnological applications.

Analytics for the modified kraft pulps

Tämän diplomityön tavoitteena on kehittää sopiva analyyttinen menetelmä muokatun kraft-sellukuidun substituutioasteen (DS) kvantitatiivista määrittämistä varten. Muokkauksella tarkoitetaan tässä yhteydessä joko kovalenttisesti tai adsorption avulla tapahtuvaa molekyylin kiinnittymistä sellukuidun pinnalle. Työn kirjallisuusosuudessa käsitellään lyhyesti eri muokkaustapoja ja yhdisteitä joiden avulla voidaan saavuttaa haluttuja ominaisuuksia sellusta valmistetuille lopputuotteille. Lisäksi kirjallisuusosuudessa käydään läpi käyttötarkoitukseen soveltuvimpia suoria ja epäsuoria analyysimenetelmiä. Analyysimenetelmistä kaikkein lupaavimpia testattiin työn kokeellisessa osassa. Diplomityön kokeellisessa osassa keskityttiin kehittämään muokatulle sellulle kvantitatiivista menetelmää DS:n määrittämiseksi Fourier-muunnos infrapuna-vaimennettu kokonaisheijastus (FTIR-ATR) spektrometrillä. Kirjallisuuskatsauksessa ei löytynyt yhtään dokumentoitua tutkimusta, jossa FTIR-ATR menetelmää olisi käytetty muokatun sellukuidun kvantitatiiviseen tutkimukseen. Muiden analyysimenetelmien, kuten alkuaineanalyysin, termogravimetrisen analyysin (TGA) ja valomikroskopian avulla pyrittiin tuottamaan lisätietoa muokkauksesta. Kvantitatiivisen FTIR-ATR menetelmän kehitykseen käytetyt muokatut sellukuidut olivat selluloosa-asetaattia ja selluloosa betainaattia. Saatujen tulosten perusteella muokattujen sulfiitti- ja kraft sellukuitujen DS:n kvantitatiivinen määrittäminen on mahdollista FTIR-ATR menetelmällä. Vähäinen kalibrointipisteiden määrä vaikeutti tarkan analyysimenetelmän tekemistä. Kehitetyn menetelmän suurimpina ongelmina olivat kiinteiden näytteiden heterogeenisyys sekä mahdollisten epäpuhtauksien tunnistaminen. Jatkotutkimusten avulla kehitettyä menetelmää on kuitenkin mahdollista käyttää muokattujen sellukuitujen jatkuvaan analysointiin selluteollisuudessa.