Modulating the electronic structure of amino acids: interaction of model lewis acids with anthranilic acid

On the basis of theoretical B3LYP calculations, Yáñez and co-workers (J. Chem. Theory Comput. 2012, 8, 2293) illustrated that beryllium ions are capable of significantly modulating (changing) the electronic structures of imidazole. In this computational organic chemistry study, the interaction of this β-amino acid and five model Lewis acids (BeF1+, Be2+, AlF2(1+), AlF2+, and Al3+) were investigated. Several aspects were addressed: natural bond orbitals, including second order perturbation analysis of intra-molecular charge delocalization and the natural population analysis atomic charges; molecular geometries; selected infrared stretching frequencies (C-N, C-O, and N-H), and selected ¹H-NMR chemical shifts. The data illustrate that this interaction can weaken the H-O bond and goes beyond strengthening the intra-molecular hydrogen bond (N...H-O) to cause a spontaneous transfer of the proton to the nitrogen atom in five cases generating zwitterion structures. Many new features are observed. Most importantly, the zwitterion structures include a stabilizing hydrogen bond (N-H...O) that varies in relative strength according to the Lewis acid. These findings explain the experimental observations of α-amino acids (for example: J. Am. Chem. Soc. 2001, 123, 3577) and are the first reported fundamental electronic structure characterization of β-amino acids in zwitterion form.

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.

Use of pyrosequencing to identify streptococci and to detect mutations causing antimicrobial resistance

Rapid identification and resistance determination of pathogens in clinical specimens is vital for accurate treatment and monitoring of infectious diseases. Antimicrobial drug resistance is increasing globally and healthcare settings are facing this cost-intensive and even life-threatening problem. The incidence of resistant pathogens in Finland has remained relatively steady and manageable at least for the time being. DNA sequencing is the gold standard method for genotyping, mutation analysis, and identification of bacteria. Due to significant cost decrease in recent years, this technique is available to many research and clinical laboratories. Pyrosequencing technique, a rapid real-time DNA sequencing method especially suitable for analyzing fairly short stretches of DNA, was used in this study. Due to its robustness and versatility, pyrosequencing was applied in this study for identification of streptococci and detection of certain mutations causing antimicrobial resistance in different bacteria. Certain streptococcal species such as S. pneumoniae and S. pyogenes are significantly important clinical pathogens. S. pneumoniae causes e.g. pneumonia and otitis media and is one of the most important community-acquired pathogens. S. pyogenes, also known as group A streptococcus, causes e.g. angina and erysipelas. In contrast, the socalled alpha-haemolytic streptococci, such as S. mitis and S. oralis, belong to the normal microbiota, which are regarded to be non-pathogenic and are nearly impossible to identify by phenotypic methods. In this thesis, a pyrosequencing method was developed for identification of streptococcal species based on the 16S rRNA sequences. Almost all streptococcal species could be differentiated from one another by the developed method, including S. pneumoniae from its close relatives S. mitis and S. oralis . New resistance genes and their variants are constantly discovered and reported. In this study, new methods for detecting certain mutations causing macrolide resistance or extended spectrum beta-lactamase (ESBL) phenotype were developed. These resistance detection approaches are not only suitable for surveillance of mechanisms causing antimicrobial resistance but also for routine analysis of clinical samples particularly in epidemic settings. In conclusion, pyrosequencing was found to be an accurate, versatile, cost-effective, and rapid DNA sequencing method that is especially suitable for mutation analysis of short DNA fragments and identification of certain bacteria.

Antimicrobial Resistance in Campylobacter jejuni and Campylobacter coli

Antimicrobial Resistance in Campylobacter jejuni and Campylobacter coli Campylobacters are a common cause of bacterial gastroenteritis worldwide, with Campylobacter jejuni and C. coli being the most common species isolated in human infections. If antimicrobial treatment is required, the drugs of choice at the moment are the macrolides and fluoroquinolones. In this thesis, the in vitro resistance profiles of the C. jejuni and C. coli strains were evaluated with emphasis on multidrug resistance. The aim was also to evaluate the different resistance mechanisms against the macrolides. Further, the disk diffusion method was compared to agar dilution method and its repeatability was evaluated, since it has been widely used for the susceptibility testing of campylobacters. The results of the present study showed that resistance to the fluoroquinolones is common in strains isolated from Finnish patients, but resistance to the macrolides is still rare. Multidrug resistance was associated with resistance to both ciprofloxacin and erythromycin. Among the available per oral drugs, least resistance was observed to coamoxiclav There was no resistance to the carbapenems. Sitafloxacin and tigecycline were in vitro highly effective towards Campylobacter species. A point mutation A2059G of the 23S rRNA gene was the main mechanism behind the macrolide resistance, whereas the efflux pumps did not seem to play an important role when a strain had A2059G mutation. A five amino acids insertion, which has not been described previously, in the ribosomal protein L22 of one highly-resistant C. jejuni strain without mutation in the 23S rRNA gene was also detected. Concerning the disk diffusion method, there was variation in the repeatability In conclusion, macrolides still appear to be the first-choice alternative for suspected Campylobacter enteritis. The in vitro susceptibilities found suggest that co-amoxiclav might be a candidate for clinical trials on campylobacteriosis, but in life-threatening situations, a carbapenem may be the drug of choice. More studies are needed on whether the disk diffusion test method could be improved or whether all susceptibilities of campylobacters should be done using a MIC based method.

Transcriptional Profiling of Organ‐Specific Autoimmunity in Human

Our understanding of the pathogenesis of organ‐specific autoinflammation has been restricted by limited access to the target organs. Peripheral blood, however, as a preferred transportation route for immune cells, provides a window to assess the entire immune system throughout the body. Transcriptional profiling with RNA stabilizing blood collection tubes reflects in vivo expression profiles at the time the blood is drawn, allowing detection of the disease activity in different samples or within the same sample over time. The main objective of this Ph.D. study was to apply gene‐expression microarrays in the characterization of peripheral blood transcriptional profiles in patients with autoimmune diseases. To achieve this goal a custom cDNA microarray targeted for gene‐expression profiling of human immune system was designed and produced. Sample collection and preparation was then optimized to allow gene‐expression profiling from whole‐blood samples. To overcome challenges resulting from minute amounts of sample material, RNA amplification was successfully applied to study pregnancy related immunosuppression in patients with multiple sclerosis (MS). Furthermore, similar sample preparation was applied to characterize longitudinal genome‐wide expression profiles in children with type 1 diabetes (T1D) associated autoantibodies and eventually clinical T1D. Blood transcriptome analyses, using both the ImmunoChip cDNA microarray with targeted probe selection and genome‐wide Affymetrix U133 Plus 2.0 oligonucleotide array, enabled monitoring of autoimmune activity. Novel disease related genes and general autoimmune signatures were identified. Notably, down‐regulation of the HLA class Ib molecules in peripheral blood was associated with disease activity in both MS and T1D. Taken together, these studies demonstrate the potential of peripheral blood transcriptional profiling in biomedical research and diagnostics. Imbalances in peripheral blood transcriptional activity may reveal dynamic changes that are relevant for the disease but might be completely missed in conventional cross‐sectional studies.

Koulutetut maahanmuuttajat työmarkkinoilla. Tutkimus korkeakoulututkinnon suorittaneiden maahanmuuttajien työllistymisestä ja työurien alusta Suomessa

Maahanmuuttajien määrä on Suomessa merkittävämmin lisääntynyt vasta 1990- ja 2000-luvuilla. Vuonna 2010 Suomessa asui lähes 170 000 ulkomaan kansalaista. Tavallisimmin Suomeen muutetaan avioliiton, paluumuuton tai pakolaisuuden vuoksi. Pieni, joskin kasvava joukko muuttaa työn tai opiskelun vuoksi. Myös kansalaisuuksien, koulutustaustojen, ammattien jne. kirjo muuttajien joukossa on suuri. Ulkomaan kansalaisten lisääntyessä Suomessa on jouduttu kohtaamaan monenlaisia maahanmuuttoon liittyviä haasteita, joista työllistymiseen liittyvät kysymykset eivät ole vähäisimpiä. Tutkimuksessa tarkastellaan lähtömaassaan korkeakoulututkinnon suorittaneiden maahanmuuttajien työllistymistä ja työuran alkua Suomessa. Tutkimuksen tarkoituksena on selvittää, miten korkeakoulutetut maahanmuuttajat ovat Suomessa työllistyneet, minkälaisia heidän työuriensa alut ovat Suomessa olleet ja miten he ovat onnistuneet uudessa maassa hyödyntämään lähtömaassa hankkimaansa koulutusta. Lisäksi tutkimuksessa tarkastellaan, miten maahanmuuttajien lähtömaan erilaiset elämäntilanteet, olosuhteet ja valinnat ovat vaikuttaneet työuran muotoutumiseen Suomessa. Tutkimuksen aineisto muodostuu kysely- sekä haastatteluaineistosta. Kyselyaineiston (n=99) tarkoituksena on luoda määrällistä kuvaa korkeakoulutettujen maahanmuuttajien työllistymisestä Suomessa. Numerotietojen taakse jää kuitenkin näkymättömiin tieto maahanmuuttajien yksilöllisistä kokemuksista liittyen työllistymiseen ja työuran muotoutumiseen uudessa maassa. Toisena aineistona hyödynnettävän elämäkerrallisen haastatteluaineiston (n=20) kautta on mahdollista tehdä näkyväksi ne tutkittavien yksilölliset työ- ja koulutusuraan liittyvät valinnat, joita maahanmuuttajat ovat tehneet niin lähtömaassa kuin Suomessa sekä ne tilanteet ja olosuhteet, joissa tutkittavat ovat lähtömaassa eläneet ja joiden pohjalta he ovat tulleet Suomeen ja Suomen työmarkkinoille. Aineistoissa mukana olevat maahanmuuttajat olivat pääosin avioliiton, paluumuuton sekä pakolaisuuden vuoksi Suomeen tulleita. Vain muutama oli tullut työn vuoksi. Maahanmuuttajien työmarkkina-asemaa selitetään usein maahanmuuttajien resursseilla, kuten kielitaidolla, koulutuksella, työkokemuksella, sosiaalisten suhteiden ja verkostojen laadulla ja määrällä jne. Myös maahanmuuttajiin kohdistuvilla syrjivillä ja ennakkoluuloisilla asenteilla on keskeinen merkitys työllistymisessä. Koulutuksen ollessa yksi keskeisimmistä työmarkkina-asemaa määräävistä tekijöistä, tulisi koulutettujen maahanmuuttajien sijoittua hankitun tutkinnon oikeuttamiin tehtäviin. Tutkimuksessa kuitenkin havaittiin, että työllistyminen oli maahanmuuttajilla vaikeaa hyvästä koulutuksesta huolimatta. Kyselyaineistoon vastanneista vain muutama (6 %) oli työssä heti Suomeen muuttovuoden lopussa, kolme vuotta Suomessa asuttuaan työssä oli runsas kolmannes (35 %) ja aineistonkeruuhetkellä eli vuonna 2004 työssä oli 38 % vastaajista. Työsuhteet olivat tutkittavilla useimmiten määräaikaisia ja kestoltaan lyhyitä. Lisäksi työurat koostuivat runsaasta työttömyydestä sekä koulutukseen osallistumisesta. Myönteistä kuitenkin oli, että mikäli korkeakoulutetut maahanmuuttajat onnistuivat Suomessa työllistymään, vastasi työ usein joko kokonaan tai ainakin osittain hankittua korkeakoulututkintoa. Korkeakoulutettujen maahanmuuttajien työuran alut Suomessa voidaan tyypitellä kolmeen ryhmään, joista kukin jakaantui vielä kahteen alaryhmään siten, että kaiken kaikkiaan saatiin kuusi erilaista työuran alun tyyppiä: koulutusta vastaava vakaa ja vakiintuva ura, koulutusta osittain vastaava sekaura ja laskeva ura sekä koulutusta vastaamaton sisääntuloura ja työttömän ura . Haastatteluaineiston kautta tarkastellaan korkeakoulutettujen maahanmuuttajien yksilöllisiä elämänuria lähtien liikkeelle korkeakoulutettujen maahanmuuttajien lähtömaassa tekemistä ura- ja ammatinvalinnoista jatkuen Suomeen muuton kautta aina työuran muotoutumiseen Suomessa. Haastatteluja toisistaan erottelevina keskeisinä teemoina olivat toisaalta pärjääminen Suomessa ja suomalaisilla työmarkkinoilla toisaalta elämän muotoutuminen lähtömaassa ja nimenomaan siellä tehdyt ura- ja ammatinvalinnat ja niihin liittyvät kokemukset ja elämäntilanteet. Näiden kriteerien pohjalta aineisto jakaantui kolmeen ryhmään, jotka nimettiin pärjääjiksi, harhailijoiksi ja sinnittelijöiksi. Pärjääjien kertomukset muotoutuivat tietyllä tavalla myönteisen kehän kautta: niin lähtömaassa tehdyt ammatinvalinnat kuin työuran muotoutuminen Suomessa tapahtuivat suhteellisen vaivattomasti. Useimmiten työt Suomessa vastasivat lähtömaassa hankittua koulutusta. Omiin uravalintoihin oltiin myöhemmin myös tyytyväisiä. Harhailijoille oman paikan löytyminen oli puolestaan hankalampaa. Leimallista tälle ryhmälle oli tietynlainen valintojen vaikeus sekä tyytymättömyys omiin aikaisemmin tehtyihin ratkaisuihin. Jotkut harmittelivat nuorena tekemiään uravalintoja niin, että päättivät Suomessa hankkia kokonaan uuden ammatin. Muutto Suomeen merkitsi useimmille ammatillisen aseman laskua. Sinnittelijät kertoivat jo lähtökohdiltaan kahteen muuhun ryhmään nähden hyvin erilaista tarinaa. Tämän ryhmän lähes koko elämä lähtömaassa oli sodan ja levottomuuksien sävyttämää. Tämä näkyi myös ammatinvalinnassa: opiskelupaikka oli saatettu valita esimerkiksi sen perusteella, missä oli milloinkin turvallista opiskella. Myös Suomeen muutto erosi kahdesta aikaisemmasta ryhmästä, sillä lähtö entisestä kotimaasta oli tapahtunut usein hyvinkin yllättäen vailla etukäteissuunnittelua tilanteiden kärjistyttyä nopeasti. Suomessa työelämään pääseminen oli kaikille sinnittelijöille vaikeaa ja haastatteluhetkellä useilla vielä hyvin alkutekijöissä. Hyväkään koulutus ei aina takaa maahanmuuttajille työtä uudessa maassa, sillä hankittua tutkintoa ja osaamista ei ole helppo siirtää maasta toiseen. Pahimmassa tapauksessa vieraassa maassa suoritettu korkeakoulututkinto voi kokonaan mitätöityä uudessa maassa ja korkeakoulututkinnon myötä hankittu osaaminen menettää täysin arvonsa. Kyse on niin yksilön kuin yhteiskunnankin resurssien tuhlaamisesta tilanteessa, jossa maassa pysyvästi asuvat koulutetut maahanmuuttajat työskentelevät tavalla tai toisella koulutustaan vastaamattomissa epävakaissa töissä, työmarkkinoiden laitamilla tai ovat kokonaan työmarkkinoiden ulkopuolella.

Start-up of horizontal anaerobic reactors with sludge blanket and fixed bed for wastewater treatment from coffee processing by wet method

In this study it was evaluated the start-up procedures of anaerobic treatment system with three horizontal anaerobic reactors (R1, R2 and R3), installed in series, with volume of 1.2 L each. R1 had sludge blanket, and R2 and R3 had half supporter of bamboo and coconut fiber, respectively. As an affluent, it was synthesized wastewater from mechanical pulping of the coffee fruit by wet method, with a mean value of total chemical oxygen demand (CODtotal) of 16,003 mg L-1. The hydraulic retention time (HRT) in each reactor was 30 h. The volumetric organic loading (VOL) applied in R1 varied from 8.9 to 25.0 g of CODtotal (L d)-1. The mean removal efficiencies of CODtotal varied from 43 to 97% in the treatment system (R1+R2+R3), stabilizing above 80% after 30 days of operation. The mean content of methane in the biogas were of 70 to 76%, the mean volumetric production was 1.7 L CH4 (L reactor d)-1 in the system, and the higher conversions were around at 0.20 L CH4 (g CODremoved)-1 in R1 and R2. The mean values of pH in the effluents ranged from 6.8 to 8.3 and the mean values of total volatile acids remained below 200 mg L-1 in the effluent of R3. The concentrations of total phenols of the affluent ranged from 45 to 278 mg L-1, and the mean removal efficiency was of 52%. The start-up of the anaerobic treatment system occurred after 30 days of operation as a result of inoculation with anaerobic sludge with active microbiota.

Molecular Systematics of Noctuoidea (Insecta, lepidoptera)

In this thesis, I conduct a series of molecular systematic studies on the large phytophagous moth superfamily Noctuoidea (Insecta, Lepidoptera) to clarify deep divergences and evolutionary affinities of the group, based on material from every zoogeographic region of the globe. Noctuoidea are the most speciose radiations of butterflies and moths on earth, comprising about a quarter of all lepidopteran diversity. The general aim of these studies was to apply suitably conservative genetic markers (DNA sequences of mitochondrial—mtDNA—and nuclear gene— nDNA—regions) to reconstruct, as the initial step, a robust skeleton phylogenetic hypothesis for the superfamily, then build up robust phylogenetic frameworks for those circumscribed monophyletic entities (i.e., families), as well as clarifying the internal classification of monophyletic lineages (subfamilies and tribes), to develop an understanding of the major lineages at various taxonomic levels within the superfamily Noctuoidea, and their inter-relationships. The approaches applied included: i) stabilizing a robust family-level classification for the superfamily; ii) resolving the phylogeny of the most speciose radiation of Noctuoidea: the family Erebidae; iii) reconstruction of ancestral feeding behaviors and evolution of the vampire moths (Erebidae, Calpinae); iv) elucidating the evolutionary relationships within the family Nolidae and v) clarifying the basal lineages of Noctuidae sensu stricto. Thus, in this thesis I present a wellresolved molecular phylogenetic hypothesis for higher taxa of Noctuoidea consisting of six strongly supported families: Oenosandridae, Notodontidae, Euteliidae, Erebidae, Nolidae, and Noctuidae. The studies in my thesis highlight the importance of molecular data in systematic and phylogenetic studies, in particular DNA sequences of nuclear genes, and an extensive sampling strategy to include representatives of all known major lineages of entire world fauna of Noctuoidea from every biogeographic region. This is crucial, especially when the model organism is as species-rich, highly diverse, cosmopolitan and heterogeneous as the Noctuoidea, traits that represent obstacles to the use of morphology at this taxonomic level.

Production of Mg(OH)2 from Mg-silicate rock for CO2 mineral sequestration

Sequestration of carbon dioxide in mineral rocks, also known as CO2 Capture and Mineralization (CCM), is considered to have a huge potential in stabilizing anthropogenic CO2 emissions. One of the CCM routes is the ex situ indirect gas/sold carbonation of reactive materials, such as Mg(OH)2, produced from abundantly available Mg-silicate rocks. The gas/solid carbonation method is intensively researched at Åbo Akademi University (ÅAU ), Finland because it is energetically attractive and utilizes the exothermic chemistry of Mg(OH)2 carbonation. In this thesis, a method for producing Mg(OH)2 from Mg-silicate rocks for CCM was investigated, and the process efficiency, energy and environmental impact assessed. The Mg(OH)2 process studied here was first proposed in 2008 in a Master’s Thesis by the author. At that time the process was applied to only one Mg-silicate rock (Finnish serpentinite from the Hitura nickel mine site of Finn Nickel) and the optimum process conversions, energy and environmental performance were not known. Producing Mg(OH)2 from Mg-silicate rocks involves a two-staged process of Mg extraction and Mg(OH)2 precipitation. The first stage extracts Mg and other cations by reacting pulverized serpentinite or olivine rocks with ammonium sulfate (AS) salt at 400 - 550 oC (preferably < 450 oC). In the second stage, ammonia solution reacts with the cations (extracted from the first stage after they are leached in water) to form mainly FeOOH, high purity Mg(OH)2 and aqueous (dissolved) AS. The Mg(OH)2 process described here is closed loop in nature; gaseous ammonia and water vapour are produced from the extraction stage, recovered and used as reagent for the precipitation stage. The AS reagent is thereafter recovered after the precipitation stage. The Mg extraction stage, being the conversion-determining and the most energy-intensive step of the entire CCM process chain, received a prominent attention in this study. The extraction behavior and reactivity of different rocks types (serpentinite and olivine rocks) from different locations worldwide (Australia, Finland, Lithuania, Norway and Portugal) was tested. Also, parametric evaluation was carried out to determine the optimal reaction temperature, time and chemical reagent (AS). Effects of reactor types and configuration, mixing and scale-up possibilities were also studied. The Mg(OH)2 produced can be used to convert CO2 to thermodynamically stable and environmentally benign magnesium carbonate. Therefore, the process energy and life cycle environmental performance of the ÅAU CCM technique that first produces Mg(OH)2 and the carbonates in a pressurized fluidized bed (FB) were assessed. The life cycle energy and environmental assessment approach applied in this thesis is motivated by the fact that the CCM technology should in itself offer a solution to what is both an energy and environmental problem. Results obtained in this study show that different Mg-silicate rocks react differently; olivine rocks being far less reactive than serpentinite rocks. In summary, the reactivity of Mg-silicate rocks is a function of both the chemical and physical properties of rocks. Reaction temperature and time remain important parameters to consider in process design and operation. Heat transfer properties of the reactor determine the temperature at which maximum Mg extraction is obtained. Also, an increase in reaction temperature leads to an increase in the extent of extraction, reaching a maximum yield at different temperatures depending on the reaction time. Process energy requirement for producing Mg(OH)2 from a hypothetical case of an iron-free serpentine rock is 3.62 GJ/t-CO2. This value can increase by 16 - 68% depending on the type of iron compound (FeO, Fe2O3 or Fe3O4) in the mineral. This suggests that the benefit from the potential use of FeOOH as an iron ore feedstock in iron and steelmaking should be determined by considering the energy, cost and emissions associated with the FeOOH by-product. AS recovery through crystallization is the second most energy intensive unit operation after the extraction reaction. However, the choice of mechanical vapor recompression (MVR) over the “simple evaporation” crystallization method has a potential energy savings of 15.2 GJ/t-CO2 (84 % savings). Integrating the Mg(OH)2 production method and the gas/solid carbonation process could provide up to an 25% energy offset to the CCM process energy requirements. Life cycle inventory assessment (LCIA) results show that for every ton of CO2 mineralized, the ÅAU CCM process avoids 430 - 480 kg CO2. The Mg(OH)2 process studied in this thesis has many promising features. Even at the current high energy and environmental burden, producing Mg(OH)2 from Mg-silicates can play a significant role in advancing CCM processes. However, dedicated future research and development (R&D) have potential to significantly improve the Mg(OH)2 process performance.

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.

The Role of an Oncoprotein CIP2A in Breast Cancer

Cancerous inhibitor of PP2A (CIP2A) is an oncoprotein expressed in several human cancer types. Previously, CIP2A has been shown to promote proliferation of cancer cells. Mechanistically, CIP2A is known to inhibit activity of a tumor suppressor protein phosphatase 2A (PP2A) towards an oncoprotein MYC, further stabilizing MYC in human cancer. However, the molecular mechanisms how CIP2A expression is induced during cellular transformation are not well known. Also, expression, functional role and clinical relevance of CIP2A in breast cancer had not been studied before. The results of this PhD thesis work demonstrate that CIP2A is highly expressed in human breast cancer, and that high expression of CIP2A in tumors is a poor prognostic factor in a subset of breast cancer patients. CIP2A expression correlates with inactivating mutations of tumor suppressor p53 in human cancer. Notably, we demonstrate that p53 inactivation up-regulates CIP2A expression via increased expression of an oncogenic transcription factor E2F1. Moreover, CIP2A promotes expression of E2F1, and this novel positive feedback loop between E2F1 and CIP2A is demonstrated to regulate sensitivity to both p53-dependent and -independent senescence induction in breast cancer cells. Importantly, in a CIP2A deficient breast cancer mouse model, abrogation of CIP2A attenuates mammary tumor formation and progression with features of E2F1 inhibition and induction of senescence. Furthermore, we demonstrate that CIP2A expression defines the cellular response to a senescence-inducing chemotherapy in breast cancer. Taken together, these results demonstrate that CIP2A is an essential promoter of breast cancer tumor growth by inhibiting senescence. Finally, this study implicates inhibition of CIP2A as a promising therapy target for breast cancer.

Numerical study of Petrov-Galerkin formulations for the shallow water wave equations

The behavior of Petrov-Galerkin formulations for shallow water wave equations is evaluated numerically considering typical one-dimensional propagation problems. The formulations considered here use stabilizing operators to improve classical Galerkin approaches. Their advantages and disadvantages are pointed out according to the intrinsic time scale (free parameter) which has a particular importance in this kind of problem. The influence of the Courant number and the performance of the formulation in dealing with spurious oscillations are adressed.

The European Union and Assembling Biofuel Development – topological investigations concerning the associations between law, policy and space

Biofuels for transport are a renewable source of energy that were once heralded as a solution to multiple problems associated with poor urban air quality, the overproduction of agricultural commodities, the energy security of the European Union (EU) and climate change. It was only after the Union had implemented an incentivizing framework of legal and political instruments for the production, trade and consumption of biofuels that the problems of weakening food security, environmental degradation and increasing greenhouse gases through land-use changes began to unfold. In other words, the difference between political aims for why biofuels are promoted and their consequences has grown – which is also recognized by the EU policy-makers. Therefore, the global networks of producing, trading and consuming biofuels may face a complete restructure if the European Commission accomplishes its pursuit to sideline crop-based biofuels after 2020. My aim with this dissertation is not only to trace the manifold evolutions of the instruments used by the Union to govern biofuels but also to reveal how this evolution has influenced the dynamics of biofuel development. Therefore, I study the ways the EU’s legal and political instruments of steering biofuels are coconstitutive with the globalized spaces of biofuel development. My analytical strategy can be outlined through three concepts. I use the term ‘assemblage’ to approach the operations of the loose entity of actors and non-human elements that are the constituents of multi-scalar and -sectorial biofuel development. ‘Topology’ refers to the spatiality of this European biofuel assemblage and its parts whose evolving relations are treated as the active constituents of space, instead of simply being located in space. I apply the concept of ‘nomosphere’ to characterize the framework of policies, laws and other instruments that the EU applies and construes while attempting to govern biofuels. Even though both the materials and methods vary in the independent articles, these three concepts characterize my analytical strategy that allows me to study law, policy and space associated with each other. The results of my examinations underscore the importance of the instruments of governance of the EU constituting and stabilizing the spaces of producing and, on the other hand, how topological ruptures in biofuel development have enforced the need to reform policies. This analysis maps the vast scope of actors that are influenced by the mechanism of EU biofuel governance and, what is more, shows how they are actively engaging in the Union’s institutional policy formulation. By examining the consequences of fast biofuel development that are spatially dislocated from the established spaces of producing, trading and consuming biofuels such as indirect land use changes, I unfold the processes not tackled by the instruments of the EU. Indeed, it is these spatially dislocated processes that have pushed the Commission construing a new type of governing biofuels: transferring the instruments of climate change mitigation to land-use policies. Although efficient in mitigating these dislocated consequences, these instruments have also created peculiar ontological scaffolding for governing biofuels. According to this mode of governance, the spatiality of biofuel development appears to be already determined and the agency that could dampen the negative consequences originating from land-use practices is treated as irrelevant.

Hintariskeiltä suojautuminen viljakaupassa - viljatermiinit ja niiden käyttö Suomessa

Tämän tutkielman tarkoituksena on selvittää, miten viljatermiinejä käytetään Suomessa hintariskeiltä suojautumisen välineenä. Tutkimus on tehty viljantuottajan näkökulmasta. Tutkimusaineistona on käytetty aiheesta aiemmin tehtyä tutkimuskirjallisuutta ja varta vasten kerättyä haastatteluaineistoa. Tuottajien mielestä hintavaihteluilla oli suuri vaikutus tuotannon tulokseen ja kaikki haastatellut olivat miettineet keinoja hintavaihtelujen vaikutusten pienentämiseksi. Termiinikaupan käyttäminen viljan myynnissä on kuitenkin Suomessa melko harvinainen ilmiö. Aiempien tutkimustulosten perusteella ja kerättyyn haastatteluaineistoon perustuen vaikuttaisi siltä, että termiinikaupan vähäiseen käyttöön Suomessa ovat vaikuttaneet ainakin maataloustukien runsas osuus tilojen liikevaihdosta, tiloilla harjoitettava muu ansiotoiminta, sekä runsaat satovaihtelut. Haastatellut tuottajat pyrkivät suojautumaan hintavaihteluilta lähinnä viljaa varastoiden ja koettamalla valita tuotantoon mahdollisimman kannattavia kasveja, joilla hintavaihtelu oli vähäistä. Termiinejä tarjoavat viljantuottajille useat eri viljanostajat. Käytettäessä termiinejä säännöllisesti ja johdonmukaisesti, on termiinikaupalla tuloja tasaava vaikutus. Sen käyttö ei kuitenkaan takaa parempaa tulosta kuin käteiskauppakaan, vaan hyötynä on nimenomaan ennustettavuuden lisääntyminen ja epävarmuuden väheneminen tulovaihteluiden pienentymisen myötä. Termiinikaupasta aiheutuu tuottajalle myös kuluja ja siihen pitäisikin suhtautua kuten vakuutukseen.

pH titration of native and unfolded ß-trypsin: evaluation of the D D G0 titration and the carboxyl pK values

The stabilizing free energy of ß-trypsin was determined by hydrogen ion titration. In the pH range from 3.0 to 7.0, the change in free energy difference for the stabilization of the native protein relative to the unfolded one (D D G0 titration) was 9.51 ± 0.06 kcal/mol. An isoelectric point of 10.0 was determined, allowing us to calculate the Tanford and Kirkwood electrostatic factor w. This factor presented a nonlinear behavior and indicated more than one type of titratable carboxyl groups in ß-trypsin. In fact, one class of carboxyl group with a pK = 3.91 ± 0.01 and another one with a pK = 4.63 ± 0.03 were also found by hydrogen ion titration of the protein in the folded state