Negative feedback regulation of calcineurin-dependent Prz1 transcription factor by the CaMKK-CaMK1 axis in fission yeast

Calcium signals trigger the translocation of the Prz1 transcription factor from the cytoplasm to the nucleus. The process is regulated by the calciumactivated phosphatase calcineurin, which activates Prz1 thereby maintaining active transcription during calcium signalling. When calcium signalling ceases, Prz1 is inactivated by phosphorylation and exported to the cytoplasm. In budding yeast and mammalian cells, different kinases have been reported to counter calcineurin activity and regulate nuclear export. Here, we show that the Ca2+/calmodulin-dependent kinase Cmk1 is first phosphorylated and activated by the newly identified kinase CaMKK2 homologue, Ckk2, in response to Ca2+. Then, active Cmk1 binds, phosphorylates and inactivates Prz1 transcription activity whilst at the same time cmk1 expression is enhanced by Prz1 in response to Ca2+. Furthermore, Cdc25 phosphatase is also phosphorylated by Cmk1, inducing cell cycle arrest in response to an increase in Ca2+. Moreover, cmk1 deletion shows a high tolerance to chronic exposure to Ca2+, due to the lack of cell cycle inhibition and elevated Prz1 activity. This work reveals that Cmk1 kinase activated by the newly identified Ckk2 counteracts calcineurin function by negatively regulating Prz1 activity which in turn is involved in activating cmk1 gene transcription. These results are the first insights into Cmk1 and Ckk2 function in Schizosaccharomyces pombe.

Dominant-Negative Effects of Adult-Onset Huntingtin Mutations Alter the Division of Human Embryonic Stem Cells-Derived Neural Cells.

Mutations of the huntingtin protein (HTT) gene underlie both adult-onset and juvenile forms of Huntington's disease (HD). HTT modulates mitotic spindle orientation and cell fate in mouse cortical progenitors from the ventricular zone. Using human embryonic stem cells (hESC) characterized as carrying mutations associated with adult-onset disease during pre-implantation genetic diagnosis, we investigated the influence of human HTT and of an adult-onset HD mutation on mitotic spindle orientation in human neural stem cells (NSCs) derived from hESCs. The RNAi-mediated silencing of both HTT alleles in neural stem cells derived from hESCs disrupted spindle orientation and led to the mislocalization of dynein, the p150Glued subunit of dynactin and the large nuclear mitotic apparatus (NuMA) protein. We also investigated the effect of the adult-onset HD mutation on the role of HTT during spindle orientation in NSCs derived from HD-hESCs. By combining SNP-targeting allele-specific silencing and gain-of-function approaches, we showed that a 46-glutamine expansion in human HTT was sufficient for a dominant-negative effect on spindle orientation and changes in the distribution within the spindle pole and the cell cortex of dynein, p150Glued and NuMA in neural cells. Thus, neural derivatives of disease-specific human pluripotent stem cells constitute a relevant biological resource for exploring the impact of adult-onset HD mutations of the HTT gene on the division of neural progenitors, with potential applications in HD drug discovery targeting HTT-dynein-p150Glued complex interactions.

Quantitative performance of a quadrupole-orbitrap-MS in targeted LC-MS determinations of small molecules.

High-resolution mass spectrometry (HRMS) has been associated with qualitative and research analysis and QQQ-MS with quantitative and routine analysis. This view is now challenged and for this reason, we have evaluated the quantitative LC-MS performance of a new high-resolution mass spectrometer (HRMS), a Q-orbitrap-MS, and compared the results obtained with a recent triple-quadrupole MS (QQQ-MS). High-resolution full-scan (HR-FS) and MS/MS acquisitions have been tested with real plasma extracts or pure standards. Limits of detection, dynamic range, mass accuracy and false positive or false negative detections have been determined or investigated with protease inhibitors, tyrosine kinase inhibitors, steroids and metanephrines. Our quantitative results show that today's available HRMS are reliable and sensitive quantitative instruments and comparable to QQQ-MS quantitative performance. Taking into account their versatility, user-friendliness and robustness, we believe that HRMS should be seen more and more as key instruments in quantitative LC-MS analyses. In this scenario, most targeted LC-HRMS analyses should be performed by HR-FS recording virtually "all" ions. In addition to absolute quantifications, HR-FS will allow the relative quantifications of hundreds of metabolites in plasma revealing individual's metabolome and exposome. This phenotyping of known metabolites should promote HRMS in clinical environment. A few other LC-HRMS analyses should be performed in single-ion-monitoring or MS/MS mode when increased sensitivity and/or detection selectivity will be necessary.

Presence of Chlamydiales DNA in samples negative by broad-range bacterial 16S rRNA PCRs: new insights into chlamydial pathogenic role.

Since routine eubacterial 16S rRNA PCR does not amplify members of the Chlamydiales order, we tested all samples received in our laboratory during a 10 months period using a pan-Chlamydiales real-time PCR. 3 of 107 samples (2.8%) revealed to be positive, suggesting a role of some Chlamydiales in the pathogenesis of chronic bronchial stenosis or bronchial stenosis superinfection and as agents of orthopaedic prosthesis infections.

Gram-negative flagella glycosylation

Protein glycosylation had been considered as an eccentricity of a few bacteria. However, through advances in analytical methods and genome sequencing, it is now established that bacteria possess both N-linked and O-linked glycosylation pathways. Both glycosylation pathways can modify multiple proteins, flagellins from Archaea and Eubacteria being one of these. Flagella O-glycosylation has been demonstrated in many polar flagellins from Gram-negative bacteria and in only the Gram-positive genera Clostridium and Listeria. Furthermore, O-glycosylation has also been demonstrated in a limited number of lateral flagellins. In this work, we revised the current advances in flagellar glycosylation from Gram-negative bacteria, focusing on the structural diversity of glycans, the O-linked pathway and the biological function of flagella glycosylation.

Threat Simulation- The Function of Dreaming?

Dreaming is a pure form of phenomenality, created by the brain untouched by external stimulation or behavioral activity, yet including a full range of phenomenal contents. Thus, it has been suggested that the dreaming brain could be used as a model system in a biological research program on consciousness (Revonsuo, 2006). In the present thesis, the philosophical view of biological realism is accepted, and thus, dreaming is considered as a natural biological phenomenon, explainable in naturalistic terms. The major theoretical contribution of the present thesis is that it explores dreaming from a multidisciplinary perspective, integrating information from various fields of science, such as dream research, consciousness research, evolutionary psychology, and cognitive neuroscience. Further, it places dreaming into a multilevel framework, and investigates the constitutive, etiological, and contextual explanations for dreaming. Currently, the only theory offering a full multilevel explanation for dreaming, that is, a theory including constitutive, etiological, and contextual level explanations, is the Threat Simulation Theory (TST) (Revonsuo, 2000a; 2000b). The empirical significance of the present thesis lies in the tests conducted to test this specific theory put forth to explain the form, content, and biological function of dreaming. The first step in the empirical testing of the TST was to define exact criteria for what is a ‘threatening event’ in dreams, and then to develop a detailed and reliable content analysis scale with which it is possible to empirically explore and quantify threatening events in dreams. The second step was to seek answers to the following questions derived from the TST: How frequent threatening events are in dreams? What kind of qualities these events have? How threatening events in dreams relate to the most recently encoded or the most salient memory traces of threatening events experienced in waking life? What are the effects of exposure to severe waking life threat on dreams? The results reveal that threatening events are relatively frequent in dreams, and that the simulated threats are realistic. The most common threats include aggression, are targeted mainly against the dream self, and include simulations of relevant and appropriate defensive actions. Further, real threat experiences activate the threat simulation system in a unique manner, and dream content is modulated by the activation of long term episodic memory traces with highest negative saliency. To sum up, most of the predictions of the TST tested in this thesis received considerable support. The TST presents a strong argument that explains the specific design of dreams as threat simulations. The TST also offers a plausible explanation for why dreaming would have been selected for: because dreaming interacted with the environment in such a way that enhanced fitness of ancestral humans. By referring to a single threat simulation mechanism it furthermore manages to explain a wide variety of dream content data that already exists in the literature, and to predict the overall statistical patterns of threat content in different samples of dreams. The TST and the empirical tests conducted to test the theory are a prime example of what a multidisciplinary approach to mental phenomena can accomplish. Thus far, dreaming seems to have always resided in the periphery of science, never regarded worth to be studied by the mainstream. Nevertheless, when brought to the spotlight, the study of dreaming can greatly benefit from ideas in diverse branches of science. Vice versa, knowledge learned from the study of dreaming can be applied in various disciplines. The main contribution of the present thesis lies in putting dreaming back where it belongs, that is, into the spotlight in the cross-road of various disciplines.

Estudo comparativo da seletividade de um método cinético-catalítico: a determinação de Cr(VI) pela oxidação da o-dianisidina pelo peróxido de hidrogênio

Selectivity studies for the determination of Cr(VI) using the catalytic oxidation of the o-dianisidine by hydrogen peroxide showed two distincts situations. In the first, when interferents were studied by a univariate procedure, Cr(III) and Cu(II) cause serious interferences even at the 2:1 proportion, relative to Cr(VI), while Fe(III) interfered at the 15:1 ratio and EDTA at the 10:1 ratio. On the other hand, when a multivariate investigation was performed, Cr(III) did not present any significant principal effects and its significant interaction effects were negative, in contrast to EDTA, that presented positive interaction effects although, like Cr(III), did not show significant interaction effects. In view of the interferent's action it become necessary to separate Cr(VI) by extraction with methylisobutylketone in a chloridric acid medium before its determination in vegetals and in wastewater from a cellulose industry samples. Using this procedure, the method precision is ±0,5% at the 10 ng/mL Cr(VI) concentration level. The detection and quantification limits, calculated by means of absorbance measurements of ten replicates of blank reagents were 1,1 and 3,2 ng/mL, respectively. The results obtained with real samples showed a relative standard deviation between 1,2% and 3,0% relative to their reference values.

The grammar of psychosis

This thesis is concerned with the philosophical grammar of certain psychiatric concepts, which play a central role in delineating the field of psychiatric work. The concepts studied are ‘psychosis’, ‘delusion’, ‘person’, ‘understanding’ and ‘incomprehensibility’. The purpose of this conceptual analysis is to provide a more perspicuous view of the logic of these concepts, how psychiatric work is constituted in relation to them, and what this tells us about the relationships between the conceptual and the empirical in psychiatric concepts. The method used in the thesis is indebted primarily to Ludwig Wittgenstein’s conception of philosophy, where we are urged to look at language uses in relation to practices in order to obtain a clearer overview of practices of interest; this will enable us to resolve the conceptual problems related to these practices. This questioning takes as its starting point the concept of psychosis, a central psychiatric concept during the twentieth century. The conceptual analysis of ‘psychosis’ shows that the concept is logically dependent on the concepts of ‘understanding’ and ‘person’. Following the lead found in this analysis, the logic of person-concepts in psychiatric discourse is analysed by a detailed textual analysis of a psychiatric journal article. The main finding is the ambiguous uses of ‘person’, enabling a specifically psychiatric form of concern in human affairs. The grammar of ‘understanding’ is then tackled from the opposite end, by exploring the logic of the concept of ‘incomprehensibility’. First, by studying the DSM-IV definition of delusion it is shown that its ambiguities boil down to the question of whether psychiatric practice is better accounted for in terms of the grammar of ‘incorrectness’ or ‘incomprehensibility’. Second, the grammar of ‘incomprehensibility’ is further focused on by introducing the distinction between positive and negative conceptions of ‘incomprehensibility’. The main finding is that this distinction has wide-ranging implications for our understanding of psychiatric concepts. Finally, some of the findings gained in these studies are ‘put into practice’ in studying the more practical question of the conceptual and ethical problems associated with the concept of ‘prodromal symptom of schizophrenia’ and the agenda of early detection and intervention in schizophrenia more generally.

Seurakunnan strategiatyö - haaste vai mahdollisuus?

Tutkimuksen tarkoituksena on selvittää miten seurakuntien strategiaprosessista saadaan toimiva. Asian selvittämiseksi tutkittiin, miten seurakunnissa strategian laadintaprosessi nyt toteutetaan ja miten sitä johdetaan. Tarkastelunäkökulmina on käytetty strategialähtöistä organisaatiota sekä tietoperustaista strategianäkemystä. Tutkimuksen empiirinen osuus toteutettiin nettikyselynä ja se kohdistettiin otokseen valittujen seurakuntien kirkkoherroille. Vastauksia kyselyyn saatiin 61 kpl. Tutkimus toteutettiin kvantitatiivisena. Tutkimustulokseksi saatiin, että tärkein seurakunnan strategiaprosessin toimivuutta edistävä tekijä on johdon asenteisiin vaikuttaminen. Tämän vaikuttamisen kautta edistetään seurakunnan strategian laadintatyöskentelyä, sen johtamista sekä strategiatyön jatkuvuutta seurakunnassa. Saadut vastaukset osoittivat selkeästi myös seurakuntien halun saada lisää koulutusta ja tukea strategiatyöskentelyn parantamiseksi. Tarkasteltaessa saatuja vastauksia kokonaisuutena, havaittiin että 64 % vastaajaseurakunnista sekä asenteet että olosuhteet ovat myönteisiä strategiatyöskentelyä kohtaan. Vain 8,7 % vastaajista asenteet strategiatyötä kohtaan olivat välinpitämättömät tai kielteiset. Koulutuksen ja tuen avulla voidaan parhaiten lisätä tietoutta strategian laadinnasta ja näin edistää seurakunnan strategiatyön menestyksellisyyttä.

Osakeperusteinen palkitseminen: maksuttoman osakkeen hyödyntäminen kannustinjärjestelmissä

Tutkielman tavoitteena on selvittää osakeyhtiön palkitsemisjärjestelmiin liittyviä kysymyksiä päämies–agenttisuhteiden ja osakeyhtiöoikeudellisten periaatteiden kautta. Tutkielmassa selvitetään, kuinka maksuttomia osakkeita voidaan hyödyntää julkisen osakeyhtiön palkitsemisjärjestelmissä ja mitä haasteita ja mahdollisuuksia niiden käyttöön liittyy. Tutkimusmetodi työssä on lainopillinen ja aihetta lähestytään luontevasti kauppatieteellisen ja oikeustieteellisen näkökulman yhdistävän oikeustaloustieteen kautta. Ensisijaisena aineistona tutkielmassa käytetään voimassaolevaa lainsäädäntöä valmisteluaineistoineen ja näkökulmaa syvennetään aiemmin voimassa olleen lainsäädännön tarkastelulla. Toissijaisena aineistona tutkielmassa käytetään sekä eri asiantuntijoiden oikeudellista kirjallisuutta että palkitsemisen asiantuntijoiden laatimaa kirjallisuutta Suomesta ja muista länsimaista. Teoreettista työtä on syvennetty asiantuntijahaastattelulla ja yhtiöiden sijoittajatiedosta saatavalla informaatiolla. Merkittävin osakeyhtiön johdon ja yhtiön välisen suhteen tehokkuutta alentava seikka on päämies–agenttisuhteen valvontaongelma. Tämä liittyy päämies - agenttisuhteeseen, joka ilmenee negatiivisesti yhtiön ja yhtiön johdon intressien ristiriitatilanteissa. Valvontaongelma voi aiheuttaa ylimääräisiä transaktiokustannuksia ja tämä näkyy yhtiön toiminnan tehokkuuden laskemisena. Päämies–agenttisuhteen ratkaisuna lainsäädäntö ja erilaiset valvonnan keinot ovat tehottomia ja nykyisen yhtiöoikeudellisen ajattelun vastaisia. Tehokkaimmin valvontaongelma saadaan ratkaistua erilaisin kannustimin tapahtuvalla johdon ohjauksella. Osakesidonnainen palkitseminen on suosituin johdon ja yhtiön intressien yhdistämisen keino. Osakesidonnainen palkitseminen on parhaimmillaan eri osapuolien näkökulmasta sitouttavaa ja kannustavaa mutta osakepalkitsemisen käyttöön liittyy myös riskejä. Eräs keskeisistä yhtiöoikeudellisista periaatteista on yhdenvertaisuus, jota saatetaan loukata eri palkitsemisjärjestelmiä käytettäessä. Varojen jakoon ja järjestelmien rahoitukseen liittyy niin ikään riskejä, jotka saattavat vaarantaa järjestelmän onnistumisen. Liian avokätiset palkkiojärjestelmät taas saattavat aiheuttaa yhtiön eri sidosryhmien piirissä tyytymättömyyttä joka taas alentaa järjestelmän tehokkuutta. Yhtiön johto vastaa yhtiön strategian toteutumisesta ja linjaa Corporate Governance käytännön mukaisesti johdon palkitsemisen tavat. Yhtiön johto on kuitenkin päätöksistään vastuussa yhtiön residuaalioikeuden omaaville päämiehille, eli osakkeenomistajille. Vaikka yhtiön johto vastaa viime kädessä yhtiön toiminnasta, sen on huomioitava toiminnassaan yhtiön toiminnan tarkoitus ja sitä kautta yksittäisen osakkeenomistajan etu. Vaikka osakeyhtiössä toteutettaisiin enemmistöomistajan valitsemaa toimintalinjaa, osakeyhtiön yhdenvertaisuusperiaate korostaa juuri vähemmistöosakkeenomistajan asemaa. Johdon fidusiaaristen velvoitteiden voidaankin nähdä korostuvan johdon suhteessa vähemmistöosakkeenomistajaan. Tämä on huomioitava myös palkitsemisjärjestelmissä. Johdon palkitsemisjärjestelmien suunnittelussa ja toteutuksessa on suunnattava keskeinen huomio sen tavoitteiden toteutumiseen, eli yksittäisen osakkeenomistajan omistuksen arvon kasvattamiseen pidemmällä aikavälillä, pitäen huolta osakeyhtiön kantavista periaatteista.