Redox-linked proton transfer by cytochrome c oxidase

The respiratory chain is found in the inner mitochondrial membrane of higher organisms and in the plasma membrane of many bacteria. It consists of several membrane-spanning enzymes, which conserve the energy that is liberated from the degradation of food molecules as an electrochemical proton gradient across the membrane. The proton gradient can later be utilized by the cell for different energy requiring processes, e.g. ATP production, cellular motion or active transport of ions. The difference in proton concentration between the two sides of the membrane is a result of the translocation of protons by the enzymes of the respiratory chain, from the negatively charged (N-side) to the positively charged side (P-side) of the lipid bilayer, against the proton concentration gradient. The endergonic proton transfer is driven by the flow of electrons through the enzymes of the respiratory chain, from low redox-potential electron donors to acceptors of higher potential, and ultimately to oxygen. Cytochrome c oxidase is the last enzyme in the respiratory chain and catalyzes the reduction of dioxygen to water. The redox reaction is coupled to proton transport across the membrane by a yet unresolved mechanism. Cytochrome c oxidase has two proton-conducting pathways through which protons are taken up to the interior part of the enzyme from the N-side of the membrane. The K-pathway transfers merely substrate protons, which are consumed in the process of water formation at the catalytic site. The D-pathway transfers both substrate protons and protons that are pumped to the P-side of the membrane. This thesis focuses on the role of two conserved amino acids in proton translocation by cytochrome c oxidase, glutamate 278 and tryptophan 164. Glu278 is located at the end of the D-pathway and is thought to constitute the branching point for substrate and pumped protons. In this work, it was shown that although Glu278 has an important role in the proton transfer mechanism, its presence is not an obligatory requirement. Alternative structural solutions in the area around Glu278, much like the ones present in some distantly related heme-copper oxidases, could in the absence of Glu278 support the formation of a long hydrogen-bonded water chain through which proton transfer from the D-pathway to the catalytic site is possible. The other studied amino acid, Trp164, is hydrogen bonded to the ∆-propionate of heme a3 of the catalytic site. Mutation of this amino acid showed that it may be involved in regulation of proton access to a proton acceptor, a pump site, from which the proton later is expelled to the P-side of the membrane. The ion pair that is formed by the ∆-propionate of heme a3 and arginine 473 is likely to form a gate-like structure, which regulates proton mobility to the P-side of the membrane. The same gate may also be part of an exit path through which water molecules produced at the catalytically active site are removed towards the external side of the membrane. Time-resolved optical and electrometrical experiments with the Trp164 to phenylalanine mutant revealed a so far undetected step in the proton pumping mechanism. During the A to PR transition of the catalytic cycle, a proton is transferred from Glu278 to the pump site, located somewhere in the vicinity of the ∆-propionate of heme a3. A mechanism for proton pumping by cytochrome c oxidase is proposed on the basis of the presented results and the mechanism is discussed in relation to some relevant experimental data. A common proton pumping mechanism for all members of the heme-copper oxidase family is moreover considered.

A quantum chemical investigation of the metal centres in cytochrome c oxidase

Quantum effects are often of key importance for the function of biological systems at molecular level. Cellular respiration, where energy is extracted from the reduction of molecular oxygen to water, is no exception. In this work, the end station of the electron transport chain in mitochondria, cytochrome c oxidase, is investigated using quantum chemical methodology. Cytochrome c oxidase contains two haems, haem a and haem a3. Haem a3, with its copper companion, CuB, is involved in the final reduction of oxygen into water. This binuclear centre receives the necessary electrons from haem a. Haem a, in turn, receives its electrons from a copper ion pair in the vicinity, called CuA. Density functional theory (DFT) has been used to clarify the charge and spin distributions of haem a, as well as changes in these during redox activity. Upon reduction, the added electron is shown to be evenly distributed over the entire haem structure, important for the accommodation of the prosthetic group within the protein. At the same time, the spin distribution of the open-shell oxidised state is more localised to the central iron. The exact spin density distribution has been disputed in the literature, however, different experiments indicating different distributions of the unpaired electron. The apparent contradiction is shown to be due to the false assumption of a unit amount of unpaired electron density; in fact, the oxidised state has about 1.3 unpaired electrons. The validity of the DFT results have been corroborated by wave function based coupled cluster calculations. Point charges, for use in classical force field based simulations, have been parameterised for the four metal centres, using a newly developed methodology. In the procedure, the subsystem for which point charges are to be obtained, is surrounded by an outer region, with the purpose of stabilising the inner region, both electronically and structurally. Finally, the possibility of vibrational promotion of the electron transfer step between haem a and a3 has been investigated. Calculating the full vibrational spectra, at DFT level, of a combined model of the two haems, revealed several normal modes that do shift electron density between the haems. The magnitude of the shift was found to be moderate, at most. The proposed mechanism could have an assisting role in the electron transfer, which still seems to be dominated by electron tunnelling.

Theoretical Studies on Coupled Electron and Proton Transfer in Cytochrome c Oxidase

The complexity of life is based on an effective energy transduction machinery, which has evolved during the last 3.5 billion years. In aerobic life, the utilization of the high oxidizing potential of molecular oxygen powers this machinery. Oxygen is safely reduced by a membrane bound enzyme, cytochrome c oxidase (CcO), to produce an electrochemical proton gradient over the mitochondrial or bacterial membrane. This gradient is used for energy-requiring reactions such as synthesis of ATP by F0F1-ATPase and active transport. In this thesis, the molecular mechanism by which CcO couples the oxygen reduction chemistry to proton-pumping has been studied by theoretical computer simulations. By building both classical and quantum mechanical model systems based on the X-ray structure of CcO from Bos taurus, the dynamics and energetics of the system were studied in different intermediate states of the enzyme. As a result of this work, a mechanism was suggested by which CcO can prevent protons from leaking backwards in proton-pumping. The use and activation of two proton conducting channels were also enlightened together with a mechanism by which CcO sorts the chemical protons from pumped protons. The latter problem is referred to as the gating mechanism of CcO, and has remained a challenge in the bioenergetics field for more than three decades. Furthermore, a new method for deriving charge parameters for classical simulations of complex metalloenzymes was developed.

Slogans of Change : Three Outlooks on Finnish Television

The study Slogans of Change. Three Outlooks on Finnish Television Contents is concerned with alleged changes of television contents during the 1990s and 2000s, such as dumbing down, tabloidisation, entertainisation , and the like. Specifically, the focus is on the ways these changes might manifest in Finnish television. The aim of the study has been threefold: 1. To operationalise public and academic discussions about changes via specific slogans emerging from the debates; 2. Consequently, to study the slogans empirically and reflect on the findings with earlier research, including studies on institutional and audience-related aspects; 3. Finally, to suggest what the findings might mean regarding discussions about television s role, and what kinds of slogans or concepts might best serve future discussions and research. The empirical outlooks presented in this study offer analyses with three different sets of opposing slogans of change. The outlooks also follow three different traditions of the study of television. The first outlook focuses on quantity, as it gives a longitudinal (1993-2004), macro-level view on programme structures. The methodological approach is derived from media economic and policy studies. The claims that frame the analysis are convergence versus diversification of programme structures. The second outlook provides quantitative and qualitative views on the characteristics and quality  the term signifying essence as well as worth  of Finnish television journalism during sample weeks from the years 2002 and 2003. This outlook follows the traditions of quantitative content analysis found in journalism studies coupled with descriptive qualitative content analyses. The slogans reflected in this section are the lightening or widening of journalism. The third outlook narrows down the material and focuses at a micro-level on form; that is, communicative conventions in a small array of selected programmes in 1993, 2000 and during 2002-2004. The analyses have been inspired by the method of conversation analysis of verbal interaction, and coupled with qualitative close readings, with the focus of different communicative situations in the programmes. The catchphrases employed in this part are emotainment versus democratainment, coupled with more specific claims of discursive hybridisation and conversationalisation. The findings depict that, empirically, changes in Finnish television contents are not clear linear trends and cannot easily be moulded into neat slogans. The quantitative outlook on programme output during 1993-2004 depicts a tendency towards differentiation of channels, paving the way for the multi-channel digital system. The change in programme structures, however, is not dramatic on the level of total output. The second outlook suggests that the dualistic concepts, such as the pair information-entertainment, are not sufficient in understanding the array and changes of programmes that could be called journalism. The outlook on communicative conventions highlights hybridisation in the manner of television talk and its relation to broader debates on contents. Despite the three dissimilar empirical approaches, unifying aspects emerge. The outlooks suggest, albeit in different ways, tendencies toward distinction and polarisation. This study proposes that in order to facilitate a more nuanced understanding of the changes in television contents, dualistic slogans should be replaced with a multi-dimensional understanding of the concept of diversity.

Cystatin C, a measure of renal function, as prognostic risk marker in acute heart failure : Studies on the cardiorenal syndrome

Acute heart failure (AHF) is a complex syndrome associated with exceptionally high mortality. Still, characteristics and prognostic factors of contemporary AHF patients have been inadequately studied. Kidney function has emerged as a very powerful prognostic risk factor in cardiovascular disease. This is believed to be the consequence of an interaction between the heart and kidneys, also termed the cardiorenal syndrome, the mechanisms of which are not fully understood. Renal insufficiency is common in heart failure and of particular interest for predicting outcome in AHF. Cystatin C (CysC) is a marker of glomerular filtration rate with properties making it a prospective alternative to the currently used measure creatinine for assessment of renal function. The aim of this thesis is to characterize a representative cohort of patients hospitalized for AHF and to identify risk factors for poor outcome in AHF. In particular, the role of CysC as a marker of renal function is evaluated, including examination of the value of CysC as a predictor of mortality in AHF. The FINN-AKVA (Finnish Acute Heart Failure) study is a national prospective multicenter study conducted to investigate the clinical presentation, aetiology and treatment of, as well as concomitant diseases and outcome in, AHF. Patients hospitalized for AHF were enrolled in the FINN-AKVA study, and mortality was followed for 12 months. The mean age of patients with AHF is 75 years and they frequently have both cardiovascular and non-cardiovascular co-morbidities. The mortality after hospitalization for AHF is high, rising to 27% by 12 months. The present study shows that renal dysfunction is very common in AHF. CysC detects impaired renal function in forty percent of patients. Renal function, measured by CysC, is one of the strongest predictors of mortality independently of other prognostic risk markers, such as age, gender, co-morbidities and systolic blood pressure on admission. Moreover, in patients with normal creatinine values, elevated CysC is associated with a marked increase in mortality. Acute kidney injury, defined as an increase in CysC within 48 hours of hospital admission, occurs in a significant proportion of patients and is associated with increased short- and mid-term mortality. The results suggest that CysC can be used for risk stratification in AHF. Markers of inflammation are elevated both in heart failure and in chronic kidney disease, and inflammation is one of the mechanisms thought to mediate heart-kidney interactions in the cardiorenal syndrome. Inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) correlate very differently to markers of cardiac stress and renal function. In particular, TNF-α showed a robust correlation to CysC, but was not associated with levels of NT-proBNP, a marker of hemodynamic cardiac stress. Compared to CysC, the inflammatory markers were not strongly related to mortality in AHF. In conclusion, patients with AHF are elderly with multiple co-morbidities, and renal dysfunction is very common. CysC demonstrates good diagnostic properties both in identifying impaired renal function and acute kidney injury in patients with AHF. CysC, as a measure of renal function, is also a powerful prognostic marker in AHF. CysC shows promise as a marker for assessment of kidney function and risk stratification in patients hospitalized for AHF.

Framtidens television. Vissa marknadsförings- och upphovsrättsliga spörsmål

Den här boken handlar om framtidens television och vissa marknadsförings- och upphovsrättsliga spörsmål som aktualiseras i samband med nya former av televisionssändningar. TV-tekniken har utvecklats mycket under de senaste åren, och det är framförallt sändningstekniken som har genomgått de största förändringarna. Tyngdpunkten i detta arbete har lagts på förmedling av TV-sändningar över Internet med hjälp av P2P-tekniken (peer-to-peer) och vilka marknadsförings- och upphovsrättsliga spörsmål detta aktualiserar. Den nya förmedlingstekniken möjliggör en rad nya marknadsföringsmetoder, och i boken behandlas hur dessa metoder förhåller sig till nuvarande marknadsföringsrättsliga reglering. Förmedling av upphovsrättsskyddat material över Internet har inneburit ett flertal reformer av gällande upphovsrättslagstiftning i syfte att stärka rättsinnehavarnas ensamrätt. En viktig fråga som boken behandlar är hur gällande upphovsrättslagstiftning skall tolkas i förhållande till nya distributionsformer över Internet. I boken tas även upp frågor i anknytning till problemet med att en stor del av det material som förmedlas över Internet sker utan rättsinnehavarens samtycke, vilket innebär en ekonomisk förlust för rättsinnehavaren. I boken framläggs alternativa lösningar för hur förmedlingen över Internet kan tilldela rättsinnehavaren ekonomisk ersättning, trots att förmedlingen är avgiftsfri.

Uhrihaastatteluista poliitikkojen kovisteluun : Tutkimus Jokelan ja Kauhajoen koulusurmien tv-uutisoinnin kerrontatavoista

Medialla on valta päättää, miten väkivallasta kerrotaan, ja miten kriisit esitetään. Median kerrontatavoilla on suuri vaikutus siihen, miten katastrofi näyttäytyy tavalliselle kansalaiselle. Keskityn tutkielmassani siihen, millaisia kerrontatapoja Jokelan ja Kauhajoen koulusurmien tv-uutisointiin on rakennettu. Selvitän, mitä eroja ja yhtäläisyyksiä uutiskerronnassa oli kahden koulusurmatapauksen välillä sekä eri tv-kanavien välillä. Tarkastelen myös, mitkä toimijat korostuvat uutisten eri kerrontatavoissa. Laadullisen tutkimusotteen piirissä metodologinen tutkimustapani on representaatioanalyysi. Tutkielman lähtökohtana on malli katastrofiuutisoinnin kerrontatavoista. Gert Z. Nordström (1996) on kehittänyt mallin alun perin kuva-analyysiä varten. Myöhemmin sitä on sovellettu mediatekstien analyysiin (Huhtala jaa Hakala 2007). Tässä tutkielmassa sovellan mallia audiovisuaalisen kerronnan analyysiin. Tutkimusaineistoni koostuu television uutislähetyksistä seitsemän päivän ajalta sekä Jokelan että Kauhajoen tapahtumien jälkeen neljällä valtakunnallisella tv-kanavalla. Analyysimallin mukaisesti koulusurmat esitetään televisiossa käyttäen eeppistä, dramaattista, lyyristä ja didaktista kerrontatapaa. Lisäksi kyseessä olleesta tv-aineistosta nousi viides kerronnan luokka, kriittinen kerronta. Eeppisellä kerronnalla tarkoitetaan tyypillistä raportoivaa ja referoivaa uutiskerrontaa. Dramaattista kerrontaa käytetään raportoitaessa suoraan tapahtumapaikalta. Lyyrinen kerronta kuvaa tunteita. Didaktisessa kerronnassa pohditaan, mitä tapahtuneesta voi oppia. Kriittisen kerronnan keinoin vaaditaan vastuunottoa tapahtuneesta. Tv-uutisten kerrontatavoissa näyttäisi korostuvan eeppisen kerronnan määrä. Kauhajoen ja Jokelan uutiskerronnat kuitenkin erosivat toisistaan niin, että eeppinen kerronta lisääntyi entisestään Kauhajoen uutisoinnissa. Myös kriittinen kerronta korostui Kauhajoen uutisoinnissa. Jokelan uutisoinnissa esiintyi puolestaan enemmän dramaattista, lyyristä ja didaktista kerrontaa. MTV3-kanavalla käytettiin muita kanavia vähemmän eeppistä kerrontaa. Didaktista kerrontaa esiintyi MTV3:lla muita kanavia reilusti enemmän molempien koulusurmatapausten yhteydessä. Lisäksi MTV3-kanavalla esiintyi eniten kriittistä kerrontaa Kauhajoen uutisoinnissa. YLEn ja Nelosen kerrontatapojen esiintyminen oli lähes samankaltaista sekä Jokelan että Kauhajoen uutisoinnissa. Jokelan uutisoinnissa Nelonen käytti kuitenkin merkittävästi enemmän dramaattista kerrontaa YLEen verrattuna. Jokelan uutisoinnissa toimijoina korostuivat ampuja, ja uhrit, kun taas Kauhajoella päätoimijoita olivat poliisi ja poliitikot. Jokelan tv-uutisoinnissa sekä uhrit että ampuja olivat kolmen keskeisimmän toimijan joukossa useassa kerrontatavan luokassa. Kauhajoen uutisoinnissa puolestaan uhrit eivät olleet missään kerrontatavan luokassa kolmen keskeisimmän toimijan joukossa, ja ampujakin vain yhdessä luokassa. Eeppisen ja kriittisen kerrontatavan korostuminen sekä poliitikkojen keskeisyys toimijoina Kauhajoen tv-uutisoinnissa kertoo siitä, että Jokelaan nähden Kauhajoen koulusurmien uutisointi oli selvästi yhteiskuntakeskeisempää. Jokelan uutisointi painottui enemmän yksittäisen tapahtuman traagisuuteen sekä henkilökohtaloihin.

Protein Kinase C and Anaplastic Lymphoma Kinase Targeted Compounds

The protein kinases (PKs) belong to the largest single family of enzymes, phosphotransferases, which catalyze the phosphorylation of other enzymes and proteins and function primarily in signal transduction. Consequently, PKs regulate cell mechanisms such as growth, differentiation, and proliferation. Dysfunction of these cellular mechanisms may lead to cancer, a major predicament in health care. Even though there is a range of clinically available cancer-fighting drugs, increasing number of cancer cases and setbacks such as drug resistance, constantly keep cancer research active. At the commencement of this study an isophthalic acid derivative had been suggested to bind to the regulatory domain of protein kinase C (PKC). In order to investigate the biological effects and structure-activity relationships (SARs) of this new chemical entity, a library of compounds was synthesized. The best compounds induced apoptosis in human leukemia HL-60 cells and were not cytotoxic in Swiss 3T3 fibroblasts. In addition, the best apoptosis inducers were neither cytotoxic nor mutagenic. Furthermore, results from binding affinity assays of PKC isoforms revealed the pharmacophores of these isophthalic acid derivatives. The best inhibition constants of the tested compounds were measured to 210 nM for PKCα and to 530 nM for PKCδ. Among natural compounds targeting the regulatory domain of PKC, the target of bistramide A has been a matter of debate. It was initially found to activate PKCδ; however, actin was recently reported as the main target. In order to clarify and to further study the biological effects of bistramide A, the total syntheses of the natural compound and two isomers were performed. Biological assays of the compounds revealed accumulation of 4n polyploid cells as the primary mode of action and the compounds showed similar overall antiproliferative activities. However, each compound showed a distinct distribution of antimitotic effect presumably via actin binding, proapoptotic effect presumably via PKCδ, and pro-differentiation effect as evidenced by CD11b expression. Furthermore, it was shown that the antimitotic and proapoptotic effects of bistramide A were not secondary effects of actin binding but independent effects. The third aim in this study was to synthesize a library of a new class of urea-based type II inhibitors targeted at the kinase domain of anaplastic lymphoma kinase (ALK). The best compounds in this library showed IC50 values as low as 390 nM for ALK while the initial low cellular activities were successfully increased even by more than 70 times for NPM-ALK- positive BaF3 cells. More importantly, selective antiproliferative activity on ALK-positive cell lines was achieved; while the best compound affected the BaF3 and SU-DHL-1 cells with IC50 values of 0.5 and 0.8 μM, respectively, they were less toxic to the NPM-ALK-negative human leukemic cells U937 (IC50 = 3.2 μM) and BaF3 parental cells (IC50 = 5.4 μM). Furthermore, SAR studies of the synthesized compounds revealed functional groups and positions of the scaffold, which enhanced the enzymatic and cellular activities.