Direct electron transfer to cytochrome c oxidase investigated by electrochemistry and time-resolved surface-enhanced infrared absorption spectroscopy

Cytochrom c Oxidase (CcO), der Komplex IV der Atmungskette, ist eine der Häm-Kupfer enthaltenden Oxidasen und hat eine wichtige Funktion im Zellmetabolismus. Das Enzym enthält vier prosthetische Gruppen und befindet sich in der inneren Membran von Mitochondrien und in der Zellmembran einiger aerober Bakterien. Die CcO katalysiert den Elektronentransfer (ET) von Cytochrom c zu O2, wobei die eigentliche Reaktion am binuklearen Zentrum (CuB-Häm a3) erfolgt. Bei der Reduktion von O2 zu zwei H2O werden vier Protonen verbraucht. Zudem werden vier Protonen über die Membran transportiert, wodurch eine elektrochemische Potentialdifferenz dieser Ionen zwischen Matrix und Intermembranphase entsteht. Trotz ihrer Wichtigkeit sind Membranproteine wie die CcO noch wenig untersucht, weshalb auch der Mechanismus der Atmungskette noch nicht vollständig aufgeklärt ist. Das Ziel dieser Arbeit ist, einen Beitrag zum Verständnis der Funktion der CcO zu leisten. Hierzu wurde die CcO aus Rhodobacter sphaeroides über einen His-Anker, der am C-Terminus der Untereinheit II angebracht wurde, an eine funktionalisierte Metallelektrode in definierter Orientierung gebunden. Der erste Elektronenakzeptor, das CuA, liegt dabei am nächsten zur Metalloberfläche. Dann wurde eine Doppelschicht aus Lipiden insitu zwischen die gebundenen Proteine eingefügt, was zur sog. proteingebundenen Lipid-Doppelschicht Membran (ptBLM) führt. Dabei musste die optimale Oberflächenkonzentration der gebundenen Proteine herausgefunden werden. Elektrochemische Impedanzspektroskopie(EIS), Oberflächenplasmonenresonanzspektroskopie (SPR) und zyklische Voltammetrie (CV) wurden angewandt um die Aktivität der CcO als Funktion der Packungsdichte zu charakterisieren. Der Hauptteil der Arbeit betrifft die Untersuchung des direkten ET zur CcO unter anaeroben Bedingungen. Die Kombination aus zeitaufgelöster oberflächenverstärkter Infrarot-Absorptionsspektroskopie (tr-SEIRAS) und Elektrochemie hat sich dafür als besonders geeignet erwiesen. In einer ersten Studie wurde der ET mit Hilfe von fast scan CV untersucht, wobei CVs von nicht-aktivierter sowie aktivierter CcO mit verschiedenen Vorschubgeschwindigkeiten gemessen wurden. Die aktivierte Form wurde nach dem katalytischen Umsatz des Proteins in Anwesenheit von O2 erhalten. Ein vier-ET-modell wurde entwickelt um die CVs zu analysieren. Die Methode erlaubt zwischen dem Mechanismus des sequentiellen und des unabhängigen ET zu den vier Zentren CuA, Häm a, Häm a3 und CuB zu unterscheiden. Zudem lassen sich die Standardredoxpotentiale und die kinetischen Koeffizienten des ET bestimmen. In einer zweiten Studie wurde tr-SEIRAS im step scan Modus angewandt. Dafür wurden Rechteckpulse an die CcO angelegt und SEIRAS im ART-Modus verwendet um Spektren bei definierten Zeitscheiben aufzunehmen. Aus diesen Spektren wurden einzelne Banden isoliert, die Veränderungen von Vibrationsmoden der Aminosäuren und Peptidgruppen in Abhängigkeit des Redoxzustands der Zentren zeigen. Aufgrund von Zuordnungen aus der Literatur, die durch potentiometrische Titration der CcO ermittelt wurden, konnten die Banden versuchsweise den Redoxzentren zugeordnet werden. Die Bandenflächen gegen die Zeit aufgetragen geben dann die Redox-Kinetik der Zentren wieder und wurden wiederum mit dem vier-ET-Modell ausgewertet. Die Ergebnisse beider Studien erlauben die Schlussfolgerung, dass der ET zur CcO in einer ptBLM mit größter Wahrscheinlichkeit dem sequentiellen Mechanismus folgt, was dem natürlichen ET von Cytochrom c zur CcO entspricht.

Untersuchungen zur Strahldynamik am Harmonischen Doppelseitigen Mikrotron von MAMI-C

Das Institut für Kernphysik der Universität Mainz betreibt seit 1990 eine weltweit einzigartige Beschleunigeranlage für kern- und teilchenphysikalische Experimente – das Mainzer Mikrotron (MAMI-B). Diese Beschleunigerkaskade besteht aus drei Rennbahn-Mikrotrons (RTMs) mit Hochfrequenzlinearbeschleunigern bei 2.45 GHz, mit denen ein quasi kontinuierlicher Elektronenstrahl von bis zu 100 μA auf 855MeV beschleunigt werden kann.rnrnIm Jahr 1999 wurde die Umsetzung der letzten Ausbaustufe – ein Harmonisches Doppelseitiges Mikrotron (HDSM, MAMI-C) – mit einer Endenergie von 1.5 GeV begonnen. Die Planung erforderte einige mutige Schritte, z.B. Umlenkmagnete mit Feldgradient und ihren daraus resultierenden strahloptischen Eigenschaften, die einen großen Einfluss auf die Longitudinaldynamik des Beschleunigers haben. Dies erforderte die Einführung der „harmonischen“ Betriebsweise mit zwei Frequenzen der zwei Linearbeschleuniger.rnrnViele Maschinenparameter (wie z.B. HF-Amplituden oder -Phasen) wirken direkt auf den Beschleunigungsprozess ein, ihre physikalischen Größen sind indes nicht immer auf einfache Weise messtechnisch zugänglich. Bei einem RTM mit einer verhältnismäßig einfachen und wohldefinierten Strahldynamik ist das im Routinebetrieb unproblematisch, beim HDSM hingegen ist schon allein wegen der größeren Zahl an Parametern die Kenntnis der physikalischen Größen von deutlich größerer Bedeutung. Es gelang im Rahmen dieser Arbeit, geeignete Methoden der Strahldiagnose zu entwickeln, mit denen diese Maschinenparameter überprüft und mit den Planungsvorgaben verglichen werden können.rnrnDa die Anpassung des Maschinenmodells an eine einzelne Phasenmessung aufgrund der unvermeidlichen Messfehler nicht immer eindeutige Ergebnisse liefert, wird eine Form der Tomographie verwendet. Der longitudinale Phasenraum wird dann in Form einer Akzeptanzmessung untersucht. Anschließend kann ein erweitertes Modell an die gewonnene Datenvielfalt angepasst werden, wodurch eine größere Signifikanz der Modellparameter erreicht wird.rnrnDie Ergebnisse dieser Untersuchungen zeigen, dass sich der Beschleuniger als Gesamtsystem im Wesentlichen wie vorhergesagt verhält und eine große Zahl unterschiedlicher Konfigurationen zum Strahlbetrieb möglich sind – im Routinebetrieb wird dies jedoch vermieden und eine bewährte Konfiguration für die meisten Situationen eingesetzt. Das führt zu einer guten Reproduzierbarkeit z.B. der Endenergie oder des Spinpolarisationswinkels an den Experimentierplätzen.rnrnDie Erkenntnisse aus diesen Untersuchungen wurden teilweise automatisiert, so dass nun den Operateuren zusätzliche und hilfreiche Diagnose zur Verfügung steht, mit denen der Maschinenbetrieb noch zuverlässiger durchgeführt werden kann.

Implementazione Libreria C per il Multiple Testing correction

Nell'era genomica moderna, la mole di dati generata dal sequenziamento genetico è diventata estremamente elevata. L’analisi di dati genomici richiede l’utilizzo di metodi di significatività statistica per quantificare la robustezza delle correlazioni individuate nei dati. La significatività statistica ci permette di capire se le relazioni nei dati che stiamo analizzando abbiano effettivamente un peso statistico, cioè se l’evento che stiamo analizzando è successo “per caso” o è effettivamente corretto pensare che avvenga con una probabilità utile. Indipendentemente dal test statistico utilizzato, in presenza di test multipli di verifica (“Multiple Testing Hypothesis”) è necessario utilizzare metodi per la correzione della significatività statistica (“Multiple Testing Correction”). Lo scopo di questa tesi è quello di rendere disponibili le implementazioni dei più noti metodi di correzione della significatività statistica. È stata creata una raccolta di questi metodi, sottoforma di libreria, proprio perché nel panorama bioinformatico moderno non è stato trovato nulla del genere.

Functional Imaging of Pheochromocytoma with Ga-DOTATOC and C-HED in a Genetically Defined Rat Model of Multiple Endocrine Neoplasia

Rats affected by the MENX multitumor syndrome develop pheochromocytoma (100%). Pheochromocytomas are uncommon tumors and animal models are scarce, hence the interest in MENX rats to identify and preclinically evaluate novel targeted therapies. A prerequisite for such studies is a sensitive and noninvasive detection of MENXassociated pheochromocytoma. We performed positron emission tomography (PET) to determine whether rat pheochromocytomas are detected by tracers used in clinical practice, such as 68Ga-DOTATOC (somatostatin analogue) or (11)C-Hydroxyephedrine (HED), a norepinephrine analogue. We analyzed four affected and three unaffected rats. The PET scan findings were correlated to histopathology and immunophenotype of the tumors, their proliferative index, and the expression of genes coding for somatostatin receptors or the norepinephrine transporter. We observed that mean 68Ga-DOTATOC standard uptake value (SUV) in adrenals of affected animals was 23.3 ± 3.9, significantly higher than in control rats (15.4 ± 7.9; P = .03). The increase in mean tumor-to-liver ratio of (11)C-HED in the MENX-affected animals (1.6 ± 0.5) compared to controls (0.7 ± 0.1) was even more significant (P = .0016). In a unique animal model, functional imaging depicting two pathways important in pheochromocytoma biology discriminated affected animals from controls, thus providing the basis for future preclinical work with MENX rats.

Secondary collisions revisited: real-world crash data and relationship to crash test criteria

Objectives: Previous research conducted in the late 1980s suggested that vehicle impacts following an initial barrier collision increase severe occupant injury risk. Now over 25years old, the data are no longer representative of the currently installed barriers or the present US vehicle fleet. The purpose of this study is to provide a present-day assessment of secondary collisions and to determine if current full-scale barrier crash testing criteria provide an indication of secondary collision risk for real-world barrier crashes. Methods: To characterize secondary collisions, 1,363 (596,331 weighted) real-world barrier midsection impacts selected from 13years (1997-2009) of in-depth crash data available through the National Automotive Sampling System (NASS) / Crashworthiness Data System (CDS) were analyzed. Scene diagram and available scene photographs were used to determine roadside and barrier specific variables unavailable in NASS/CDS. Binary logistic regression models were developed for second event occurrence and resulting driver injury. To investigate current secondary collision crash test criteria, 24 full-scale crash test reports were obtained for common non-proprietary US barriers, and the risk of secondary collisions was determined using recommended evaluation criteria from National Cooperative Highway Research Program (NCHRP) Report 350. Results: Secondary collisions were found to occur in approximately two thirds of crashes where a barrier is the first object struck. Barrier lateral stiffness, post-impact vehicle trajectory, vehicle type, and pre-impact tracking conditions were found to be statistically significant contributors to secondary event occurrence. The presence of a second event was found to increase the likelihood of a serious driver injury by a factor of 7 compared to cases with no second event present. The NCHRP Report 350 exit angle criterion was found to underestimate the risk of secondary collisions in real-world barrier crashes. Conclusions: Consistent with previous research, collisions following a barrier impact are not an infrequent event and substantially increase driver injury risk. The results suggest that using exit-angle based crash test criteria alone to assess secondary collision risk is not sufficient to predict second collision occurrence for real-world barrier crashes.

Electromyographic activity of the cervical flexor muscles in patients with temporomandibular disorders while performing the craniocervical flexion test: a cross-sectional study

Most patients with temporomandibular disorders (TMD) have been shown to have cervical spine dysfunction. However, this cervical dysfunction has been evaluated only qualitatively through a general clinical examination of the cervical spine.

Is there an optimal scan time for 6-[F-18]fluoro-L-DOPA PET in pheochromocytomas and paragangliomas?

To define the appropriate scan time for fluorine-18-labeled dihydroxyphenylalanine (F-18 DOPA) PET in oncological imaging of pheochromocytomas and paragangliomas.

Congenital hypothyroidism in a kitten resulting in decreased IGF-I concentration and abnormal liver function tests

A 7-month-old male kitten was presented with chronic constipation and retarded growth. Clinical examination revealed disproportional dwarfism with mild skeletal abnormalities and a palpable thyroid gland. The presumptive diagnosis of congenital hypothyroidism was confirmed by low serum total thyroxine (tT(4)) concentration prior to and after the administration of thyroid stimulation hormone (TSH), increased endogenous TSH concentration and abnormal thyroid scintigraphic scan. The kitten had abnormal liver function tests and decreased insulin-like growth factor 1 (IGF-1) concentration, both of which returned to normal in correspondence with an improvement of the clinical signs after 6 weeks of thyroxine therapy. Congenital hypothyroidism is a rare disease that may present with considerable variation in clinical manifestation. In cases in which clinical signs are ambiguous, disorders such as portosystemic shunt and hyposomatotropism have to be taken into account as differential diagnosis. As hypothyroidism may be associated with abnormal liver function tests and low IGF-1 concentrations, test results have to be interpreted carefully.

Current patch test results with the European baseline series and extensions to it from the 'European Surveillance System on Contact Allergy' network, 2007-2008

The pattern of contact sensitization to the supposedly most important allergens assembled in the baseline series differs between countries, presumably at least partly because of exposure differences. Objectives. To describe the prevalence of contact sensitization to allergens tested in consecutive patients in the years 2007 and 2008, and to discuss possible differences.

Impact of the availability of an influenza virus rapid antigen test on diagnostic decision making in a pediatric emergency department

OBJECTIVES: Fever is one of the most commonly seen symptoms in the pediatric emergency department. The objective of this study was to observe how the rapid testing for influenza virus impacts on the management of children with fever. METHODS: We performed a review of our pediatric emergency department records during the 2008/2009 annual influenza season. The BinaxNow Influenza A+B test was performed on patients with the following criteria: age 1.0 to 16.0 years, fever greater than 38.5 °C, fever of less than 96 hours' duration after the onset of clinical illness, clinical signs compatible with acute influenza, and nontoxic appearance. Additional laboratory tests were performed at the treating physician's discretion. RESULTS: The influenza rapid antigen test was performed in 192 children. One hundred nine (57%) were influenza positive, with the largest fraction (101 patients) positive for influenza A. The age distribution did not differ between children with negative and positive test results (mean, 5.3 vs. 5.1 years, not statistically significant). A larger number of diagnostic tests were performed in the group of influenza-negative patients. Twice as many complete blood counts, C-reactive protein determinations, lumbar punctures, and urinalyses were ordered in the latter group. CONCLUSIONS: Rapid diagnosis of influenza in the pediatric emergency department affects the management of febrile children as the confirmation of influenza virus infection decreases additional diagnostic tests ordered.

The Permanent Income Hypothesis: A Test on Czech Privatisation

Since the late eighties, economists have been regarding the transition from command to market economies in Central and Eastern Europe with intense interest. In addition to studying the transition per se, they have begun using the region as a testing ground on which to investigate the validity of certain classic economic propositions. In his research, comprising three articles written in English and totalling 40 pages, Mr. Hanousek uses the so-called "Czech national experiment" (voucher privatisation scheme) to test the permanent income hypothesis (PIH). He took as his inspiration Kreinin's recommendation: "Since data concerning the behaviour of windfall income recipients is relatively scanty, and since such data can constitute an important test of the permanent income hypothesis, it is of interest to bring to bear on the hypothesis whatever information is available". Mr. Hanousek argues that, since the transfer of property to Czech citizens from 1992 to 1994 through the voucher scheme was not anticipated, it can be regarded as windfall income. The average size of the windfall was more than three month's salary and over 60 percent of the Czech population received this unexpected income. Furthermore, there are other reasons for conducting such an analysis in the Czech Republic. Firstly, the privatisation process took place quickly. Secondly, both the economy and consumer behaviour have been very stable. Thirdly, out of a total population of 10 million Czech citizens, an astonishing 6 million, that is, virtually every household, participated in the scheme. Thus Czech voucher privatisation provides a sample for testing the PIH almost equivalent to a full population, thus avoiding problems with the distribution of windfalls. Compare this, for instance with the fact that only 4% of the Israeli urban population received personal restitution from Germany, while the number of veterans who received the National Service Life Insurance Dividends amounted to less than 9% of the US population and were concentrated in certain age groups. But to begin with, Mr. Hanousek considers the question of whether the public percieves the transfer from the state to individual as an increase in net wealth. It can be argued that the state is only divesting itself of assets that would otherwise provide a future source of transfers. According to this argument, assigning these assets to individuals creates an offsetting change in the present value of potential future transfers so that individuals are no better off after the transfer. Mr. Hanousek disagrees with this approach. He points out that a change in the ownership of inefficient state-owned enterprises should lead to higher efficiency, which alone increases the value of enterprises and creates a windfall increase in citizens' portfolios. More importantly, the state and individuals had very different preferences during the transition. Despite government propaganda, it is doubtful that citizens of former communist countries viewed government-owned enterprises as being operated in the citizens' best interest. Moreover, it is unlikely that the public fully comprehended the sophisticated links between the state budget, state-owned enterprises, and transfers to individuals. Finally, the transfers were not equal across the population. Mr. Hanousek conducted a survey on 1263 individuals, dividing them into four monthly earnings categories. After determining whether the respondent had participated in the voucher process, he asked those who had how much of what they received from voucher privatisation had been (a) spent on goods and services, (b) invested elsewhere, (c) transferred to newly emerging pension funds, (d) given to a family member, and (e) retained in their original form as an investment. Both the mean and the variance of the windfall rise with income. He obtained similar results with respect to education, where the mean (median) windfall for those with a basic school education was 13,600 Czech Crowns (CZK), a figure that increased to 15,000 CZK for those with a high school education without exams, 19,900 CZK for high school graduates with exams, and 24,600 CZK for university graduates. Mr. Hanousek concludes that it can be argued that higher income (and better educated) groups allocated their vouchers or timed the disposition of their shares better. He turns next to an analysis of how respondents reported using their windfalls. The key result is that only a relatively small number of individuals reported spending on goods. Overall, the results provide strong support for the permanent income hypothesis, the only apparent deviation being the fact that both men and women aged 26 to 35 apparently consume more than they should if the windfall were annuitised. This finding is still fully consistent with the PIH, however, if this group is at a stage in their life-cycle where, without the windfall, they would be borrowing to finance consumption associated with family formation etc. Indeed, the PIH predicts that individuals who would otherwise borrow to finance consumption would consume the windfall up to the level equal to the annuitised fraction of the increase in lifetime income plus the full amount of the previously planned borrowing for consumption. Greater consumption would then be financed, not from investing the windfall, but from avoidance of future repayment obligations for debts that would have been incurred without the windfall.

Molecular characterization of the equine collagen, type IX, alpha 2 (COL9A2) gene on horse chromosome 2p16-->p15

The mammalian collagen, type IX, alpha 2 gene (COL9A2) encodes the alpha-2 chain of type IX collagen and is located on horse chromosome 2p16-->p14 harbouring a quantitative trait locus for osteochondrosis. We isolated a bacterial artificial chromosome (BAC) clone containing the equine COL9A2 gene and determined the complete genomic sequence of this gene. Cloning and characterization of equine COL9A2 revealed that the equine gene consists of 32 exons spanning approximately 15 kb. The COL9A2 transcript encodes a single protein of 688 amino acids. Thirty two single nucleotide polymorphisms (SNPs) equally distributed in the gene were detected in a mutation scan of eight unrelated Hanoverian warmblood stallions, including one SNP that affects the amino acid sequence of COL9A2. Comparative analyses between horse, human, mouse and rat indicate that the chromosomal location of equine COL9A2 is in agreement with known chromosomal synteny relationships. The comparison of the gene structure and transcript revealed a high degree of conservation towards the other mammalian COL9A2 genes. We chose three informative SNPs for association and linkage disequilibrium tests in three to five paternal half-sib families of Hanoverian warmblood horses consisting of 44 to 75 genotyped animals. The test statistics did not reach the significance threshold of 5% and so we could not show an association of COL9A2 with equine osteochondrosis.