Analyse und Identifikation des quasistationären Verhaltens der Drehmomentübertragung von automatisierten Reibungskupplungen in Kraftfahrzeugen

Die meisten, praxisrelevanten Konzepte zur Regelung von automatisierten Reibungskupplungen in Kraftfahrzeugen stellen eine Kombination aus Vorsteuerung und Regelung dar. Mit Hilfe der Vorsteuerung wird der Hauptanteil der erforderlichen Kupplungsstellgröße gebildet. Die unterlagerte Regelung dient im Wesentlichen dem Ausgleich von Ungenauigkeiten der Vorsteuerung. Infolgedessen spielt das zur Vorsteuerung verwendete Modell des Kupplungsverhaltens eine sehr wichtige Rolle und entscheidet maßgeblich über Schaltkomfort und Verschleißverhalten einer Reibungskupplung während des Fahrbetriebs. Im Bereich automatisierter Kupplungen werden als Vorsteuerungsmodelle überwiegend Kupplungskennlinien eingesetzt. Eine Kupplungskennlinie beschreibt dabei den quasistationären Zusammenhang zwischen der Stellgröße der Kupplung und dem übertragbaren Kupplungsmoment. Abhängig von verschiedensten Einflussfaktoren unterliegt die Kupplungskennlinie während des Fahrbetriebs sowohl dauerhaften als auch temporären Änderungen. Um die erforderliche Regelgüte des Gesamtregelkreises zu gewährleisten, ist es erforderlich, Änderungen des Kupplungsverhaltens schnell zu erkennen und die Kupplungskennlinie entsprechend anzupassen. Zur Anpassung der Kupplungskennlinie an die aktuelle Betriebssituation werden in dieser Arbeit ausgewählte Methoden der Systemidentifikation eingesetzt. Zur Approximation der Kupplungskennlinie wird ein neues Kennlinienmodell eingesetzt. Dieses Modell zeichnet sich durch zwei wesentliche Merkmale, seine analytische und parametrische Form sowie die physikalisch-geometrische Interpretierbarkeit der Modellparameter, aus. Das erste Merkmal ermöglicht den Einsatz von ableitungsbasierter Least-Squares Methoden der zur Ermittlung unbekannter Modellparameter. Diese Methoden sind konzipiert zur Verarbeitung von verrauschten Signalen und bitten eine sehr hohe Robustheit der Kennlinienidentifikation unter realen Bedingungen. Das zweite Merkmal ermöglicht eine separate Modifikation einzelner Modellparameter unter Berücksichtigung ihrer Änderungsdynamik bzw. ihrer Zeitvarianz während des Fahrbetriebs. Die Untersuchungen der Kennlinienidentifikation erfolgen zunächst in der Simulation. Dadurch können einzelne Effekte der Drehmomentübertragung ab- bzw. zugeschaltet und deren Auswirkung auf den Identifikationsvorgang gezielt analysiert werden. Anschließend erfolgen ausführliche Untersuchungen der Kennlinienidentifikation anhand von realen Fahrzeugmessungen. Die erzielten Ergebnisse belegen zum einen eine hohe Performance der vorgestellten Methodik zur Identifikation von Kupplungskennlinien. Zum anderen decken sie Potentiale für weiterführende Arbeiten im Bereich der Identifikation des Übertragungsverhaltens von Reibungskupplungen auf.Many practice-relevant concepts for controlling automated friction clutches in motor vehicles are designed as a combination of feed forward and feedback control. The main amount of the actuating variable required for the clutch engagement is calculated by the feed forward path. The task of the feedback path is to compensate for the uncertainness of the feed forward path. Therefore, the model used for the feed forward path is very important for the shift comfort as well as for the wear of the clutch system during operation. In the field of automated clutches, the clutch characteristic is typically used as the feed forward model. A clutch characteristic represents the relationship between the actuating variable and the torque transferred through the clutch. Dependent on various factors, the clutch characteristic changes both temporarily and permanently during the operation. To ensure the requirements for the clutch control, it is necessary to track the relevant changes of the behavior of the clutch system, as well as to adapt the clutch characteristic accordingly. In this work, preselected identification methods are used for the adaption of the clutch characteristic on the actual operating point. Thereby, the approximation of the clutch characteristic is carried out using an innovative model. This model is characterized by two essential properties, these are the analytically and parametrically model form, as well as the physically and geometrically interpretability of the model parameter. The first property allows using derivative-based identification methods for determination of unknown parameters of the characteristic model. These methods provide a high robustness of the identification of the clutch characteristics under realistically operating conditions because they are designed for processing signals corrupted by noise. The second property of the new characteristic model allows an identification of the model parameter under consideration their change dynamics and time variance respectively. In the first step, the identification of the clutch characteristic was investigated in simulation. This allows activating or deactivating of diverse effects of the torque transfer through the clutch, with the goal, to analyze the influence of these effects on the identification procedure. In the last step, the investigation of the identification procedure was carried out using real measurement data. On the one hand, the experimental results show a high performance of the presented method for identification of clutch characteristics. On the other hand, potential for advanced research works in the field of identification of transmission behavior of friction clutches was detected

Massively Parallel Interrogation of Aptamer Sequence, Structure and Function

BACKGROUND: Optimization of high affinity reagents is a significant bottleneck in medicine and the life sciences. The ability to synthetically create thousands of permutations of a lead high-affinity reagent and survey the properties of individual permutations in parallel could potentially relieve this bottleneck. Aptamers are single stranded oligonucleotides affinity reagents isolated by in vitro selection processes and as a class have been shown to bind a wide variety of target molecules. METHODOLOGY/PRINCIPAL FINDINGS: High density DNA microarray technology was used to synthesize, in situ, arrays of approximately 3,900 aptamer sequence permutations in triplicate. These sequences were interrogated on-chip for their ability to bind the fluorescently-labeled cognate target, immunoglobulin E, resulting in the parallel execution of thousands of experiments. Fluorescence intensity at each array feature was well resolved and shown to be a function of the sequence present. The data demonstrated high intra- and inter-chip correlation between the same features as well as among the sequence triplicates within a single array. Consistent with aptamer mediated IgE binding, fluorescence intensity correlated strongly with specific aptamer sequences and the concentration of IgE applied to the array. CONCLUSION AND SIGNIFICANCE: The massively parallel sequence-function analyses provided by this approach confirmed the importance of a consensus sequence found in all 21 of the original IgE aptamer sequences and support a common stem:loop structure as being the secondary structure underlying IgE binding. The microarray application, data and results presented illustrate an efficient, high information content approach to optimizing aptamer function. It also provides a foundation from which to better understand and manipulate this important class of high affinity biomolecules

APTASENSORS FOR BIOSECURITY APPLICATIONS

Nucleic acid aptamers have found steadily increased utility and application steadily over the last decade. In particular, aptamers have been touted as a valuable complement to and in some cases replacement for antibodies due to their structural and functional robustness as well as their ease in generation and synthesis. They are thus attractive for biosecurity applications, e.g. pathogen detection, and are especially well suited since their in vitro generation process does not require infection of any host systems. Herein we provide a brief overview of the aptamers generated against biopathogens over the last few years. In addition, a few recently described detection platforms using aptamers (aptasensors) and potentially suitable for biosecurity applications will be discussed

Heightened sense for sensing: recent advances in pathogen immunoassay sensing platforms

As part of its own defense mechanism, most bacteria have developed an innate ability to enable toxic secretion to ward off potential predators or invaders. However, this naturally occurring process has been abused since over production of the bacteria's toxin molecules could render them as potential bioweapons. As these processes (also known as ''black biology'') can be clandestinely performed in a laboratory, the threat of inflicting enormous potential damage to a nation's security and economy is invariably clear and present. Thus, efficient detection of these biothreat agents in a timely and accurate manner is highly desirable. A wealth of publications describing various pathogen immuno-sensing advances has appeared over the last few years, and it is not the intent of this review article to detail each reported approach. Instead, we aim to survey a few recent highlights in hopes of providing the reader an overall sense of the breath of these sensing systems and platforms. Antigen targets are diverse and complex as they encompass proteins, whole viruses, and bacterial spores. The signaling processes for these reported immunoassays are usually based on colorimetric, optical, or electrochemical changes. Of equal interest is the type of platform in which the immunoassay can be performed. A few platforms suitable for pathogen detection are described

A role for hydrophobicity in a Diels–Alder reaction catalyzed by pyridyl-modified RNA

New classes of RNA enzymes or ribozymes have been obtained by in vitro evolution and selection of RNA molecules. Incorporation of modified nucleotides into the RNA sequence has been proposed to enhance function. DA22 is a modified RNA containing 5-(4-pyridylmethyl) carboxamide uridines, which has been selected for its ability to promote a Diels–Alder cycloaddition reaction. Here, we show that DA_TR96, the most active member of the DA22 RNA sequence family, which was selected with pyridyl-modified nucleotides, accelerates a cycloaddition reaction between anthracene and maleimide derivatives with high turnover. These widely used reactants were not used in the original selection for DA22 and yet here they provide the first demonstration of DA_TR96 as a true multiple-turnover catalyst. In addition, the absence of a structural or essential kinetic role for Cu2+, as initially postulated, and nonsequence-specific hydrophobic interactions with the anthracene substrate have led to a reevaluation of the pyridine modification's role. These findings broaden the catalytic repertoire of the DA22 family of pyridyl-modified RNAs and suggest a key role for the hydrophobic effect in the catalytic mechanism

Polymerase-directed synthesis of C5-ethynyl locked nucleic acids

Modified nucleic acids have considerable potential in nanobiotechnology for the development of nanomedicines and new materials. Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far and we herein for the first time report the enzymatic incorporation of LNA-U and C5-ethynyl LNA-U nucleotides into oligonucleotides. Phusion High Fidelity and KOD DNA polymerases efficiently incorporated LNA-U and C5-ethynyl LNA-U nucleotides into a DNA strand and T7 RNA polymerase successfully accepted the LNA-U nucleoside 5′-triphosphate as substrate for RNA transcripts

Aptamer-based multiplexed proteomic technology for biomarker discovery

Interrogation of the human proteome in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 [mu]L of serum or plasma). Our current assay allows us to measure ~800 proteins with very low limits of detection (1 pM average), 7 logs of overall dynamic range, and 5% average coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding DNA aptamer concentration signature, which is then quantified with a DNA microarray. In essence, our assay takes advantage of the dual nature of aptamers as both folded binding entities with defined shapes and unique sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to discover unique protein signatures characteristic of various disease states. More generally, we describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine

Three critical hydrogen bonds determine the catalytic activity of the Diels–Alderase ribozyme

Compared to protein enzymes, our knowledge about how RNA accelerates chemical reactions is rather limited. The crystal structures of a ribozyme that catalyzes Diels–Alder reactions suggest a rich tertiary architecture responsible for catalysis. In this study, we systematically probe the relevance of crystallographically observed ground-state interactions for catalytic function using atomic mutagenesis in combination with various analytical techniques. The largest energetic contribution apparently arises from the precise shape complementarity between transition state and catalytic pocket: A single point mutant that folds correctly into the tertiary structure but lacks one H-bond that normally stabilizes the pocket is completely inactive. In the rate-limiting chemical step, the dienophile is furthermore activated by two weak H-bonds that contribute ∼7–8 kJ/mol to transition state stabilization, as indicated by the 25-fold slower reaction rates of deletion mutants. These H-bonds are also responsible for the tight binding of the Diels–Alder product by the ribozyme that causes product inhibition. For high catalytic activity, the ribozyme requires a fine-tuned balance between rigidity and flexibility that is determined by the combined action of one inter-strand H-bond and one magnesium ion. A sharp 360° turn reminiscent of the T-loop motif observed in tRNA is found to be important for catalytic function

A multifunctional bioconjugate module for versatile photoaffinity labeling and click chemistry of RNA

A multifunctional reagent based on a coumarin scaffold was developed for derivatization of naive RNA. The alkylating agent N3BC [7-azido-4-(bromomethyl)coumarin], obtained by Pechmann condensation, is selective for uridine. N3BC and its RNA conjugates are pre-fluorophores which permits controlled modular and stepwise RNA derivatization. The success of RNA alkylation by N3BC can be monitored by photolysis of the azido moiety, which generates a coumarin fluorophore that can be excited with UV light of 320 nm. The azidocoumarin-modified RNA can be flexibly employed in structure-function studies. Versatile applications include direct use in photo-crosslinking studies to cognate proteins, as demonstrated with tRNA and RNA fragments from the MS2 phage and the HIV genome. Alternatively, the azide function can be used for further derivatization by click-chemistry. This allows e.g. the introduction of an additional fluorophore for excitation with visible light