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Making glucose oxidase fir for biofuel cell applications by directed protein evolution
No full textInspired by nature Darwinian evolution, directed evolution involves mutation,recombination and screening or selection to accumulate the mutants required to achieve significant change in protein function. The gene encoding gox harboring á-pheromone signal sequence from Aspergillus niger was expressed as secretary product in yeast Saccharomyces cerevisiae. Error prone PCR has been used to generate mutant libraries of GOx by adjusting the concentration of MnCl2. 4000 mutants have been screened for catalytic activity and thermostability. One mutant (G4) showed two-fold higher activity than WT and an enhanced thermostability. For the screening of mutants, two novel and reliable assays were developed for medium-throughput screening. Glucose oxidase detection assay (GODA) and ferroecenemethanol based assay were developed for improving GOx properties by directed protein evolution. GODA is a reaction product detection assay based on coupled enzymatic reactions leading to NADPH formation which is recorded at 340 nm. The main advantage of the assay is that it detects the production of D-gluconolactone instead of the side-product hydrogen peroxide and enables to improve bioelectrochemical properties of GOx. Ferrocenemethanol based assay can directly screen electron transfer between oxidized form of ferrocenemethanol and reduced form of GOx by monitoring colorimetric development. It is a simple, less expensive and rapid assay, more importantly; it is insensitive to nature electron acceptor dioxygen in pH 8
Modeling Fundamental Geo-Raster Operations with Array Algebra
No full textRaster image data is the most voluminous data type encountered in remote sensing applications. With its multidimensional, gridded nature, the raster data structure is found to be very similar to the structures treated in OLAP (Online Analytical Processing); further, striking similarities can be observed in the mathematical modeling of operations in both domains. However, while in both remote sensing / imaging and OLAP there exists a host of research on analysis and extraction techniques, there is no cross-fertilization to the best of our knowledge.
One particular technique known in OLAP for speeding up complex database queries on very large datacubes is pre-aggregation, i.e., materialization of earlier results for subsequent use in same or similar requests. We feel that pre-aggregation can be advantageously transposed into the remote sensing / imaging area to speed up raster aggregation operations such as image scaling.
In this report we present fundamental operations on raster image data, model them using an algebraic framework, and classify them based on their structural properties. We then identify a set of operations requiring aggregation (summarization) of data. These operations are the potential candidates for support by OLAP pre-aggregation algorithms, which is our next research step
Overview of Reservoir Recipes
No full textEcho State Networks (ESNs) and Liquid State Machines (LSMs) introduced a simple new paradigm in artificial recurrent neural network (RNN) training, where an RNN (the reservoir) is generated randomly and only a readout is trained. The paradigm, becoming known as reservoir computing, made RNNs accessible for practical applications as never before and outperformed classical fully trained RNNs in many tasks. The latter, however, does not imply that random reservoirs are optimal, but rather that adequate training methods for them are yet to be developed. Thus much of the current research in reservoir computing is done on reservoir adaptation, redefining the paradigm as using different methods for training the reservoir and the readout. This report motivates the new definition of the paradigm and surveys the reservoir generation/adaptation techniques, offering a natural conceptual classification which transcends boundaries of the current "brand-names" of reservoir methods. The survey focuses more on methods relevant to practical applications of RNNs rather than modeling biological brains
Norm observable operator models
No full textObservable operator models (OOMs), a recently developed matrix model class of stochastic processes [6], possesses several advantages over hidden Markov models (HMMs). Nevertheless, there is a critical issue, the negative probability problem (NPP), which remains unsolved in OOMs theory; and which has heavily prevented it from being an alternative to HMMs in practice. To avoid the NPP we introduce in this report a variation of OOM, the norm observable operator models (norm-OOMs).
Like OOMs, norm-OOMs describe stochastic processes also using linear observable operators. But norm-OOMs differ from OOMs in that they employ a nonlinear function acting on the state vectors, instead of the linear one used by OOMs, to compute probabilities. Under this nonlinear map, the family of all probability distributions can be embedded into a special inner product space. This provides novel insights into the relationship between the stochastic processes theory and linear algebra; and enables us to study stochastic processes by concepts and methods from linear algebra, a more convenient field of mathematics.
In this report the basic theory of norm-OOMs is set up; an iterative method for learning norm-OOMs is developed based upon the maximum likelihood (ML) principle; the advantages and limitations of norm-OOMs are discussed; and some problems for future investigation are outlined
Support size conditions for time-frequency representations on finite Abelian groups
No full textThe uncertainty principle for functions on finite Abelian groups provides us with lower bounds on the cardinality of the support of Fourier transforms of functions of small support. We discuss novel results in this realm and generalize these to obtain results relating the support sizes of functions and their short-time Fourier transforms. We then apply these results to construct a class of equal norm tight Gabor frames that are maximally robust to erasures. We discuss consequences of our findings to the theory of recovering and storing signals with sparse time-frequency representations
Establishment of an in vitro vesible generation assay to investigate intracellular trafficking of MHC class I molecules
No full textMajor histocompatibility complex (MHC) class I molecules bind viral peptides inside the Endoplasmic Reticulum (ER). The complex of class I molecule and peptide only is able to reach the Golgi apparatus and the plasma membrane where it is presented to cytotoxic T cells (CTL). Peptide-receptive MHC class I molecules are kept inside the cell under normal conditions to avoid binding of extracellular peptides and the killing of an innocent cell. How this differentiation is achieved and where does it take place? Are peptide-receptive class I molecules strictly retained inside the ER or are they able to cycle between ER and ERGIC? To address these questions, a new experimental system was first established in yeast and further adopted to mammalian cells, which enables the generation and isolation of ER-derived COPII vesicles in vitro from perforated cells. Analysis of vesicular contents by Immunoblot revealed that overexpressed as well as endogenous class I molecules can enter COPII vesicles in potentially peptide-receptive forms. ER escape and retrieval mechanisms therefore contribute to intracellular retention and transport regulation of peptide-receptive class I molecules suggesting that their quality control and peptide loading may even take place in post-ER compartments
Performative politics and petrified image : the Mao cult during China's cultural revolution
No full textThe modern personality cult, the godlike glorification of a political leader with mass medial techniques supported by excessive popular worship, appears to be a nearly universal feature of the 20th century with leader cults spreading from Albania to Zimbabwe. While the Stalin cult proved to be most influential in providing a blueprint for other socialist leader cults, none was to rival the intensity and scope of the cult of Mao Zedong during the Cultural Revolution (1966-1976). Contrary to claims of the Chinese Communist Party that emphasize the traditional nature of the cult or functionalist approaches that reduce it to mere social engineering, I propose to interpret the leader cult as a medium of symbolic power, as the creation of a communicative space that affected both the leadership's performative politics and the populace's adaptation of its directives. Socialist leader cults, unlike their fascist counterparts, relied less on the leader's fervent rhetoric than on the distribution and collective study of his printed works. Intended as a loyalty-creating device to ease the identification with the Communist Movement for its often illiterate supporters, the cult merged the success of the revolution with the fate of a single person. Controlling the leader's powerful image and sayings thus became a formidable task, as the gap between the petrified public image of an omniscient, unmoved mover and day to day politics paved the way for varying interpretations on all levels of society. Perhaps never have these tendencies become more obvious then during the Cultural Revolution, when cult-building came to serve most different purposes oscillating between manipulation, ritual obedience and cult anarchy. Like a magnifying glass, the cultural revolutionary Mao cult presents us with the unique possibility of studying the workings, consequences and defects of personality cults, offering a comparison with the experiences of other countries up to the present day
Time Varying Narrowband Communications Channels: Analysis and Implementation
No full textWe derive and describe a Matlab implementation of an efficient numerical algorithm for the analysis of certain classes of Hilbert-Schmidt operators that naturally occur in models of wireless radio and sonar communications channels.
A common short-time model of these channels writes the channel output as a weighted superposition of time- and frequency shifted copies of the transmitted signal, where the weight function is often called the spreading function of the channel operator.
It is often believed that a good channel model must allow for spreading functions containing Dirac delta distributions. However, we show that many narrow-band finite lifelength channels such as wireless radio communications can be well modelled by smooth and compactly supported spreading functions.
We derive a fast algorithm for computing the matrix representation of such operators with respect to well time-frequency localized Gabor bases, such as pulse shaped OFDM bases. Hereby we use a minimum of approximations, simplifications, and assumptions on the channel. The primary intended target application is comparisons of how different parameter settings and pulse shapes affect the diagonalization properties of an OFDM system acting on a given channel.
Finally, we provide and demonstrate the use of a Matlab implementation that is fast enough for the number of carrier frequencies typically in use today.
A shortened and improved version of the main results in this report is published in the journal paper [GP07], which excludes some discussions, proofs and the software documentation contained here
Peptide-dependent trafficking of MHC class I molecules
No full textI have investigated the trafficking of peptide-receptive MHC class I molecules using confocal fluorescence microscopy. I have found that both peptide-free and -bound forms can leave the endoplasmic reticulum and become transported to the cis-Golgi, from where only those forms that are bound to high-affinity peptides can proceed to the cell surface, while peptide-receptive forms are retrieved to the ER. I have then checked different peptides in their ability to move class I to the cell surface, and I have found that the carboxy terminus of the peptide is important for the export of the class I to the cell surface. I have also investigated the effect of tapasin and calreticulin, components of the peptide loading complex, on the trafficking of class I. In both tapasin-negative and calreticulin-negative cell lines, class I remains intracellular. Interestingly, introduction of high-affinity peptides can partially overcome this defect in both cell lines. That confirms the role of tapasin and calreticulin in the exchange of the peptides bound to class I from low-affinity to higher-affinity peptides
Improving glucose oxidase properties by directed evolution
No full textThe goal of my PhD project was to improve properties of glucose oxidase (GOx) for its putative use in the anodic compartment of miniaturized biofuel cell (BFC) for human in-body applications. The method used for achieving the goal was directed protein evolution using yeast Saccharomyces cerevisiae as host for functional GOx expression. Diversity at gene level was obtained by epPCR (0.05 mM Mn2+) and saturation mutagenesis in position T30. Shuttle vector pYES2 (GAL1 promoter) and S. cerevisiae were used for enzyme library expression. Construction of the mutagenic library in E. coli was performed with a ligase-free method. An ABTS assay based high-throughput screening system was developed for solid-phase prescreening and liquid-phase screening of the mutant libraries. A mutant L(n14)P T30V I94V with increased substrate specificity toward ß-D-glucose (Km: from 21.3 mM to 13.7 mM) was found. A possible explanation for the observed improvement could be a reorientation of the isoalloxazine ring of the FAD resulting in a better accommodation of the substrate inside the binding site. Reorientation of the isoalloxazine ring of the FAD is promoted by the disappearance of a hydrogen bond between the protein backbone and the noncovalently bound FAD. Despite of the increased catalytic efficiency, other properties of L(n14)P T30V I94V mutant such as thermal stability and pH activity profile remained unchanged. In perspective, rspective, engineering of GOx by directed evolution will likely enable the development of BFCs driving implantable device such as pacemaker and insulin pump. Such devices might become important for the rapidly growing field of personalized medicine. The established genetic system and optimized screening assay present for the first time a successful directed evolution to tailor properties of GOx in yeast