Analysis of the influence of epitope flanking regions on MHC class I restricted antigen presentation

Peptides presented by MHC class I molecules for CTL recognition are derived mainly from cytosolic proteins. For antigen presentation on the cell surface, epitopes require correct processing by cytosolic and ER proteases, efficient TAP transport and MHC class I binding affinity. The efficiency of epitope generation depends not only on the epitope itself, but also on its flanking regions. In this project, the influence of the C-terminal region of the model epitope SIINFEKL (S8L) from chicken ovalbumin (aa 257-264) on antigen processing has been investigated. S8L is a well characterized epitope presented on the murine MHC class I molecule, H-2Kb. The Flp-In 293Kb cell line was transfected with different constructs each enabling the expression of the S8L sequence with different defined C-terminal flanking regions. The constructs differed at the two first C-terminal positions after the S8L epitope, so called P1’ and P2’. At these sites, all 20 amino acids were exchanged consecutively and tested for their influence on H-2Kb/S8L presentation on the cell surface of the Flp-In 293Kb cells. The detection of this complex was performed by immunostaining and flow cytometry. The prevailing assumption is that proteasomal cleavages are exclusively responsible for the generation of the final C-termini of CTL epitopes. Nevertheless, recent publications showed that TPPII (tripeptidyl peptidase II) is required for the generation of the correct C-terminus of the HLA-A3-restricted HIV epitope Nef(73-82). With this background, the dependence of the S8L generation on proteasomal cleavage of the designed constructs was characterized using proteasomal inhibitors. The results obtained indicate that it is crucial for proteasomal cleavage, which amino acid is flanking the C-terminus of an epitope. Furthermore, partially proteasome independent S8L generation from specific S8L-precursor peptides was observed. Hence, the possibility of other existing endo- or carboxy-peptidases in the cytosol that could be involved in the correct trimming of the C-terminus of antigenic peptides for MHC class I presentation was investigated, performing specific knockdowns and using inhibitors against the target peptidases. In parallel, a purification strategy to identify the novel peptidase was established. The purified peaks showing an endopeptidase activity were further analyzed by mass spectrometry and some potential peptidases (like e.g. Lon) were identified, which have to be further characterized.

Top-down influences on lexical selection during spoken word production: A 4T fMRI investigation of refractory effects in picture naming

Spoken word production is assumed to involve stages of processing in which activation spreads through layers of units comprising lexical-conceptual knowledge and their corresponding phonological word forms. Using high-field (4T) functional magnetic resonance imagine (fMRI), we assessed whether the relationship between these stages is strictly serial or involves cascaded-interactive processing, and whether central (decision/control) processing mechanisms are involved in lexical selection. Participants performed the competitor priming paradigm in which distractor words, named from a definition and semantically related to a subsequently presented target picture, slow picture-naming latency compared to that with unrelated words. The paradigm intersperses two trials between the definition and the picture to be named, temporally separating activation in the word perception and production networks. Priming semantic competitors of target picture names significantly increased activation in the left posterior temporal cortex, and to a lesser extent the left middle temporal cortex, consistent with the predictions of cascaded-interactive models of lexical access. In addition, extensive activation was detected in the anterior cingulate and pars orbitalis of the inferior frontal gyrus. The findings indicate that lexical selection during competitor priming is biased by top-down mechanisms to reverse associations between primed distractor words and target pictures to select words that meet the current goal of speech.

Geospatial Data Indexing Analysis and Visualization via Web Services with Autonomic Resource Management

With the exponential growth of the usage of web-based map services, the web GIS application has become more and more popular. Spatial data index, search, analysis, visualization and the resource management of such services are becoming increasingly important to deliver user-desired Quality of Service. First, spatial indexing is typically time-consuming and is not available to end-users. To address this, we introduce TerraFly sksOpen, an open-sourced an Online Indexing and Querying System for Big Geospatial Data. Integrated with the TerraFly Geospatial database [1-9], sksOpen is an efficient indexing and query engine for processing Top-k Spatial Boolean Queries. Further, we provide ergonomic visualization of query results on interactive maps to facilitate the user’s data analysis. Second, due to the highly complex and dynamic nature of GIS systems, it is quite challenging for the end users to quickly understand and analyze the spatial data, and to efficiently share their own data and analysis results with others. Built on the TerraFly Geo spatial database, TerraFly GeoCloud is an extra layer running upon the TerraFly map and can efficiently support many different visualization functions and spatial data analysis models. Furthermore, users can create unique URLs to visualize and share the analysis results. TerraFly GeoCloud also enables the MapQL technology to customize map visualization using SQL-like statements [10]. Third, map systems often serve dynamic web workloads and involve multiple CPU and I/O intensive tiers, which make it challenging to meet the response time targets of map requests while using the resources efficiently. Virtualization facilitates the deployment of web map services and improves their resource utilization through encapsulation and consolidation. Autonomic resource management allows resources to be automatically provisioned to a map service and its internal tiers on demand. v-TerraFly are techniques to predict the demand of map workloads online and optimize resource allocations, considering both response time and data freshness as the QoS target. The proposed v-TerraFly system is prototyped on TerraFly, a production web map service, and evaluated using real TerraFly workloads. The results show that v-TerraFly can accurately predict the workload demands: 18.91% more accurate; and efficiently allocate resources to meet the QoS target: improves the QoS by 26.19% and saves resource usages by 20.83% compared to traditional peak load-based resource allocation.

Automatic top-down processing explains common left occipito-temporal responses to visual words and objects.

Previous studies have demonstrated that a region in the left ventral occipito-temporal (LvOT) cortex is highly selective to the visual forms of written words and objects relative to closely matched visual stimuli. Here, we investigated why LvOT activation is not higher for reading than picture naming even though written words and pictures of objects have grossly different visual forms. To compare neuronal responses for words and pictures within the same LvOT area, we used functional magnetic resonance imaging adaptation and instructed participants to name target stimuli that followed briefly presented masked primes that were either presented in the same stimulus type as the target (word-word, picture-picture) or a different stimulus type (picture-word, word-picture). We found that activation throughout posterior and anterior parts of LvOT was reduced when the prime had the same name/response as the target irrespective of whether the prime-target relationship was within or between stimulus type. As posterior LvOT is a visual form processing area, and there was no visual form similarity between different stimulus types, we suggest that our results indicate automatic top-down influences from pictures to words and words to pictures. This novel perspective motivates further investigation of the functional properties of this intriguing region.

Blocking Top-down awareness of Kanizsa Figures: An ERP Investigation into Bottom-up Processing of Illusory Contours

Neural models of the processing of illusory contour (ICs) diverge from one another in terms of their emphasis on bottom-up versus top-down constituents. The current study uses a dichoptic fusion paradigm to block top-down awareness of ICs in order to examine possible bottom-up effects. Group results indicate that the N170 ERP component is particularly sensitive to ICs at central occipital sites when top-down awareness of the stimulus is permitted. Furthermore, single-subject statistics reveal that the IC N170 ERP effect is highly variable across individuals in terms of timing and topographical spread. The results suggest that the ubiquitous N170 effect to ICs found in the literature depends, at least in part, on participants’ awareness of the stimulus. Therefore a strong bottom-up model of IC processing at the time of the N170 is unlikely.

Top-K Query Processing in Edge-Labeled Graph Data

Edge-labeled graphs have proliferated rapidly over the last decade due to the increased popularity of social networks and the Semantic Web. In social networks, relationships between people are represented by edges and each edge is labeled with a semantic annotation. Hence, a huge single graph can express many different relationships between entities. The Semantic Web represents each single fragment of knowledge as a triple (subject, predicate, object), which is conceptually identical to an edge from subject to object labeled with predicates. A set of triples constitutes an edge-labeled graph on which knowledge inference is performed. Subgraph matching has been extensively used as a query language for patterns in the context of edge-labeled graphs. For example, in social networks, users can specify a subgraph matching query to find all people that have certain neighborhood relationships. Heavily used fragments of the SPARQL query language for the Semantic Web and graph queries of other graph DBMS can also be viewed as subgraph matching over large graphs. Though subgraph matching has been extensively studied as a query paradigm in the Semantic Web and in social networks, a user can get a large number of answers in response to a query. These answers can be shown to the user in accordance with an importance ranking. In this thesis proposal, we present four different scoring models along with scalable algorithms to find the top-k answers via a suite of intelligent pruning techniques. The suggested models consist of a practically important subset of the SPARQL query language augmented with some additional useful features. The first model called Substitution Importance Query (SIQ) identifies the top-k answers whose scores are calculated from matched vertices' properties in each answer in accordance with a user-specified notion of importance. The second model called Vertex Importance Query (VIQ) identifies important vertices in accordance with a user-defined scoring method that builds on top of various subgraphs articulated by the user. Approximate Importance Query (AIQ), our third model, allows partial and inexact matchings and returns top-k of them with a user-specified approximation terms and scoring functions. In the fourth model called Probabilistic Importance Query (PIQ), a query consists of several sub-blocks: one mandatory block that must be mapped and other blocks that can be opportunistically mapped. The probability is calculated from various aspects of answers such as the number of mapped blocks, vertices' properties in each block and so on and the most top-k probable answers are returned. An important distinguishing feature of our work is that we allow the user a huge amount of freedom in specifying: (i) what pattern and approximation he considers important, (ii) how to score answers - irrespective of whether they are vertices or substitution, and (iii) how to combine and aggregate scores generated by multiple patterns and/or multiple substitutions. Because so much power is given to the user, indexing is more challenging than in situations where additional restrictions are imposed on the queries the user can ask. The proposed algorithms for the first model can also be used for answering SPARQL queries with ORDER BY and LIMIT, and the method for the second model also works for SPARQL queries with GROUP BY, ORDER BY and LIMIT. We test our algorithms on multiple real-world graph databases, showing that our algorithms are far more efficient than popular triple stores.

Multi-modal summarization of key events and top players in sports tournament videos

To detect and annotate the key events of live sports videos, we need to tackle the semantic gaps of audio-visual information. Previous work has successfully extracted semantic from the time-stamped web match reports, which are synchronized with the video contents. However, web and social media articles with no time-stamps have not been fully leveraged, despite they are increasingly used to complement the coverage of major sporting tournaments. This paper aims to address this limitation using a novel multimodal summarization framework that is based on sentiment analysis and players' popularity. It uses audiovisual contents, web articles, blogs, and commentators' speech to automatically annotate and visualize the key events and key players in a sports tournament coverage. The experimental results demonstrate that the automatically generated video summaries are aligned with the events identified from the official website match reports.

Enhanced processing of SPOT multispectral satellite imagery for environmental monitoring and modelling

The Taita Hills in southeastern Kenya form the northernmost part of Africa’s Eastern Arc Mountains, which have been identified by Conservation International as one of the top ten biodiversity hotspots on Earth. As with many areas of the developing world, over recent decades the Taita Hills have experienced significant population growth leading to associated major changes in land use and land cover (LULC), as well as escalating land degradation, particularly soil erosion. Multi-temporal medium resolution multispectral optical satellite data, such as imagery from the SPOT HRV, HRVIR, and HRG sensors, provides a valuable source of information for environmental monitoring and modelling at a landscape level at local and regional scales. However, utilization of multi-temporal SPOT data in quantitative remote sensing studies requires the removal of atmospheric effects and the derivation of surface reflectance factor. Furthermore, for areas of rugged terrain, such as the Taita Hills, topographic correction is necessary to derive comparable reflectance throughout a SPOT scene. Reliable monitoring of LULC change over time and modelling of land degradation and human population distribution and abundance are of crucial importance to sustainable development, natural resource management, biodiversity conservation, and understanding and mitigating climate change and its impacts. The main purpose of this thesis was to develop and validate enhanced processing of SPOT satellite imagery for use in environmental monitoring and modelling at a landscape level, in regions of the developing world with limited ancillary data availability. The Taita Hills formed the application study site, whilst the Helsinki metropolitan region was used as a control site for validation and assessment of the applied atmospheric correction techniques, where multiangular reflectance field measurements were taken and where horizontal visibility meteorological data concurrent with image acquisition were available. The proposed historical empirical line method (HELM) for absolute atmospheric correction was found to be the only applied technique that could derive surface reflectance factor within an RMSE of < 0.02 ps in the SPOT visible and near-infrared bands; an accuracy level identified as a benchmark for successful atmospheric correction. A multi-scale segmentation/object relationship modelling (MSS/ORM) approach was applied to map LULC in the Taita Hills from the multi-temporal SPOT imagery. This object-based procedure was shown to derive significant improvements over a uni-scale maximum-likelihood technique. The derived LULC data was used in combination with low cost GIS geospatial layers describing elevation, rainfall and soil type, to model degradation in the Taita Hills in the form of potential soil loss, utilizing the simple universal soil loss equation (USLE). Furthermore, human population distribution and abundance were modelled with satisfactory results using only SPOT and GIS derived data and non-Gaussian predictive modelling techniques. The SPOT derived LULC data was found to be unnecessary as a predictor because the first and second order image texture measurements had greater power to explain variation in dwelling unit occurrence and abundance. The ability of the procedures to be implemented locally in the developing world using low-cost or freely available data and software was considered. The techniques discussed in this thesis are considered equally applicable to other medium- and high-resolution optical satellite imagery, as well the utilized SPOT data.

Mechanisms for alphavirus nonstructural polyprotein processing

Replication and transcription of the RNA genome of alphaviruses relies on a set of virus-encoded nonstructural proteins. They are synthesized as a long polyprotein precursor, P1234, which is cleaved at three processing sites to yield nonstructural proteins nsP1, nsP2, nsP3 and nsP4. All the four proteins function as constitutive components of the membrane-associated viral replicase. Proteolytic processing of P1234 polyprotein is precisely orchestrated and coordinates the replicase assembly and maturation. The specificity of the replicase is also controlled by proteolytic cleavages. The early replicase is composed of P123 polyprotein intermediate and nsP4. It copies the positive sense RNA genome to complementary minus-strand. Production of new plus-strands requires complete processing of the replicase. The papain-like protease residing in nsP2 is responsible for all three cleavages in P1234. This study addressed the mechanisms of proteolytic processing of the replicase polyprotein in two alphaviruses Semliki Forest virus (SFV) and Sindbis virus (SIN) representing different branches of the genus. The survey highlighted the functional relation of the alphavirus nsP2 protease to the papain-like enzymes. A new structural motif the Cys-His catalytic dyad accompanied with an aromatic residue following the catalytic His was described for nsP2 and a subset of other thiol proteases. Such an architecture of the catalytic center was named the glycine specificity motif since it was implicated in recognition of a specific Gly residue in the substrate. In particular, the presence of the motif in nsP2 makes the appearance of this amino acid at the second position upstream of the scissile bond a necessary condition for the cleavage. On top of that, there were four distinct mechanisms identified, which provide affinity for the protease and specifically direct the enzyme to different sites in the P1234 polyprotein. Three factors RNA, the central domain of nsP3 and the N-terminus of nsP2 were demonstrated to be external modulators of the nsP2 protease. Here I suggest that the basal nsP2 protease specificity is inherited from the ancestral papain-like enzyme and employs the recognition of the upstream amino acid signature in the immediate vicinity of the scissile bond. This mechanism is responsible for the efficient processing of the SFV nsP3/nsP4 junction. I propose that the same mechanism is involved in the cleavage of the nsP1/nsP2 junction of both viruses as well. However, in this case it rather serves to position the substrate, whereas the efficiency of the processing is ensured by the capability of nsP2 to cut its own N-terminus in cis. Both types of cleavages are demonstrated here to be inhibited by RNA, which is interpreted as impairing the basal papain-like recognition of the substrate. In contrast, processing of the SIN nsP3/nsP4 junction was found to be activated by RNA and additionally potentiated by the presence of the central region of nsP3 in the protease. The processing of the nsP2/nsP3 junction in both viruses occurred via another mechanism, requiring the exactly processed N-terminus of nsP2 in the protease and insensitive to RNA addition. Therefore, the three processing events in the replicase polyprotein maturation are performed via three distinct mechanisms in each of two studied alphaviruses. Distinct sets of conditions required for each cleavage ensure sequential maturation of P1234 polyprotein: nsP4 is released first, then the nsP1/nsP2 site is cut in cis, and liberation of the nsP2 N-terminus activates the cleavage of the nsP2/nsP3 junction at last. The first processing event occurs differently in SFV and SIN, whereas the subsequent cleavages are found to be similar in the two viruses and therefore, their mechanisms are suggested to be conserved in the genus. The RNA modulation of the alphavirus nonstructural protease activity, discovered here, implies bidirectional functional interplay between the alphavirus RNA metabolism and protease regulation. The nsP2 protease emerges as a signal transmitting moiety, which senses the replication stage and responds with proteolytic cleavages. A detailed hypothetical model of the alphavirus replicase core was inferred from the data obtained in the study. Similar principles in replicase organization and protease functioning are expected to be employed by other RNA viruses.

Interfacial Phenomena in Pharmaceutical Low Moisture Content Powder Processing

Powders are essential materials in the pharmaceutical industry, being involved in majority of all drug manufacturing. Powder flow and particle size are central particle properties addressed by means of particle engineering. The aim of the thesis was to gain knowledge on powder processing with restricted liquid addition, with a primary focus on particle coating and early granule growth. Furthermore, characterisation of this kind of processes was performed. A thin coating layer of hydroxypropyl methylcellulose was applied on individual particles of ibuprofen in a fluidised bed top-spray process. The polymeric coating improved the flow properties of the powder. The improvement was strongly related to relative humidity, which can be seen as an indicator of a change in surface hydrophilicity caused by the coating. The ibuprofen used in the present study had a d50 of 40 μm and thus belongs to the Geldart group C powders, which can be considered as challenging materials in top-spray coating processes. Ibuprofen was similarly coated using a novel ultrasound-assisted coating method. The results were in line with those obtained from powders coated in the fluidised bed process mentioned above. It was found that the ultrasound-assisted method was capable of coating single particles with a simple and robust setup. Granule growth in a fluidised bed process was inhibited by feeding the liquid in pulses. The results showed that the length of the pulsing cycles is of importance, and can be used to adjust granule growth. Moreover, pulsed liquid feed was found to be of greater significance to granule growth in high inlet air relative humidity. Liquid feed pulsing can thus be used as a tool in particle size targeting in fluidised bed processes and in compensating for changes in relative humidity of the inlet air. The nozzle function of a two-fluid external mixing pneumatic nozzle, typical for small scale pharmaceutical fluidised bed processes, was studied in situ in an ongoing fluidised bed process with particle tracking velocimetry. It was found that the liquid droplets undergo coalescence as they proceed away from the nozzle head. The coalescence was expected to increase droplet speed, which was confirmed in the study. The spray turbulence was studied, and the results showed turbulence caused by the event of atomisation and by the oppositely directed fluidising air. It was concluded that particle tracking velocimetry is a suitable tool for in situ spray characterisation. The light transmission through dense particulate systems was found to carry information on particle size and packing density as expected based on the theory of light scattering by solids. It was possible to differentiate binary blends consisting of components with differences in optical properties. Light transmission showed potential as a rapid, simple and inexpensive tool in characterisation of particulate systems giving information on changes in particle systems, which could be utilised in basic process diagnostics.

Binaural Signal Processing Motivated Generalized Analytic Signal Construction and AM-FM Demodulation

Binaural hearing studies show that the auditory system uses the phase-difference information in the auditory stimuli for localization of a sound source. Motivated by this finding, we present a method for demodulation of amplitude-modulated-frequency-modulated (AM-FM) signals using a ignal and its arbitrary phase-shifted version. The demodulation is achieved using two allpass filters, whose impulse responses are related through the fractional Hilbert transform (FrHT). The allpass filters are obtained by cosine-modulation of a zero-phase flat-top prototype halfband lowpass filter. The outputs of the filters are combined to construct an analytic signal (AS) from which the AM and FM are estimated. We show that, under certain assumptions on the signal and the filter structures, the AM and FM can be obtained exactly. The AM-FM calculations are based on the quasi-eigenfunction approximation. We then extend the concept to the demodulation of multicomponent signals using uniform and non-uniform cosine-modulated filterbank (FB) structures consisting of flat bandpass filters, including the uniform cosine-modulated, equivalent rectangular bandwidth (ERB), and constant-Q filterbanks. We validate the theoretical calculations by considering application on synthesized AM-FM signals and compare the performance in presence of noise with three other multiband demodulation techniques, namely, the Teager-energy-based approach, the Gabor's AS approach, and the linear transduction filter approach. We also show demodulation results for real signals.