Interaction and cystogenesis of Toxoplasma gondii within skeletal muscle cells in vitro

Infection by the protozoan parasite Toxoplasma gondii is widely prevalent in humans and animals. To prevent human infection, all meat should be well cooked before consumption, since the parasite is present in skeletal muscle. In this context, the use of skeletal muscle cells (SkMCs) as a cellular model opens up new approaches to investigate T. gondii-host cell interactions. Immunofluorescent detection of proteins that are stage-specific for bradyzoites indicated that complete cystogenesis of T. gondii in in vitro cultures of SkMCs occurs after 96 h of infection. Ultrastructural analysis showed that, after 48 h of interaction, there were alterations on the parasitophorous vacuole membrane, including greater thickness and increased electron density at the inner face of the membrane. The present study demonstrates the potential use of primary cultures of SkMCs to evaluate different molecular aspects of T. gondii invasion and cystogenesis and presents a promising in vitro model for the screening of drug activities toward tissue cysts and bradyzoites.

Interaction of Trichomonas vaginalis and Tritrichomonas foetus with keratin: an important role in parasite infection

Trichomonas vaginalis and Tritrichomonas foetus are human and bovine parasites, respectively, that provoke the sexually transmitted disease trichomoniasis. These extracellular parasites adhere to the host epithelial cell surface. Although mucinases and proteases have been described as important proteins for parasite adhesion to epithelial cells, no studies have examined the role of the keratin molecules that cornify the vaginal epithelium. Here, we investigated the interaction of T. vaginalis and T. foetus with human keratin in vitro; additionally, adherence assays were performed in cattle with T. foetus to elucidate whether trichomonads were able to interact with keratin in vivo. We demonstrated that both T. vaginalisand T. foetusinteracted directly with keratin. Additionally, the trichomonads ingested and digested keratin, shedding new light on the Trichomonas infection process.

Disruption of GIP/GIPR axis in human adipose tissue is linked to obesity and insulin resistance.

CONTEXT Glucose-dependent insulinotropic peptide (GIP) has a central role in glucose homeostasis through its amplification of insulin secretion; however, its physiological role in adipose tissue is unclear. OBJECTIVE Our objective was to define the function of GIP in human adipose tissue in relation to obesity and insulin resistance. DESIGN GIP receptor (GIPR) expression was analyzed in human sc adipose tissue (SAT) and visceral adipose (VAT) from lean and obese subjects in 3 independent cohorts. GIPR expression was associated with anthropometric and biochemical variables. GIP responsiveness on insulin sensitivity was analyzed in human adipocyte cell lines in normoxic and hypoxic environments as well as in adipose-derived stem cells obtained from lean and obese patients. RESULTS GIPR expression was downregulated in SAT from obese patients and correlated negatively with body mass index, waist circumference, systolic blood pressure, and glucose and triglyceride levels. Furthermore, homeostasis model assessment of insulin resistance, glucose, and G protein-coupled receptor kinase 2 (GRK2) emerged as variables strongly associated with GIPR expression in SAT. Glucose uptake studies and insulin signaling in human adipocytes revealed GIP as an insulin-sensitizer incretin. Immunoprecipitation experiments suggested that GIP promotes the interaction of GRK2 with GIPR and decreases the association of GRK2 to insulin receptor substrate 1. These effects of GIP observed under normoxia were lost in human fat cells cultured in hypoxia. In support of this, GIP increased insulin sensitivity in human adipose-derived stem cells from lean patients. GIP also induced GIPR expression, which was concomitant with a downregulation of the incretin-degrading enzyme dipeptidyl peptidase 4. None of the physiological effects of GIP were detected in human fat cells obtained from an obese environment with reduced levels of GIPR. CONCLUSIONS GIP/GIPR signaling is disrupted in insulin-resistant states, such as obesity, and normalizing this function might represent a potential therapy in the treatment of obesity-associated metabolic disorders.

Interaction of cationic ultrasmall superparamagnetic iron oxide nanoparticles with human melanoma cells.

Ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) are currently under development for the intracellular delivery of therapeutics. However, the mechanisms of cellular uptake and the cellular reaction to this uptake, independent of therapeutics, are not well defined. The interactions of biocompatible cationic aminoUSPIONs with human cells was studied in 2D and 3D cultures using biochemical and electron microscopy techniques. AminoUSPIONs were internalized by human melanoma cells in 2D and 3D cultures. Uptake was clathrin mediated and the particles localized in lysosomes, inducing activation of the lysosomal cathepsin D and decreasing the expression of the transferrin receptor in human melanoma cells and/or skin fibroblasts. AminoUSPIONs deeply invaded 3D spheroids of human melanoma cells. Thus, aminoUSPIONs can invade tumors and their uptake by human cells induces cell reaction.

Development and validation of AMANDA, a new algorithm for selecting highly relevant regions in Molecular Interaction Fields

Descriptors based on Molecular Interaction Fields (MIF) are highly suitable for drug discovery, but their size (thousands of variables) often limits their application in practice. Here we describe a simple and fast computational method that extracts from a MIF a handful of highly informative points (hot spots) which summarize the most relevant information. The method was specifically developed for drug discovery, is fast, and does not require human supervision, being suitable for its application on very large series of compounds. The quality of the results has been tested by running the method on the ligand structure of a large number of ligand-receptor complexes and then comparing the position of the selected hot spots with actual atoms of the receptor. As an additional test, the hot spots obtained with the novel method were used to obtain GRIND-like molecular descriptors which were compared with the original GRIND. In both cases the results show that the novel method is highly suitable for describing ligand-receptor interactions and compares favorably with other state-of-the-art methods.

Different internalization properties of the alpha1a- and alpha1b-adrenergic receptor subtypes: the potential role of receptor interaction with beta-arrestins and AP50.

The internalization properties of the alpha1a- and alpha1b-adrenergic receptors (ARs) subtypes transiently expressed in human embryonic kidney (HEK) 293 cells were compared using biotinylation experiments and confocal microscopy. Whereas the alpha1b-AR displayed robust agonist-induced endocytosis, the alpha1a-AR did not. Constitutive internalization of the alpha1a-AR was negligible, whereas the alpha1b-AR displayed significant constitutive internalization and recycling. We investigated the interaction of the alpha1-AR subtypes with beta-arrestins 1 and 2 as well as with the AP50 subunit of the clathrin adaptor complex AP2. The results from both coimmunoprecipitation experiments and beta-arrestin translocation assays indicated that the agonistinduced interaction of the alpha1a-AR with beta-arrestins was much weaker than that of the alpha1b-AR. In addition, the alpha1a-AR did not bind AP50. The alpha1b-AR mutant M8, lacking the main phosphorylation sites in the receptor C tail, was unable to undergo endocytosis and was profoundly impaired in binding beta-arrestins despite its binding to AP50. In contrast, the alpha1b-AR mutant DeltaR8, lacking AP50 binding, bound beta-arrestins efficiently, and displayed delayed endocytosis. RNA interference showed that beta-arrestin 2 plays a prominent role in alpha1b-AR endocytosis. The findings of this study demonstrate differences in internalization between the alpha1a- and alpha1b-AR and provide evidence that the lack of significant endocytosis of the alpha1a-AR is linked to its poor interaction with beta-arrestins as well as with AP50. We also provide evidence that the integrity of the phosphorylation sites in the C tail of the alpha1b-AR is important for receptor/beta-arrestin interaction and that this interaction is the main event triggering receptor internalization.

Disentangling in vivo the effects of iron content and atrophy on the ageing human brain.

Evidence from magnetic resonance imaging (MRI) studies shows that healthy aging is associated with profound changes in cortical and subcortical brain structures. The reliable delineation of cortex and basal ganglia using automated computational anatomy methods based on T1-weighted images remains challenging, which results in controversies in the literature. In this study we use quantitative MRI (qMRI) to gain an insight into the microstructural mechanisms underlying tissue ageing and look for potential interactions between ageing and brain tissue properties to assess their impact on automated tissue classification. To this end we acquired maps of longitudinal relaxation rate R1, effective transverse relaxation rate R2* and magnetization transfer - MT, from healthy subjects (n=96, aged 21-88 years) using a well-established multi-parameter mapping qMRI protocol. Within the framework of voxel-based quantification we find higher grey matter volume in basal ganglia, cerebellar dentate and prefrontal cortex when tissue classification is based on MT maps compared with T1 maps. These discrepancies between grey matter volume estimates can be attributed to R2* - a surrogate marker of iron concentration, and further modulation by an interaction between R2* and age, both in cortical and subcortical areas. We interpret our findings as direct evidence for the impact of ageing-related brain tissue property changes on automated tissue classification of brain structures using SPM12. Computational anatomy studies of ageing and neurodegeneration should acknowledge these effects, particularly when inferring about underlying pathophysiology from regional cortex and basal ganglia volume changes.

Evaluation of uptake and transport of ultrasmall superparamagnetic iron oxide nanoparticles by human brain-derived endothelial cells.

Aim: Ultrasmall superparamagnetic iron oxide nanoparticles (USPIO-NPs) are under development for imaging and drug delivery; however, their interaction with human blood-brain barrier models is not known. Materials & Methods: The uptake, reactive oxygen species production and transport of USPIO-NPs across human brain-derived endothelial cells as models of the blood-brain tumor barrier were evaluated for either uncoated, oleic acid-coated or polyvinylamine-coated USPIO-NPs. Results: Reactive oxygen species production was observed for oleic acid-coated and polyvinylamine-coated USPIO-NPs. The uptake and intracellular localization of the iron oxide core of the USPIO-NPs was confirmed by transmission electron microscopy. However, while the uptake of these USPIO-NPs by cells was observed, they were neither released by nor transported across these cells even in the presence of an external dynamic magnetic field. Conclusion: USPIO-NP-loaded filopodia were observed to invade the polyester membrane, suggesting that they can be transported by migrating angiogenic brain-derived endothelial cells.

Disrupting the EMMPRIN (CD147)-cyclophilin A interaction reduces infarct size and preserves systolic function after myocardial ischemia and reperfusion.

Objective-Inflammation and proteolysis crucially contribute to myocardial ischemia and reperfusion injury. The extracellular matrix metalloproteinase inducer EMMPRIN (CD147) and its ligand cyclophilin A (CyPA) may be involved in both processes. The aim of the study was to characterize the role of the CD147 and CyPA interplay in myocardial ischemia/reperfusion (I/R) injury.Methods and Results-Immunohistochemistry showed enhanced expression of CD147 and CyPA in myocardial sections from human autopsies of patients who had died from acute myocardial infarction and from mice at 24 hours after I/R. At 24 hours and 7 days after I/R, the infarct size was reduced in CD147(+/-) mice vs CD147(+/+) mice (C57Bl/6), in mice (C57Bl/6) treated with monoclonal antibody anti-CD147 vs control monoclonal antibody, and in CyPA(-/-) mice vs CyPA(+/+) mice (129S6/SvEv), all of which are associated with reduced monocyte and neutrophil recruitment at 24 hours and with a preserved systolic function at 7 days. The combination of CyPA(-/-) mice with anti-CD147 treatment did not yield further protection compared with either inhibition strategy alone. In vitro, treatment with CyPA induced monocyte chemotaxis in a CD147-and phosphatidylinositol 3-kinase-dependent manner and induced monocyte rolling and adhesion to endothelium (human umbilical vein endothelial cells) under flow in a CD147-dependent manner.Conclusion-CD147 and its ligand CyPA are inflammatory mediators after myocardial ischemia and reperfusion and represent potential targets to prevent myocardial I/R injury.

Human leukocyte antigen class II variants and adult-onset asthma: does occupational allergen exposure play a role?

Recently, a locus centred on rs9273349 in the HLA-DQ region emerged from genome-wide association studies of adult-onset asthma. We aimed to further investigate the role of human leukocyte antigen (HLA) class II in adult-onset asthma and a possible interaction with occupational exposures. We imputed classical HLA-II alleles from 7579 single-nucleotide polymorphisms in 6025 subjects (1202 with adult-onset asthma) from European cohorts: ECRHS, SAPALDIA, EGEA and B58C, and from surveys of bakers and agricultural workers. Based on an asthma-specific job-exposure matrix, 2629 subjects had ever been exposed to high molecular weight (HMW) allergens. We explored associations between 23 common HLA-II alleles and adult-onset asthma, and tested for gene-environment interaction with occupational exposure to HMW allergens. Interaction was also tested for rs9273349. Marginal associations of classical HLA-II alleles and adult-onset asthma were not statistically significant. Interaction was detected between the DPB1*03:01 allele and exposure to HMW allergens (p = 0.009), in particular to latex (p = 0.01). In the unexposed group, the DPB1*03:01 allele was associated with adult-onset asthma (OR 0.67, 95%CI 0.53-0.86). HMW allergen exposures did not modify the association of rs9273349 with adult-onset asthma. Common classical HLA-II alleles were not marginally associated with adult-onset asthma. The association of latex exposure and adult-onset asthma may be modified by DPB1*03:01.

Search Interfaces on the Web: Querying and Characterizing

Current-day web search engines (e.g., Google) do not crawl and index a significant portion of theWeb and, hence, web users relying on search engines only are unable to discover and access a large amount of information from the non-indexable part of the Web. Specifically, dynamic pages generated based on parameters provided by a user via web search forms (or search interfaces) are not indexed by search engines and cannot be found in searchers’ results. Such search interfaces provide web users with an online access to myriads of databases on the Web. In order to obtain some information from a web database of interest, a user issues his/her query by specifying query terms in a search form and receives the query results, a set of dynamic pages that embed required information from a database. At the same time, issuing a query via an arbitrary search interface is an extremely complex task for any kind of automatic agents including web crawlers, which, at least up to the present day, do not even attempt to pass through web forms on a large scale. In this thesis, our primary and key object of study is a huge portion of the Web (hereafter referred as the deep Web) hidden behind web search interfaces. We concentrate on three classes of problems around the deep Web: characterization of deep Web, finding and classifying deep web resources, and querying web databases. Characterizing deep Web: Though the term deep Web was coined in 2000, which is sufficiently long ago for any web-related concept/technology, we still do not know many important characteristics of the deep Web. Another matter of concern is that surveys of the deep Web existing so far are predominantly based on study of deep web sites in English. One can then expect that findings from these surveys may be biased, especially owing to a steady increase in non-English web content. In this way, surveying of national segments of the deep Web is of interest not only to national communities but to the whole web community as well. In this thesis, we propose two new methods for estimating the main parameters of deep Web. We use the suggested methods to estimate the scale of one specific national segment of the Web and report our findings. We also build and make publicly available a dataset describing more than 200 web databases from the national segment of the Web. Finding deep web resources: The deep Web has been growing at a very fast pace. It has been estimated that there are hundred thousands of deep web sites. Due to the huge volume of information in the deep Web, there has been a significant interest to approaches that allow users and computer applications to leverage this information. Most approaches assumed that search interfaces to web databases of interest are already discovered and known to query systems. However, such assumptions do not hold true mostly because of the large scale of the deep Web – indeed, for any given domain of interest there are too many web databases with relevant content. Thus, the ability to locate search interfaces to web databases becomes a key requirement for any application accessing the deep Web. In this thesis, we describe the architecture of the I-Crawler, a system for finding and classifying search interfaces. Specifically, the I-Crawler is intentionally designed to be used in deepWeb characterization studies and for constructing directories of deep web resources. Unlike almost all other approaches to the deep Web existing so far, the I-Crawler is able to recognize and analyze JavaScript-rich and non-HTML searchable forms. Querying web databases: Retrieving information by filling out web search forms is a typical task for a web user. This is all the more so as interfaces of conventional search engines are also web forms. At present, a user needs to manually provide input values to search interfaces and then extract required data from the pages with results. The manual filling out forms is not feasible and cumbersome in cases of complex queries but such kind of queries are essential for many web searches especially in the area of e-commerce. In this way, the automation of querying and retrieving data behind search interfaces is desirable and essential for such tasks as building domain-independent deep web crawlers and automated web agents, searching for domain-specific information (vertical search engines), and for extraction and integration of information from various deep web resources. We present a data model for representing search interfaces and discuss techniques for extracting field labels, client-side scripts and structured data from HTML pages. We also describe a representation of result pages and discuss how to extract and store results of form queries. Besides, we present a user-friendly and expressive form query language that allows one to retrieve information behind search interfaces and extract useful data from the result pages based on specified conditions. We implement a prototype system for querying web databases and describe its architecture and components design.

Towards a lightweight adaptative multicomponential affective recognition system

As part of the Affective Computing research field, the development of automatic affective recognition systems can enhance human-computer interactions by allowing the creation of interfaces that react to the user's emotional state. To that end, this Master Thesis brings affect recognition to nowadays most used human computer interface, mobile devices, by developing a facial expression recognition system able to perform detection under the difficult conditions of viewing angle and illumination that entails the interaction with a mobile device. Moreover, this Master Thesis proposes to combine emotional features detected from expression with contextual information of the current situation, to infer a complex and extensive emotional state of the user. Thus, a cognitive computational model of emotion is defined that provides a multicomponential affective state of the user through the integration of the detected emotional features into appraisal processes. In order to account for individual differences in the emotional experience, these processes can be adapted to the culture and personality of the user.

User Experience of Geocaching and Its Application to Tourism and Education

Advances in technology have provided new ways of using entertainment and game technology to foster human interaction. Games and playing with games have always been an important part of people’s everyday lives. Traditionally, human-computer interaction (HCI) research was seen as a psychological cognitive science focused on human factors, with engineering sciences as the computer science part of it. Although cognitive science has made significant progress over the past decade, the influence of people’s emotions on design networks is increasingly important, especially when the primary goal is to challenge and entertain users (Norman 2002). Game developers have explored the key issues in game design and identified that the driving force in the success of games is user experience. User-centered design integrates knowledge of users’ activity practices, needs, and preferences into the design process. Geocaching is a location-based treasure hunt game created by a community of players. Players use GPS (Global Position System) technology to find “treasures” and create their own geocaches; the game can be developed when the players invent caches and used more imagination to creations the caches. This doctoral dissertation explores user experience of geocaching and its applications in tourism and education. Globally, based on the Geocaching.com webpage, geocaching has been played about 180 countries and there are more than 10 million registered geocachers worldwide (Geocaching.com, 25.11.2014). This dissertation develops and presents an interaction model called the GameFlow Experience model that can be used to support the design of treasure hunt applications in tourism and education contexts. The GameFlow Model presents and clarifies various experiences; it provides such experiences in a real-life context, offers desirable design targets to be utilized in service design, and offers a perspective to consider when evaluating the success of adventure game concepts. User-centered game designs have adapted to human factor research in mainstream computing science. For many years, the user-centered design approach has been the most important research field in software development. Research has been focusing on user-centered design in software development such as office programs, but the same ideas and theories that will reflect the needs of a user-centered research are now also being applied to game design (Charles et al. 2005.) For several years, we have seen a growing interest in user experience design. Digital games are experience providers, and game developers need tools to better understand the user experience related to products and services they have created. This thesis aims to present what the user experience is in geocaching and treasure hunt games and how it can be used to develop new concepts for the treasure hunt. Engineers, designers, and researchers should have a clear understanding of what user experience is, what its parts are, and most importantly, how we can influence user satisfaction. In addition, we need to understand how users interact with electronic products and people, and how different elements synergize their experiences. This doctoral dissertation represents pioneering work on the user experience of geocaching and treasure hunt games in the context of tourism and education. The research also provides a model for game developers who are planning treasure hunt concepts.

Cognitive performance of young and elderly subjects on the free word recall memory test: effect of presentation order on recall order

The influence of aging on memory has been extensively studied, but the importance of short-term memory and recall sequence has not. The objective of the current study was to examine the recall order of words presented on lists and to determine if age affects recall sequence. Physically and psychologically healthy male subjects were divided into two groups according to age, i.e., 23 young subjects (20 to 30 years) and 50 elderly subjects (60 to 70 years) submitted to the Wechsler Adult Intelligence Scale-Revised and the free word recall test. The order of word presentation significantly affected the 3rd and 4th words recalled (P < 0.01; F = 14.6). In addition, there was interaction between the presentation order and the type of list presented (P < 0.05; F = 9.7). Also, both groups recalled the last words presented from each list (words 13-15) significantly more times 3rd and 4th than words presented in all remaining positions (P < 0.01). The order of word presentation also significantly affected the 5th and 6th words recalled (P = 0.05; F = 7.5) and there was a significant interaction between the order of presentation and the type of list presented (P < 0.01; F = 20.8). The more developed the cognitive functions, resulting mainly from formal education, the greater the cognitive reserve, helping to minimize the effects of aging on the long-term memory (episodic declarative).

Novel Interaction Techniques for Mobile Augmented Reality applications. A Systematic Literature Review

This study reviews the research on interaction techniques and methods that could be applied in mobile augmented reality scenarios. The review is focused on themost recent advances and considers especially the use of head-mounted displays. Inthe review process, we have followed a systematic approach, which makes the reviewtransparent, repeatable, and less prone to human errors than if it was conducted in amore traditional manner. The main research subjects covered in the review are headorientation and gaze-tracking, gestures and body part-tracking, and multimodality– as far as the subjects are related to human-computer interaction. Besides these,also a number of other areas of interest will be discussed.