Formal Concept Analysis for the Interpretation of Relational Learning applied on 3D Protein-Binding Sites

International audienceInductive Logic Programming (ILP) is a powerful learning method which allows an expressive representation of the data and produces explicit knowledge. However, ILP systems suffer from a major drawback as they return a single theory based on heuristic user-choices of various parameters, thus ignoring potentially relevant rules. Accordingly, we propose an original approach based on Formal Concept Analysis for effective interpretation of reached theories with the possibility of adding domain knowledge. Our approach is applied to the characterization of three-dimensional (3D) protein-binding sites which are the protein portions on which interactions with other proteins take place. In this context, we define a relational and logical representation of 3D patches and formalize the problem as a concept learning problem using ILP. We report here the results we obtained on a particular category of protein-binding sites namely phosphorylation sites using ILP followed by FCA-based interpretation

The use of novel techniques to study the roles of cytokines in joint pain and inflammation

Rheumatoid arthritis (RA) is a common, chronic, autoimmune, inflammatory disease characterized by persistent synovitis that results in the progressive destruction ofjoints. The cellular and molecular basis of the inflammation is complex and multifactorial. During the progression of the disease many types of cells are activated, which in turn secrete a variety of mediators, including cytokines, which initiate and perpetuate the disease.Rat adjuvant-induced unilateral arthritis is a well established RA disease model and use of this model has facilitated the understanding of the pathology of joint inflammation. The model closely mimics the pathology of human RA, including histopathological changes, cell infiltration, as well as hypersensitivity and swelling of the joint. Measurements of spontaneous pain and hypersensitivity states are assessed in this model. However, no objective measure ofjoint hypersensitivity is used to assess experimental arthritic joint pain in laboratory rodents. To that end, the pressure application device (PAD) was developed to align pre-clinical measures to those used clinically and help the translation of animal studies to human conditions. PAD was able to detect FCA-induced hypersensitivity in mice and rats, observed as a decrease in limb withdrawal thresholds (LWTs) of around 60% and 40% respectively, compared with basal levels in normal joints. PAD subsequently detected prednisolone analgesia in both species, which was abolished after dosing ceased. PAD also showed significant reversal of evoked mechanical hypersensitivity in arthritic animals treated with morphine or celecoxib, which was comparable to that measured by the weight distribution readout. PAD provides a novel, accurate behavioural tool for detecting localised primary mechanical hypersensitivity in two animal models of chronic inflammatory joint pain.The infiltration of cells and release of inflammatory proteins in the synovial tissue and joint space is a key characteristic of synovitis. Measuring the levels of these in the synovial fluid can provide information about the underlying pathophysiology of joint v disease. Furthermore changes occurring in the synovial fluid can be used as biomarkers of disease; therefore the joint perfusion method was developed to evaluate the inflammatory protein and cell content of rat knee joints, to further validate the adjuvant-induced arthritis model, as well as to determine the effects of inflammatory insults or the effect of anti¬ inflammatory, analgesic or anti-rheumatic drugs. This technique proved to be reliable and consistent when perfusing the joint cavity, and regular volumes of sample were easily collected. This technique is therefore a valuable addition to protocols which use homogenates of entire joints to assess inflammatory mediator content.The temporal expression patterns of cytokines and inflammatory cells in the knee joints of rats following induction of arthritis were determined using the novel perfusion technique. Cytokine expression altered over time as arthritis progressed from the acute to the more "chronic" phase. The proportion of inflamed joints that contained detectable levels of each mediator measured was significantly increased during the study. This suggests that it may be the presence of the protein, even at low levels, that is important for the development and maintenance of joint inflammation and hypersensitivity. In addition, significant correlations between measures ofjoint swelling or mechanical hypersensitivity and levels of cytokines in inflamed joints were seen. Prednisolone did not affect the absolute levels of cytokines in inflamed joints, although it reduced the percentage of inflamed joints that contained detectable levels of ILla and IL6. This suggests that the steroid appears to have an all-or-none effect in terms of cytokine expression levels in this study.The roles of ILip and IL6 in joint pain and inflammation were assessed. The contribution of the activity of primary afferent fibres to joint pain and hypersensitivity after administration of intra-articular ILip or IL6 was investigated by recording action potentials from primary afferent nerves innervating the knee joint. IL1 p caused a transient increase in the frequency of basal neural discharge by 88% within three hours. It also decreased the threshold of mechanical stimulation required to evoke neural activity by 50% between one and four hours after injection. In contrast, IL6 did not affect the frequency of basal neural discharge or the mechanical threshold. Neither ILiß nor IL6 affected the neural discharge frequency to mechanical stimulation above the threshold. The induction of basal neural activity resembles the occurrence of spontaneous pain during inflammation, such as that measured by the incapacitance tester as a result of intra-articular IL1ß or IL6. A reduction in the LWT, measured by PAD, following ILiß or IL6 occurred within a few hours, similar to the decrease in the mechanical threshold to von Frey hairs in primary afferents after ILiß, as a result of neuronal sensitization. Although ILiß or IL6 did not cause swelling of the joint, they did induce mechanical hypersensitivity within a couple of hours, which lasted for up to four days. Intra-articular IL1ß or IL6 had no effect on joint structure, bone or cartilage. ILiß and IL6 evoked increases in the expression of ILip, IL6 and TNFa within the first eight hours, and additionally elevated levels of ILla, IL2, IL4 (IL6-treated only) and IL10 (ILiß-treated only) from day one post administration. ILiß also resulted in recruitment of inflammatory cells into the synovial cavity one day after administration.In conclusion, this study has developed and validated two novel techniques to study experimental joint pain and inflammation in rodents; the behavioural measure of joint mechanical hypersensitivity, PAD; and the joint perfusion technique to assess inflammatory mediator and cell content of synovial fluid. These methods have been used alongside other techniques to show the temporal cytokine expression patterns during adjuvant-induced arthritis and the relationship of these to swelling and hypersensitivity of the joint. The roles of ILip and IL6 in evoking joint pain and hypersensitivity were also investigated. This data supports the hypothesis that ILip and IL6 are directly involved in the development of joint pain, but cannot alone elicit swelling or joint damage at doses sufficient to evoke hypersensitivity. Furthermore, similarities between this animal model of joint disease and human RA have been demonstrated that further validate the model as a valuable pre-clinical tool to study the inflammatory process of human RA. Moreover, consolidation of these similarities helps improve the confidence of novel drug screening using this model prior to use in the clinic

Advanced Mathematical Modelling of Pancreatic β-Cells

Insulin-secreting pancreatic $\beta$-cells are responsible for maintaining the whole body glucose homeostasis. Dysfunction or loss of $\beta$-cell mass results in impaired insulin secretion and, in some cases, diabetes. Many of the factors that influence $\beta$-cell function or insulin exocytosis, however, are not fully understood. To support the investigation, mathematical models have been developed and used to design experiments. In this dissertation, we present the Integrated Oscillator Model (IOM) that is one of the mathematical models used for the investigation of the mechanism behind the bursting activity that underlies intracellular Ca$^{2+}$ oscillations and pulsatile insulin secretion. The IOM describes the interaction of the cellular electrical activity and intracellular Ca$^{2+}$ with glucose metabolism via numerous feedforward and feedback pathways. These interactions, in turn, produce metabolic oscillations with a sawtooth or pulsatile time course, reflecting different oscillation mechanisms. We determine conditions favorable to each type of oscillations, and show that the model accounts for key experimental findings of $\beta$-cell activity. We propose several extensions of the model to include all the main elements involved in the insulin secretion. The latest and most sophisticated model describes the complex metabolism in the mitochondria and the several biological processes in the insulin exocytosis cascade. The model, also, captures the changes in the $\beta$-cell activity and the resulting amount of secreted insulin in response to different concentrations of glucose in the blood. The model predictions, in agreement with findings reported in the experimental literature, show an increase of insulin secretion when the glucose level is high and a basal-low insulin concentration when the glucose level decreases. Finally, we use the new model to simulate the interaction among $\beta$-cells (through gap junction) within the same islet. The simulations show that the electrical coupling is sufficient to synchronize the $\beta$-cells within an islet. We also show that the amplitude of the oscillations in the insulin secretion rate is bigger when the $\beta$-cells synchronize. This suggests a more efficient secretion of insulin in the bloodstream when the cells burst in unison, as it has been observed experimentally

Studies on the regulatory mechanisms of Fcγ receptor function

Receptors for immunoglobulins (Fc receptors) play a central role during an immune response, as they mediate the specific recognition of antigens of almost infinite diversity by leukocytes, thereby linking the innate with the adaptive branches of immunity. This thesis undertook a series of studies to further investigate the role of Fc receptors in innate immune responses, using in vitro approaches to examine functional activity and underlying regulatory mechanisms.FcγRIla (CD32a) is a member of the Fcγ receptor family and mediates binding of multivalent IgG. Although CD32a is expressed by a number of myeloid cell types, including neutrophils, macrophages, eosinophils and monocytes, we observed that ligand binding to this receptor was suppressed, as evidenced by the low levels of CD32a-mediated IgG binding to these cells. We therefore aimed to determine the mechanisms that account for the myeloid-specific suppression of CD32a ligand binding. A series of experiments were performed to examine the effects of proteases and sialylation of CD32a in the regulation of IgG binding. In addition, the role of association of CD32a with plasma membrane microdomains rich in cholesterol and sphingolipids in the regulation of IgG binding to CD32a was examined. These membrane microdomains, also termed lipid rafts, have previously been reported to be essential for efficient receptor signalling. Chemical disruption of lipid raft structure by depletion or sequestration of membrane cholesterol greatly inhibited CD32a-mediated IgG binding, strongly implicating lipid rafts in the control of CD32a function. To further investigate this suggestion, specific CD32a mutants were generated, which would be predicted to have altered association with lipid rafts. Both these mutants showed reduced association with lipid rafts (A224S and C241A) and displayed decreased levels of IgG binding compared with wild type CD32a. Additionally, we generated a chimaeric CD32a receptor containing a glycophosphatidylinositol (GPI] lipid anchor consensus sequence, which would constitutively associate with lipid rafts. GPI-anchored CD32a exhibited increased capacity for IgG binding compared with the full-length transmembrane CD32a. Our findings clearly suggest a major role for lipid rafts in the regulation of IgG binding and more specifically, that suppression of CD32a-mediated IgG binding in myeloid cells is achieved by receptor exclusion from lipid raft membrane microdomainsThis thesis also describes an investigation of the association of Fcγ receptor genetic variants with idiopathic pulmonary fibrosis (IPF) susceptibility and progression. In particular, two allelic variants of CD32a (H131/R131) and CD16 (NA1/NA2) were examined that confer altered IgG binding and may therefore contribute to disease pathogenesis. Copy number variation of the FCGR3B gene was also determined using a quantitative PCR-based approach. Susceptibility to IPF was found to be associated with the NA1 allele of FcγRIIIb and increased FCGR3B copy number. In addition, IPF disease severity at disease presentation and progression over a 1 2-month period following diagnosis was found to be linked to the FcγRIIa H131 variant. These results support the involvement of Fcγ-mediated interactions in IPF and reveal a novel role of Fcγ receptors in IPF disease pathogenesis and progression

Nanomaterials for Biomedical and Biotechnological Applications

The need for constant improvement to reach a high standard of safety and to make nanomaterials accessible for marketing has generated a considerable number of scientific papers that highlight new important aspects to be considered, such as synthesis, stability, biocompatibility, and easy manipulation. In order to provide a comprehensive update on the latest discoveries concerning nanomaterials, this reprint presents 14 scientific papers, 10 research articles and 4 reviews, that deal with biomedical and biotechnological applications of nanomaterials

Breeding Innovations in Underutilized Temperate Fruit Trees

The recent growing interest in minor species (i.e., fig, pomegranate, feijoa, etc.) has recently driven new research on breeding and genetics to address producer and consumer traits. Since these species have received little attention from the scientific community, they were less improved via conventional breeding, and lacked detailed genomic information on important traits. This lack of data, together with a general poor genetic knowledge of these species, has limited a wider cultivation of varieties with improved characteristics. For these reasons, and with the objective to increase the interest of scientists, farmers, and consumers for these fruits, this Research Topic “Breeding Innovations in Underutilized Temperate Fruit Trees” comprises biochemical, morphological, and genetic studies on some minor species regarding fruit trait variation, resistance, evolution, or sex determination. In this context, Marcotuli et al. examined the mechanisms behind the bud evolution toward breba or main crop in fig (Ficus carica L.), since this aspect remains unclear. The X-ray images of buds showed a great structural similarity between breba and the main crop during the initial stages of development, but breba inflorescence differentiation was completed in two seasons whereas that of main crop started at the end of winter and was completed within 2–3 months in the same season. The higher expression of floral homeotic protein AGAMOUS in breba compared to the main crop may indicate a role of these fruits on staminate flowers’ production for pollination of the main crop, as profichi act in the caprifig. Within the same species (Ficus carica L.) and for sexual determination, Ikegami et al. analyzed the FcRAN1 gene (during a breeding program for the selection of female plants) strongly associated with the sex phenotype. A male-biased segregation ratio distortion was obtained in 12 F1 populations, suggesting some genetic factor(s) affecting it. A comparison between the annotated genes and the genes required for normal embryo or gametophyte development and function identified several candidate genes responsible for the segregation distortion in fig. Following the same topic, Wang et al. hypothesized an early sex-identification method to improve breeding efficiency. The use of a deletion as a forward primer, a newly established AG-Marker, was as accurate as the RAN1-Marker, and provided the identification of male plants, giving new clues to understanding Ficus sex determination. Moving toward another attracting species, i.e., pomegranate (Punica granatum L.), Trainin et al. investigated the black peel color of some pomegranate varieties. Biochemical analysis revealed that delphinidin is highly abundant in the peel of black varieties and the pattern of anthocyanin accumulation is different from that of other pomegranates with red or pink colors of the peel. Genetic analysis of an F2 population segregating for the black phenotype revealed that it is determined by a single recessive gene. Pomegranate was also studied by Goudappa Patil et al. with regards to the SSR of “Tunisia” pomegranate variety. There was a positive trend in chromosome length and the SSR abundance, as marker density, enhanced with a shorter chromosome length. Examination of the distribution of SSR motif types within a chromosome suggested the abundance of hexanucleotide repeats in each chromosome followed by dinucleotides. A comprehensive set of highly polymorphic genome-wide SSRs was successively developed and tested. These chromosome-specific SSRs could serve as a powerful genomic tool to leverage future genetic studies, germplasm management, and genomics-assisted breeding in pomegranate varieties. Some evolutionary aspects of pear were investigated by Kumar et al. who put a light on runs of homozygosity (ROH) in self-incompatible plants, in particular Asian pears, European pears, and interspecific hybrids using genotyping-by-sequencing. The observed ROH patterns suggested that systematic breeding of European pears would have started earlier than Asian pears. Fruit trait variation in Persian walnut (Juglans regia L.) was addressed by Bernard et al. who conducted a genome-wide association study (GWAS) using multi-locus models in a panel of 170 accessions of J. regia to elucidate the genetic determinants of fruit quality traits in walnut toward the breeding of new varieties. The authors proposed several candidate genes involved in nut characteristics, such as a gene coding for a beta-galactosidase linked to several size-related traits and known to also be involved in fruit development in other species. With regards of fruit traits, Kyratzis et al. investigated the germplasm of an ancient species, the carob (Ceratonia siliqua L.), on the island of Cyprus. The domestic germplasm varies both in terms of pod morphology and composition, reflecting the genetic and physiological characteristics of both grafted and non-grafted accessions, and possibly the impact of agro-environmental conditions. Morphological traits, such as seeds-to-pod weight ratio, pod width, and thickness, were principally under genetic control. Contrarily, chemical compounds, particularly total phenolic content, including condensed tannins, in vitro antioxidant capacity, and to a lesser extent gallic acid, organic acids, sugars (glucose and fructose), and minerals were more under agro-environmental control. In the Southern Hemisphere, Quezada et al. worked on feijoa (Acca sellowiana Berg.), a fruit tree species native to Uruguay and Brazil. A high-density composite genetic linkage map of feijoa was constructed using two genetically populations. Genotyping by sequencing (GBS) approach was successfully applied for developing single nucleotide polymorphism (SNP) markers. They used both the reference genome of the closely related species Eucalyptus grandis and a de novo pipeline to construct a composite map. A novel approach for the construction of composite maps where the meiosis information of individuals of two connected populations is captured in a single estimator is described. The topic of resistance was carried out by Alves et al. in order to find sources of genetic resistance to Huanglongbing (HLB)-associated “Candidatus Liberibacter asiaticus” (Las), one of the most destructive diseases of citrus. Some genotypes from subtribe Citrinae, sexually incompatible but graft-compatible with Citrus, may provide new rootstocks able to restrict bacterial titer in the canopy. Authors tested for Las resistance a wide collection of graft-compatible Citrinae species using an aggressive and consistent challenge-inoculation and evaluation procedure. Eremocitrus glauca and Papua/New Guinea Microcitrus species as well as their hybrids resulted in full resistance, opening the way for using these underutilized genotypes as Las resistance sources in breeding programs

Analysis of genetic polymorphisms as risk factors for Aggressive Periodontitis.

This PhD consisted of a series of studies aiming at detecting genetic risk factors for Aggressive Periodontitis (AgP). AgP is a destructive disease of the periodontium affecting around 1% of the population and leading to early tooth loss. Microbiological and environmental factors are thought to act on a genetically susceptible host to determine AgP. We conducted a case-control association study on 224 AgP patients (both Generalised AgP and Localised AgP) and 231 healthy controls to detect differences in genotype distributions of 13 single nucleotide polymorphisms (SNPs). The selected SNPs included FcR and FPR, NADPH oxidase, IL-6, TNF-a and VDR polymorphisms. Further studies on subsets of patients were conducted to detect associations between these SNPs and classical features of AgP: disease severity, familial aggregation, presence of periodontopathogenic bacteria and neutrophil hyperactivity. The NADPH p22phox 242 polymorphism was associated with the AgP trait and with disease severity. The IL-6 -174 SNP was associated with LAgP and with increased detection of periodontopathogenic bacteria. The FcyRIIIb NA polymorphism was associated with GAgP, while FcyR haplotypes were linked with AgP in Blacks and FcyRIIa was associated with familial aggregation of the AgP phenotype. The VDR Taq-I polymorphism showed a trend for association with AgP in smokers. The overall results of the study provide two possible pathogenic pathways leading to AgP: one is mediated through an excessive inflammatory response triggered by the presence of specific bacteria in individuals with hyper-responsive genotypes (NADPH p22phox 242 T allele, FcyRIIIb NA1 homozygosity, IL-6 -174 G homozygosity) the second is initiated by an increased susceptibility to bacterial colonization (FcyRIIa R homozygosity). In conclusion, this study supports the importance of genetic factors in Aggressive Periodontitis and hypothesizes possible pathogenic mechanisms