EPIPOX:immunoinformatic characterization of the shared T-cell epitome between variola virus and related pathogenic orthopoxviruses

Concerns that variola viruses might be used as bioweapons have renewed the interest in developing new and safer smallpox vaccines. Variola virus genomes are now widely available, allowing computational characterization of the entire T-cell epitome and the use of such information to develop safe and yet effective vaccines. To this end, we identified 124 proteins shared between various species of pathogenic orthopoxviruses including variola minor and major, monkeypox, cowpox, and vaccinia viruses, and we targeted them for T-cell epitope prediction. We recognized 8,106, and 8,483 unique class I and class II MHC-restricted T-cell epitopes that are shared by all mentioned orthopoxviruses. Subsequently, we developed an immunological resource, EPIPOX, upon the predicted T-cell epitome. EPIPOX is freely available online and it has been designed to facilitate reverse vaccinology. Thus, EPIPOX includes key epitope-focused protein annotations: time point expression, presence of leader and transmembrane signals, and known location on outer membrane structures of the infective viruses. These features can be used to select specific T-cell epitopes suitable for experimental validation restricted by single MHC alleles, as combinations thereof, or by MHC supertypes.

Identification, distribution, and probability of infection in sympatric gastropods by digenean parasites at Pa-hay-okee, Everglades National Park using molecules and morphology

In this study, I determined the identity, taxonomic placement, and distribution of digenetic trematodes parasitizing the snails Pomacea paludosa and Planorbella duryi at Pa-hay-okee, Everglades National Park. I also characterized temporal and geographic variation in the probability of parasite infection for these snails based on two years of sampling. Although studies indicate that digenean parasites may have important effects both on individual species and the structure of communities, there have been no studies of digenean parasitism on snails within the Everglades ecosystem. For example, the endangered Everglade Snail Kite, a specialist that feeds almost exclusively on Pomacea paludosa, and is known to be a definitive host of digenean parasites, may suffer direct and indirect effects from consumption of parasitized apple snails. Therefore, information on the diversity and abundance of parasites harbored in snail populations in the Everglades should be of considerable interest for management and conservation of wildlife. Juvenile digeneans (cercariae) representing 20 species were isolated from these two snails, representing a quadrupling of the number of species known. Species were characterized based on morphological, morphometric, and sequence data (18S rDNA, COI, and ITS). Species richness of shed cercariae from P. duryi was greater than P. paludosa, with 13 and 7 species respectively. These species represented 14 families. P. paludosa and P. duryi had no digenean species in common. Probability of digenean infection was higher for P. duryi than P. paludosa and adults showed a greater risk of infection than juveniles for both of these snails. Planorbella duryi showed variation in probability of infection between sampling sites and hydrological seasons. The number of unique combinations of multi-species infections was greatest among P. duryi individuals, while the overall percentage of multi-species infections was greatest in P. paludosa. Analyses of six frequently-observed multiple infections from P. duryi suggest the presence of negative interactions, positive interactions, and neutral associations between larval digeneans. These results should contribute to an understanding of the factors controlling the abundance and distribution of key species in the Everglades ecosystem and may in particular help in the management and recovery planning for the Everglade Snail Kite.

Stray dogs in Bucharest, and the political economy of Romanian socialism. From hunting the “enemies” of Stalinist economy, to poisoning urban parasites, and the rise of urban anarchy: 1950s-1990s.

My paper discusses three different ways in which stray dogs have been intertwined with ideologies of economic and urban development in Romania. I categorize results from archival and ethnographic research under three major time periods: early socialism, late socialism, and post-socialism. During early socialism stray dogs were seen to be damaging the soviet economy by killing species that humans could also hunt, like rabbits. During late socialism, stray dogs appeared as the enemies of the communist city, and the department of urban sanitation was given orders to poison dogs with strychnine. Finally, the increasing number of stray dogs in Bucharest after the collapse of communism was seen as a direct result of former communist demolitions, and was also taken as a sign of the collapsing state. Through such examples my paper discusses how the state and particular population groups have seen dogs as parts of an unwanted and dangerous nature, rather than a species that needs to be protected. I argue that distinctions of nature and culture have served discourses of civilization and the view of Bucharest as a model socialist, and then European city. Throughout my paper I juxtapose the treatment of stray dogs with other, more “valued” urban natures like the protection of parks, the wide-spread hobby of pigeon breeding during socialist years, the most recent debate on saving the rural area of Rosia Montana from non-environmentally friendly methods of gold extraction, and the current trend of healthy eating and living.

Structural and Biochemical Dissection of the Trehalose Biosynthetic Complex in Pathogenic Fungi

Trehalose is a non-reducing disaccharide essential for pathogenic fungal survival and virulence. The biosynthesis of trehalose requires the trehalose-6-phosphate synthase, Tps1, and trehalose-6-phosphate phosphatase, Tps2. More importantly, the trehalose biosynthetic pathway is absent in mammals, conferring this pathway as an ideal target for antifungal drug design. However, lack of germane biochemical and structural information hinders antifungal drug design against these targets.

In this dissertation, macromolecular X-ray crystallography and biochemical assays were employed to understand the structures and functions of proteins involved in the trehalose biosynthetic pathway. I report here the first eukaryotic Tps1 structures from Candida albicans (C. albicans) and Aspergillus fumigatus (A. fumigatus) with substrates or substrate analogs. These structures reveal the key residues involved in substrate binding and catalysis. Subsequent enzymatic assays and cellular assays highlight the significance of these key Tps1 residues in enzyme function and fungal stress response. The Tps1 structure captured in its transition-state with a non-hydrolysable inhibitor demonstrates that Tps1 adopts an “internal return like” mechanism for catalysis. Furthermore, disruption of the trehalose biosynthetic complex formation through abolishing Tps1 dimerization reveals that complex formation has regulatory function in addition to trehalose production, providing additional targets for antifungal drug intervention.

I also present here the structure of the Tps2 N-terminal domain (Tps2NTD) from C. albicans, which may be involved in the proper formation of the trehalose biosynthetic complex. Deletion of the Tps2NTD results in a temperature sensitive phenotype. Further, I describe in this dissertation the structures of the Tps2 phosphatase domain (Tps2PD) from C. albicans, A. fumigatus and Cryptococcus neoformans (C. neoformans) in multiple conformational states. The structures of the C. albicans Tps2PD -BeF3-trehalose complex and C. neoformans Tps2PD(D24N)-T6P complex reveal extensive interactions between both glucose moieties of the trehalose involving all eight hydroxyl groups and multiple residues of both the cap and core domains of Tps2PD. These structures also reveal that steric hindrance is a key underlying factor for the exquisite substrate specificity of Tps2PD. In addition, the structures of Tps2PD in the open conformation provide direct visualization of the conformational changes of this domain that are effected by substrate binding and product release.

Last, I present the structure of the C. albicans trehalose synthase regulatory protein (Tps3) pseudo-phosphatase domain (Tps3PPD) structure. Tps3PPD adopts a haloacid dehydrogenase superfamily (HADSF) phosphatase fold with a core Rossmann-fold domain and a α/β fold cap domain. Despite lack of phosphatase activity, the cleft between the Tps3PPD core domain and cap domain presents a binding pocket for a yet uncharacterized ligand. Identification of this ligand could reveal the cellular function of Tps3 and any interconnection of the trehalose biosynthetic pathway with other cellular metabolic pathways.

Combined, these structures together with significant biochemical analyses advance our understanding of the proteins responsible for trehalose biosynthesis. These structures are ready to be exploited to rationally design or optimize inhibitors of the trehalose biosynthetic pathway enzymes. Hence, the work described in this thesis has laid the groundwork for the design of Tps1 and Tps2 specific inhibitors, which ultimately could lead to novel therapeutics to treat fungal infections.

Diffusible Signal Factor (DSF)-dependent quorum sensing in pathogenic bacteria and its exploitation for disease control

Cell-to-cell signals of the Diffusible Signal Factor (DSF) family are cis-2-unsaturated fatty acids of differing chain length and branching pattern. DSF signalling has been described in diverse bacteria to include plant and human pathogens where it acts to regulate functions such as biofilm formation, antibiotic tolerance and the production of virulence factors. DSF family signals can also participate in interspecies signalling with other bacteria and interkingdom signaling such as with the yeast Candida albicans. Interference with DSF signalling may afford new opportunities for the control of bacterial disease. Such strategies will depend in part on detailed knowledge of the molecular mechanisms underlying the processes of signal synthesis, perception and turnover. Here, I review both recent progress in understanding DSF signalling at the molecular level and prospects for translating this knowledge into approaches for disease control.

Fork lengths, stomach contents and parasites of ninespine stickleback and arctic char in Iqalugaajuruluit Lake, Canada

Stable d13C and d15N isotopes, diet and parasites demonstrated that the prey consumed by ninespine stickleback Pungitius pungitius in a small lake on Baffin Island changed during the summer and also revealed intraspecific variation in their ecological niche. In July, there were differences in the diets of male and female ninespine stickleback as indicated by the stable isotopes, differences corroborated by the data on diet composition and the parasite fauna. Differences suggested that the sexes occupied different habitats during spawning. During July, females utilise the shallower littoral areas consuming zooplankton and benthic organisms, while males occupy deeper areas of the littoral zone feeding mainly on pelagic zooplankton. Parasite data support these observations as males had higher infections of copepod-transmitted parasites than females. There appeared to be no segregation of resources between males and females in late August, although the diet of both male and female ninespine stickleback shifted towards more benthic organisms, compared with July. Differences in d13C isotope, diet composition and infections of co-occurring parasites demonstrated that sympatric ninespine stickleback and Arctic char Salvelinus alpinus captured in the littoral zone occupied separate niches. Ninespine stickleback preyed mainly on zooplankton and chironomids, while Arctic char consumed a greater variety of prey items, including zooplankton and larger-sized prey such as insects and ninespine stickleback. The multifaceted approach improved our understanding of the trophic ecology of ninespine stickleback in southern Baffin Island and quantified resource use and dietary overlap with Arctic char.

Identification of potentially pathogenic variants in candidate breast and ovarian cancer susceptibility genes in BRCAX patients

Mutations within the BRCA1 and BRCA2 genes account for approximately 20% of hereditary breast cancers, with a further 10%–15% being attributable to rare mutations in moderate-risk genes and common variants in low-risk genes. The genes harbouring mutations in the remaining ∼65% of hereditary breast cancers are unknown. The identification of mutation carriers in hereditary breast and ovarian cancer (hboc) families is critical for determining who is most at risk of developing the disease and therefore who should be offered risk-reducing procedures or more intensive screening, or both.

Many of the high- and moderate-risk genes for hereditary breast cancers encode proteins that work in concert to maintain genomic stability and in dna damage signalling and repair. A novel BRCA1 protein complex identified within the research group whose target genes are involved in dna repair provided novel candidates for hboc susceptibility genes. These 12 candidate genes were sequenced in a cohort of 675 affected individuals from the Kathleen Cunningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) with hereditary breast or ovarian cancer, but with no mutations in known susceptibility genes (BRCAx patients). This analysis identified 20 individuals (each from a different BRCAx family) with different potentially pathogenic variants across 6 of the candidate hboc susceptibility genes. The family members of each BRCAx index case were tested for the presence of the specific mutation identified in the proband to examine segregation with disease. To further expand on the potential role of the novel candidate hboc susceptibility genes identified in this study, the genetic variation of a second cohort of 520 Northern Irish BRCAx patients is being characterized using a 61-gene panel.

A Loop-mediated Isothermal Amplification Method for Rapid Direct Detection and Differentiation of Non-pathogenic and Verocytotoxigenic Escherichia coli in Beef and Bovine Faeces

The aim of this study was to develop a multiplex loop-mediated isothermal amplification (LAMP) method capable of detecting Escherichia coli generally and verocytotoxigenic E. coli (VTEC) specifically in beef and bovine faeces. The LAMP assay developed was highly specific (100%) and able to distinguish between E. coli and VTEC based on the amplification of the phoA, and stx1 and/or stx2 genes, respectively. In the absence of an enrichment step, the limit of detection 50% (LOD50) of the LAMP assay was determined to be 2.83, 3.17 and 2.83-3.17 log CFU/g for E. coli with phoA, stx1 and stx2 genes, respectively, when artificially inoculated minced beef and bovine faeces were tested. The LAMP calibration curves generated with pure cultures, and spiked beef and faeces, suggested that the assay had good quantification capability. Validation of the assay, performed using retail beef and bovine faeces samples, demonstrated good correlation between counts obtained by the LAMP assay and by a conventional culture method, but suggested the possibility of false negative LAMP results for 12.5-14.7% of samples tested. The multiplex LAMP assay developed potentially represents a rapid alternative to culture for monitoring E.coli levels in beef or faeces and it would provide additional information on the presence of VTEC. However, some further optimisation is needed to improve detection sensitivity.

Endonuclease Controlled Aggregation of Gold Nanoparticles for the Ultrasensitive Detection of Pathogenic Bacterial DNA

The development of an ultrasensitive biosensor for the low-cost and on-site detection of pathogenic DNA could transform detection capabilities within food safety, environmental monitoring and clinical diagnosis. Herein, we present an innovative approach exploiting endonuclease-controlled aggregation of plasmonic gold nanoparticles (AuNPs) for label-free and ultrasensitive detection of bacterial DNA. The method utilizes RNA-functionalized AuNPs which form DNA-RNA heteroduplex structures through specific hybridization with target DNA. Once formed, the DNA-RNA heteroduplex is susceptible to RNAse H enzymatic cleavage of the RNA probe, allowing the target DNA to liberate and hybridize with another RNA probe. This continuously happens until all of the RNA probes are cleaved, leaving the nanoparticles unprotected and thus aggregated upon exposure to a high electrolytic medium. The assay is ultrasensitive, allowing the detection of target DNA at femtomolar level by simple spectroscopic analysis (40.7 fM and 2.45 fM as measured by UV-vis and dynamic light scattering (DLS), respectively). The target DNA spiked food matrix (chicken meat) is also successfully detected at a concentration of 1.2 pM (by UV-vis) or 18.0 fM (by DLS). In addition to the ultra-high sensitivity, the total analysis time of the assay is less than 3 hours, thus demonstrating its practicality for food analysis.

Pathogenic effect of microorganisms on loggerhead eggs

[EN] Different types of fungi and bacteria have been isolated from hatched and non-hatched as well as failed and non-failed eggs in natural sea turtles nests (Marco et al. 2006, Phillott and Parmenter, 2001, Phillott et al. 2001). Microbiota infections are common in artificial incubation activities and they seem to have an important negative impact on embryo development (Phillott, 2002). However, no clear evidences of their pathogenic effects have been described. The aim of this study was to investigate whether fungi and bacteria represent pathogenic agents to sea turtle eggs, and to assess whether there exists a specific period during incubation in which eggs are more susceptible to microorganisms.

Next-Generation Sequencing of Hereditary Hemochromatosis-Related Genes: Novel Likely Pathogenic Variants Found in the Portuguese Population

Hereditary hemochromatosis (HH) is an autosomal recessive disorder characterized by excessive iron absorption resulting in pathologically increased body iron stores. It is typically associated with common HFE gene mutation (p.Cys282Tyr and p.His63Asp). However, in Southern European populations up to one third of HH patients do not carry the risk genotypes. This study aimed to explore the use of next-generation sequencing (NGS) technology to analyse a panel of iron metabolism-related genes (HFE, TFR2, HJV, HAMP, SLC40A1, and FTL) in 87 non-classic HH Portuguese patients. A total of 1241 genetic alterations were detected corresponding to 53 different variants, 13 of which were not described in the available public databases. Among them, five were predicted to be potentially pathogenic: three novel mutations in TFR2 [two missense (p.Leu750Pro and p.Ala777Val) and one intronic splicing mutation (c.967-1G>C)], one missense mutation in HFE (p.Tyr230Cys), and one mutation in the 5'-UTR of HAMP gene (c.-25G>A). The results reported here illustrate the usefulness of NGS for targeted iron metabolism-related gene panels, as a likely cost-effective approach for molecular genetics diagnosis of non-classic HH patients. Simultaneously, it has contributed to the knowledge of the pathophysiology of those rare iron metabolism-related disorders.

Natural colonization of loggerhead turtle eggs by the pathogenic fungus Fusarium oxysporum

[EN] Sea turtle nests are exposed to different environmental risks that may affect their hatching success. Human exploitation, predation by wild or domestic animals, nest flooding or severe beach erosion or accession are common causes of egg mortality. However, there is very little information about the impact of microorganisms on turtle eggs. We analyzed loggerhead turtle eggs from Boavista Island (Republic of Cabo Verde) which were incubated under different environmental conditions in order to evaluate the presence and impact of fungus. We have isolated Fusarium oxysporum from dead and live eggs after three days of incubation.

Potential pathogenic fungi assessment through molecular biology in cork industry

Portugal has been the world leader in the cork sectr in terms of exports, employing ten thousands of workers. In this working activity, the permanent contact with cork may lead to the exposure to fungi raising concerns as occupational hazards in cork industry. A study was developed aiming at assessing fungal contamination due to Aspergillus fumigatus complex and Penicillium glabrum complex by molecular methods in three cork industries in the outskirt of Lisbon city. The chosen fungal species are the ones most frequently associated with respiratory problems in workers from these industries.

Apport d'un programme de génétique à une filière de production aquacole : l'exemple de l'ostréiculture

Two oyster species are currently present along the French coasts : the indigenous European flat oyster (Ostrea edulis), and the Pacific cupped oyster (Crassostrea gigas), that has been introduced from Japan since the beginning of the 70ies. The flat oyster successively suffered from two protozoan diseases during the 60ies and its production decreased from 20 000 tons/year by that time to 1 500 tons/year nowadays. Consequently, the oyster production is principally (99%) based upon the Pacific oyster species with approximately 150 000 tons/year among which 90% are grown from the natural spat. However, the hatchery production of this species is developing and was estimated to 400 to 800 millions spat in 2002. Moreover, strengthened relationships between IFREMER and the 5 commercial hatcheries, that all joined the SYSAAF (Union of the French poultry, shellfish and fish farming selectors), allow to plan for new genetic breeding programs. At the end of the 80ies, IFREMER initiated a genetic breeding program for the resistance of the European flat oyster to the bonamiosis, and obtained strains more tolerant to this disease. After two generations of massal selection, molecular markers had identified a reduced genetic basis in this program. It was then reoriented to an intra-familial selection. However, we were confronted to a zootechnic problem to manage such a scheme and we compromised by an intra-cohorts of families selection scheme managed using molecular markers. The program has now reached the transfer level with experimentation at a professional scale. Concerning the Pacific cupped oyster, and in parallel with the obtaining and the study of polyploids, performance of different Asian cupped oyster strains were compared to the one introduced in France thirty years ago and currently suffering from summer mortalities. The local strain exhibited better performance, certainly based upon a good local adaptation. In other respects, although early growth is a relevant criteria for selection for growth to commercial stage, it is not to be privileged in the context of an oyster producing region with a limited food availability. Contrary, the spat summer mortality became a priority for numerous teams (genetic, physiology, pathology, ecology,...) joined in the MOREST program. The first results showed important survival differences between fullsib and halsib families. They indicate a genetic determinism to this character "survival" and promote for its selection.