Hazelnut Allergens: Molecular Characterisation, Detection and Clinical Relevance

In the last years, special attention has been devoted to food-induced allergies, from which hazelnut allergy is highlighted. Hazelnut is one of the most commonly consumed tree nuts, being largely used by the food industry in a wide variety of processed foods. It has been regarded as a food with potential health benefits, but also as a source of allergens capable of inducing mild to severe allergic reactions in sensitised individuals. Considering the great number of reports addressing hazelnut allergens, with an estimated increasing trend, this review intends to assemble all the relevant information available so far on the main issues: prevalence of tree nut allergy, clinical threshold levels, molecular characterisation of hazelnut allergens (Cor a 1, Cor a 2, Cor a 8, Cor a 9, Cor a 10, Cor a 11, Cor a 12, Cor a 14 and Cor a TLP) and their clinical relevance, and methodologies for hazelnut allergen detection in foods. A comprehensive overview on the current data about the molecular characterisation of hazelnut allergens is presented, relating biochemical classification and biological function with clinical importance. Recent advances on hazelnut allergen detection methodologies are summarised and compared, including all the novel protein- and DNA-based approaches.

Frequency and molecular characterisation of Entamoeba histolytica , Entamoeba dispar , Entamoeba moshkovskii , and Entamoeba hartmanni in the context of water scarcity in northeastern Brazil

This study aimed to estimate the frequency, associated factors, and molecular characterisation of Entamoeba histolytica , Entamoeba dispar, Entamoeba moshkovskii , and Entamoeba hartmanni infections. We performed a survey (n = 213 subjects) to obtain parasitological, sanitation, and sociodemographic data. Faecal samples were processed through flotation and centrifugation methods. E. histolytica, E. dispar, E. moshkovskii, and E. hartmanni were identified by nested-polymerase chain reaction (PCR). The overall prevalence of infection was 22/213 (10.3%). The infection rate among subjects who drink rainwater collected from roofs in tanks was higher than the rate in subjects who drink desalinated water pumped from wells; similarly, the infection rate among subjects who practice open defecation was significantly higher than that of subjects with latrines. Out of the 22 samples positive for morphologically indistinguishable Entamoeba species, the differentiation by PCR was successful for 21. The species distribution was as follows: 57.1% to E. dispar, 23.8% to E. histolytica, 14.3% to E. histolytica and E. dispar, and 4.8% E. dispar and E. hartmanni. These data suggest a high prevalence of asymptomatic infection by the group of morphologically indistinguishable Entamoeba histolytica/dispar/moshkovskii complex and E. hartmanni species. In this context of water scarcity, the sanitary and socioenvironmental characteristics of the region appear to favour transmission.

Characterisation of six novel A-subunit mutations leading to congenital factor XIII deficiency and molecular analysis of the first diagnosed patient with this rare bleeding disorder

In 1960, the first case report on factor XIII deficiency was published describing a seven-year-old Swiss boy with a so far unknown bleeding disorder. Today, more than 60 mutations in the factor XIIIA- and B-subunit genes are known leading to congenital factor XIII deficiency. In the present study, we describe six novel mutations in the factor XIII A-subunit gene. Additionally, we present the molecular characterisation of the first described patient with congenital factor XIII deficiency. The six novel mutations include a small deletion, Glu202 delG, leading to a premature stop codon and truncation of the protein, and a splice site mutation at the exon 10/intron 10 boundary, +1G/A, giving rise to an incorrect spliced mRNA lacking exons 10 and 11. The remaining four mutations are characterised by the single amino acid changes Met159Arg, Gly215Arg, Trp375Cys, and His716Arg, and were expressed in COS-1 cells. Antigen levels and activity of the mutants were significantly reduced compared to the wild-type. The patient described in 1960 also shows a single amino acid change, Arg77Cys. Structural analysis of all mutant enzymes suggests several mechanisms leading to destabilisation of the protein.

Clinical and molecular characterization of Brazilian families with von Hippel-Lindau disease: a need for delineating genotype-phenotype correlation

von Hippel-Lindau (VHL) disease is an autosomal dominant hereditary cancer syndrome that predisposes to the development of a variety of benign and malignant tumours, especially cerebellar haemangioblastomas, retinal angiomas and clear-cell renal cell carcinomas (RCC). The etiology and manifestations are due to germline and somatic mutations in the VHL tumour suppressor gene. VHL disease is classified into type 1 and type 2, showing a clear genotype-phenotype correlation, as type 2 is associated with phaeochromocytoma and essentially caused by missense mutations. The aim of this study is to characterize the phenotype and genotype of families with VHL disease. Eighteen of twenty patients from ten unrelated families underwent genetic testing, nine of them fulfilled VHL disease criteria and one had an apparently sporadic cerebellar haemangioblastoma. Four different germline mutations in the VHL gene were identified: c.226_228delTTC (p.Phe76del); c.217C > T (p.Gln73X); IVS1-1 G > A and IVS2-1 G > C. The first three mutations were associated with type 1 disease and the last one with type 2B, which had never been identified in the germline. The transcriptional processing of a novel splice-site mutation was characterised. Three type 1 VHL families showed large deletions of the VHL gene, two of them encompassed the FANCD2/C3orf10 genes and were not associated with renal lesions. We also suggest that such families should be subclassified according to the risk of RCC and the extent of the VHL gene deletions. This study highlights the need for a through clinical and molecular characterisation of families with VHL disease to better delineate its genotype-phenotype correlation.

Molecular identification of Sporothrix species involved in the first familial outbreak of sporotrichosis in the state of Espírito Santo, southeastern Brazil

Sporotrichosis is a widespread subcutaneous mycosis caused by the dimorphic fungi now known as the Sporothrix schenckii complex. This complex is comprised of at least six species, including Sporothrix albicans, Sporothrix brasiliensis, Sporothrix globosa, Sporothrix luriei, Sporothrix mexicana and S. schenckii. Cases of sporotrichosis have significantly increased in Brazil over the past decade, especially in the state of Rio de Janeiro (RJ), where an epidemic among cat owners has been observed. The zoonotic transmission from cats to humans suggests a common source of infection and indicates that animals can act as vectors. We performed a molecular characterisation of samples collected during the first outbreak of familial sporotrichosis caused by S. brasiliensis in the state of Espírito Santo, Brazil. These results represent the first description of such an outbreak outside the endemic area of zoonotic sporotrichosis in RJ.

Characterisation of divergent flavivirus NS3 and NS5 protein sequences detected in Rhipicephalus microplus ticks from Brazil

Transcripts similar to those that encode the nonstructural (NS) proteins NS3 and NS5 from flaviviruses were found in a salivary gland (SG) complementary DNA (cDNA) library from the cattle tick Rhipicephalus microplus.Tick extracts were cultured with cells to enable the isolation of viruses capable of replicating in cultured invertebrate and vertebrate cells. Deep sequencing of the viral RNA isolated from culture supernatants provided the complete coding sequences for the NS3 and NS5 proteins and their molecular characterisation confirmed similarity with the NS3 and NS5 sequences from other flaviviruses. Despite this similarity, phylogenetic analyses revealed that this potentially novel virus may be a highly divergent member of the genus Flavivirus. Interestingly, we detected the divergent NS3 and NS5 sequences in ticks collected from several dairy farms widely distributed throughout three regions of Brazil. This is the first report of flavivirus-like transcripts inR. microplus ticks. This novel virus is a potential arbovirus because it replicated in arthropod and mammalian cells; furthermore, it was detected in a cDNA library from tick SGs and therefore may be present in tick saliva. It is important to determine whether and by what means this potential virus is transmissible and to monitor the virus as a potential emerging tick-borne zoonotic pathogen.

Molecular genetic approaches to the biology of the moss "Physcomitrella patens"

La mousse haplobiontique Physcomitrella patens est utilisée comme système génétique modèle pour l'étude du développement des plantes. Cependant, l'absence d'un protocole efficace de transformation a constitué jusqu'à présent un gros désavantage méthodologique pour le développement futur de ce système expérimental. Les résultats présentés dans le premier chapitre relatent la mise au point d'un protocole de transformation basé sur la technique de transfert direct de gènes dans des protoplastes par précipitation au PEG. Un essai d'expression transitoire de gènes a été mis au point. Ce protocole a été adapté afin de permettre l'introduction in vivo d'anticorps dans des protoplastes. Le protocole modifié permet d'introduire simultanément du DNA et des IgG dans les cellules, et nous avons démontré que ces anticorps peuvent inactiver spécifiquement le produit d'un gène co-introduit (GUS), ainsi que certaines protéines impliquées dans des processus cellulaires (tubuline). Cet essai, baptisé "essai transitoire d'immuno-inactivation in vivo", devrait être directement applicable à d'autres protoplastes végétaux, et permettre l'élaboration de nouvelles stratégies dans l'étude de processus cellulaires. Le second chapitre est consacré aux expériences de transformation de la mousse avec des gènes conférant une résistance à des antibiotiques. Nos résultats démontrent que l'intégration de gènes de résistance dans le génome de P. patens est possible, mais que cet événement est rare. Il s'agit là néanmoins de la première démonstration d'une transformation génétique réussie de cet organisme. L'introduction de gènes de résistance aux antibiotiques dans les protoplastes de P. patens génère à haute fréquence des clones résistants instables. Deux classes de clones instables ont été identifiés. La caractérisation phénotypique, génétique et moléculaire de ces clones suggère fortement que les séquences transformantes sont concaténées pour former des structures de haut poids moléculaire, et que ces structures sont efficacement répliquées et maintenues dans les cellules résistantes en tant qu'éléments génétiques extrachromosomaux. Ce type de transformation nous permet d'envisager des expériences permettant l'identification des séquences génomiques impliquées dans la replication de l'ADN de mousse. Plusieurs lignées transgéniques ont été retransformées avec des plasmides portant des séquences homologues aux séquences intégrées dans le génome, mais conférant une résistance à un autre antibiotique. Les résultats présentés dans le troisième chapitre montrent que les fréquences de transformation intégrative dans les lignées transgéniques sont 10 fois plus élevées que dans la lignée sauvage, et que cette augmentation est associée à une coségrégation des gènes de résistance dans la plupart des clones testés. Ces résultats génétiques indiquent que l'intégration de séquences d'ADN étranger dans le génome de P. patens a lieu en moyenne 10 fois plus fréquemment par recombinaison homologue que par intégration aléatoire. Ce rapport homologue/aléatoire est 10000 fois supérieur aux rapports obtenus avec d'autres plantes, et fournit l'outil indispensable à la réalisation d'expériences de génétique inverse dans cet organisme à haplophase dominante. THESIS SUMMARY The moss Physcomitrella patens is used as a model genetic system to study plant development, taking advantage of the fact that the haploid gametophyte dominates in its life cycle. But further development of this model system was hampered by the lack of a protocol allowing the genetic transformation of this plant. We have developed a transformation protocol based on PEG-mediated direct gene transfer to protoplasts. Our data demonstrate that this procedure leads to the establishment of an efficient transient gene expression assay. A slightly modified protocol has been developed allowing the in vivo introduction of antibodies in moss protoplasts. Both DNA and IgGs can be loaded simultaneously, and specific antibodies can immunodeplete the product of an expression cassette (GUS) as well as proteins involved in cellular processes (tubulins). This assay, named transient in vivo immunodepletion assay, should be applicable to other plant protoplasts, and offers new approaches to study cellular processes. Transformations have been performed with bacterial plasmids carrying antibiotic resistance expression cassette. Our data demonstrate that integrative transformation occurs, but at low frequencies. This is the first demonstration of a successful genetic transformation of mosses. Resistant unstable colonies are recovered at high frequencies following transformation, and two different classes of unstable clones have been identified. Phenotypical, genetic and molecular characterisation of these clones strongly suggests that bacterial plasmids are concatenated to form high molecular arrays which are efficiently replicated and maintained as extrachromosomal elements in the resistant cells. Replicative transformation in P. patens should allow the design of experiments aimed at the identification of genomic sequences involved in moss DNA replication. Transgenic strains have been retransformed with bacterial plasmids carrying sequences homologous to the integrated transloci, but conferring resistance to another antibiotic. Our results demonstrate an order of magnitude increase of integrative transformation frequencies in transgenic strains as compared to wild-type, associated with cosegregation of the resistance genes in most of these double resistant transgenic strains. These observations provide strong genetic evidence that gene targeting occurs about ten times more often than random integration in the genome of P. patens. Such ratio of targeted to random integration is about 10 000 times higher than previous reports of gene targeting in plants, and provides the essential requirement for the development of efficient reverse genetics in the haplodiplobiontic P. patens.

Caracterização molecular de isolados de Paracoccidioides brasiliensis obtidos de Dasypus novemcinctus (tatu) e sua correlação com virulência e origem geográfica

Pós-graduação em Patologia - FMB

Characterisation of chlorate reduction in the haloarchaeon Haloferax mediterranei

Background: Haloferax mediterranei is a denitrifying haloarchaeon using nitrate as a respiratory electron acceptor under anaerobic conditions in a reaction catalysed by pNarGH. Other ions such as bromate, perchlorate and chlorate can also be reduced. Methods: Hfx. mediterranei cells were grown anaerobically with nitrate as electron acceptor and chlorate reductase activity measured in whole cells and purified nitrate reductase. Results: No genes encoding (per)chlorate reductases have been detected either in the Hfx. mediterranei genome or in other haloarchaea. However, a gene encoding a chlorite dismutase that is predicted to be exported across the cytoplasmic membrane has been identified in Hfx. mediterranei genome. Cells did not grow anaerobically in presence of chlorate as the unique electron acceptor. However, cells anaerobically grown with nitrate and then transferred to chlorate-containing growth medium can grow a few generations. Chlorate reduction by the whole cells, as well as by pure pNarGH, has been characterised. No clear chlorite dismutase activity could be detected. Conclusions: Hfx. mediterranei pNarGH has its active site on the outer-face of the cytoplasmic membrane and reacts with chlorate and perchlorate. Biochemical characterisation of this enzymatic activity suggests that Hfx. mediterranei or its pure pNarGH could be of great interest for waste water treatments or to better understand biological chlorate reduction in early Earth or Martian environments. General significance: Some archaea species reduce (per)chlorate. However, results here presented as well as those recently reported by Liebensteiner and co-workers [1] suggest that complete perchlorate reduction in archaea follows different rules in terms of biological reactions.

Focused molecular analysis of small cell lung cancer: feasibility in routine clinical practice.

Background: There is an urgent need to identify molecular signatures in small cell lung cancer (SCLC) that may select patients who are likely to respond to molecularly targeted therapies. In this study, we investigate the feasibility of undertaking focused molecular analyses on routine diagnostic biopsies in patients with SCLC.

Methods: A series of histopathologically confirmed formalin-fixed, paraffin-embedded SCLC specimens were analysed for epidermal growth factor receptors (EGFR), KRAS, NRAS and BRAF mutations, ALK gene rearrangements and MET amplification. EGFR and KRAS mutation testing was evaluated using real time polymerase chain reaction (RT-PCR cobas®), BRAF and NRAS mutations using multiplex PCR and capillary electrophoresis-single strand conformation analysis, and ALK and MET aberrations with fluorescent in situ hybridization. All genetic aberrations detected were validated independently.

Results: A total of 105 patients diagnosed with SCLC between July 1990 and September 2006 were included. 60 (57 %) patients had suitable tumour tissue for molecular testing. 25 patients were successfully evaluated for all six pre-defined molecular aberrations. Eleven patients failed all molecular analysis. No mutations in EGFR, KRAS and NRAS were detected, and no ALK gene rearrangements or MET gene amplifications were identified. A V600E substitution in BRAF was detected in a Caucasian male smoker diagnosed with SCLC with squamoid and glandular features.

Conclusion: The paucity of patients with sufficient tumour tissue, quality of DNA extracted and low frequency of aberrations detected indicate that alternative molecular characterisation approaches are necessary, such as the use of circulating plasma DNA in patients with SCLC.

Development and characterisation of a neurogenic model of brain cancer

Primary glioblastoma (GB), the most common and aggressive adult brain tumour, is refractory to conventional therapies and characterised by poor prognosis. GB displays striking cellular heterogeneity, with a sub-population, called Glioblastoma Stem Cells (GSCs), intrinsically resistant to therapy, hence the high rate of recurrence. Alterations of the tumour suppressor gene PTEN are prevalent in primary GBM, resulting in the inhibition of the polarity protein Lgl1 due to aPKC hyperactivation. Dysregulation of this molecular axis is one of the mechanisms involved in GSC maintenance. After demonstrating that the PTEN/aPKC/Lgl axis is conserved in Drosophila, I deregulated it in different cells populations of the nervous system in order to individuate the cells at the root of neurogenic brain cancers. This analysis identified the type II neuroblasts (NBs) as the most sensitive to alterations of this molecular axis. Type II NBs are a sub-population of Drosophila stem cells displaying a lineage similar to that of the mammalian neural stem cells. Following aPKC activation in these stem cells, I obtained an adult brain cancer model in Drosophila that summarises many phenotypic traits of human brain tumours. Fly tumours are indeed characterised by accumulation of highly proliferative immature cells and keep growing in the adult leading the affected animals to premature death. With the aim to understand the role of cell polarity disruption in this tumorigenic process I carried out a molecular characterisation and transcriptome analysis of brain cancers from our fly model. In summary, the model I built and partially characterised in this thesis work may help deepen our knowledge on human brain cancers by investigating many different aspects of this complicate disease.

Human pigmentation genes: identification, structure and consequences of polymorphic variation

The synthesis of the visible pigment melanin by the melanocyte cell is the basis of the human pigmentary system, those genes directing the formation, transport and distribution of the specialised melanosome organelle in which melanin accumulates can legitimately be called pigmentation genes. The genes involved in this process have been identified through comparative genomic studies of mouse coat colour mutations and by the molecular characterisation of human hypopigmentary genetic diseases such as OCA1 and OCA2. The melanocyte responds to the peptide hormones a-MSH or ACTH through the MC1R G-protein coupled receptor to stimulate melanin production through induced maturation or switching of melanin type. The pheomelanosome, containing the key enzyme of the pathway tyrosinase, produces light red/yellowish melanin, whereas the eumelanosome produces darker melanins via induction of additional TYRP1, TYRP2, SILV enzymes, and the P-protein. Intramelanosomal pH governed by the P-protein may act as a critical determinant of tyrosinase enzyme activity to control the initial step in melanin synthesis or TYRP complex formation to facilitate melanogenesis and melanosomal maturation. The search for genetic variation in these candidate human pigmentation genes in various human populations has revealed high levels of polymorphism in the MC1R locus, with over 30 variant alleles so far identified. Functional correlation of MC1R alleles with skin and hair colour provides evidence that this receptor molecule is a principle component underlying normal human pigment variation. (C) 2001 Elsevier Science B.V. All rights reserved.

Distinction between familial and sporadic forms of colorectal cancer showing DNA microsatellite instability

Attempts to classify colorectal cancer into subtypes based upon molecular characterisation are overshadowed by the classical stepwise model in which the adenoma-carcinoma sequence serves as the morphological counterpart. Clarity is achieved when cancers showing DNA microsatellite instability (MSI) are distinguished as sporadic MSI-low (MSI-L), sporadic MSI-high (MSI-H) and hereditary non-polyposis colorectal cancer (HNPCC). Divergence of the 'methylator' pathway into MSI-L and MSI-H is at least partly determined by the respective silencing of MGMT and hMLH1. Multiple differences can be demonstrated between sporadic and familial (HNPCC) MSI-H colorectal cancer with respect to early mechanisms, evolution, molecular characterisation, demographics and morphology. By acknowledging the existence of multiple pathways, rapid advances in the fields of basic and translational research will occur and this will lead to improved strategies for the prevention, early detection and treatment of colorectal cancer. (C) 2002 Elsevier Science Ltd. All rights reserved.