Diagnostic value of H3F3A mutations in giant cell tumour of bone compared to osteoclast-rich mimics

Driver mutations in the two histone 3.3 (H3.3) genes, H3F3A and H3F3B, were recently identified by whole genome sequencing in 95% of chondroblastoma (CB) and by targeted gene sequencing in 92% of giant cell tumour of bone (GCT). Given the high prevalence of these driver mutations, it may be possible to utilise these alterations as diagnostic adjuncts in clinical practice. Here, we explored the spectrum of H3.3 mutations in a wide range and large number of bone tumours (n 5 412) to determine if these alterations could be used to distinguish GCT from other osteoclast-rich tumours such as aneurysmal bone cyst, nonossifying fibroma, giant cell granuloma, and osteoclast-rich malignant bone tumours and others. In addition, we explored the driver landscape of GCT through whole genome, exome and targeted sequencing (14 gene panel). We found that H3.3 mutations, namely mutations of glycine 34 in H3F3A, occur in 96% of GCT. We did not find additional driver mutations in GCT, including mutations in IDH1, IDH2, USP6, TP53. The genomes of GCT exhibited few somatic mutations, akin to the picture seen in CB. Overall our observations suggest that the presence of H3F3A p.Gly34 mutations does not entirely exclude malignancy in osteoclast-rich tumours. However, H3F3A p.Gly34 mutations appear to be an almost essential feature of GCT that will aid pathological evaluation of bone tumours, especially when confronted with small needle core biopsies. In the absence of H3F3A p.Gly34 mutations, a diagnosis of GCT should be made with caution.

Role on serine protease inhibitors in the pathogenisis of Steinernema carpocapsae

Tese de Doutoramento, Biologia (Biologia Celular e Molecular), 18 de Novembro de 2013, Universidade dos Açores.

Potencial da diversidade microbiana dos Açores na saúde : screening, purificação e caracterização de uma enzima trombolítica

Dissertação de Mestrado, Mestrado em Ciências Biomédicas, 8 de Maio de 2015, Universidade dos Açores.

Celiac disease diagnosis and gluten-free food analytical control

Celiac disease (CD) is an autoimmune enteropathy, characterized by an inappropriate T-cell-mediated immune response to the ingestion of certain dietary cereal proteins in genetically susceptible individuals. This disorder presents environmental, genetic, and immunological components. CD presents a prevalence of up to 1% in populations of European ancestry, yet a high percentage of cases remain underdiagnosed. The diagnosis and treatment should be made early since untreated disease causes growth retardation and atypical symptoms, like infertility or neurological disorders. The diagnostic criteria for CD, which requires endoscopy with small bowel biopsy, have been changing over the last few decades, especially due to the advent of serological tests with higher sensitivity and specificity. The use of serological markers can be very useful to rule out clinical suspicious cases and also to help monitor the patients, after adherence to a gluten-free diet. Since the current treatment consists of a life-long glutenfree diet, which leads to significant clinical and histological improvement, the standardization of an assay to assess in an unequivocal way gluten in gluten-free foodstuff is of major importance.

Molecular epidemiology of Aspergillus collected from cystic fibrosis patients

Background - Aspergillus respiratory infection is a common complication in cystic fibrosis (CF) and is associated with loss of pulmonary function and allergic disease. Methods - Fifty-three Aspergillus isolates recovered from CF patients were identified to species by Internal Transcribed Spacer Region (ITS), β-tubulin, and calmodulin sequencing. Results - Three species complexes (Terrei, Nigri, and Fumigati) were found. Identification to species level gave a single Aspergillus terreus sensu stricto, one Aspergillus niger sensu stricto and 51 Aspergillus fumigatus sensu stricto isolates. No cryptic species were found. Conclusions - To our knowledge, this is the first prospective study of Aspergillus species in CF using molecular methods. The paucity of non-A. fumigatus and of cryptic species of A. fumigatus suggests a special association of A. fumigatus sensu stricto with CF airways, indicating it likely displays unique characteristics making it suitable for chronic residence in that milieu. These findings could refine an epidemiologic and therapeutic approach geared to this pathogen.

Determination of ochratoxin A in bread: evaluation of microwave-assisted extraction using an orthogonal composite design coupled with response surface methodology

An analytical method using microwave-assisted extraction (MAE) and liquid chromatography (LC) with fluorescence detection (FD) for the determination of ochratoxin A (OTA) in bread samples is described. A 24 orthogonal composite design coupled with response surface methodology was used to study the influence of MAE parameters (extraction time, temperature, solvent volume, and stirring speed) in order to maximize OTA recovery. The optimized MAE conditions were the following: 25 mL of acetonitrile, 10 min of extraction, at 80 °C, and maximum stirring speed. Validation of the overall methodology was performed by spiking assays at five levels (0.1–3.00 ng/g). The quantification limit was 0.005 ng/g. The established method was then applied to 64 bread samples (wheat, maize, and wheat/maize bread) collected in Oporto region (Northern Portugal). OTAwas detected in 84 % of the samples with a maximum value of 2.87 ng/g below the European maximum limit established for OTA in cereal products of 3 ng/g.

Specific label-free and real-time detection of oxidized low density lipoprotein (oxLDL) using an immunosensor with three monoclonal antibodies

Increased levels of plasma oxLDL, which is the oxidized fraction of Low Density Lipoprotein (LDL), are associated with atherosclerosis, an inflammatory disease, and the subsequent development of severe cardiovascular diseases that are today a major cause of death in modern countries. It is therefore important to find a reliable and fast assay to determine oxLDL in serum. A new immunosensor employing three monoclonal antibodies (mAbs) against oxLDL is proposed in this work as a quick and effective way to monitor oxLDL. The oxLDL was first employed to produce anti-oxLDL monoclonal antibodies by hybridoma cells that were previously obtained. The immunosensor was set-up by selfassembling cysteamine (Cyst) on a gold (Au) layer (4 mm diameter) of a disposable screen-printed electrode. Three mAbs were allowed to react with N-hydroxysuccinimide (NHS) and ethyl(dimethylaminopropyl)carbodiimide (EDAC), and subsequently incubated in the Au/Cys. Albumin from bovine serum (BSA) was immobilized further to ensure that other molecules apart from oxLDL could not bind to the electrode surface. All steps were followed by various characterization techniques such as electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV). The analytical operation of the immunosensor was obtained by incubating the sensing layer of the device in oxLDL for 15 minutes, prior to EIS and SWV. This was done by using standard oxLDL solutions prepared in foetal calf serum, in order to simulate patient's plasma with circulating oxLDL. A sensitive response was observed from 0.5 to 18.0 mg mL 1 . The device was successfully applied to determine the oxLDL fraction in real serum, without prior dilution or necessary chemical treatment. The use of multiple monoclonal antibodies on a biosensing platform seemed to be a successful approach to produce a specific response towards a complex multi-analyte target, correlating well with the level of oxLDL within atherosclerosis disease, in a simple, fast and cheap way.

Detecção e Quantificação de Neurónios em C.elegans

Nesta dissertação é apresentado um estudo dos sistemas de processamento automático de imagem em contexto de um problema relacionado com a individualização de neurónios em imagens da nematoda C. elegans durante estudos relacionados com a doença de Parkinson. Apresenta-se uma breve introdução à anatomia do verme, uma introdução à doença de Parkinson e uso do C. elegans em estudos relacionados e também é feita a análise de artigos em contexto de processamento de imagem para contextualizar a situação atual de soluções para o problema de extração de características e regiões específicas. Neste projeto é desenvolvida uma pipeline com o auxilio do software CellProfiler para procurar uma resposta para o problema em questão.

In what extent can a segmented packaging make children choose healthier breakfast cereals?

Field lab in marketing: Children consumer behaviour

The retinoid-related orphan receptor RORα promotes keratinocyte differentiation via FOXN1.

RORα is a retinoid-related orphan nuclear receptor that regulates inflammation, lipid metabolism, and cellular differentiation of several non-epithelial tissues. In spite of its high expression in skin epithelium, its functions in this tissue remain unclear. Using gain- and loss-of-function approaches to alter RORα gene expression in human keratinocytes (HKCs), we have found that this transcription factor functions as a regulator of epidermal differentiation. Among the 4 RORα isoforms, RORα4 is prominently expressed by keratinocytes in a manner that increases with differentiation. In contrast, RORα levels are significantly lower in skin squamous cell carcinoma tumors (SCCs) and cell lines. Increasing the levels of RORα4 in HKCs enhanced the expression of structural proteins associated with early and late differentiation, as well as genes involved in lipid barrier formation. Gene silencing of RORα impaired the ability of keratinocytes to differentiate in an in vivo epidermal cyst model. The pro-differentiation function of RORα is mediated at least in part by FOXN1, a well-known pro-differentiation transcription factor that we establish as a novel direct target of RORα in keratinocytes. Our results point to RORα as a novel node in the keratinocyte differentiation network and further suggest that the identification of RORα ligands may prove useful for treating skin disorders that are associated with abnormal keratinocyte differentiation, including cancer.

Promise for plant pest control: root-associated pseudomonads with insecticidal activities.

Insects are an important and probably the most challenging pest to control in agriculture, in particular when they feed on belowground parts of plants. The application of synthetic pesticides is problematic owing to side effects on the environment, concerns for public health and the rapid development of resistance. Entomopathogenic bacteria, notably Bacillus thuringiensis and Photorhabdus/Xenorhabdus species, are promising alternatives to chemical insecticides, for they are able to efficiently kill insects and are considered to be environmentally sound and harmless to mammals. However, they have the handicap of showing limited environmental persistence or of depending on a nematode vector for insect infection. Intriguingly, certain strains of plant root-colonizing Pseudomonas bacteria display insect pathogenicity and thus could be formulated to extend the present range of bioinsecticides for protection of plants against root-feeding insects. These entomopathogenic pseudomonads belong to a group of plant-beneficial rhizobacteria that have the remarkable ability to suppress soil-borne plant pathogens, promote plant growth, and induce systemic plant defenses. Here we review for the first time the current knowledge about the occurrence and the molecular basis of insecticidal activity in pseudomonads with an emphasis on plant-beneficial and prominent pathogenic species. We discuss how this fascinating Pseudomonas trait may be exploited for novel root-based approaches to insect control in an integrated pest management framework.

Recueil de chants royaux, ballades et rondeaux, composés au puy de Rouen ; précédé d'un prologue adressé à Guillaume « Cretin » par « JAQUES LE LYEUR ».

Contient : 1 « Le riche don d'amoureuse mercy ». « Me NICOLE OSMONT » ; 2 « En vray amour il n'est riens impossible ». « Me JAQUES LE LYEUR » ; 3 « L'arbre de vie en l'isle fortunée ». « Me JEHAN ALYNE » ; 4 « Le sainct desert plain de manne angelique ». « Damp NICOLE LESCARRE » ; 5 « Le grant decret d'auctorité divine ». « Me JAQUES FILLASTRE » ; 6 « Le restaurant qui pour mort rend la vie ». « COURNILLE, alias TOURMENTE » ; 7 « Romme où se tient le sainct pape Innocent ». « Damp NICOLE LESCARRE » ; 8 « Le bien de paix aux humains necessaire ». « Me NICOLE DU PUYS » ; 9 « Dessus la loy d'humaine congnoissance ». « Me YSAMBERT BUSQUET » ; 10 « Femme parfaicte en nature imparfaicte ». « Me GUILLAUME THIBAULD » ; 11 « La souveraine en parfaicte beaulté ». « PIERRE CRIGNON » ; 12 « Ung corps parfaict sus ordre elementaire ». « Me JAQUES LE LYEUR » ; 13 « Au fondz d'yver printemps qui rend verdure ». « Me INNOCENT TOURMENTE » ; 14 « La purité du feu elementaire ». « Me P. AVRIL » ; 15 « La digne couche où le roy reposa ». « Me CLEMENT MAROT » ; 16 « Harnoys d'espreuve au puissant roy de gloire ». « PIERRES CRIGNON » ; 17 « Le regne franc de la loy tributaire ». « Me GUILLAUME THIBAULD » ; 18 « Saine et entiere en note et escripture ». « Me CHARLES DE SAINCT GERMAIN » ; 19 « Femme en la loy et hors la loy conceue ». « Me JO. DE BEAUVOYS » ; 20 « Selon ton nom louenge te soit faicte ». « Le general DE CAEN » ; 21 « Au grant festin du cereal convive ». « Me THOMAS LE PREVOST » ; 22 « La saincte Bible où verité repose ». « Me GUILLAUME THYBAULD » ; 23 « Pour le tout beau conceue toute belle ». « Me JEHAN DE BEAUVOYS » ; 24 « Fille obtenant la grace de son pere ». « Me ADAM LAIR » ; 25 « Le noble cueur commencement de vie ». « Me JAQUES LE LYEUR » ; 26 « Triumphanment la victoire obtenue ». « GUILLAUME DE SEVYNGUEHEN » ; 27 « Le feu d'amour pour reschauffer le monde ». « Me NICOLE DU PUYS » ; 28 « Du faulx serpent la puissance a destruicte ». « Me JAQUES LE LIEUR » ; 29 « Vray Mythridat contre mordz de vipere ». « Me INNOCENT TOURMENTE » ; 1 « Toute belle en ame et corps nect ». « Me JAQUES LE LYEUR » ; 2 « La bouge plaine de salutz ». « DUPUYS » ; 3 « La main armée aux ennemys ». « Damp NICOLE LESCARRE » ; 4 « La terre rendent bled de grace ». « Me GUILLAUME THIBAULT » ; 5 « Le nombre d'or de l'an de grace ». « Me THOMAS LEPREVOST » ; 6 « Victoire sur mes ennemys ». « Me JAQUES LELYEUR » ; 7 « Pour les siens poison importable ». « Me REUIL DOUBLET » ; 8 « Entre deux vertes une meure ». « Me THOMAS LEPREVOST » ; 9 « Entre deux vertes une meure ». « Damp NICOLE LESCARRE » ; 10 « Entre imparfaictz toute parfaicte ». « Me GUILLAUME DE SEVYNGUEHEN » ; 11 « Il n'est à Dieu rien impossible ». « Me GUILLAUME THYBAULT » ; 12 « Toutes à l'oeil, mais une au cueur ». « Me THOMAS LEPREVOST » ; 13 « Grace en toy par divin plaisir ». « Damp NICOLLE LESCARRE » ; 14 « Conception plaine de grace » ; 15 « Juste balence et loyal poix ». « Damp NICOLE LESCARRE » ; 16 « La haulte tour de fortitude ». « LESCARRE » ; 17 « La franche terre du grand roy ». « JEHAN PARMENTIER » ; 18 « D'azur à trois fleurs de lys d'or ». « PIERRE CRIGNON » ; 19 « Pour humains lyez deslier ». « Me JAQUES LE LIEUR » ; 20 « Corde l'homme et Dieu accordant ». « LIEUR » ; 21 « Ne craignez plus, la beste est prise ». « Me JAQUES LE LIEUR » ; 1 « Seule sans cy ». « Me JAQUES LE LIEUR » ; 2 « Par devolut ». « Me PIERRES AVRIL » ; 3 « Le povre Adam ». « Me NICOLE DU PUYS » ; 4 « Fors vous ». « Me NICOLE OSMONT » ; 5 « En fleur et fruict ». « Me JEHAN ALYNE » ; 6 « En unité ». « Me PIERRES AVRIL » ; 7 « Comme nature ». « Me CLEMENT MAROT » ; 8 « Grace sur grace » « Me THOMAS LEPREVOST » ; 9 « En mon concept ». « Me INNOCENT TOURMENTE » ; 10 « Ou bien de Dieu ». « GUILLAUME DE SEVYNGUEHEN » ; 11 « Une et non plus ». « Me THOMAS LEPREVOST » ; 12 « En tout honneur ». « Me PIERRES AVRIL » ; 13 « Pour moy sans plus ». « Me GUILLAUME TYBAULD » ; 14 « Grace nous vient ». « Damp NICOLE LESCARRE » ; 15 « Par mon cher filz ». « Me GUILLAUME THIBAULD » ; 16 « Mauldict serpent ». « AVRIL » ; 17 « Voulez vous myeulx ». « Me JEHAN BROYSE » ; 18 « Nonobstant loy ». « G. DE SEVYNGUEHEN » ; 19 « Au gré d'amour ». « Me GUILLAUME TYBAULD » ; 20 « Est ce bien faict ». « L'abbé DE SAINCT VANDRILLE » ; 21 « Hors paradis ». « Me JAQUES LE LYEUR » ; 1 « Deploracion de l'acteur » ; 2 « Espitre », par « LE LIEUR »

Studies on oxamyl : analytical method development and investigation of fate in peach seedlings and corn seeds

A high performance liquid chromatographic method employing two columns connected in series and separated~y·a.switching valve has been developed for the analysis of the insecticide/ nematicide oxamyl (methyl-N' ,N'-dimethyl-N-[(methylcarbamoyl) oxy]-l-thiooxarnimidate) and two of its metabolites. A variation of this method involving two reverse phase columns was employed to monitor the persistence and translocation of oxamyl in treated peach seedlings. It was possible to simultaneously analyse for oxamyl and its corresponding oxime (methyl-N',N'-dimethyl-N-hydroxy-l-thiooxamimidate}, a major metabolite of oxamyl in plants, without prior cleanup of the samples. The method allowed detection of 0.058 pg oxamyl and 0.035 p.g oxime. On treated peach leaves oxamyl was found to dissipate rapidly during the first two-week period, followed by a period of slow decomposition. Movement of oxamyl or its oxime did not occur in detectable quantities to untreated leaves or to the root or soil. A second variation of the method which employed a size exclusion column as·the first column and a reverse phase column as the second was used to monitor the degradation of oxamyl in treated, planted corn seeds and was suitable for simultaneous analysis of oxamyl, its oxime and dimethylcyanoformamide (DMCF), a metabolite of oxamyl. The method allowed detection of 0.02 pg oxamyl, 0.02 p.g oxime and 0.005 pg DMCF. Oxamyl was found to persist for a period of 5 - 6 weeks, which is long enough to permit oxamyl seedtreatment to be considered as a potential means of protecting young corn plants from nematode attack. Decomposition was found to be more rapid in unsterilized soil than in sterililized soil. DMCF was found to have a nematostatic effect at high concentrations ( 2,OOOpprn), but at lower concentrations no effect on nematode mobility was observed. Oxamyl, on the other hand, was found to reduce the mobility of nematodes at concentrations down to 4 ppm.

The determination of sinigrin in Brassica, and investigations into the use of allyl-isothiocyanate as a nematicide

The goal of this thesis was to study factors related to the development of Brassica juncea as a sustainable nematicide. Brassica juncea is characterized by the glycoside (glucosinolate) sinigrin. Various methods were developed for the determination of sinigrin in Brassica juncea tissue extracts. Sinigrin concentrations in plant tissues at various stages of growth were monitored. Sinigrin enzymatically breaks down into allylisothiocyanate (AITC). AITC is unstable in aqueous solution and degradation was studied in water and in soil. Finally, the toxicity of AITC against the root-lesion nematode (Pratylenchus penetrans) was determined. A method was developed to extract sinigrin from whole Brassica j uncea tissues. The optimal time of extraction wi th boiling phosphate buffer (0.7mM, pH=6.38) and methanol/water (70:30 v/v) solutions were both 25 minutes. Methanol/water extracted 13% greater amount of sinigrin than phosphate buffer solution. Degradation of sinigrin in boiling phosphate buffer solution (0.13%/minute) was similar to the loss of sinigrin during the extraction procedure. The loss of sinigrin from boiling methanol/water was estimated to be O.Ol%/minute. Brassica juncea extract clean up was accomplished by an ion-pair solid phase extraction (SPE) method. The recovery of sinigrin was 92.6% and coextractive impurities were not detected in the cleaned up extract. Several high performance liquid chromatography (HPLC) methods were developed for the determination of sinigrin. All the developed methods employed an isocratic mobile phase system wi th a low concentration of phosphate buffer solution, ammonium acetate solution or an ion-pair reagent solution. A step gradient system was also developed. The method involved preconditioning the analytical column with phosphate buffer solution and then switching the mobile phase to 100% water after sample injection.Sinigrin and benzyl-glucosinolate were both studied by HPLC particle beam negative chemical ionization mass spectrometry (HPLCPB- NCI-MS). Comparison of the mass spectra revealed the presence of fragments arising from the ~hioglucose moiety and glucosinolate side-chain. Variation in the slnlgrin concentration within Brassica juncea plants was studied (Domo and Cutlass cuItivars). The sinigrin concentration in the top three leaves was studied during growth of each cultivar. For Cutlass, the minimum (200~100~g/g) and maximum (1300~200~g/g) concentrations were observed at the third and seventh week after planting, respectively. For Domo, the minimum (190~70~g/g) and maximum (1100~400~g/g) concentrations were observed at the fourth and eighth week after planting, respectively. The highest sinigrin concentration was observed in flower tissues 2050±90~g/g and 2300±100~g/g for Cutlass and Domo cultivars, respectively. Physical properties of AITC were studied. The solubility of AITC in water was determined to be approximately 1290~g/ml at 24°C. An HPLC method was developed for the separation of degradation compounds from aqueous AITC sample solutions. Some of the degradation compounds identified have not been reported in the literature: allyl-thiourea, allyl-thiocyanate and diallyl-sulfide. In water, AITC degradation to' diallyl-thiourea was favored at basic pH (9.07) and degradation to diallyl-sulfide was favored at acidic pH (4 . 97). It wap necessary to amend the aqueous AITC sample solution with acetonitrile ?efore injection into the HPLC system. The acetonitrile amendment considerably improved AITC recovery and the reproducibility of the results. The half-life of aqueous AITC degradation at room temperature did not follow first-order kinetics. Beginning with a 1084~g/ml solution, the half-life was 633 hours. Wi th an ini tial AITC concentration of 335~g/ml the half-life was 865 hours. At 35°C the half-life AITC was 76+4 hours essentially independent of the iiisolution pH over the range of pH=4.97 to 9.07 (1000~g/ml). AITC degradation was also studied in soil at 35°C; after 24 hours approximately 75% of the initial AITC addition was unrecoverable by water extraction. The ECso of aqueous AITC against the root-lesion nematode (Pratylenchus penetrans) was determined to be approximately 20~g/ml at one hour exposure of the nematode to the test solution. The toxicological study was also performed with a myrosinase treated Brassica juncea extract. Myrosinase treatment of the Brassica juncea extract gave nearly quantitative conversion of sinigrin into AITC. The myrosinase treated extract was of the same efficacy as an aqueous AITC solution of equivalent concentration. The work of this thesis was focused upon understanding parameters relevant to the development of Brassica juncea as a sustainable nematicide. The broad range of experiments were undertaken in support of a research priority at Agriculture and Agri-Food Canada.

Canadian Shredded Wheat Company Fonds

In 1893 Attorney Henry Drushel Perky and William H. Ford invented a machine for the preparation of cereals for food. Perky soon realized that the actual cereal biscuits were more popular than the machines and he opened Shredded Wheat plants in Massachusetts and Niagara Falls, New York. In 1904, the Niagara Falls, Canada plant was opened. These factories also served as tourist attractions. In 1907, one hundred thousand people visited the plants on both sides of the border. In 1928, the company was sold to The National Biscuit Company and the product name changed to Nabisco Shredded Wheat. The name of the plant was changed to Nabisco Foods in 1956 to reflect the variety of foods that were being produced at that time. In 1985, Nabisco was purchased by J.R. Reynolds thus forming RJR Nabisco. In 1994, RJR sold its breakfast cereal business to Kraft Foods and the international licenses to General Mills. Shredded Wheat is now integrated into the Post Foods portfolio. with information from: Niagara Falls Canada a History by The Kiwanis Club of Stamford, Ontario Inc. and Business Source Complete