Whole exome sequencing is an efficient and sensitive method for detection of germline mutations in patients with phaeochromcytomas and paragangliomas

Background Genetic testing is recommended when the probability of a disease-associated germline mutation exceeds 10%. Germline mutations are found in approximately 25% of individuals with phaeochromcytoma (PCC) or paraganglioma (PGL); however, genetic heterogeneity for PCC/PGL means many genes may require sequencing. A phenotype-directed iterative approach may limit costs but may also delay diagnosis, and will not detect mutations in genes not previously associated with PCC/PGL. Objective To assess whether whole exome sequencing (WES) was efficient and sensitive for mutation detection in PCC/PGL. Methods Whole exome sequencing was performed on blinded samples from eleven individuals with PCC/PGL and known mutations. Illumina TruSeq™ (Illumina Inc, San Diego, CA, USA) was used for exome capture of seven samples, and NimbleGen SeqCap EZ v3.0 (Roche NimbleGen Inc, Basel, Switzerland) for five samples (one sample was repeated). Massive parallel sequencing was performed on multiplexed samples. Sequencing data were called using Genome Analysis Toolkit and annotated using annovar. Data were assessed for coding variants in RET, NF1, VHL, SDHD, SDHB, SDHC, SDHA, SDHAF2, KIF1B, TMEM127, EGLN1 and MAX. Target capture of five exome capture platforms was compared. Results Six of seven mutations were detected using Illumina TruSeq™ exome capture. All five mutations were detected using NimbleGen SeqCap EZ v3.0 platform, including the mutation missed using Illumina TruSeq™ capture. Target capture for exons in known PCC/PGL genes differs substantially between platforms. Exome sequencing was inexpensive (<$A800 per sample for reagents) and rapid (results <5 weeks from sample reception). Conclusion Whole exome sequencing is sensitive, rapid and efficient for detection of PCC/PGL germline mutations. However, capture platform selection is critical to maximize sensitivity.

Preliminary investigations into the use of a functionalised polymer to reduce diffusion in Fricke gel dosimeters

Purpose A modification of the existing PVA-​FX hydrogel has been made to investigate the use of a functionalised polymer in a Fricke gel dosimetry system to decrease Fe3+ diffusion. Methods The chelating agent, xylenol orange, was chem. bonded to the gelling agent, polyvinyl alc. (PVA) to create xylenol orange functionalised PVA (XO-​PVA)​. A gel was created from the XO-​PVA (20​% w​/v) with ferrous sulfate (0.4 mM) and sulfuric acid (50 mM)​. Results This resulted in an optical d. dose sensitivity of 0.014 Gy-​1, an auto-​oxidn. rate of 0.0005 h-​1, and a diffusion rate of 0.129 mm2 h-​1; an 8​% redn. compared to the original PVA-​FX gel, which in practical terms adds approx. 1 h to the time span between irradn. and accurate read-​out. Conclusions Because this initial method of chem. bonding xylenol orange to polyvinyl alc. has inherently low conversion, the improvement on existing gel systems is minimal when compared to the drawbacks. More efficient methods of functionalising polyvinyl alc. with xylenol orange must be developed for this system to gain clin. relevance.

Genetic Mechanisms of Tumourigenesis in von Hippel-Lindau-Associated Tumours with the Emphasis on Capillary Hemangioblastoma

The von Hippel-lindau (VHL) disease is a dominantly inherited neoplastic disorder which predisposes patients to multiple tumours including capillary haemangioblastomas (CHBs), pheochromocytomas (PCCs), renal cell carcinomas (RCCs). CHBs are the most common manifestations of VHL disease, occurring sporadically or as a manifestation of VHL disease. Inactivation of the VHL gene at 3p25-26 is believed to cause both familial and sporadic VHL-associated tumours and germ-line mutation of the VHL gene have been detected in 100% of the CHBs studied. However, a limited number of sporadic CHBs, PCCs display VHL inactivation. Other molecular alterations involved in tumourigenesis of sporadic CHBs, PCCs remain largely unknown. The purpose of the present work was to search for genetic alterations, or other mechanisms of inactivation, in addition to the VHL gene, that may be important in the development of VHL-associated tumours. Though less satisfactory than cure, prevention and early detection are the most promising and feasible means reducing cancer morbidity and mortality. This work is based on the view that increasing knowledge about the molecular events underlying tumour development will eventually aid in early detection and lead to improved treatment. We evaluated a large set of VHL-associated patients, searched for a clinical and radiologic signs of the disease. We succesfully performed a germ-line mutation analysis and characterised three patient groups, VHL, suspect VHL and sporadic, a germ-line mutation analysis revealed a 50% mutation rate only in the VHL groups, no sporadic or suspect cases displayed any mutation. We also utilized comparative genomic hybridization (CGH) to screen for DNA copy number changes in both sporadic and VHL-associated CHB. Our analysis revealed (27%) DNA copy number losses. The most common finding was loss of chromosomal arm 6q, seen in (23%) cases, No differences were noted between VHL-associated and sporadic tumours. Furthermore a loss of heterozygosity (LOH) study on chromosome 3p and 6q was done with the purpose to determine allele losses not observable by CGH, and to uncover the location of putative tumour suppressor genes important in CHB and PCC tumourigenesis. We identified loss of chromosome 6q and a minimal deleted area at 6q23-24 in CHBs. We also showed LOH at 6q23-24 in PCCs and identified the ZAC1 (6q24-25) as a candidate gene, ZAC1 is a maternally imprinted tumour suppressor gene with anti proliferative properties. To study further the role of ZAC inactivation in CHBs, we investigated LOH, promoter hypermethylation and expression status of the ZAC1 gene in mainly sporadic CHBs. Our LOH analysis revealed that the majority of the tumours with allele loss. The gene promoter methylation analysis similarly detected predominance of the methylated ZAC sequence in almost all tumours. Immunohistochemistry exhibited a strongly reduced expression of ZAC in stromal cells of all CHBs studied. Our current results indicate that the absence of the unmethylated, ZAC1 promoter sequence was highly concurrent with LOH for the ZAC1 region or 6q loss. This observation together with lack of ZAC expression, points to preferential loss of the non imprinted, expressed ZAC allele in CHB, in summary, our series of studies reveal a new chromosomal region 6q, emphasizes the importance of ZAC1 gene in the development of CHB and PCC, particularly in non-VHL associated cases.

Genetic variation in the LDL receptor-related protein 5 (LRP5) gene : Association with bone health and metabolic parameters

Bone mass accrual and maintenance are regulated by a complex interplay between genetic and environmental factors. Recent studies have revealed an important role for the low-density lipoprotein receptor-related protein 5 (LRP5) in this process. The aim of this thesis study was to identify novel variants in the LRP5 gene and to further elucidate the association of LRP5 and its variants with various bone health related clinical characteristics. The results of our studies show that loss-of-function mutations in LRP5 cause severe osteoporosis not only in homozygous subjects but also in the carriers of these mutations, who have significantly reduced bone mineral density (BMD) and increased susceptibility to fractures. In addition, we demonstrated for the first time that a common polymorphic LRP5 variant (p.A1330V) was associated with reduced peak bone mass, an important determinant of BMD and osteoporosis in later life. The results from these two studies are concordant with results seen in other studies on LRP5 mutations and in association studies linking genetic variation in LRP5 with BMD and osteoporosis. Several rare LRP5 variants were identified in children with recurrent fractures. Sequencing and multiplex ligation-dependent probe amplification (MLPA) analyses revealed no disease-causing mutations or whole-exon deletions. Our findings from clinical assessments and family-based genotype-phenotype studies suggested that the rare LRP5 variants identified are not the definite cause of fractures in these children. Clinical assessments of our study subjects with LPR5 mutations revealed an unexpectedly high prevalence of impaired glucose tolerance and dyslipidaemia. Moreover, in subsequent studies we discovered that common polymorphic LRP5 variants are associated with unfavorable metabolic characteristics. Changes in lipid profile were already apparent in pre-pubertal children. These results, together with the findings from other studies, suggest an important role for LRP5 also in glucose and lipid metabolism. Our results underscore the important role of LRP5 not only in bone mass accrual and maintenance of skeletal health but also in glucose and lipid metabolism. The role of LRP5 in bone metabolism has long been studied, but further studies with larger study cohorts are still needed to evaluate the specific role of LRP5 variants as metabolic risk factors.

Synthesis Of (+/-)-2-Isopropyl-5,6-Dimethylbicyclo[3.2.1]Oct-6-En-8-One

Methanolic hydrogen chloride cyclization of the triketone 8, prepared from the Mannich base 7 and 2-methylcyclopentane-1,3-dione, gives ketones 9 and 10. NaBH4 reduction of 9 followed by Grignard reaction with CH3MgI affords the diol 12. Catalytic hydrogenation of 12 followed by PCC oxidation yields the ketoalcohol 13. Dehydration of 13 with SOCl2/pyridine results in a 1:1 mixture of the endo-14 and exo-15 olefins, separated by chromatography.

Synthesis Of (+/-)-Beta-Cuparenone Via 3-Oxa-Beta-Cuparenone

Grignard reaction followed by ozonolysis, or ozonolysis followed by Grignard reaction on the pentenoate 8, generates the diol 9. Cyclodehydration of 9 leads to the 3-oxacuparene (6), whereas PCC oxidation furnishes the 3-oxa-beta-cuparenone (7). Methanesulfonic acid-P2O5 transforms 7 into cyclopentenones 4, 5, known precursors to beta-cuparenone (3), and the naphthalenone 14.

Bi-directional Switch Commutation for a Resonant Matrix Converter supplying a Contactless Energy Transmission System

This paper describes a bi-directional switch commutation strategy for a resonant matrix converter loaded with a contactless energy transmission system. Due to the different application compared to classical 3 phase to 3 phase matrix converters supplying induction machines a new investigation of possible commutation principles is necessary. The paper therefore compares the full bridge series-resonant converter with the 3 phase to 2 phase matrix converter. From the commutation of the full bridge series-resonant converter, conditions for the bi-directional switch commutation are derived. One of the main benefits of the derived strategy is the minimization of commutation steps, which is independent from the load current sign.

An approach toward the synthesis of PPAP natural product garsubellin A: construction of the tricyclic core

In a study directed toward the bioactive natural product garsubellin A, an expedient route to the bicyclo 3.3.1]nonan-9-one bearing tricyclic core, with a bridgehead anchored tetrahydrofuran ring, is delineated. The approach emanating from commercially available dimedone involved a DIBAL-H mediated retro aldol/re-aldol cyclization cascade and a PCC mediated oxidative cyclization as the key steps. (C) 2013 Elsevier Ltd. All rights reserved.

Evaluación de los anabólicos de crecimiento implix, ganamax, ralgro en novillos (finalización) en el trópico seco de Nicaragua

Con el propósito de evaluar el efecto de tres promotores de crecimiento Implix, Ralqro, Ganamax y un qrupo Testiqo (4 tratamientos), sobre variables de rendimiento en novillos de repasto Criollos x Brahaman con niveles de encaste desconocido, en el trópico seco de Nicaragua (Nandaime) 20 novillos fueron distribuidos al azar, 5 novillos por tratamiento. El estudio tuvo una duración de 3 meses, tiempo en el cual los animales fueron Implantados con los anabólicos antes mencionados. Las variables estudiadas fueron ganancia media diaria (GMD), peso de la canal (PCC), peso a los 90 días (P90d). Los datos obtenidos para determinar estas variables fueron peso inicial, peso a los 15 días, peso final. Los datos obtenidos para determinar estas variables se sometieron a un análisis de varianza y luego a una separación de medios a través de la prueba de DUNCAN. Los tratamientos no tuvieron efecto (P>0.05) sobre la características física de la canal; sin embargo se obtuvo mayor porcentaje de carne maqra y menos grasa en los animales implantados. Los resultados de las variables en estudio presentaron los siguientes valores: Ganamax (T1)tuvo una GMD de 380 grs, peso a los 90 días 392 kq y rendimiento en canal caliente de 194 kq, sequido de Implix (T2l con GMD 360 grs, peso 90 días de 380 kq y rendimiento en canal caliente 189 kg, el tratamiento Ralqro (T3) tuvo un peso a los 90 días de 375 kq obteniendo un peso final igual que el tratamiento Testigo, GMD 260 grs para Ralgro y 300 grs para el tratamiento Testigo, un rendimiento en canal caliente 186 kq para e1 tratamiento Ralqro Y 185 kq para el tratamiento Testigo.

Evaluacion de riesgos y puntos criticos de control de riesgos (HACCP) en el Matadero de Carne Bovina Nuevo Carnic

La realización de este trabajo investigativo tuvo lugar en un importante matadero del país, en el que se toman las medidas HACCP que los países extranjeros piden como requisitos para exportarles, los problemas sanitarios de la carne dependen, en primer lugar, del estado sanitario del animal y además, de la higiene con la que se realizan todas las operaciones hasta que el alimento llega al consumidor. Sin embargo han aparecido nuevos riesgos asociados al consumo de carne, más difíciles de controlar mediante una inspección tradicional, como las encefalopatías espongiformes, las intoxicaciones por E. coli O157, la presencia de residuos tóxicos, etc., y que están obligando a modificar los métodos tradicionales de inspección. Por lo que en este trabajo investigación se dispuso realizar la Evaluación de Riesgos y Puntos Críticos de Control (HACCP), en el Matadero de Carne Bovina Nuevo Carnic, donde por un periodo de seis meses se desarrollo la inspección detallada en diferentes puntos del proceso de matanza, permitiéndose con la inspección, determinar los puntos críticos que deben tener mayor control que garantice un producto inocuo y seguro para el consumidor. Encontrándose que los Puntos Críticos que contemplan en el plan HACCP de la empresa Nuevo Carnic son PCC-1 Evisceración, PCC-2 lavado antimicrobiano, PCC-3 enfriamiento, de estos puntos el 1 y 2 caen en constantes desviaciones y no se toman medidas correctivas adecuadas. También encontramos que se hace necesario incluir en el plan HACCP de Nuevo Carnic el PCC-4 empaque y PCC-5 almacenamiento para asegurar un producto inocuo para el consumidor, por tanto consideramos que con este trabajo se puede a portar al lector las medidas correctivas al seguimiento y control de los Puntos Críticos de Control en los mataderos que aplican el sistema HACCP o deseen hacerlo.

Copia de una carta remitida por el obispo Mateo Múgica al Presidente del Gobierno Vasco en el exilio.

Fecha: 19-3-1946 (>1970 copia) / Unidad de instalación: Carpeta 45 - Expediente 2-24 / Nº de pág.: 3 (mecanografiadas)

The Object Agreement Restriction

[EN]This paper deals with the so-called Person Case Constraint (Bonet, 1991), a universal constraint blocking accusative clitics and object agreement morphemes other than third person when a dative is inserted in the same clitic/agreement cluster. The aim of this paper is twofold. First, we argue that the scope of the PCC is considerably broader than assumed in previous work, and that neither its formulation in terms of person (1st/2nd vs. 3rd)-case (accusative vs. dative) restrictions nor its morphological nature are part of the right descriptive generalization.We present evidence (i) that the PCC is triggered by the presence of an animacy feature in the object’s agreement set; (ii) that it is not case dependent, also showing up in languages that lack dative case; and (iii) that it is not morphologically bound. Second, we argue that the PCC, even if it is modified accordingly, still puts together two different properties of the agreement system that should be set apart: (i) a cross linguistic sensitivity of object agreement to animacy and (ii) a similarly widespread restriction on multiple object agreement observed crosslinguistically. These properties lead us to propose a new generalization, the Object Agreement Constraint (OAC): if the verbal complex encodes object agreement, no other argument can be licensed through verbal agreement.

Discontinuous growth mechanisms of mode II crack under high-speed impact conditions

A recoverable plate impact testing technology has been used for studying the growth mechanisms of mode II crack. The results show that interactions of microcracks ahead of a crack tip cause the crack growth unsteadily. Failure mode transitions of materials were observed. Based on the observations, a discontinuous crack growth model was established. Analysis shows that the shear crack grows unsteady as the growth speed is between the Rayleigh wave speed c(R) and the shear wave speed c(s); however, when the growth speed approaches root 2c(s), the crack grows steadily. The transient microcrack growth makes the main crack speed to jump from subsonic to intersonic and the steady growth of all the sub-cracks leads the main crack to grow stably at an intersonic speed.

Developing peptide based capture agents for diagnostics and therapeutics

Iterative in situ click chemistry (IISCC) is a robust general technology for development of high throughput, inexpensive protein detection agents. In IISCC, the target protein acts as a template and catalyst, and assembles its own ligand from modular blocks of peptides. This process of ligand discovery is iterated to add peptide arms to develop a multivalent ligand with increased affinity and selectivity. The peptide based protein capture agents (PCC) should ideally have the same degree of selectivity and specificity as a monoclonal antibody, along with improved chemical stability. We had previously reported developing a PCC agent against bovine carbonic anhydrase II (bCAII) that could replace a polyclonal antibody. To further enhance the affinity or specificity of the PCC agent, I explore branching the peptide arms to develop branched PCC agents against bCAII. The developed branched capture agents have two to three fold higher affinities for the target protein. In the second part of my thesis, I describe the epitope targeting strategy, a strategy for directing the development of a peptide ligand against specific region or fragment of the protein. The strategy is successfully demonstrated by developing PCC agents with low nanomolar binding affinities that target the C-terminal hydrophobic motif of Akt2 kinase. One of the developed triligands inhibits the kinase activity of Akt. This suggests that, if targeted against the right epitope, the PCC agents can also influence the functional properties of the protein. The exquisite control of the epitope targeting strategy is further demonstrated by developing a cyclic ligand against Akt2. The cyclic ligand acts as an inhibitor by itself, without any iteration of the ligand discovery process. The epitope targeting strategy is a cornerstone of the IISCC technology and opens up new opportunities, leading to the development of protein detection agents and of modulators of protein functions.