Functional validation of LexADBDGim chimeras in saccharomyces cerevisiae

GimC/Prefoldin is a hetero-oligomeric complex involved in cytoskeleton biogenesis. In order to identify by two-hybrid system targets that directly interact with Gims and support the stress phenotypes, this work aimed the functional validation of all Gims in saccharomyces cerevisiae.

Computational identification and functional validation of regulatory motifs in cartilage-expressed genes.

Chondrocyte gene regulation is important for the generation and maintenance of cartilage tissues. Several regulatory factors have been identified that play a role in chondrogenesis, including the positive transacting factors of the SOX family such as SOX9, SOX5, and SOX6, as well as negative transacting factors such as C/EBP and delta EF1. However, a complete understanding of the intricate regulatory network that governs the tissue-specific expression of cartilage genes is not yet available. We have taken a computational approach to identify cis-regulatory, transcription factor (TF) binding motifs in a set of cartilage characteristic genes to better define the transcriptional regulatory networks that regulate chondrogenesis. Our computational methods have identified several TFs, whose binding profiles are available in the TRANSFAC database, as important to chondrogenesis. In addition, a cartilage-specific SOX-binding profile was constructed and used to identify both known, and novel, functional paired SOX-binding motifs in chondrocyte genes. Using DNA pattern-recognition algorithms, we have also identified cis-regulatory elements for unknown TFs. We have validated our computational predictions through mutational analyses in cell transfection experiments. One novel regulatory motif, N1, found at high frequency in the COL2A1 promoter, was found to bind to chondrocyte nuclear proteins. Mutational analyses suggest that this motif binds a repressive factor that regulates basal levels of the COL2A1 promoter.

Functional validation of MB-OFDM system using HW-in the loop

Functional validation of complex digital systems is a hard and critical task in the design flow. In particular, when dealing with communication systems, like Multiband Orthogonal Frequency Division Multiplexing Ultra Wideband (MB-OFDM UWB), the design decisions taken during the process have to be validated at different levels in an easy way. In this work, a unified algorithm-architecture-circuit co-design environment for this type of systems, to be implemented in FPGA, is presented. The main objective is to find an efficient methodology for designing a configurable optimized MB-OFDM UWB system by using as few efforts as possible in verification stage, so as to speed up the development period. Although this efficient design methodology is tested and considered to be suitable for almost all types of complex FPGA designs, we propose a solution where both the circuit and the communication channel are tested at different levels (algorithmic, RTL, hardware device) using a common testbench.

Functional brain perfusion evaluation with Arterial Spin Labeling at 3 Tesla

Dissertation submitted in Faculdade de Ciências e Tecnologia of Universidade Nova de Lisboa for the degree of Master of Biomedical Engineering

A highly expressed miR-101 isomiR is a functional silencing small RNA

Background MicroRNAs (miRNAs) are short non-coding regulatory RNAs that control gene expression usually producing translational repression and gene silencing. High-throughput sequencing technologies have revealed heterogeneity at length and sequence level for the majority of mature miRNAs (IsomiRs). Most isomiRs can be explained by variability in either Dicer1 or Drosha cleavage during miRNA biogenesis at 5" or 3" of the miRNA (trimming variants). Although isomiRs have been described in different tissues and organisms, their functional validation as modulators of gene expression remains elusive. Here we have characterized the expression and function of a highly abundant miR-101 5"-trimming variant (5"-isomiR-101). Results The analysis of small RNA sequencing data in several human tissues and cell lines indicates that 5"-isomiR-101 is ubiquitously detected and a highly abundant, especially in the brain. 5"- isomiR-101 was found in Ago-2 immunocomplexes and complementary approaches showed that 5"-isomiR-101 interacted with different members of the silencing (RISC) complex. In addition, 5"-isomiR-101 decreased the expression of five validated miR-101 targets, suggesting that it is a functional variant. Both the binding to RISC members and the degree of silencing were less efficient for 5"-isomiR-101 compared with miR-101. For some targets, both miR-101 and 5"-isomiR-101 significantly decreased protein expression with no changes in the respective mRNA levels. Although a high number of overlapping predicted targets suggest similar targeted biological pathways, a correlation analysis of the expression profiles of miR-101 variants and predicted mRNA targets in human brains at different ages, suggest specific functions for miR-101- and 5"-isomiR-101. Conclusions These results suggest that isomiRs are functional variants and further indicate that for a given miRNA, the different isomiRs may contribute to the overall effect as quantitative and qualitative fine-tuners of gene expression.

Validation of evacuation models

Too often, validation of computer models is considered as a "once and forget" task. In this paper a systematic and graduated approach to evacuation model validation is suggested. This involves, (i) component testing, (ii) functional validation, (iii) qualitative validation and (iv) quantitative validation. Viewed in this manner, validation is considered an on-going activity and an integral part of the life cycle of the software. While the first three components of the validation protocol pose little or no significant problems, the task of quantitative validation poses a number of challenges, the most significant being a shortage of suitable experimental data. Finally, the validation protocol used in the development of the EXODUS suite of evacuation models is examined.

In silico adjuvant design and validation

Adjuvants are substances that boost the protective immune response to vaccine antigens. The majority of known adjuvants have been identified through the use of empirical approaches. Our aim was to identify novel adjuvants with well-defined cellular and molecular mechanisms by combining a knowledge of immunoregulatory mechanisms with an in silico approach. CD4 + CD25 + FoxP3 + regulatory T cells (Tregs) inhibit the protective immune responses to vaccines by suppressing the activation of antigen presenting cells such as dendritic cells (DCs). In this chapter, we describe the identification and functional validation of small molecule antagonists to CCR4, a chemokine receptor expressed on Tregs. The CCR4 binds the chemokines CCL22 and CCL17 that are produced in large amounts by activated innate cells including DCs. In silico identified small molecule CCR4 antagonists inhibited the migration of Tregs both in vitro and in vivo and when combined with vaccine antigens, significantly enhanced protective immune responses in experimental models.

Analyse génétique moléculaire du gène de la voie non-canonique Frizzled/Dishevelled PRICKLE1 dans les anomalies du tube neural chez l’humain

La voie de la polarité planaire cellulaire (PCP), aussi connue sous le nom de la voie non-canonique du Frizzled/Dishevelled, contrôle le processus morphogénétique de l'extension convergente (CE) qui est essentiel pour la gastrulation et la formation du tube neural pendant l'embryogenèse. La signalisation du PCP a été récemment associée avec des anomalies du tube neural (ATN) dans des modèles animaux et chez l'humain. Prickle1 est une protéine centrale de la voie PCP, exprimée dans la ligne primitive et le mésoderme pendant l'embryogenèse de la souris. La perte ou le gain de fonction de Prickle1 mène à des mouvements de CE fautifs chez le poisson zèbre et la grenouille. PRICKLE1 interagit directement avec deux autres membres de la voie PCP, Dishevelled et Strabismus/Vang. Dans notre étude, nous avons investigué le rôle de PRICKLE1 dans l'étiologie des ATN dans une cohorte de 810 patients par le re-séquençage de son cadre de lecture et des jonctions exon-intron. Le potentiel pathogénique des mutations ainsi identifiées a été évalué par des méthodes bioinformatiques, suivi par une validation fonctionnelle in vivo dans un système poisson zèbre. Nous avons identifié dans notre cohorte un total de 9 nouvelles mutations dont sept: p.Ile69Thr, p.Asn81His, p.Thr275Met, p.Arg682Cys et p.Ser739Phe, p.Val550Met et p.Asp771Asn qui affectent des acides aminés conservés. Ces mutations ont été prédites in silico d’affecter la fonction de la protéine et sont absentes dans une large cohorte de contrôles de même origine ethnique. La co-injection de ces variantes avec le gène prickle1a de type sauvage chez l’embryon de poisson zèbre a démontré qu’une mutation, p.Arg682Cys, modifie dans un sens négatif le phénotype du défaut de la CE produit par pk1 de type sauvage. Notre étude démontre que PK1 peut agir comme facteur prédisposant pour les ATN chez l’humain et élargit encore plus nos connaissances sur le rôle des gènes de la PCP dans la pathogenèse de ces malformations.

Identification et caractérisation d’une souris mutante Skam26Jus comme un nouveau modèle des anomalies du tube neural

Les anomalies du tube neural (ATN) sont des malformations congénitales très fréquentes chez l’humain en touchant 1-2 nouveau-nés sur 1000 naissances. Elles résultent d’une fermeture incomplète du tube neural lors de l’embryogenèse. L’étiologie des ATN est complexe impliquant des facteurs environnementaux et des facteurs génétiques. La souris représente un outil puissant afin de mieux comprendre la génétique des ATN. Particulièrement, la souris modèle a impliqué fortement la voie de la polarité cellulaire planaire (PCP) dans ces malformations. Dans cette étude, nous avons identifié et caractérisé une nouvelle souris mutante, Skam26Jus dans le but d’identifier un nouveau gène causant les ATN. Skam26Jus a été générée par l’agent mutagène N-Ethyl-N-Nitrosuera. Cette souris est caractérisée par une queue en forme de boucle ou de crochet, soit un phénotype associé aux ATN. La complémentation génétique de la souris Skam26Jus avec une souris mutante d’un gène de la voie PCP Vangl2 (Looptail) a montré une interaction génétique entre le gène muté chez Skam26Jus et Vangl2, suggérant que ces deux gènes fonctionnent dans des voies de signalisation semblables ou parallèles. Un total de 50% des embryons doubles hétérozygotes avec un phénotype de la queue présentent un spina bifida. La cartographie par homozygotie du génome entier suivie par un clonage positionnel a permis d’identifier Lrp6 comme le gène muté chez Skam26Jus. Une mutation homozygote, p.Ile681Arg, a été identifiée dans Lrp6 chez les souris ayant une queue en boucle/crochet. Cette mutation était absente dans 30 souches génétiques pures indiquant que cette mutation est spécifique au phénotype observé. Une étude de phénotype-génotype évalue la pénétrance à 53 % de la mutation Ile681Arg. Lrp6 est connu pour activer la voie canonique Wnt/β-caténine et inhiber la voie non canonique Wnt/PCP. Le séquençage de la région codante et de la jonction exon-intron de LRP6 chez 268 patients a mené à l’identification de quatre nouvelles rares mutations faux sens absentes chez 272 contrôles et de toutes les bases de données publiques. Ces mutations sont p.Tyr306His ; p.Tyr373Cys ; p.Val1386Ile; p.Tyr1541Cys et leur pathogénicité prédite in silico indiquent que p.Val1386Ile est bénigne, et que p.Tyr306Hiset p.Tyr373Cys et p.Tyr1541Cys sont i possiblement dommageables. Les mutations p.Tyr306His, p.Tyr373Cys et p.Tyr1541Cys ont affecté l’habilité de LRP6 d’activer la voie Wnt/β-caténine en utilisant le système rapporteur luciférase de pTOPflash. Nos résultats suggèrent que LRP6 joue un rôle dans le développement des ATN chez une petite fraction de patients ayant une ATN. Cette étude présente aussi Skam26Jus comme un nouveau modèle pour étudier les ATN chez l’humain et fournit un outil important pour comprendre les mécanismes moléculaires à l’origine des A TN.

Estimating hardness of eucalyptus wood with a portable hardness tester

More than 80% of the 29600 km of the Brazilian railroad mesh employs wooden sleepers. The problem of hard availability of native wood for this purpose leads to the alternative use of reforestation species to produce sleepers. Considering the great difficulty to, in field condition, evaluate characteristics that are of major importance to define its suitability to sleeper production the Research Group on Forest Products from FCA/UNESP - Brazil had developed equipment for field evaluation of hardness in wood - Portable Hardness Tester. This paper reports the functional validation tests, performed with different species of Eucalyptus. Results revealed the equipment great functionality, easy-to-use characteristics and applicability to Eucalyptus wood. Moderate to strong relationships between laboratory and validated values of hardness were found. The best validation model was obtained using the data provided by the experimental dispositive 3 (R2=0.74 and SSE= 7.71 kJ/m2) while the experimental dispositive 1 gave the worse validation (R2=0.55 and SSE= 13.46 kJ/m2).

MicroRNoma do carcinoma de pâncreas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A coding variant in RARG confers susceptibility to anthracycline-induced cardiotoxicity in childhood cancer

Anthracyclines are used in over 50% of childhood cancer treatment protocols, but their clinical usefulness is limited by anthracycline-induced cardiotoxicity (ACT) manifesting as asymptomatic cardiac dysfunction and congestive heart failure in up to 57% and 16% of patients, respectively. Candidate gene studies have reported genetic associations with ACT, but these studies have in general lacked robust patient numbers, independent replication or functional validation. Thus, the individual variability in ACT susceptibility remains largely unexplained. We performed a genome-wide association study in 280 patients of European ancestry treated for childhood cancer, with independent replication in similarly treated cohorts of 96 European and 80 non-European patients. We identified a nonsynonymous variant (rs2229774, p.Ser427Leu) in RARG highly associated with ACT (P = 5.9 × 10(-8), odds ratio (95% confidence interval) = 4.7 (2.7-8.3)). This variant alters RARG function, leading to derepression of the key ACT genetic determinant Top2b, and provides new insight into the pathophysiology of this severe adverse drug reaction.

Whole Exome Sequencing widens the genetic landscape of Hereditary Optic Neuropathies

Hereditary optic neuropathies (HON) are a genetic cause of visual impairment characterized by degeneration of retinal ganglion cells. The majority of HON are caused by pathogenic variants in mtDNA genes and in gene OPA1. However, several other genes can cause optic atrophy and can only be identified by high throughput genetic analysis. Whole Exome Sequencing (WES) is becoming the primary choice in rare disease molecular diagnosis, being both cost effective and informative. We performed WES on a cohort of 106 cases, of which 74 isolated ON patients (ON) and 32 syndromic ON patients (sON). The total diagnostic yield amounts to 27%, slightly higher for syndromic ON (31%) than for isolated ON (26%). The majority of genes found are related to mitochondrial function and already reported for harbouring HON pathogenic variants: ACO2, AFG3L2, C19orf12, DNAJC30, FDXR, MECR, MTFMT, NDUFAF2, NDUFB11, NDUFV2, OPA1, PDSS1, SDHA, SSBP1, and WFS1. Among these OPA1, ACO2, and WFS1 were confirmed as the most relevant genetic causes of ON. Moreover, several genes were identified, especially in sON patients, with direct impairment of non-mitochondrial molecular pathways: from autophagy and ubiquitin system (LYST, SNF8, WDR45, UCHL1), to neural cells development and function (KIF1A, GFAP, EPHB2, CACNA1A, CACNA1F), but also vitamin metabolism (SLC52A2, BTD), cilia structure (USH2A), and nuclear pore shuttling (NUTF2). Functional validation on yeast model was performed for pathogenic variants detected in MECR, MTFMT, SDHA, and UCHL1 genes. For SDHA and UCHL1 also muscle biopsy and fibroblast cell lines from patients were analysed, pointing to possible pathogenic mechanisms that will be investigated in further studies. In conclusion, WES proved to be an efficient tool when applied to our ON cohort, for both common disease-genes identification and novel genes discovery. It is therefore recommended to consider WES in ON molecular diagnostic pipeline, as for other rare genetic diseases.

Pathogenetic mechanisms of gastrointestinal symptoms in a mouse model of Fabry disease: insights on the microbiota-gut-brain axis

Fabry disease (FD), X-linked metabolic disorder caused by a deficiency in α-galactosidase A activity, leads to the accumulation of glycosphingolipids, mainly Gb3 and lyso-Gb3, in several organs. Gastrointestinal (GI) symptoms are among the earliest and most common, strongly impacting patients’ quality of life. However, the origin of these symptoms and the exact mechanisms of pathogenesis are still poorly understood, thus the pressing need to improve their knowledge. Here we aimed to evaluate whether a FD murine model (α-galactosidase A Knock-Out) captures the functional GI issues experienced by patients. In particular, the potential mechanisms involved in the development and maintenance of GI symptoms were explored by looking at the microbiota-gut-brain axis involvement. Moreover, we sought to examine the effects of lyso-Gb3 on colonic contractility and the intestinal epithelium and the enteric nervous system, which together play important roles in regulating intestinal ion transport and fluid and electrolyte homeostasis. Fabry mice revealed visceral hypersensitivity and a diarrhea-like phenotype accompanied by anxious-like behavior and reduced locomotor activity. They reported also an imbalance of SCFAs and an early compositional and functional dysbiosis of the gut microbiota, which partly persisted with advancing age. Moreover, overexpression of TRPV1 was found in affected mice, and partial alteration of TRPV4 and TRPA1 as well, identifying them as possible therapeutic targets. The Ussing chamber results after treatment with lyso-Gb3 showed an increase in Isc (likely mediated by HCO3- ions movement) which affects neuron-mediated secretion, especially capsaicin- and partly veratridine-mediated. This first characterization of gut-brain axis dysfunction in FD mouse provides functional validation of the model, suggesting new targets and possible therapeutic approaches. Furthermore, lyso-Gb3 is confirmed to be not only a marker for the diagnosis and follow-up of FD but also a possible player in the alteration of the FD colonic ion transport process.

Selection And Validation Of Reference Genes For Functional Studies In The Calliphoridae Family.

The genera Cochliomyia and Chrysomya contain both obligate and saprophagous flies, which allows the comparison of different feeding habits between closely related species. Among the different strategies for comparing these habits is the use of qPCR to investigate the expression levels of candidate genes involved in feeding behavior. To ensure an accurate measure of the levels of gene expression, it is necessary to normalize the amount of the target gene with the amount of a reference gene having a stable expression across the compared species. Since there is no universal gene that can be used as a reference in functional studies, candidate genes for qPCR data normalization were selected and validated in three Calliphoridae (Diptera) species, Cochliomyia hominivorax Coquerel, Cochliomyia macellaria Fabricius, and Chrysomya albiceps Wiedemann . The expression stability of six genes ( Actin, Gapdh, Rp49, Rps17, α -tubulin, and GstD1) was evaluated among species within the same life stage and between life stages within each species. The expression levels of Actin, Gapdh, and Rp49 were the most stable among the selected genes. These genes can be used as reliable reference genes for functional studies in Calliphoridae using similar experimental settings.