Thalidomide and birth defects

Apologies to the many papers we were unable to cite, due to space constraints. We thank Lynda Erskine, Shaunna Beedie and Chris Mahony for helpful discussions. Lucas Rosa Fraga is funded by a PhD scholarship from the Science without Borders program - CNPq Brazil - INAGEMP/ Grant CNPq 573993/2008-4. Alex J. Diamond is funded by a BBSRC DTP PhD Scholarship.

Anticancer Properties of a Novel Class of Tetrafluorinated Thalidomide Analogues

Date of Acceptance: 28/07/2015 The authors thank Scott McMenemy for carrying out preliminary, early studies looking at effects of Gu compounds upon chicken embryology, as well as Charles D. Crowe, Jeffrey E. Roth, and Adam C. Rolt for critical comments on the article. fli1:EGFP zebrafish were obtained from the Zebrafish International Research Center (27). mpo:GFP zebrafish [also termed Tg(MPO:GFP)114] zebrafish were obtained from Dr. Stephen Renshaw, University of Sheffield (Sheffield, South Yorkshire, UK; ref. 29).

Thalidomide and birth defects

Apologies to the many papers we were unable to cite, due to space constraints. We thank Lynda Erskine, Shaunna Beedie and Chris Mahony for helpful discussions. Lucas Rosa Fraga is funded by a PhD scholarship from the Science without Borders program - CNPq Brazil - INAGEMP/ Grant CNPq 573993/2008-4. Alex J. Diamond is funded by a BBSRC DTP PhD Scholarship.

Anticancer Properties of a Novel Class of Tetrafluorinated Thalidomide Analogues

Date of Acceptance: 28/07/2015 The authors thank Scott McMenemy for carrying out preliminary, early studies looking at effects of Gu compounds upon chicken embryology, as well as Charles D. Crowe, Jeffrey E. Roth, and Adam C. Rolt for critical comments on the article. fli1:EGFP zebrafish were obtained from the Zebrafish International Research Center (27). mpo:GFP zebrafish [also termed Tg(MPO:GFP)114] zebrafish were obtained from Dr. Stephen Renshaw, University of Sheffield (Sheffield, South Yorkshire, UK; ref. 29).

Implication de la convertase NARC-1 / PCSK9 au cours de la différenciation neuroectodermale

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Interpreting Human Genetic Variation through Genomics and In Vivo Models

Improvements in genomic technology, both in the increased speed and reduced cost of sequencing, have expanded the appreciation of the abundance of human genetic variation. However the sheer amount of variation, as well as the varying type and genomic content of variation, poses a challenge in understanding the clinical consequence of a single mutation. This work uses several methodologies to interpret the observed variation in the human genome, and presents novel strategies for the prediction of allele pathogenicity.

Using the zebrafish model system as an in vivo assay of allele function, we identified a novel driver of Bardet-Biedl Syndrome (BBS) in CEP76. A combination of targeted sequencing of 785 cilia-associated genes in a cohort of BBS patients and subsequent in vivo functional assays recapitulating the human phenotype gave strong evidence for the role of CEP76 mutations in the pathology of an affected family. This portion of the work demonstrated the necessity of functional testing in validating disease-associated mutations, and added to the catalogue of known BBS disease genes.

Further study into the role of copy-number variations (CNVs) in a cohort of BBS patients showed the significant contribution of CNVs to disease pathology. Using high-density array comparative genomic hybridization (aCGH) we were able to identify pathogenic CNVs as small as several hundred bp. Dissection of constituent gene and in vivo experiments investigating epistatic interactions between affected genes allowed for an appreciation of several paradigms by which CNVs can contribute to disease. This study revealed that the contribution of CNVs to disease in BBS patients is much higher than previously expected, and demonstrated the necessity of consideration of CNV contribution in future (and retrospective) investigations of human genetic disease.

Finally, we used a combination of comparative genomics and in vivo complementation assays to identify second-site compensatory modification of pathogenic alleles. These pathogenic alleles, which are found compensated in other species (termed compensated pathogenic deviations [CPDs]), represent a significant fraction (from 3 – 10%) of human disease-associated alleles. In silico pathogenicity prediction algorithms, a valuable method of allele prioritization, often misrepresent these alleles as benign, leading to omission of possibly informative variants in studies of human genetic disease. We created a mathematical model that was able to predict CPDs and putative compensatory sites, and functionally showed in vivo that second-site mutation can mitigate the pathogenicity of disease alleles. Additionally, we made publically available an in silico module for the prediction of CPDs and modifier sites.

These studies have advanced the ability to interpret the pathogenicity of multiple types of human variation, as well as made available tools for others to do so as well.

Lysosomal Disorders Drive Susceptibility to Tuberculosis by Compromising Macrophage Migration.

A zebrafish genetic screen for determinants of susceptibility to Mycobacterium marinum identified a hypersusceptible mutant deficient in lysosomal cysteine cathepsins that manifests hallmarks of human lysosomal storage diseases. Under homeostatic conditions, mutant macrophages accumulate undigested lysosomal material, which disrupts endocytic recycling and impairs their migration to, and thus engulfment of, dying cells. This causes a buildup of unengulfed cell debris. During mycobacterial infection, macrophages with lysosomal storage cannot migrate toward infected macrophages undergoing apoptosis in the tuberculous granuloma. The unengulfed apoptotic macrophages undergo secondary necrosis, causing granuloma breakdown and increased mycobacterial growth. Macrophage lysosomal storage similarly impairs migration to newly infecting mycobacteria. This phenotype is recapitulated in human smokers, who are at increased risk for tuberculosis. A majority of their alveolar macrophages exhibit lysosomal accumulations of tobacco smoke particulates and do not migrate to Mycobacterium tuberculosis. The incapacitation of highly microbicidal first-responding macrophages may contribute to smokers' susceptibility to tuberculosis.

Direct In Vivo Manipulation and Imaging of Calcium Transients in Neutrophils Identify a Critical Role for Leading-Edge Calcium Flux.

Calcium signaling has long been associated with key events of immunity, including chemotaxis, phagocytosis, and activation. However, imaging and manipulation of calcium flux in motile immune cells in live animals remain challenging. Using light-sheet microscopy for in vivo calcium imaging in zebrafish, we observe characteristic patterns of calcium flux triggered by distinct events, including phagocytosis of pathogenic bacteria and migration of neutrophils toward inflammatory stimuli. In contrast to findings from ex vivo studies, we observe enriched calcium influx at the leading edge of migrating neutrophils. To directly manipulate calcium dynamics in vivo, we have developed transgenic lines with cell-specific expression of the mammalian TRPV1 channel, enabling ligand-gated, reversible, and spatiotemporal control of calcium influx. We find that controlled calcium influx can function to help define the neutrophil's leading edge. Cell-specific TRPV1 expression may have broad utility for precise control of calcium dynamics in other immune cell types and organisms.

Characterizing the Role of the Previously Undescribed Protein Caskin2 in Vascular Biology

Maintenance of vascular homeostasis is an active process that is dependent on continuous signaling by the quiescent endothelial cells (ECs) that line mature vessels. Defects in vascular homeostasis contribute to numerous disorders of significant clinical impact including hypertension and atherosclerosis. The signaling pathways that are active in quiescent ECs are distinct from those that regulate angiogenesis but are comparatively poorly understood. Here we demonstrate that the previously uncharacterized scaffolding protein Caskin2 is a novel regulator of EC quiescence and that loss of Caskin2 in mice results in elevated blood pressure at baseline. Caskin2 is highly expressed in ECs from various vascular beds both in vitro and in vivo. When adenovirally expressed in vitro, Caskin2 inhibits EC proliferation and migration but promotes survival during hypoxia and nutrient deprivation. Likewise, loss of Caskin2 in vivo promotes increased vascular branching and permeability in mouse and zebrafish models. Caskin2 knockout mice are born in normal Mendelian ratios and appear grossly normal during early adulthood. However, they have consistently elevated systolic and diastolic blood pressure at baseline and significant context-dependent abnormalities in systemic metabolism (e.g., body weight, fat deposition, and glucose homeostasis). Although the precise molecular mechanisms of these effects remain unclear, we have shown that Caskin2 interacts with several proteins known to have important roles in endothelial biology and cardiovascular disease including the serine/threonine phosphatase PP1, the endothelial receptor Tie1, and eNOS, which is a critical regulator of vascular homeostasis. Ongoing work seeks to further characterize the functions of Caskin2 and its mechanisms of action with a focus on how Caskin2-mediated regulation of endothelial phenotype relates to its systemic effects on cardiovascular and metabolic function.

Implication de la convertase NARC-1 / PCSK9 au cours de la différenciation neuroectodermale

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Molecular docking and geographical information systems as tools to assess the potential impact of veterinary medicines on non-target organisms and the environment

Veterinary medicines (VMs) from agricultural industry can enter the environment in a number of ways. This includes direct exposure through aquaculture, accidental spillage and disposal, and indirect entry by leaching from manure or runoff after treatment. Many compounds used in animal treatments have ecotoxic properties that may have chronic or sometimes lethal effects when they come into contact with non-target organisms. VMs enter the environment in mixtures, potentially having additive effects. Traditional ecotoxicology tests are used to determine the lethal and sometimes reproductive effects on freshwater and terrestrial organisms. However, organisms used in ecotoxicology tests can be unrepresentative of the populations that are likely to be exposed to the compound in the environment. Most often the tests are on single compound toxicity but mixture effects may be significant and should be included in ecotoxicology testing. This work investigates the use, measured environmental concentrations (MECs) and potential impact of sea lice treatments on salmon farms in Scotland. Alternative methods for ecotoxicology testing including mixture toxicity, and the use of in silico techniques to predict the chronic impact of VMs on different species of aquatic organisms were also investigated. The Scottish Environmental Protection Agency (SEPA) provided information on the use of five sea lice treatments from 2008-2011 on Scottish salmon farms. This information was combined with the recently available data on sediment MECs for the years 2009-2012 provided by SEPA using ArcGIS 10.1. In depth analysis of this data showed that from a total of 55 sites, 30 sites had a MEC higher than the maximum allowable concentration (MAC) as set out by SEPA for emamectin benzoate and 7 sites had a higher MEC than MAC for teflubenzuron. A number of sites that were up to 16 km away from the nearest salmon farm reported as using either emamectin benzoate or teflubenzuron measured positive for the two treatments. There was no relationship between current direction and the distribution of the sea lice treatments, nor was there any evidence for alternative sources of the compounds e.g. land treatments. The sites that had MECs higher than the MAC could pose a risk to non-target organisms and disrupt the species dynamics of the area. There was evidence that some marine protected sites might be at risk of exposure to these compounds. To complement this work, effects on acute mixture toxicity of the 5 sea lice treatments, plus one major metabolite 3-phenoxybenzoic acid (3PBA), were measured using an assay using the bioluminescent bacteria Aliivibrio fischeri. When exposed to the 5 sea lice treatments and 3PBA A. fischeri showed a response to 3PBA, emamectin benzoate and azamethiphos as well as combinations of the three. In order to establish any additive effect of the sea lice treatments, the efficacy of two mixture prediction equations, concentration addition (CA) and independent action ii(IA) were tested using the results from single compound dose response curves. In this instance IA was the more effective prediction method with a linear regression confidence interval of 82.6% compared with 22.6% of CA. In silico molecular docking was carried out to predict the chronic effects of 15 VMs (including the five used as sea lice control). Molecular docking has been proposed as an alternative screening method for the chronic effects of large animal treatments on non-target organisms. Oestrogen receptor alpha (ERα) of 7 non-target bony fish and the African clawed frog Xenopus laevis were modelled using SwissModel. These models were then ‘docked’ to oestradiol, the synthetic oestrogen ethinylestradiol, two known xenoestrogens dichlorodiphenyltrichloroethane (DDT) and bisphenol A (BPA), the antioestrogen breast cancer treatment tamoxifen and 15 VMs using Auto Dock 4. Based on the results of this work, four VMs were identified as being possible xenoestrogens or anti-oestrogens; these were cypermethrin, deltamethrin, fenbendazole and teflubenzuron. Further investigation, using in vitro assays, into these four VMs has been suggested as future work. A modified recombinant yeast oestrogen screen (YES) was attempted using the cDNA of the ERα of the zebrafish Danio rerio and the rainbow trout Oncorhynchus mykiss. Due to time and difficulties in cloning protocols this work was unable to be completed. Use of such in vitro assays would allow for further investigation of the highlighted VMs into their oestrogenic potential. In conclusion, VMs used as sea lice treatments, such as teflubenzuron and emamectin benzoate may be more persistent and have a wider range in the environment than previously thought. Mixtures of sea lice treatments have been found to persist together in the environment, and effects of these mixtures on the bacteria A. fischeri can be predicted using the IA equation. Finally, molecular docking may be a suitable tool to predict chronic endocrine disrupting effects and identify varying degrees of impact on the ERα of nine species of aquatic organisms.

Exploration génétique de l'hypothyroïdie congénitale par dysgénésie thyroïdienne

L'hypothyroïdie congénitale par dysgénésie thyroïdienne (HCDT, ectopie dans plus de 80 %) a une prévalence de 1 cas sur 4000 naissances vivantes. L’HCDT est la conséquence d'une défaillance de la thyroïde embryonnaire à se différencier, à se maintenir ou à migrer vers sa localisation anatomique (partie antérieure du cou), qui aboutit à une absence totale de la thyroïde (athyréose) ou à une ectopie thyroïdienne (linguale ou sublinguale). Les HCDT sont principalement non-syndromiques (soit 98% des cas sont non-familiale), ont un taux de discordance de 92% chez les jumeaux monozygotes, et ont une prédominance féminine et ethnique (i.e., Caucasienne). La majorité des cas d’HCDT n’a pas de cause connue, mais est associée à un déficit sévère en hormones thyroïdiennes (hypothyroïdie). Des mutations germinales dans les facteurs de transcription liés à la thyroïde (NKX2.1, FOXE1, PAX8, NKX2.5) ont été identifiées dans seulement 3% des patients atteints d’HCDT sporadiques et l’analyse de liaisons exclue ces gènes dans les rares familles multiplex avec HCDT. Nous supposons que le manque de transmission familiale claire d’HCDT peut résulter de la nécessité d’au moins deux « hits » génétiques différents dans des gènes importants pour le développement thyroïdien. Pour répondre au mieux nos questions de recherche, nous avons utilisé deux approches différentes: 1) une approche gène candidat, FOXE1, seul gène impliqué dans l’ectopie dans le modèle murin et 2) une approche en utilisant les techniques de séquençage de nouvelle génération (NGS) afin de trouver des variants génétiques pouvant expliquer cette pathologie au sein d’une cohorte de patients avec HCDT. Pour la première approche, une étude cas-contrôles a été réalisée sur le promoteur de FOXE1. Il a récemment été découvert qu’une région du promoteur de FOXE1 est différentiellement méthylée au niveau de deux dinucléotides CpG consécutifs, définissant une zone cruciale de contrôle de l’expression de FOXE1. L’analyse d’association basée sur les haplotypes a révélé qu’un haplotype (Hap1: ACCCCCCdel1C) est associé avec le HCDT chez les Caucasiens (p = 5x10-03). Une réduction significative de l’activité luciférase est observée pour Hap1 (réduction de 68%, p<0.001) comparé au promoteur WT de FOXE1. Une réduction de 50% de l’expression de FOXE1 dans une lignée de cellules thyroïdienne humaine est suffisante pour réduire significativement la migration cellulaire (réduction de 55%, p<0.05). Un autre haplotype (Hap2: ACCCCCCC) est observé moins fréquemment chez les Afro-Américain comparés aux Caucasiens (p = 1.7x10-03) et Hap2 diminue l’activité luciférase (réduction de 26%, p<0.001). Deux haplotypes distincts sont trouvés fréquemment dans les contrôles Africains (Black-African descents). Le premier haplotype (Hap3: GTCCCAAC) est fréquent (30.2%) chez les contrôles Afro-Américains comparés aux contrôles Caucasiens (6.3%; p = 2.59 x 10-9) tandis que le second haplotype (Hap4: GTCCGCAC) est trouvé exclusivement chez les contrôles Afro-Américains (9.4%) et est absent chez les contrôles Caucasiens (P = 2.59 x 10-6). Pour la deuxième approche, le séquençage de l’exome de l’ADN leucocytaire entre les jumeaux MZ discordants n’a révélé aucune différence. D'où l'intérêt du projet de séquençage de l’ADN et l’ARN de thyroïdes ectopiques et orthotopiques dans lesquelles de l'expression monoallélique aléatoire dans a été observée, ce qui pourrait expliquer comment une mutation monoallélique peut avoir des conséquences pathogéniques. Finalement, le séquençage de l’exome d’une cohorte de 36 cas atteints d’HCDT a permis d’identifier de nouveaux variants probablement pathogéniques dans les gènes récurrents RYR3, SSPO, IKBKE et TNXB. Ces quatre gènes sont impliqués dans l’adhésion focale (jouant un rôle dans la migration cellulaire), suggérant un rôle direct dans les défauts de migration de la thyroïde. Les essais de migration montrent une forte diminution (au moins 60% à 5h) de la migration des cellules thyroïdiennes infectées par shRNA comparés au shCtrl dans 2 de ces gènes. Des zebrafish KO (-/- et +/-) pour ces nouveaux gènes seront réalisés afin d’évaluer leur impact sur l’embryologie de la thyroïde.

Development and regeneration of neuronal circuits in the vertebrate retina

Thesis (Ph.D.)--University of Washington, 2016-08

Identificação do transcrito CYP1A no peixe Phalloceros caudimaculatus e resposta para a exposição a beta-naftoflavona e amostras ambientais de sedimento

Contaminantes orgânicos, como os hidrocarbonetos policíclicos aromáticos (HPAs), podem atingir corpos da água e possuem potencial para causar efeitos tóxicos em organismos. A exposição aos HPAs causa indução nos níveis de citocromo P450 1A (CYP1A) em peixes, e portanto, é utilizado como um biomarcador de contaminação ambiental. O guarú Phalloceros caudimaculatus ocorre naturalmente em ambientes aquáticos dulcícolas e mixohalinos na América do Sul. O presente estudo identificou a sequência nucleotídica do transcrito CYP1A de P. caudimaculatus, que codifica uma proteína com 521 aminoácidos, e que apresenta 91% e 70% de identidade com CYP1A de killifish e paulistinha, respectivamente. A partir desta sequência foi possível realizar a avaliação dos níveis de mRNA de CYP1A deste peixe por RTq-PCR. Foi realizada uma caracterização de sua indução órgão- e tempo-dependente frente a exposição ao HPA beta-naftoflavona (BNF) e ao elutriato preparado a partir de sedimento de dois corpos da água possivelmente contaminados com HPAs. Foi constatado um aumento significativo nos níveis de mRNA de CYP1A em fígado, brânquia, intestino, cérebro, nadadeira anal de macho adultos e em alevinos na primeira hora de exposição a 1 µM de BNF, em relação ao grupo controle. O rim e as nadadeiras caudal e dorsal apresentaram indução de CYP1A após duas horas de exposição ao BNF. As maiores induções nos peixes dos grupos expostos ao BNF em relação ao controle foram de 176 no rim e 122 vezes no cérebro, observadas respectivamente após 8 e 48 horas de exposição. Os níveis de mRNA de CYP1A nos órgãos e tecidos de alevino, mantiveramse induzidos pela exposição ao BNF até o final das 96 horas de exposição. A exposição dos peixes ao elutriato produzido a partir dos sedimentos coletados em dois locais potencialmente contaminados causou indução do CYP1A no fígado de 22 e 122 vezes em relação ao controle. Os resultados demonstram que a indução de CYP1A em Phalloceros caudimaculatus ocorre em tempos curtos de exposição, além da variação de acordo com o tempo de exposição e com o órgão analisado. Além disso, foi demonstrado que tecidos externos também podem ser utilizados para tais análises e que o elutriato feito a partir de sedimento de locais que recebem descargas de contaminantes podem causar indução de CYP1A nos organismos.

Efeitos da dupla transgenia do eixo somatotrópico (GH/GHR) sobre aspectos estruturais e moleculares do sistema imunológico do peixe-zebra (Danio rerio)

O desenvolvimento de organismos transgênicos para o hormônio do crescimento (GH) tem sido considerado uma importante alternativa para o aumento nas taxas de crescimento animal. Entretanto, os efeitos do excesso do GH não se limitam aos processos do crescimento. Sistemas fisiológicos como o sistema imunológico, já demonstraram ser prejudicados pelo desbalanço dos níveis do GH. Tendo em vista a importância da geração de organismos transgênicos para o GH no âmbito da aquicultura, esforços se fazem necessários na elaboração de estratégias com o intuito de reduzir ou compensar os efeitos adicionais do excesso de GH. Nos hipotetizamos que a geração de peixes duplo transgênicos os quais superexpressem GH e GHR apenas no músculo esquelético, poderia ser uma possível alternativa para compensar os efeitos prejudiciais ocasionados pelo excesso de GH sobre os sistemas fisiológicos mantendo, ao mesmo tempo, as altas taxas de crescimento. Afim de testar esta hipótese, nós avaliamos a morfometria de órgãos imunes, como o timo, o rim cefálico e o baço; os conteúdos de células T CD3+ e CD4+ no timo e no rim anterior e a expressão de genes relacionados à imunidade. Contrariando as expectativas iniciais, os resultados revelaram que a superexpressão do GHR no músculo esquelético não é capaz de diminuir os efeitos danosos causados pelo GH no tamanho do timo e rim anterior e no conteúdo de células T CD3+ e CD4+ nestes órgãos. Inesperadamente, zebrafish transgênicos somente para o GHR revelaram prejuízos nos aspectos imunes similares aos observados aos transgênicos para GH. De forma geral, estes resultados indicam que a dupla transgenia para o GH/GHR não é capaz de atenuar os efeitos negativos causados pelo excess de GH sobre o sistema imunológico de zebrafish transgênicos. Além disso, a transgenia para genes componentes do eixo somatotrófico pode ainda reforçar os danos as funções imunes em transgênicos, não recuperando os danos causados pelo excesso de GH.