Investigating the effects of mutations causative for early-onset familial Alzheimer’s disease using zebrafish as a model organism

Development of an effective therapeutic for Alzheimer’s disease (AD) is currently a global health priority. To prevent, or at least delay, the onset of AD, we must understand the initial cellular stresses/changes that drive the disease. These initiating changes are likely subtle and occur decades before symptom onset. We cannot easily investigate these changes in humans, as pre-symptomatic brain material from individuals genetically pre-disposed to AD is inaccessible for detailed molecular analyses. For this, we must utilise animal models. The most frequently used animal models of AD are mice expressing one or more transgenes containing the sequences of human genes bearing mutations which cause AD. These transgenic models have been useful in elucidating some aspects of the pathogenic mechanisms of AD. However, they have not led to development of successful therapeutics. Mutations in a small number of genes cause early-onset familial forms of AD (EOfAD). These mutations can be introduced into the orthologous, endogenous genes of an animal (i.e. knock-in models). However, relatively few papers describe research with knock-in models. Transcriptome analysis is currently the most detailed form of molecular phenotyping and can give a largely unbiased view of the molecular state of the brains of young knock-in models of EOfAD. Surprisingly, this has not previously been performed using knock-in models of EOfAD-like mutations. To address this gap in our knowledge, the work presented in this thesis (along with previous work from the Alzheimer’s Disease Genetics Laboratory (ADGL)), describes the generation, and/or characterisation of a collection of zebrafish knock-in models of EOfAD-like mutations. The power of zebrafish as a model organism lies in this species’ ability to generate large families of synchronous siblings which can be raised together in the same tank. This has allowed the assessment of the effects of heterozygosity for EOfAD-like mutations (closely mimicking the genetic state of human EOfAD) or the effects of non-EOfAD-like mutations (such as frameshift mutations in presenilin genes) on the brain transcriptome with minimal external sources of “noise.” These analyses have revealed that the only cellular process predicted to be affected by EOfAD-like mutations in the heterozygous state, and not by non-AD-related mutations, is oxidative phosphorylation. Comparison of these transcriptomes with recent, publicly available brain transcriptomes from two knock-in mouse models of late onset AD risk alleles revealed similar affected processes, thereby supporting the findings from the zebrafish models. Preliminary non-transcriptomic characterisations of previously generated/novel zebrafish models were also performed. The effects of heterozygosity for EOfAD-like mutations on brain vasculature were assessed, as well as effects on spatial working memory. Only limited differences were observed in these studies. However, future work with greater statistical power and/or alternate study designs is recommended. Overall, the research described in this thesis demonstrates the value of unbiased, transcriptome analyses of young, knock-in animals models for understanding the early stages of AD pathogenesis.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 202

O aprendizado da leitura e da escrita pela perspectiva da neurolinguística discursiva Aprendizaje de la lectura y la escritura para concepción discursiva neurolingüística Learning of reading and writing for perspective discursive neurolinguistics

Embasado na abordagem de Neurolinguística Discursiva (1986), este artigo tem como objetivos refletir sobre as dificuldades de aprendizagem vividas pelas crianças para se alfabetizarem e contribuir para a reflexão sobre as práticas escolares que ainda se mantêm pautadas pela concepção tradicional de linguagem. Norteadas pelo conceito de interlocução, as questões relativas à entrada da criança na leitura e na escrita foram tratadas através do papel privilegiado atribuído à fala na constituição da subjetividade e a partir da perspectiva vygotskyana, que considera a leitura e a escrita como desdobramentos da relação entre a fala e o pensamento (Vygotsky, 1934)

Plantagora: Modeling Whole Genome Sequencing and Assembly of Plant Genomes

BACKGROUND: Genomics studies are being revolutionized by the next generation sequencing technologies, which have made whole genome sequencing much more accessible to the average researcher. Whole genome sequencing with the new technologies is a developing art that, despite the large volumes of data that can be produced, may still fail to provide a clear and thorough map of a genome. The Plantagora project was conceived to address specifically the gap between having the technical tools for genome sequencing and knowing precisely the best way to use them. METHODOLOGY/PRINCIPAL FINDINGS: For Plantagora, a platform was created for generating simulated reads from several different plant genomes of different sizes. The resulting read files mimicked either 454 or Illumina reads, with varying paired end spacing. Thousands of datasets of reads were created, most derived from our primary model genome, rice chromosome one. All reads were assembled with different software assemblers, including Newbler, Abyss, and SOAPdenovo, and the resulting assemblies were evaluated by an extensive battery of metrics chosen for these studies. The metrics included both statistics of the assembly sequences and fidelity-related measures derived by alignment of the assemblies to the original genome source for the reads. The results were presented in a website, which includes a data graphing tool, all created to help the user compare rapidly the feasibility and effectiveness of different sequencing and assembly strategies prior to testing an approach in the lab. Some of our own conclusions regarding the different strategies were also recorded on the website. CONCLUSIONS/SIGNIFICANCE: Plantagora provides a substantial body of information for comparing different approaches to sequencing a plant genome, and some conclusions regarding some of the specific approaches. Plantagora also provides a platform of metrics and tools for studying the process of sequencing and assembly further

Grid flexibility and patching techniques

The numerical determination of combustor flowfields is of great value to the combustor designer. An a priori knowledge of the flow behavior can speed the combustor design process and reduce the number of experimental test rigs required to arrive at an optimal design. Even 2-D steady incompressible isothermal flow predictions are of use; many codes of this kind are available, each employing different techniques to surmount the difficulties arising from the nonlinearity of the governing equations and from typically irregular combustor geometries. Mapping techniques (algebraic and elliptic PDE), and adaptive grid methods (both multi-grid and grid embedding) as applied to axisymmetric combustors are discussed

Comparison of the contributions of the nuclear and cytoplasmic compartments to global gene expression in human cells

<p>Abstract</p> <p>Background</p> <p>In the most general sense, studies involving global analysis of gene expression aim to provide a comprehensive catalog of the components involved in the production of recognizable cellular phenotypes. These studies are often limited by the available technologies. One technology, based on microarrays, categorizes gene expression in terms of the abundance of RNA transcripts, and typically employs RNA prepared from whole cells, where cytoplasmic RNA predominates.</p> <p>Results</p> <p>Using microarrays comprising oligonucleotide probes that represent either protein-coding transcripts or microRNAs (miRNA), we have studied global transcript accumulation patterns for the HepG2 (human hepatoma) cell line. Through subdividing the total pool of RNA transcripts into samples from nuclei, the cytoplasm, and whole cells, we determined the degree of correlation of these patterns across these different subcellular locations. The transcript and miRNA abundance patterns for the three RNA fractions were largely similar, but with some exceptions: nuclear RNA samples were enriched with respect to the cytoplasm in transcripts encoding proteins associated with specific nuclear functions, such as the cell cycle, mitosis, and transcription. The cytoplasmic RNA fraction also was enriched, when compared to the nucleus, in transcripts for proteins related to specific nuclear functions, including the cell cycle, DNA replication, and DNA repair. Some transcripts related to the ubiquitin cycle, and transcripts for various membrane proteins were sorted into either the nuclear or cytoplasmic fractions.</p> <p>Conclusion</p> <p>Enrichment or compartmentalization of cell cycle and ubiquitin cycle transcripts within the nucleus may be related to the regulation of their expression, by preventing their translation to proteins. In this way, these cellular functions may be tightly controlled by regulating the release of mRNA from the nucleus and thereby the expression of key rate limiting steps in these pathways. Many miRNA precursors were also enriched in the nuclear samples, with significantly fewer being enriched in the cytoplasm. Studies of mRNA localization will help to clarify the roles RNA processing and transport play in the regulation of cellular function.</p

Comparison of image analysis software packages in the assessment of adhesion of microorganisms to mucosal epithelium using confocal laser scanning microscopy

We have compared current image analysis software packages in order to find the most useful one for assessing microbial adhesion and inhibition of adhesion to tissue sections. We have used organisms of different sizes, the bacterium Helicobacter pylori and the yeast Candida albicans. Adhesion of FITC-labelled H. pylori and C. albicans was assessed by confocal microscopy. Four different Image analysis software packages, NIH-Image, IP Lab, Image Pro+, and Metamorph, were compared for their ability to quantify adhesion of the two organisms and several quantification methods were devised for each package. For both organisms, the dynamic range that could be detected by the software packages was 1×106?1×109 cells/ml. Of the four software packages tested, our results showed that Metamorph software, using our ?Region of Interest? method, with the software's ?Standard Area Method? of counting, was the most suitable for quantifying adhesion of both organisms because of its unique ability to separate clumps of microbial cells. Moreover, fewer steps were required. By pre-incubating H. pylori with the glycoconjugate Lewis b-HSA, an inhibition of binding of 48.8% was achieved using 250 ?g/ml Lewis b-HSA. The method we have devised using Metamorph software, provides a simple, quick and accurate way of quantifying adhesion and inhibition of adhesion of microbial cells to the epithelial surface of tissue sections. The method can be applied to organisms ranging in size from small bacteria to larger yeast cells

Transcriptome analysis indicates dominant effects on ribosome and mitochondrial function of a premature termination codon mutation in the zebrafish gene psen2

Published: July 13, 2020PRESENILIN 2 (PSEN2) is one of the genes mutated in early onset familial Alzheimer's disease (EOfAD). PSEN2 shares significant amino acid sequence identity with another EOfAD-related gene PRESENILIN 1 (PSEN1), and partial functional redundancy is seen between these two genes. However, the complete range of functions of PSEN1 and PSEN2 is not yet understood. In this study, we performed targeted mutagenesis of the zebrafish psen2 gene to generate a premature termination codon close downstream of the translation start with the intention of creating a null mutation. Homozygotes for this mutation, psen2S4Ter, are viable and fertile, and adults do not show any gross psen2-dependent pigmentation defects, arguing against significant loss of γ-secretase activity. Also, assessment of the numbers of Dorsal Longitudinal Ascending (DoLA) interneurons that are responsive to psen2 but not psen1 activity during embryogenesis did not reveal decreased psen2 function. Transcripts containing the S4Ter mutation show no evidence of destabilization by nonsense-mediated decay. Forced expression in zebrafish embryos of fusions of psen2S4Ter 5' mRNA sequences with sequence encoding enhanced green fluorescent protein (EGFP) indicated that the psen2S4Ter mutation permits utilization of cryptic, novel downstream translation start codons. These likely initiate translation of N-terminally truncated Psen2 proteins lacking late endosomal/lysosomal localization sequences and that obey the "reading frame preservation rule" of PRESENILIN EOfAD mutations. Transcriptome analysis of entire brains from a 6-month-old family of wild type, heterozygous and homozygous psen2S4Ter female siblings revealed profoundly dominant effects on gene expression likely indicating changes in ribosomal, mitochondrial, and anion transport functions.Haowei Jiang, Stephen Martin Pederson, Morgan Newman, Yang Dong, Karissa Barthelson, Michael Lardell

BgaA acts as an adhesin to mediate attachment of some pneumococcal strains to human epithelial cells

Streptococcus pneumoniae colonization of the respiratory tract is an essential precursor for pneumococcal disease. To colonize efficiently, bacteria must adhere to the epithelial-cell surface. S. pneumoniae possesses surface-associated exoglycosidases that are capable of sequentially deglycosylating human glycans. Two exoglycosidases, neuraminidase (NanA) and β-galactosidase (BgaA), have previously been shown to contribute to S. pneumoniae adherence to human epithelial cells, as deletion of either of these genes results in reduced adherence. It has been suggested that these enzymes may modulate adherence by cleaving sugars to reveal a receptor on host cells. Pretreatment of epithelial cells with exogenous neuraminidase restores the adherence of a nanA mutant, whereas pretreatment with β-galactosidase does not restore the adherence of a bgaA mutant. These data suggest that BgaA may not function to reveal a receptor, and implicate an alternative role for BgaA in adherence. Here we demonstrate that β-galactosidase activity is not required for BgaA-mediated adherence. Addition of recombinant BgaA (rBgaA) to adherence assays and pretreatment of epithelial cells with rBgaA both significantly reduced the level of adherence of the parental strain, but not the BgaA mutant. One possible explanation of these data is that BgaA is acting as an adhesin and that rBgaA is binding to the receptor, preventing bacterial binding. A bead-binding assay demonstrated that BgaA can bind directly to human epithelial cells, supporting the hypothesis that BgaA is an adhesin. Preliminary characterization of the epithelial-cell receptor suggests that it is a glycan in the context of a glycosphingolipid. To further establish the relevance of this adherence mechanism, we demonstrated that BgaA-mediated adherence contributed to adherence of a recent clinical isolate to primary human epithelial cells. Together, these data suggest a novel role for BgaA as an adhesin and suggest that this mechanism could contribute to adherence of at least some pneumococcal strains in vivo