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Investigating the role of eef1a2 in zebrafish as a potential disease model
Get PDFEukaryotic elongation factor (eEF1A) plays a vital role in protein synthesis. It recruits
amino-acylated tRNAs and delivers them to the ribosome during protein translation.
eEF1A is conserved throughout evolution and exists as independently encoded
isoforms in many species. In mammals, there are two isoforms: eEF1A1 and eEF1A2.
Unlike eEF1A1 which is widely expressed, expression of eEF1A2 is restricted to the
brain, heart and skeletal muscle and is upregulated during development. In mice,
homozygote deletion in Eef1a2 gene resulting in the complete loss of function of
eEF1A2 causes severe neurodegeneration, loss of muscle bulk and death by 28 days.
Recently, de novo heterozygous missense mutation in EEF1A2 has been identified in
humans which cause epilepsy, autism and intellectual disability. The main aim of this
project was to investigate the use of zebrafish as a model to better understand the role
of eEF1A2 in neurological disorders. In addition to its many advantages, the zebrafish
has been shown to be an excellent tool for in vivo drug screening. This is an attractive
attribute for our studies as regards developing treatment strategies for these disorders.
Zebrafish possess four eef1a genes: eef1a1l1, eef1a1a, eef1a1b and eef1a2 which
encodes separate highly similar proteins: eEF1A1L1, eEF1A1A, eEF1A1B and
eEF1A2 respectively. The zebrafish eEF1A2 shares a 94% sequence identity with the
mouse and human eEF1A2 at the amino acid level. In this work, characterisation of
zebrafish eEF1A genes was first carried out, as there is currently little information
available. Using conventional reverse transcriptase polymerase chain reaction (RTPCR)
and real time quantitative PCR (qPCR), I analysed the expression pattern of
eef1a genes at different embryonic stages and adult tissues. These genes were
differentially expressed with only eef1a1l1 detected at earlier developmental stages,
followed by eef1a1a and eef1a1b. Similar to mammals, eef1a2 is detected much later
(48 hpf) during development. Co-expression of eef1a mRNA was observed in the adult
tissues analysed except in liver where eef1a2 was not detected. An attempt to knock-in
one of the epilepsy causing variant, G70S into the zebrafish genome using
CRISPR/Cas9 technology was unsuccessful. However, I established two null eef1a2
mutant lines using this technology. Homozygotes from these null lines showed no
obvious phenotype and in contrast to null Eef1a2 mice, they are fertile and viable
through adulthood. No evidence of neurodegeneration was observed. These results
suggest the possibility of compensatory mechanisms activated by the other eef1a genes
to buffer the loss of eef1a2 in the mutants. However, preliminary findings suggest that
eef1a null mutation might cause zebrafish to be susceptible to PTZ-induced seizures.
Results from this work has provided vital information on functional redundancy of
eef1a genes in zebrafish and a foundation for further validation of the zebrafish as a
model system
Functional analysis of RP2 and ARL3 in X-linked retinitis pigmentosa
Get PDFRetinitis Pigmentosa (RP) is a disease of the retina, which causes progressive retinal
degeneration. X-linked RP is one of the most severe subtypes with an estimated 15%
of cases caused by mutations in RP2. RP2 functions as a GTPase Activating Protein
(GAP) for the small G protein ARL3, which is proposed to regulate the traffic of lipid-modified
proteins within photoreceptors. It is hypothesised that mutations in RP2
result in dysregulation of ARL3 and therefore protein mis-trafficking. In order to
elucidate the contribution of ARL3 dysregulation to the pathogenesis of RP, I have
established new mouse models by CRISPR-mediated genome editing. These include
an Rp2h knockout line and a line, which harbours a human pathogenic missense
mutation, E135G, which abolishes interaction with ARL3.
Furthermore, I have generated mice carrying a Q71L missense mutation in Arl3. This
mutation locks ARL3 in the active GTP-bound state, and hence is predicted to
phenocopy Rp2h knockout. Histological examination has revealed that Rp2h
knockout, Rp2h E135G and Arl3 Q71L/+ mutant animals display progressive retinal
degeneration evident from age 6 months. Arl3 Q71L/Q71L animals display retinal
degeneration at age 3 months demonstrating that elevated levels of ARL3-GTP is a
driver of retinal degeneration in mice. Immunofluorescence analysis has shown ARL3
Q71L mice, Rp2h knockout mice and Rp2h E135G/Y mice show mislocalisation of
lipid modified proteins likely driving retinal degeneration, however further analysis has
shown that these mice do not completely phenocopy each other suggesting that levels
of ARL3-GTP may not be the only mechanism contributing to retinal degeneration in
Rp2h mutant mice.
The mechanisms of RP2 regulation are not well understood; therefore to identify
potential interactors of RP2 a BIO-ID proximity labelling assay in RPE-1 cells was
performed. A top hit from this assay was palmitoyltransferase ZDHHC5. I confirmed
this interaction in cells and using a click chemistry based approach demonstrated that
it is unlikely that this enzyme functions to palmitoylate RP2. Using
immunofluorescence in HeLa cells I have shown that overexpression of ZDHHC5 can
rescue the localisation of human pathogenic RP2 mutants C3S and G2A, which are
normally mislocalised in vivo, independent of its catalytic activity. SiRNA knockdown
of ZDHHC5 in cells leads to mislocalisation of RP2 demonstrating ZDHHC5 has a role
in trafficking RP2. Results from these studies have provided new knowledge
regarding the mechanisms that cause retinal degeneration and new insights into the
potential mechanisms that regulate the trafficking of lipid-modified proteins in
photoreceptors
Genetic analysis of urinary traits in Scottish and Croatian populations and a functional study of Arl15 in the kidneys
Get PDFMagnesium is the second most abundant bivalent cation in the body and is essential for many cellular processes. Electrolyte imbalance, including changes in magnesium concentration and other clinically relevant electrolyte ratios in the body, can result in dizziness and arrhythmia. If left untreated, this can result in serious illness or even death. Renal magnesium handling plays an important role in maintaining magnesium homeostasis, however the exact biological mechanisms remain unclear.
A recent genome-wide association study (GWAS) identified an association between the ratio of urinary magnesium concentration to creatinine and variants in the ARL15 gene on chromosome 5. I expanded these analyses by performing meta-analyses for 29 urinary traits in 11,617 individuals from Scottish and Croatian populations. This identified additional genes that may be involved in these traits and confirmed that ARL15 is associated with urinary magnesium (uMg) related traits. The SNP with the most significant P value associated with uMg in the meta-analyses, rs35931, lies within an EGR1 transcription factor binding site in an enhancer region of ARL15. Individuals homozygous for the non-reference allele of rs35931 had lower urinary magnesium levels compared to individuals with the homozygous reference allele. Since ARL15 encodes a GTP-binding protein that regulates the magnesium transporter channel TRPM6, and other proteins involved in magnesium homeostasis in physiologically relevant cell lines, the hypothesis was that genetic variation affects the expression of ARL15, which, in turn modulates magnesium transport by regulating magnesium transporters. Functional studies were performed in order to investigate this and to elucidate the role of ARL15 in magnesium homeostasis both in vitro and in vivo. This confirmed that Arl15 interacts with magnesium transporters within the distal convoluted tubule segment of the kidneys. In vitro knockout models of Arl15 established using CRISPR-Cas9 gene editing in a mouse distal convoluted tubule cell-line showed significantly impaired magnesium transport. Furthermore, homozygous deletion of Arl15 is lethal at the organismal level in the mouse, highlighting the crucial role of ARL15 in health and disease
Molecular mechanisms underlying Retinitis pigmentosa type 2
Get PDFThe term “Retinitis pigmentosa” (RP) represents a group of inherited, late-onset
diseases characterised by progressive retinal degeneration due to photoreceptor
death. Mutations in the RP2 gene are found in 7-18% of patients with X-linked RP,
one of the most severe forms. The RP2 gene product is a membrane-associated
protein which encompasses two distinct domains. The N-terminal domain is well
characterised as possessing GTPase-activating protein (GAP) activity towards the
small GTPase ARL3 and thus regulate the transport of lipid-modified proteins within
the photoreceptor cell. However, it is not known if the loss of this particular function
of RP2 is the sole reason that causes the disease, while the role of the protein’s C-terminus
remains unknown. This thesis focuses on the characterisation of two novel
protein-protein interactions of RP2 with the aim to investigate novel roles of the
protein. Firstly, evidence is provided that a highly-conserved cluster of RP2 residues
that span both the N- and C-terminus participate in direct interaction with Osteoclast-stimulating
factor 1 (OSTF1). Two hypotheses are explored about the potential role
of the complex in SRC-mediated RP2 phosphorylation and the regulation of cell
motility. Secondly, the catalytic subunit of DNA-dependent protein kinase (DNA
PK) is identified as a novel interaction partner of RP2 in cultured cells. The two
proteins are shown to co-localise in the nuclear and membrane compartments of a
retinal-derived cell line and might engage in a kinase-substrate relationship. So far,
no evidence was found that RP2 participates in the canonical function of DNA PK
which is the regulation of DNA double-stranded breaks. Finally, the CRISPR/Cas9
genome editing method was applied on zebrafish embryos to generate a novel
vertebrate animal model for the loss of RP2 function. One out of three different
zebrafish lines with rp2 mutations was shown by histology to have mild late-onset
thinning of the photoreceptor outer segments. The present thesis reports previously
unexplored aspects of RP2’s function and will, therefore, contribute to understanding
the molecular mechanisms that underlie RP. Moreover, this thesis will contribute to
the discussion about the usefulness of zebrafish as an RP model
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Retinitis Pigmentosa GTPase Regulator (RPGR) protein isoforms in mammalian retina:insights into X-linked Retinitis Pigmentosa and associated ciliopathies
Get PDFMutations in the cilia-centrosomal protein Retinitis Pigmentosa GTPase Regulator (RPGR) are a frequent cause of retinal degeneration. The RPGR gene undergoes complex alternative splicing and encodes multiple protein isoforms. To elucidate the function of major RPGR isoforms (RPGR 1-19 and RPGR ORF15), we have generated isoform-specific antibodies and examined their expression and localization in the retina. Using sucrose-gradient centrifugation, immunofluorescence and co-immunoprecipitation methods, we show that RPGR isoforms localize to distinct sub-cellular compartments in mammalian photoreceptors and associate with a number of cilia-centrosomal proteins. The RCC1-like domain of RPGR, which is present in all major RPGR isoforms, is sufficient to target it to the cilia and centrosomes in cultured cells. Our findings indicate that multiple isotypes of RPGR may perform overlapping yet somewhat distinct transport-related functions in photoreceptors
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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