16 research outputs found
Molecular mechanisms underlying Retinitis pigmentosa type 2
No full textThe 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
The Sexually Dimorphic Adrenal Cortex: Implications for Adrenal Disease
No full textMany adrenocortical diseases are more prevalent in women than in men, but the reasons underlying this sex bias are still unknown. Recent studies involving gonadectomy and sex hormone replacement experiments in mice have shed some light onto the molecular basis of sexual dimorphism in the adrenal cortex. Indeed, it has been shown that gonadal hormones influence many aspects of adrenal physiology, ranging from stem cell-dependent tissue turnover to steroidogenesis and X-zone dynamics. This article reviews current knowledge on adrenal cortex sexual dimorphism and the potential mechanisms underlying sex hormone influence of adrenal homeostasis. Both topics are expected to contribute to personalized and novel therapeutic approaches in the future
ERT’s shutdown, social amnesia, and communicative entitlements
No full textThe Greek government’s decision to close ERT has been criticised in various activist channels as anti-democratic or even irrational. Yet these activists and opponents of the ERT decision are held together only by thin strands and, in truth, represent heterogenous and conflicting interests and agendas
Avatar's 'Development' Predicament
No full textThe globally-acclaimed film looks back to the past from a futuristic standpoint to simulate an archetypal moral tale of developmental inequality. Is that a good thing
Functional analysis of RP2 and ARL3 in X-linked retinitis pigmentosa
No full textRetinitis 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
Osteoclast stimulation factor 1 (Ostf1) KNOCKOUT increases trabecular bone mass in mice
No full textOsteoclast stimulation factor 1 (OSTF1) is an SH3-domain containing protein that was initially identified as a factor involved in the indirect activation of osteoclasts. It has been linked to spinal muscular atrophy in humans through its interaction with SMN1, and is one of six genes deleted in a human developmental microdeletion syndrome. To investigate the function of OSTF1, we generated an Ostf1 knockout mouse model, with exons 3 and 4 of Ostf1 replaced by a LacZ orf. Extensive X-Gal staining was performed to examine the developmental and adult expression pattern, followed by phenotyping. We show widespread expression of the gene in the vasculature of most organs and in a number of cell types in adult and embryonic mouse tissues. Furthermore, whilst SHIRPA testing revealed no behavioural defects, we demonstrate increased trabecular mass in the long bones, confirming a role for OSTF1 in bone development
Characterization of a novel RP2-OSTF1 interaction and its implication for actin remodeling
No full textRetinitis pigmentosa 2 (RP2) is the causative gene for a form of X-linked retinal degeneration. RP2 was previously shown to have GAP activity towards the small GTPase ARL3 via its N-terminus, but the function of the C-terminus remains elusive. Here, we report a novel interaction between RP2 and Osteoclast-stimulating factor 1 (OSTF1), an intracellular protein that indirectly enhances osteoclast formation and activity and is a negative regulator of cell motility. Moreover, this interaction is abolished by a human pathogenic mutation in RP2. We utilized a structure-based approach to pinpoint the binding interface to a strictly conserved cluster of residues on the surface of RP2 that spans both the C- and N- terminal domains of the protein, and which is structurally distinct from the ARL3 binding site. In addition, we show that RP2 is a positive regulator of cell motility in vitro, recruiting OSTF1 to the cell membrane and preventing its interaction with the migration regulator Myo1E.</p
Validation of the TROPOMI/S5P aerosol layer height using EARLINET lidars
No full textThe purpose of this study is to investigate the ability of the Sentinel-5P TROPOspheric Monitoring Instrument (TROPOMI) to derive accurate geometrical features of lofted aerosol layers, selecting the Mediterranean Basin as the study area. Comparisons with ground-based correlative measurements constitute a key component in the validation of passive and active satellite aerosol products. For this purpose, we use ground-based observations from quality-controlled lidar stations reporting to the European Aerosol Research Lidar Network (EARLINET). An optimal methodology for validation purposes has been developed and applied using the EARLINET optical profiles and TROPOMI aerosol products, aiming at the in-depth evaluation of the TROPOMI aerosol layer height (ALH) product for the period 2018 to 2022 over the Mediterranean Basin. Seven EARLINET stations were chosen, taking into consideration their proximity to the sea, which provided 63 coincident aerosol cases for the satellite retrievals. In the following, we present the first validation results for the TROPOMI/S5P ALH using the optimized EARLINET lidar products employing the automated validation chain designed for this purpose. The quantitative validation at pixels over the selected EARLINET stations illustrates that the TROPOMI ALH product is consistent with the EARLINET lidar products, with a high correlation coefficient RCombining double low line0.82 (RCombining double low line0.51) and a mean bias of -0.51±0.77 km and -2.27±1.17 km over ocean and land, respectively. Overall, it appears that aerosol layer altitudes retrieved from TROPOMI are systematically lower than altitudes from the lidar retrievals. High-albedo scenes, as well as low-aerosol-load scenes, are the most challenging for the TROPOMI retrieval algorithm, and these results testify to the need to further investigate the underlying cause. This work provides a clear indication that the TROPOMI ALH product can under certain conditions achieve the required threshold accuracy and precision requirements of 1 km, especially when only ocean pixels are included in the comparison analysis. Furthermore, we describe and analyse three case studies in detail, one dust and two smoke episodes, in order to illustrate the strengths and limitations of the TROPOMI ALH product and demonstrate the presented validation methodology. The present analysis provides important additions to the existing validation studies that have been performed so far for the TROPOMI S5P ALH product, which were based only on satellite-to-satellite comparisons.Atmospheric Remote Sensin
