TLE proteins in mouse embryonic stem cell self renewal and early lineage specification

TLE proteins are a closely related family of vertebrate corepressors. They have no intrinsic DNA binding ability, but are recruited as transcriptional repressors by other sequence specific proteins. TLE proteins and their homologues in other species have been implicated in many developmental processes including neurogenesis, haematopoiesis and the formation of major organs. They have also been implicated in early lineage specification in vertebrates but a direct role in this has not been found in mammals. The aim of my PhD is therefore to analyse the function of TLE proteins in early lineage specification and cell fate decisions using mouse embryonic stem cells (ESCs) as a model. The investigation of this has previously been complicated, firstly by the large array of transcription factors that TLEs interact with and secondly by redundancy between similar TLE proteins hindering loss of function approaches. To circumvent these problems, I have used two complementary experimental strategies. The first was identification of point mutations in TLE1 that affect specific classes of DNA binding. Two of these mutations L743F and R534A were of particular interest and were reversibly overexpressed in ES cells to correlate phenotypes to biochemical activity. The second strategy was the mutation of the two primary TLC genes in ES cells and early mouse embryos, TLE3 and TLE4. Complementary evidence from these approaches revealed a role for TLEs in the promotion of ES cell differentiation by repression of pluripotency/self-renewal associated genes. Additionally, neural specification was increased by TLE1 expression especially by the TLE1 point mutations, highlighting opposing roles for negative effects on mesendodermal differentiation. Early mesoderm/primitive streak was increased by loss of TLE, probably through Wnt antagonism. Anterior endoderm was increased by reduced TLE, but a critical level of TLE was still necessary and TLE1 overexpression also upregulated some anterior endoderm markers suggesting both negative and positive roles for TLE proteins in this process

The thalamus as a low pass filter: filtering at the cellular level does not equate with filtering at the network level

In the mammalian central nervous system, most sensory information passes through primary sensory thalamic nuclei, however the consequence of this remains unclear. Various propositions exist, likening the thalamus to a gate, or a high pass filter. Here, using a simple leaky integrate and fire model based on physiological parameters, we show that the thalamus behaves akin to a low pass filter. Specifically, as individual cells in the thalamus rely on consistent drive to spike, stimuli that is rapidly and continuously changing over time such that it activates sensory cells with different receptive fields are unable to drive thalamic spiking. This means that thalamic encoding is robust to sensory noise, however it induces a lag in sensory representation. Thus, the thalamus stabilizes encoding of sensory information, at the cost of response rate

Selective metal extraction by biologically produced siderophores during bioleaching from low-grade primary and secondary mineral resources

Siderophores are a class of biogenic macromolecules that have high affinities for metals in the environment, thus could be exploited for alternate sustainable metal recovery technologies. Here, we assess the role of siderophores in the extraction and complexation of metals from an iron oxide-rich metallurgical processing residue and a low-grade primary Ni ore. Evaluation of the biological siderophore production by three pseudomonads, P. fluorescens, P. azotoformans and P. putida identified that P. putida could generate the highest siderophore yield, which was characterized as a hydroxamate and catecholate mixed-type pyoverdine PyoPpC-3B. Key physicochemical parameters involved in raw siderophore mediated metal extraction were identified using a fractional factorial design of experiments (DOE) and subsequently employed in purified PyoPpC-3B leaching experiments. Further targeted experiments with hydroxamate and catecholate functional analogues of PyoPpC-3B confirmed their marked ability to competitively or selectively leach and chelate hard metal ions, including Al(OH)(4)(-), Mn2+ and Zn2+. Interestingly, complexation of Mn and Zn ions exceeded the natural affinity of pyoverdine for Fe3+, thus despite the low metal recoveries from the materials tested in this study, this work provides important new insights in siderophore-metal interactions

Selective leaching of copper and zinc from primary ores and secondary mineral residues using biogenic ammonia

With the number of easily accessible ores depleting, alternate primary and secondary sources are required to meet the increasing demand of economically important metals. Whilst highly abundant, these materials are of lower grade with respect to traditional ores, thus highly selective and sustainable metal extraction technologies are needed to reduce processing costs. Here, we investigated the metal leaching potential of biogenic ammonia produced by a ureolytic strain of Lysinibacillus sphaericus on eight primary and secondary materials, comprised of mining and metallurgical residues, sludges and automotive shredder residues (ASR). For the majority of materials, moderate to high yields (30–70%) and very high selectivity (>97% against iron) of copper and zinc were obtained with 1 mol L−1 total ammonia. Optimal leaching was achieved and further refined for the ASR in a two-step indirect leaching system with biogenic ammonia. Copper leaching was the result of local corrosion and differences in leaching against the synthetic (NH4)2CO3 control could be accounted for by pH shifts from microbial metabolism, subsequently altering free NH3 required for coordination. These results provide important findings for future sustainable metal recovery technologies from secondary materials.This work was conducted under the financial support of the Strategic Initiative Materials in Flanders (SIM) (SBO-SMART: Sustainable Metal Extraction from Tailings, grant no. HBC.2016.0456) and the European Union’s Horizon 2020 research and innovation programme, Metal Re-covery from Low-Grade Ores and Wastes Plus (METGROW+, grant no. 690088) . FV acknowledges support by the Flemish Agency for Inno-vation and Entrepreneurship (Vlaio) via a Baekeland PhD fellowship (HBC.2017.0224) and by the Research & Development Umicore Group. We would like to thank Pieter Ostermeyer and Karel Folens for assis-tance with thermodynamic modelling and CMET and ECOCHEM group members and SMART/METGROW+partners for valuable discussions throughout the projec

Evidence for a dominant-negative effect in ACTA1 nemaline myopathy caused by abnormal folding, aggregation and altered polymerization of mutant actin isoforms

We have studied a cohort of nemaline myopathy (NM) patients with mutations in the muscle α-skeletal actin gene (ACTA1). Immunoblot analysis of patient muscle demonstrates increased γ-filamin, myotilin, desmin and α-actinin in many NM patients, consistent with accumulation of Z line-derived nemaline bodies. We demonstrate that nebulin can appear abnormal secondary to a primary defect in actin, and show by isoelectric focusing that mutant actin isoforms are present within insoluble actin filaments isolated from muscle from two ACTA1 NM patients. Transfection of C2C12 myoblasts with mutant actinEGFP constructs resulted in abnormal cytoplasmic and intranuclear actin aggregates. Intranuclear aggregates were observed with V163L-, V163M- and R183G-actinEGFP constructs, and modeling shows these residues to be adjacent to the nuclear export signal of actin. V163L and V163M actin mutants are known to cause intranuclear rod myopathy, however, intranuclear bodies were not reported in patient R183G. Transfection studies in C2C12 myoblasts showed significant alterations in the ability of V136L and R183G actin mutants to polymerize and contribute to insoluble actin filaments. Thus, we provide direct evidence for a dominant-negative effect of mutant actin in NM. In vitro studies suggest that abnormal folding, altered polymerization and aggregation of mutant actin isoforms are common properties of NM ACTA1 mutants. Some of these effects are mutation-specific, and likely result in variations in the severity of muscle weakness seen in individual patients. A combination of these effects contributes to the common pathological hallmarks of NM, namely intranuclear and cytoplasmic rod formation, accumulation of thin filaments and myofibrillar disorganizatio

Bioleaching of metals from secondary materials using glycolipid biosurfactants

With the global demand for economically important metals increasing, compounded by the depletion of readily accessible ores, secondary resources and low-grade ores are being targeted to meet growing demands. Novel technologies developed within biobased industries, such as microbial biosurfactants, could be implemented to improve the sustainability of traditional hydrometallurgy techniques. This study investigates newly developed microbial biosurfactants (acidic- and bolaform glycolipids) for the leaching of metals (particularly Cu and Zn) from a suite of mine tailings, metallurgical sludges and automotive shredder residues. Generally, acidic sophorolipids were the most performant, and optimal Cu leaching was observed from a fayalite slag (27%) and a copper sulfide mine tailing (53%). Further investigation of the leached fayalite material showed that leaching was occurring from small metallic Cu droplets in this material via a corrosion-based mechanism, and/or from Cu-Pb sulfides, selective against dominant Fe-silicate matrices. This study highlights that acidic sophorolipid microbial biosurfactants have the potential to leach Cu and Zn from low-grade secondary materials. It also provides important fundamental insights into biosurfactant-metal and mineral interactions that are currently unexplored. Together, the convergence of leaching and mining industries with bio-industries can improve material recovery and will positively impact the bio- and circular economies and the environment.The authors thank Bio Base Europe Pilot plant for supplying the biosurfactants that enabled the execution of the leaching experiments. We also thank Joachim Neri, Karel Folens, Nina Ricci Nicomel and Melgü Kizilmese for their assistance during ICP-analyses