Set mapping reflection

In this note we will discuss a new reflection principle which follows from the Proper Forcing Axiom. The immediate purpose will be to prove that the bounded form of the Proper Forcing Axiom implies both that 2^omega = omega_2 and that L(P(omega_1)) satisfies the Axiom of Choice. It will also be demonstrated that this reflection principle implies that combinatorial principle Square(kappa) fails for all regular kappa > omega_1.Comment: 11 page

A toolbox of nanobodies developed and validated for use as intrabodies and nanoscale immunolabels in mammalian brain neurons.

Nanobodies (nAbs) are small, minimal antibodies that have distinct attributes that make them uniquely suited for certain biomedical research, diagnostic and therapeutic applications. Prominent uses include as intracellular antibodies or intrabodies to bind and deliver cargo to specific proteins and/or subcellular sites within cells, and as nanoscale immunolabels for enhanced tissue penetration and improved spatial imaging resolution. Here, we report the generation and validation of nAbs against a set of proteins prominently expressed at specific subcellular sites in mammalian brain neurons. We describe a novel hierarchical validation pipeline to systematically evaluate nAbs isolated by phage display for effective and specific use as intrabodies and immunolabels in mammalian cells including brain neurons. These nAbs form part of a robust toolbox for targeting proteins with distinct and highly spatially-restricted subcellular localization in mammalian brain neurons, allowing for visualization and/or modulation of structure and function at those sites

Super-resolution microscopy reveals specific recruitment of HIV-1 envelope proteins to viral assembly sites dependent on the envelope C-terminal tail

The inner structural Gag proteins and the envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) traffic independently to the plasma membrane, where they assemble the nascent virion. HIV-1 carries a relatively low number of glycoproteins in its membrane, and the mechanism of Env recruitment and virus incorporation is incompletely understood. We employed dual-color super-resolution microscopy visualizing Gag assembly sites and HIV-1 Env proteins in virus-producing and in Env expressing cells. Distinctive HIV-1 Gag assembly sites were readily detected and were associated with Env clusters that always extended beyond the actual Gag assembly site and often showed enrichment at the periphery and surrounding the assembly site. Formation of these Env clusters depended on the presence of other HIV-1 proteins and on the long cytoplasmic tail (CT) of Env. CT deletion, a matrix mutation affecting Env incorporation or Env expression in the absence of other HIV-1 proteins led to much smaller Env clusters, which were not enriched at viral assembly sites. These results show that Env is recruited to HIV-1 assembly sites in a CT-dependent manner, while Env(ΔCT) appears to be randomly incorporated. The observed Env accumulation surrounding Gag assemblies, with a lower density on the actual bud, could facilitate viral spread . Keeping Env molecules on the nascent virus low may be important for escape from the humoral immune response, while cell-cell contacts mediated by surrounding Env molecules could promote HIV-1 transmission through the virological synapse

DOPAL derived alpha-synuclein oligomers impair synaptic vesicles physiological function

Parkinson's disease is a neurodegenerative disorder characterized by the death of dopaminergic neurons and by accumulation of alpha-synuclein (aS) aggregates in the surviving neurons. The dopamine catabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is a highly reactive and toxic molecule that leads to aS oligomerization by covalent modifications to lysine residues. Here we show that DOPAL-induced aS oligomer formation in neurons is associated with damage of synaptic vesicles, and with alterations in the synaptic vesicles pools. To investigate the molecular mechanism that leads to synaptic impairment, we first aimed to characterize the biochemical and biophysical properties of the aS-DOPAL oligomers; heterogeneous ensembles of macromolecules able to permeabilise cholesterol-containing lipid membranes. aS-DOPAL oligomers can induce dopamine leak in an in vitro model of synaptic vesicles and in cellular models. The dopamine released, after conversion to DOPAL in the cytoplasm, could trigger a noxious cycle that further fuels the formation of aS-DOPAL oligomers, inducing neurodegeneration

Establishing optogenetic tools in the auditory system of the Mongolian Gerbil

The Mongolian Gerbil (Meriones unguiculatus) serves as a popular and widely used model organism for the human auditory system. Its hearing range largely overlaps with that of human’s and even extends below 1 kHz, frequencies very important for human hearing. Like humans, gerbils can localize sounds based on their interaural time difference (ITD) or interaural level difference (ILD) and also show perceptual suppression of the spatial source of reverberations (precedence effect). The auditory circuitries underlying the computation of ITDs and ILDs are very well described in the gerbil, although the exact mechanisms for the extraction of ITDs are still under debate. The contribution of the medial nucleus of the trapezoid body (MNTB) in tuning neurons sensitive to ITDs is still unclear. Similarly, the precedence effect is well known and thought to greatly facilitate listening in reverberant environments, yet the neural substrate of the precedence effect is still elusive. A circuitry that might subserve the precedence effect is hypothesized to be formed by the dorsal nucleus of the lateral lemniscus (DNLL) and the inferior colliculus (IC). However, a precise and reversible manipulation of the DNLL-IC circuitry or the ITD circuitry has not been possible due to the lack of technical means. With the advent of optogenetics, tools are becoming available that would allow to specifically activate and silence nuclei within both circuitries. Yet, transgenic lines or genetic tools are neither disposable nor established for the Mongolian Gerbil. Hence, in order to express optogenetic tools in the gerbil auditory brainstem and midbrain, a reliable and neuron specific gene delivery system needs to be established as a major prerequisite. Only when this important first step is taken, the actual optogenetical tools can be applied and tested. In this study, the first hurdle of gene delivery into the Mongolian Gerbil was successfully cleared by using recombinant adeno-associated viruses (rAAV) as vectors. Via the stereotactic injection of rAAVs into the DNLL, IC and MNTB, not only reliable and efficient transduction of neurons was achieved but also neuronal specific expression of transgenes was attained. As a second accomplishment, the channelrhodopsin mutant CatCH as well as the halorhodopsin NpHR3.0 were characterized in acute brain slices by performing whole cell patch-clamp recordings of transduced neurons. As a final step and proof of principle experiment, sound evoked neural responses in the DNLL and IC were successfully manipulated with light in vivo, as could be demonstrated by single cell extracellular recordings from anaesthetized animals. In sum, this study successfully adapted and established gene delivery and optogenetic tools in the auditory system of the Mongolian Gerbil. This represents a fully functional and highly versatile toolbox that not only paves the way to further elucidate the ITD as well as the DNLL-IC circuitry but is also applicable to other questions

Doctor of Philosophy

dissertationInjury from explosive blast is a growing public health threat worldwide with complex mechanisms and limited treatment and prevention pathways. Blast-related traumatic brain injury (bTBI) is a multimodal injury event in which cerebral blood vessels play a central role in both the mechanical and physiological response to blast loading. This dissertation seeks to define the nature of vessel injury from primary blast loading by measuring injury thresholds for vasculature in bTBI by assessing blood-brain barrier (BBB) integrity and disruption, examining which types of vessels are affected, and mapping the distribution of injury in the brain. To assess the consequences of vascular injury, we measured inflammatory changes in glial cell activity with immunohistological techniques, and evaluated changes in behavior in a rodent model of bTBI. The importance of overpressure duration and impulse are examined by performing matched assays with two distinct blast tube devices capable of producing a wide range of blast wave characteristics. Exploration in measuring changes in cerebral blood flow, blood oxygen levels, and cerebral hemorrhage is described. Our primary findings include the presence of focal deposits of IgG in the parenchymal brain tissue indicating an elevated permeability of the blood-brain barrier, a heterogeneous distribution of these lesions among various brain structures, changes in astrocyte glial fibrillary acidic protein (GFAP) expression at lesion locations, and decrease in nociception and pedal withdrawal reflex following primary blast exposure. Changes in macrophage and neural cell populations were observed using markers for IBA1, CD68, and NeuN. Injury levels between devices were broadly similar; however, some differences in both histology and behavior were seen following high-impulse blast testing. Blast injury research remains an important topic with many unanswered questions, and further effort will provide help to those afflicted and preventative protection for those at risk

SORLA regulates endosomal trafficking and oncogenic fitness of HER2

The human epidermal growth factor receptor 2 (HER2) is an oncogene targeted by several kinase inhibitors and therapeutic antibodies. While the endosomal trafficking of many other receptor tyrosine kinases is known to regulate their oncogenic signalling, the prevailing view on HER2 is that this receptor is predominantly retained on the cell surface. Here, we find that sortilin-related receptor 1 (SORLA; SORL1) co-precipitates with HER2 in cancer cells and regulates HER2 subcellular distribution by promoting recycling of the endosomal receptor back to the plasma membrane. SORLA protein levels in cancer cell lines and bladder cancers correlates with HER2 levels. Depletion of SORLA triggers HER2 targeting to late endosomal/lysosomal compartments and impairs HER2-driven signalling and in vivo tumour growth. SORLA silencing also disrupts normal lysosome function and sensitizes anti-HER2 therapy sensitive and resistant cancer cells to lysosome-targeting cationic amphiphilic drugs. These findings reveal potentially important SORLA-dependent endosomal trafficking-linked vulnerabilities in HER2-driven cancers.Peer reviewe