THz in biology and medicine: toward quantifying and understanding the interaction of millimeter- and submillimeter-waves with cells and cell processes

As the application and commercial use of millimeter- and submillimeter-wavelength radiation become more widespread, there is a growing need to understand and quantify both the coupling mechanisms and the impact of this long wavelength energy on biological function. Independent of the health impact of high doses of radio frequency (RF) energy on full organisms, which has been extensively investigated, there exists the potential for more subtle effects, which can best be quantified in studies which examine real-time changes in cellular functions as RF energy is applied. In this paper we present the first real time examination of RF induced changes in cellular activity at absorbed power levels well below the existing safe exposure limits. Fluorescence microscopy imaging of immortalized epithelial and neuronal cells in vitro indicate increased cellular membrane permeability and nanoporation after short term exposure to modest levels (10-50 mW/cm2) of RF power at 60 GHz. Sensitive patch clamp measurements on pyramidal neurons in cortical slices of neonatal rats showed a dramatic increase in cellular membrane permeability resulting either in suppression or facilitation of neuronal activity during exposure to sub-μW/cm2 of RF power at 60 GHz. Non-invasive modulation of neuronal activity could prove useful in a variety of health applications from suppression of peripheral neuropathic pain to treatment of central neurological disorders

Signal generation and storage in FRET-based nanocommunications

The paper is concerned with Forster Resonance Energy Transfer (FRET) considered as a mechanism for communication between nanodevices. Two solved issues are reported in the paper, namely: signal generation and signal storage in FRET-based nanonetworks. First, luciferase molecules as FRET transmitters which are able to generate FRET signals themselves, taking energy from chemical reactions without any external light exposure, are proposed. Second, channelrhodopsins as FRET receivers, as they can convert FRET signals into voltage, are suggested. Further, medical in-body systems where both molecule types might be successfully applied, are discussed. Luciferase-channelrhodopsin communication is modeled and its performance is numerically validated, reporting on its throughput, bit error rate, propagation delay and energy consumption

The mechanical control of nervous system development.

The development of the nervous system has so far, to a large extent, been considered in the context of biochemistry, molecular biology and genetics. However, there is growing evidence that many biological systems also integrate mechanical information when making decisions during differentiation, growth, proliferation, migration and general function. Based on recent findings, I hypothesize that several steps during nervous system development, including neural progenitor cell differentiation, neuronal migration, axon extension and the folding of the brain, rely on or are even driven by mechanical cues and forces.This work was supported by the Medical Research CouncilThis is the accepted version of the original publication available at http://dev.biologists.org/content/140/15/3069

Serotonergic neurons respond to nutrients and regulate the timing of steroid hormone biosynthesis in Drosophila

The temporal transition of development is flexibly coordinated in the context of the nutrient environment, and this coordination is essential for organisms to increase their survival fitness and reproductive success. Steroid hormone, a key player of the juvenile-to-adult transition, is biosynthesized in a nutrient-dependent manner; however, the underlying genetic mechanism remains unclear. Here we report that the biosynthesis of insect steroid hormone, ecdysteroid, is regulated by a subset of serotonergic neurons in Drosophila melanogaster. These neurons directly innervate the prothoracic gland (PG), an ecdysteroid-producing organ and share tracts with the stomatogastric nervous system. Interestingly, the projecting neurites morphologically respond to nutrient conditions. Moreover, reduced activity of the PG-innervating neurons or of ​serotonin signalling in the PG strongly correlates with a delayed developmental transition. Our results suggest that serotonergic neurons form a link between the external environment and the internal endocrine system by adaptively tuning the timing of steroid hormone biosynthesis

Multivalently binding antagonists for the neurokinin-1 receptor

The goal of this thesis was to investigate the neurokinin-1 receptor (NK1R) as a potential target site for multivalent receptor blockade. Since the NK1R is peripherally expressed on various cell types of the human body and its expression increases when an immune response occurs, this receptor seems to offer great potential for selective and specific antagonistic surface receptor targeting with a minimizing effect of inflammation and pain. In the experiments described in Chapter 3, it was found that the inflammatory factor IL-1β has a direct influence on NK1R expression levels in human U87 MG glioblastoma and MDA-MB-231 breast cancer cell lines and primary bovine chondrocytes in 2D cell culture. In addition, it could be demonstrated in 2D cell cultures that IL-1β and substance P together contribute to the regulation of the chondrocytes´ surrounding extracellular matrix (ECM) behavior by the selective regulation of matrix-metalloproteinases´ (MMPs) gene expression. In this context, it could be shown that MMP-13 in particular is regulated in a time and concentration-dependent manner by substance P and can be antagonized by the specific NK1R antagonist spantide I, which allows to assume that this intracellular signaling pathway is triggered via NK1Rs. This aspect is intriguing since the literature also mentions that MMP-13 has an impact on the progression of arthritis. In Chapter 4, fluorescent PEGylated quantum dots (QDs) were used for ligand coupling to the surface of nanoparticles and to study their interactions with NK1R positive cells. The introduction of thiol groups into cysteine-free peptide ligands is a common strategy for coupling well water-soluble ligands to maleimide functionalized nanoparticles such as QDs. In nanoparticle uptake experiments with receptor positive CHO-NK1R cells, a high unspecific nanoparticle binding for amino-PEG modified QDs was examined. However, in FACS displacement experiments with high concentrations of free competing antagonists it was shown that nanoparticle binding was inhibited. This indicated receptor mediated nanoparticle binding. Besides PEGylated QDs, branched 8-arm PEGs were used in Chapter 5 for multivalent cell interaction studies. In contrast to QDs, PEGs are classified as nontoxic biomaterials; additionally, they do not interfere with luminescence-based calcium assays. It was shown that there is a gain in affinity for 8armPEG-20k-spantide I due to multivalent receptor binding. In Chapter 6, a small molecular weight antagonist, aprepitant, was modified to make it amenable for further PEG coupling, either to branched PEGs or PEG-coated nanoparticles. It could be shown that chemical modification of the triazole group of aprepitant is possible by using a strong deprotonation reagent and a tert-Butyl-(3-bromopropyl)carbamate as an alkyl linker with Bocprotected amine functionality. Another new strategy for site-specific multivalent nanoparticle targeting is presented in the final chapter, Chapter 7. This strategy is based on an enzyme driven activation mechanism of ligands which are immobilized on the surface of nanoparticles. For these studies, the ACE driven angiotensin I to angiotensin II conversion was used and the successful conversion was checked by AT1 receptor binding studies. The results of these experiments have shown that enzymatically processed angiotensin I coated nanoparticles are able to selectively bind to AT1R positive mesangial cells

The function of NaV1.8 clusters in lipid rafts

NaV1.8 is a voltage gated sodium channel mainly expressed on the membrane of thin diameter c-fibre neurons involved in the transmission of pain signals. In these neurons NaV1.8 is essential for the propagation of action potentials. NaV1.8 is located in lipid rafts along the axons of sensory neurons and disruption of these lipid rafts leads to NaV1.8 dependant conduction failure. Using computational modelling, I show that the clustering of NaV1.8 channels in lipid rafts along the axon of thin diameter neurons is energetically advantageous and requires fewer channels to conduct action potentials. During an action potential NaV1.8 currents across the membrane in these thin axons are large enough to dramatically change the sodium ion concentration gradient and thereby void the assumptions upon which the cable equation is based. Using scanning electron microscopy NaV1.8 is seen to be clustered, as are lipid raft marker proteins, on neurites at scales below 200nm. FRET signals show that the lipid raft marker protein Flotillin is densely packed on the membrane however disruption of rafts does not reduce the FRET signal from dense protein packing. Using mass spectrometry I investigated the population of proteins found in the lipid rafts of sensory neurons. I found that the membrane pump NaK-ATPase, which restores the ion concentrations across the membrane, is also contained in lipid rafts. NaK-ATPase may help to offset concentration changes due to NaV1.8 currents enabling the repeated firing of c-fibres, which is associated with spontaneous pain in chronic pain disorders.Open Acces