How Characean algae take up needed and excrete unwanted ions – An overview explaining how insights from electrophysiology are useful to understand the ecology of aquatic macrophytes

Characean algae have been a model plant for electrophysiology for many decades, due to the large size of the internodal cells and their robust recovery from invasive manipulation. The information gained from them has provided a template for understanding the electrophysiology of many plant groups. The relative ability to take up or export ions, including nutrients and toxins, can be part of the explanation as to why certain macrophytes occur preferably in nutrient-rich or oligotrophic habitats, why some macrophytes can grow in brackish water or only in freshwater, or why growth is limited to a particular range of pH. The electrical characteristics of the macrophyte’s cells play a determining factor in these transport properties, yet electrophysiological results are seldom cited in ecological publications, perhaps due to difficulties of communication between fields with different research approaches and terminology. We here present main electrophysiological findings on the transport of ions in and out of cells, in a way that is more accessible to ecologists. We examine the mechanism by which Characean algae generate the electrical voltage difference across their membrane, its effect on the transport of ions, and the mechanisms by which ions can be moved against the gradients that determine passive movements. Finally, we use the example of salinity tolerance to show what we learn about the evolution of salt tolerance in plants by using electrophysiological techniques.publishedVersio

Master of Science

thesisTransporters must be sustained in proper working function at the plasma membrane to prevent serious harm to a cell. A dysfunctional transporter may cause leaking of ions, loss of membrane potential, disturbed cell signaling, and even cell death. Therefore, cautious monitoring of transporters occurs at the plasma membrane. A transporter which becomes unfolded or unstable is quickly endocytosed and taken to the early endosome compartment of the cell. However, these unfolded transporters are not automatically degraded, as a cell attempts to preserve them by allowing time for refolding. It is this process we refer to as quality control: where the decision is made whether to degrade the dysfunctional protein to maintain cell integrity or attempt to refold it to conserve energy. The endosome provides a safe place where this decision can be made. Properly folded and functional transporters will return to the plasma membrane to resume pumping, while those deemed dysfunctional will continue on to be degraded. In the case of the yeast uracil transporter Fur4, unfolded/destabilized Fur4 is ubiquitinated (signal for degradation added) by Rsp5, the only ubiquitin ligase known to work at the plasma membrane in yeast. After being endocytosed and brought to the early endosome, the decision to degrade or recycle Fur4 is made there. How exactly that quality control decision is made is not well understood. It is known that ubiquitination status plays a major role in that decision, and that both ubiquitination and deubiquitination can occur at the early endosome. However, studies have shown that Fur4 can recycle in spite of being in a ubiquitin tagged state in some mutant strains, suggesting the quality control decision is not so black and white. We attempt to elucidate this quality control process. Our results, and those published, have led us to a model whereby ubiquitination status is not the sole deciding factor involved in sorting at the early endosome, but where retention of ubiquitinated cargo is actually the key sorting step. Herein we describe a complex of proteins working together at the early endosome to carry out quality control

Models of natural computation : gene assembly and membrane systems

This thesis is concerned with two research areas in natural computing: the computational nature of gene assembly and membrane computing. Gene assembly is a process occurring in unicellular organisms called ciliates. During this process genes are transformed through cut-and-paste operations. We study this process from a theoretical point of view. More specifically, we relate the theory of gene assembly to sorting by reversal, which is another well-known theory of DNA transformation. In this way we obtain a novel graph-theoretical representation that provides new insights into the nature of gene assembly. Membrane computing is a computational model inspired by the functioning of membranes in cells. Membrane systems compute in a parallel fashion by moving objects, through membranes, between compartments. We study the computational power of various classes of membrane systems, and also relate them to other well-known models of computation.Netherlands Organisation for Scientific Research (NWO), Institute for Programming research and Algorithmics (IPA)UBL - phd migration 201

Validation of the suitability of mouse neuroblastoma cells in the study of chloride homeostasis using patch-clamp

Abstract. The function of the nervous system is dependent on the balance between excitatory and inhibitory activity. In the central nervous system (CNS), the homeostasis of Cl- is in a key role in modulating neuronal excitability, since the intracellular Cl- concentration ([Cl-]i) determines the postsynaptic responses mediated by GABA and glycine, the main inhibitory neurotransmitters. Deficiency in the regulation of Cl- homeostasis, that is maintained by various ion channels and transporters, is associated with many neurological disorders such as epilepsy. Hence, the understanding of the mechanisms underlying the regulation of Cl- homeostasis is important. Patch-clamp is an electrophysiological method that is used to study the electrical properties of excitable cells. It is a versatile technique, as it provides information of the concentration gradients of ions as well as ion channel kinetics. One important electrophysiological parameter describing channel function is the reversal potential of an ion (E(i)). Patch-clamp experiments can be done by using cultured cells or tissue slices, which can be genetically modified to express exogenous proteins, such as membrane transporters. There are a variety of cell lines that can be used as expression systems in patch-clamp, such as the commercial mouse neuroblastoma (N2a) cell line, that is used in the experiments in this thesis. The aim of this thesis is to validate the suitability of the N2a cell line as an expression system for E(Cl) recordings using patch-clamp. In the experiments, whole-cell configuration of the patch-clamp was used to study the endogenous voltage-gated currents that are expressed in the N2a cell line, especially in the voltage-range wherein the E(Cl) typically lies

Study of the effects of oligosaccharides in liquid cultures of penicillium chrysogenum

Oligosaccharides and polysaccharides have different effects on the morphology and production of secondary metabolites by Penicillium chrysogenum P2 (ATCC 48271). Addition Of oligosaccharides, derived from sodium alginate and locust bean gum, to submerged cultures of P. chrysogenum P2, at milligram per litre concentration (150 mgL-1), increased secondary metabolite levels and spore production, caused changes in morphology and gerRiination of spores, and affected the production of Reactive Oxygen Species. The source of the oligosaccharides controlled their effects on the cultures. Oligosaccharides when added to submerged cultures of P. chrysogenum P2 increased both penicillin G and extracellular levels of 6-aminopenicillanic acid concentrations. The oligosaccharides had no significant effects on biomass levels. Locust bean gum-derived oligosaccharides (mannan oligosaccharides, DP 5-8), showed the highest levels of enhancement in both penicillin G and 6-aminopenicillanic acid concentrations. Sodium alginate-derived oligosaccharides, (oligoguluronate, DP 7 and oligomannuronate, DP 7), also induced elicitation of penicillin G and 6-aminopenicillanic acid. Oligomannuronate was shown to be more effective than oligoguluronate. In P. chrysogenum P2 cultures mannan, oligomannuronate and oligoguluronate oligosaccharides enhanced yields of penicillin G by 101%, 78% and 59%, respectively. Addition of mannan, oligomannuronate and oligoguluronate oligosaccharides enhanced the levels of 6- aminopenicillanic acid by 39%, 26% and 19%, respectively. The addition of oligosaccharides and polysaccharides to spores of P. chrysogenum P2 in liquid medium had varying (inhibitory or stimulatory) effects on germination, germ-tube and clump development. The addition of oligosaccharides to submerged cultures of P. chrysogenum P2 showed effects on clump size and hyphal tip numbers. Mannan oligosaccharides had the greatest effect on morphology followed by oligomannuronate and oligoguluronate oligosaccharides. Oligosaccharides also speeded-up the sporulation and increased the concentration of spores of P. chrysogenum P2 in liquid cultures. Mannan oligosaccharides had the greatest effect followed by oligomannuronate and oligoguluronate oligosaccharides. 8-aminonaphthalene-1,3,6-trisulphonate-tagged oligosaccharide studies showed that the oligosaccharides pass through the cell wall of P. chrysogenum P2 suggesting a possible mechanism through modulation of gene function. The elicitation pattern was shown to be similar to untagged oligosaccharides. Oligosaccharides and polysaccharides were shown to inhibit production of Reactive Oxygen Species in P. chrysogenum P2. The highest level of inhibition was elicited by mannan followed by oligornannuronate and oligoguluronate oligosaccharides, and then locust bean gum and alginate. The results of the study showed the potential of oligosaccharides as elicitors of secondary metabolites in P. chrysogenum P2 as a filamentous fungus model. Understanding the elicitation mechanism could provide routes for ftirther exploitation of the potential of filamentous fungi in production of commercial products

Advanced analytical strategies to determine biomedical parameters for medical diagnostics, drug delivery, and therapy

Biomedical parameters are critical for diseases prognosis, diagnosis and therapy. Many research groups have dedicated their studies to develop analytical instrumentation and apply analytical methods to determine biomedical parameters that have the potential to help with disease control and increase public health. This dissertation focuses on three major aspects of analytical strategies development and applications: 1) detection of pH changes caused by nanotoxicity (induced by TiO2 nanoparticles) using newly developed micro-pH sensor, 2) quantification of renal cell carcinoma (RCC) biomarkers by high-performance liquid chromatography - tandem mass spectrometry (HPLC-MS/MS), and 3) nuclear magnetic resonance (NMR) studies of porous wall hollow glass microspheres (PWHGMs) that have the potential to be used as drug delivery carriers. Firstly, a dual-core fiber-optic pH micro-probe was developed which can be used within the biologically relevant pH range from 6.20 - 7.92 (R2 = 0.9834). Secondly, a targeted HPLC-MS/MS protocol was developed to simultaneously monitor four urinary biomarkers for RCC and applied to human urine specimen analysis. Thirdly, a vacuum-based loading system was developed to charge PWHGMs with specific materials followed by a washing procedure. Immiscible binary model systems (n-dodecane/water and chloroform/water) as well as isopropanol- acetic acid esterification and the hydrolysis of isopropyl acetate were investigated to obtain NMR evidence for material loading into PWHGMs and their subsequent release into the surrounding solutions. In addition, microspheres loaded with H2O were suspended in D2O to obtain quantitative information about the release kinetics from PWHGMs. The results demonstrate that NMR is a particularly useful tool to study developments and applications of PWHGMs in the targeted and controlled drug delivery --Abstract, page iv