Force-controlled patch clamp of beating cardiac cells.

From its invention in the 1970s, the patch clamp technique is the gold standard in electrophysiology research and drug screening because it is the only tool enabling accurate investigation of voltage-gated ion channels, which are responsible for action potentials. Because of its key role in drug screening, innovation efforts are being made to reduce its complexity toward more automated systems. While some of these new approaches are being adopted in pharmaceutical companies, conventional patch-clamp remains unmatched in fundamental research due to its versatility. Here, we merged the patch clamp and atomic force microscope (AFM) techniques, thus equipping the patch-clamp with the sensitive AFM force control. This was possible using the FluidFM, a force-controlled nanopipette based on microchanneled AFM cantilevers. First, the compatibility of the system with patch-clamp electronics and its ability to record the activity of voltage-gated ion channels in whole-cell configuration was demonstrated with sodium (NaV1.5) channels. Second, we showed the feasibility of simultaneous recording of membrane current and force development during contraction of isolated cardiomyocytes. Force feedback allowed for a gentle and stable contact between AFM tip and cell membrane enabling serial patch clamping and injection without apparent cell damage.

The regulation of mTORC2 kinase activity and complex integrity

Growth factor signaling promotes anabolic processes via activation of the PI3K-Akt kinase cascade. Deregulation of the growth factor-dependent PI3K-Akt pathway was implicated in tumorigenesis. Akt is an essential serine/threonine protein kinase that controls multiple physiological functions such as cell growth, proliferation, and survival to maintain cellular homeostasis. Recently, the mammalian Target of Rapamycin Complex 2 (mTORC2) was identified as the main Akt Ser-473 kinase, and Ser-473 phosphorylation is required for Akt hyperactivation. However, the detailed mechanism of mTORC2 regulation in response to growth factor stimulation or cellular stresses is not well understood. In the first project, we studied the regulation of the mTORC2-Akt signaling under ER stress. We identified the inactivation of mTORC2 by glycogen synthase kinase-3β (GSK-3β). Under ER stress, the essential mTORC2 component, rictor, is phosphorylated by GSK-3β at Ser-1235. This phosphorylation event results in the inhibition of mTORC2 kinase activity by interrupting Akt binding to mTORC2. Blocking rictor Ser-1235 phosphorylation can attenuate the negative impacts of GSK-3β on mTORC2/Akt signaling and tumor growth. Thus, our work demonstrated that GSK-3β-mediated rictor Ser-1235 phosphorylation in response to ER stress interferes with Akt signaling by inhibiting mTORC2 kinase activity. In the second project, I investigated the regulation of the mTORC2 integrity. We found that basal mTOR kinase activity depends on ATP level, which is tightly regulated by cell metabolism. The ATP-sensitive mTOR kinase is required for SIN1 protein phosphorylation and stabilization. SIN1 is an indispensable subunit of mTORC2 and is required for the complex assembly and mTORC2 kinase activity. Our findings reveal that mTOR-mediated phosphorylation of SIN1 is critical for maintaining complex integrity by preventing SIN1 from lysosomal degradation. In sum, our findings verify two distinct mTORC2 regulatory mechanisms via its components rictor and SIN1. First, GSK-3β-mediated rictor Ser-1235 phosphorylation results in mTORC2 inactivation by interfering its substrate binding ability. Second, mTOR-mediated Ser-260 phosphorylation of SIN1 preserves its complex integrity. Thus, these two projects provide novel insights into the regulation of mTORC2.

The Role of Type I Insulin-Like Growth Factor Receptor Signaling in Breast Cancer Brain Metastasis

Brain metastasis is a common cause of mortality in cancer patients. Approximately 20-30% of breast cancer patients acquire brain metastasis, yet potential therapeutic targets remain largely unknown. The type I insulin-like growth factor receptor (IGF- IR) is known to play a role in the progression of breast cancer and is currently being investigated in the clinical setting for various types of cancer. The present study demonstrates that the IGF-IR signaling axis is constitutively active in brain-seeking sublines of breast cancer cells, driving an increase in in vitro metastatic properties. We demonstrate that IGF-IR signaling is activated in an autocrine manner as a result of IGFBP3 overexpression in brain-seeking cells. Transient and stable knockdown of IGF-IR results in a downregulation of IGF-IR downstream signaling through phospho-AKT, as well as decreased in vitro migration and invasion of MDA- MB-231Br brain-seeking cells. Using an in vivo experimental brain metastasis model, we show that IGF-IR ablation attenuates the establishment of brain metastases and prolongs survival. Finally, we demonstrate that the malignancy of brain-seeking cells is attenuated by pharmacological inhibition with picropodophyllin, an IGF-IR-specific tyrosine kinase inhibitor. Together, our data suggest that the IGF-IR is an important mediator of brain metastasis and its ablation delays the onset of brain metastases in our model system.

Histograms of oriented gradients for fast on-board vehicle verification

Histograms of Oriented Gradients (HoGs) provide excellent results in object detection and verification. However, their demanding processing requirements bound their applicability in some critical real-time scenarios, such as for video-based on-board vehicle detection systems. In this work, an efficient HOG configuration for pose-based on-board vehicle verification is proposed, which alleviates both the processing requirements and required feature vector length without reducing classification performance. The impact on classification of some critical configuration and processing parameters is in depth analyzed to propose a baseline efficient descriptor. Based on the analysis of its cells contribution to classification, new view-dependent cell-configuration patterns are proposed, resulting in reduced descriptors which provide an excellent balance between performance and computational requirements, rendering higher verification rates than other works in the literature.

Influência da configuração da célula combustível microbiana na geração de energia elétrica a partir da degradação de compostos orgânicos

Nessa pesquisa foram testadas tanto CCM com membranas e sem membranas que tinham por característica reproduzir sistemas de tratamento de esgoto sanitário. A etapa experimental desse trabalho foi dividida entre o Brasil (ensaios com CCM sem a MTP e utilizando esgoto sanitário) e Portugal (ensaios com CCM tradicionais de uma e duas câmaras, utilizando água residuária sintética e a bactéria Lactobacillus pentosus). A execução em dois locais diferentes resultou em um maior aprofundamento e desenvolvimento da pesquisa. As CCM foram avaliadas principalmente quanto ao potencial elétrico e eficiência da degradação de compostos orgânicos (esgoto sanitário e água residuária sintética). Para os dados obtidos no Brasil, as três configurações apresentaram maior diferença na potência em função do modo de operação. A operação intermitente apresentou a maior potência (11 mW/m2) para a CCM cilíndrica de fluxo ascendente, enquanto que operação continua a maior potência (4,2 mW/m2) foi obtida para a CCM retangular de fluxo horizontal, a qual também apresentava uma maior facilidade na manutenção quanto aos eletrodos (adição/remoção). A CCM cúbica de fluxo ascendente devido a sua concepção simples demandava um sistema complementar para o aumento da remoção de DQO. Apesar da baixa potência mensurada para os ensaios realizados no Brasil há de se pontuar que os mesmos foram obtidos para reatores sem membranas e utilizando o esgoto sanitário, o qual apresentou grande sazonalidade. Para a etapa realizada em Portugal, foi possível realizar quinze diferentes ensaios e mais um ensaio específico de crescimento. A maior potência (10,37 mW/m2) foi obtida para CCM de câmara dupla operada de modo contínuo para um tempo de detenção hidráulico (TDH) de 20 horas. A maior potência obtida para a CCM de câmara única foi de 5,53 mW/m2 quando houve a adição do extrato de levedura (função teórica de mediador). A potência da CCM, na maioria das vezes, esteve relacionada à proporção de sólidos voláteis e totais, SV/ST, quantidade de bactérias, pH, características de operação e por fim a configuração da CCM. O ensaio de crescimento revelou a correlação da potência em função da quantidade de bactérias inseridas da massa do biofilme (SV) e mostra-se como uma ferramenta na avaliação da potência das CCM.

Electrochemical behaviour of activated carbons obtained via hydrothermal carbonization

Activated carbons were prepared by chemical activation of hydrochars, obtained by hydrothermal carbonisation (HTC) using low cost and abundant precursors such as rye straw and cellulose, with KOH. Hydrochars derived from rye straw were chemically activated using different KOH/precursor ratios, in order to assess the effect of this parameter on their electrochemical behaviour. In the case of cellulose, the influence of the hydrothermal carbonisation temperature was studied by fixing the activating agent/cellulose ratio. Furthermore, N-doped activated carbons were synthesised by KOH activation of hydrochars prepared by HTC from a mixture of glucose with melamine or glucosamine. In this way, N-doped activated carbons were prepared in order to evaluate the influence of nitrogen groups on their electrochemical behaviour in acidic medium. The results showed that parameters such as chemical activation or carbonisation temperature clearly affect the capacitance, since these parameters play a key role in the textural properties of activated carbons. Finally, symmetric capacitors based on activated carbon and N-doped activated carbon were tested at 1.3 V in a two-electrode cell configuration and the results revealed that N-groups improved the capacitance at high current density.

Activation of calcium-sensitive potassium channels in L6 skeletal myocytes by arginine vasopressin requires extracellular calcium

The effects of extracellular application of arginine vasopressin (AVP) upon membrane currents in L6 skeletal myocytes was investigated using the whole-cell configuration of the patch-clamp technique. At O mV AVP produced large amplitude, transient outward currents that reversed when the clamping potential was changed to -100 mV (negative to EK) The effects of alterations in the extracellular K+ concentration upon the current reversal potential suggested that the current elicited by AVP was carried mainly by K+ ions. Intracellular dialysis with 10 μM inositol 1,4,5-trisphosphate (InsP3) elicited similar currents but only in 6/14 cells. Inclusion of 5 mg ml-1 heparin in the intracellular solutions was ineffective at inhibiting the current responses to AVP. The AVP-induced current was totally abolished when the intracellular EGTA concentration was increased from 0.05 mM to 10 mM or Ca2+ was removed from the extracellular perfusing solution. These results suggest that AVP produces activation of a Ca2+-sensitive K+ conductance in L6 skeletal myocytes by a process dependent upon extracellular Ca2+ and not intracellular Ca2+ release. © 1995 Academic Press. All rights reserved.

Characterization of a volume-sensitive chloride current in rat osteoblast-like (ROS 17/2.8) cells

1. During osmotic swelling, cultured osteoblastic cells (ROS 17/2.8) exhibited activation of large amplitude Cl- currents in the whole-cell configuration of the patch-clamp technique. Effects of hypotonic shock on cell volume and membrane conductance were rapidly reversed on return to isotonic conditions. 2. Voltage command pulses in the range -80 to +50 mV produce instantaneous activation of Cl- currents. At potentials more positive than +50 mV the current exhibited time-dependent inactivation. The instantaneous current-voltage relationship was outwardly rectifying. 3. The anion permeability sequence of the induced current was SCN- (2.2) > I- (1.9) > Br- (1.5) > Cl- (1.0) > F- (0.8) > gluconate- (0.2). This corresponds to Eisenman's sequence I. 4. The volume-sensitive Cl- current was effectively inhibited by the Cl- channel blockers 4,4'-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). Outward currents were more effectively suppressed by DIDS than inward currents. The concentrations for 50% inhibition (IC50) of outward and inward currents were 81 and 298 μM, respectively. NPPB was equally effective at inhibiting outward and inward currents (IC50 of 64 μM). The current was relatively insensitive to diphenylamine-2-carboxylate (DPC), 500 μM producing only 22.5 ± 4.0% inhibition. 5. Inhibitors of protein kinase A (H-89, 1 μM) and tyrosine kinase (tyrphostin A25, 200 μM) were without effect upon activation of Cl- currents in response to hypotonic shock. Under isotonic conditions, elevation of intracellular Ca2+ by ionomycin (1 μM) or activation of protein kinase C by 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.1 μM) failed to evoke increases in basal Cl- conductance levels. 6. It is concluded that an outwardly rectifying Cl- conductance is activated upon osmotic swelling and may be involved in cell volume regulation of ROS 17/2.8 cells.

VSC topology comparison for STATCOM application under unbalanced conditions

This paper compares the performance of four different power electronic converter topologies, which have been proposed for STATCOM applications. Two of the topologies are Modular Multilevel Cascaded Converters (MMCC), whilst the remaining circuits utilize magnetic elements and an open-winding transformer configuration to combine individual power modules. It is assumed that the STATCOM has to work under unbalanced conditions, so that it delivers both positive and negative sequence currents. Simulation studies for the four topologies have been carried out using the simulation tool Saber. © 2013 IEEE.

An investigation of Hebbian phase sequences as assembly graphs

Hebb proposed that synapses between neurons that fire synchronously are strengthened, forming cell assemblies and phase sequences. The former, on a shorter scale, are ensembles of synchronized cells that function transiently as a closed processing system; the latter, on a larger scale, correspond to the sequential activation of cell assemblies able to represent percepts and behaviors. Nowadays, the recording of large neuronal populations allows for the detection of multiple cell assemblies. Within Hebb's theory, the next logical step is the analysis of phase sequences. Here we detected phase sequences as consecutive assembly activation patterns, and then analyzed their graph attributes in relation to behavior. We investigated action potentials recorded from the adult rat hippocampus and neocortex before, during and after novel object exploration (experimental periods). Within assembly graphs, each assembly corresponded to a node, and each edge corresponded to the temporal sequence of consecutive node activations. The sum of all assembly activations was proportional to firing rates, but the activity of individual assemblies was not. Assembly repertoire was stable across experimental periods, suggesting that novel experience does not create new assemblies in the adult rat. Assembly graph attributes, on the other hand, varied significantly across behavioral states and experimental periods, and were separable enough to correctly classify experimental periods (Naïve Bayes classifier; maximum AUROCs ranging from 0.55 to 0.99) and behavioral states (waking, slow wave sleep, and rapid eye movement sleep; maximum AUROCs ranging from 0.64 to 0.98). Our findings agree with Hebb's view that assemblies correspond to primitive building blocks of representation, nearly unchanged in the adult, while phase sequences are labile across behavioral states and change after novel experience. The results are compatible with a role for phase sequences in behavior and cognition.

An investigation of Hebbian phase sequences as assembly graphs

Hebb proposed that synapses between neurons that fire synchronously are strengthened, forming cell assemblies and phase sequences. The former, on a shorter scale, are ensembles of synchronized cells that function transiently as a closed processing system; the latter, on a larger scale, correspond to the sequential activation of cell assemblies able to represent percepts and behaviors. Nowadays, the recording of large neuronal populations allows for the detection of multiple cell assemblies. Within Hebb's theory, the next logical step is the analysis of phase sequences. Here we detected phase sequences as consecutive assembly activation patterns, and then analyzed their graph attributes in relation to behavior. We investigated action potentials recorded from the adult rat hippocampus and neocortex before, during and after novel object exploration (experimental periods). Within assembly graphs, each assembly corresponded to a node, and each edge corresponded to the temporal sequence of consecutive node activations. The sum of all assembly activations was proportional to firing rates, but the activity of individual assemblies was not. Assembly repertoire was stable across experimental periods, suggesting that novel experience does not create new assemblies in the adult rat. Assembly graph attributes, on the other hand, varied significantly across behavioral states and experimental periods, and were separable enough to correctly classify experimental periods (Naïve Bayes classifier; maximum AUROCs ranging from 0.55 to 0.99) and behavioral states (waking, slow wave sleep, and rapid eye movement sleep; maximum AUROCs ranging from 0.64 to 0.98). Our findings agree with Hebb's view that assemblies correspond to primitive building blocks of representation, nearly unchanged in the adult, while phase sequences are labile across behavioral states and change after novel experience. The results are compatible with a role for phase sequences in behavior and cognition.

Nano-Engineering of Densely Packed Electrochemical Energy Storage Architectures by Atomic Layer Deposition

Nanostructures are highly attractive for future electrical energy storage devices because they enable large surface area and short ion transport time through thin electrode layers for high power devices. Significant enhancement in power density of batteries has been achieved by nano-engineered structures, particularly anode and cathode nanostructures spatially separated far apart by a porous membrane and/or a defined electrolyte region. A self-aligned nanostructured battery fully confined within a single nanopore presents a powerful platform to determine the rate performance and cyclability limits of nanostructured storage devices. Atomic layer deposition (ALD) has enabled us to create and evaluate such structures, comprised of nanotubular electrodes and electrolyte confined within anodic aluminum oxide (AAO) nanopores. The V2O5- V2O5 symmetric nanopore battery displays exceptional power-energy performance and cyclability when tested as a massively parallel device (~2billion/cm2), each with ~1m3 volume (~1fL). Cycled between 0.2V and 1.8V, this full cell has capacity retention of 95% at 5C rate and 46% at 150C, with more than 1000 charge/discharge cycles. These results demonstrate the promise of ultrasmall, self-aligned/regular, densely packed nanobattery structures as a testbed to study ionics and electrodics at the nanoscale with various geometrical modifications and as a building block for high performance energy storage systems[1, 2]. Further increase of full cell output potential is also demonstrated in asymmetric full cell configurations with various low voltage anode materials. The asymmetric full cell nanopore batteries, comprised of V2O5 as cathode and prelithiated SnO2 or anatase phase TiO2 as anode, with integrated nanotubular metal current collectors underneath each nanotubular storage electrode, also enabled by ALD. By controlling the amount of lithium ion prelithiated into SnO2 anode, we can tune full cell output voltage in the range of 0.3V and 3V. This asymmetric nanopore battery array displays exceptional rate performance and cyclability. When cycled between 1V and 3V, it has capacity retention of approximately 73% at 200C rate compared to 1C, with only 2% capacity loss after more than 500 charge/discharge cycles. With increased full cell output potential, the asymmetric V2O5-SnO2 nanopore battery shows significantly improved energy and power density. This configuration presents a more realistic test - through its asymmetric (vs symmetric) configuration – of performance and cyclability in nanoconfined environment. This dissertation covers (1) Ultra small electrochemical storage platform design and fabrication, (2) Electron and ion transport in nanostructured electrodes inside a half cell configuration, (3) Ion transport between anode and cathode in confined nanochannels in symmetric full cells, (4) Scale up energy and power density with geometry optimization and low voltage anode materials in asymmetric full cell configurations. As a supplement, selective growth of ALD to improve graphene conductance will also be discussed[3]. References: 1. Liu, C., et al., (Invited) A Rational Design for Batteries at Nanoscale by Atomic Layer Deposition. ECS Transactions, 2015. 69(7): p. 23-30. 2. Liu, C.Y., et al., An all-in-one nanopore battery array. Nature Nanotechnology, 2014. 9(12): p. 1031-1039. 3. Liu, C., et al., Improving Graphene Conductivity through Selective Atomic Layer Deposition. ECS Transactions, 2015. 69(7): p. 133-138.

Improvement of carbon materials performance by nitrogen functional groups in electrochemical capacitors in organic electrolyte at severe conditions

N-doped activated carbon fibers have been synthesized by using chemically polymerized aniline as source of nitrogen. Commercial activated carbon fibers (A20) were chemically modified with a thin film of polyaniline (PANI) inside the microporosity of the carbon fibers. The modified activated carbon fibers were carbonized at 600 and 800 °C, respectively. In this way, activated carbon fibers modified with surface nitrogen species were prepared in order to analyze their influence in the performance of electrochemical capacitors in organic electrolyte. Symmetric capacitors were made of activated carbon fibers and N-doped activated carbon fibers and tested in a two-electrode cell configuration, using triethylmethylammonium tetrafluoroborate/propylene carbonate (TEMA-BF4/PC) as electrolyte. The effect of nitrogen species in the degradation or stabilization of the capacitor has been analyzed through floating durability tests using a high voltage charging (3.2 V). The results show higher stabilizing effect in carbonized samples (N-ACF) than in non-carbonized samples and pristine activated carbon fibers, which is attributed to the presence of aromatic nitrogen group, especially positively charged N-functional groups.

Thermal characterisation of doped InP using photoacoustic technique

An open cell photoacoustic configuration has been employed to evaluate the thermal diffusivity of pure InP as well as InP doped with sulphur and iron. Chopped optical radiation at 488 nm from an Ar-ion laser has been used to excite photoacoustic signals which been detected by a sensitive electret microphone. Thermal diffusivity value have been calculated from phase versus chopping frequency plots. Doped sample are found to show a reduced value for thermal diffusivity in comparison with intrinsically pure sample. The results have been interpreted in terms of the mechanisms of heat generation and transmission in semiconductors.

A collagen network phase improves cell seeding of open-pore structure scaffolds under perfusion

Scaffolds with open-pore morphologies offer several advantages in cell-based tissue engineering, but their use is limited by a low cell seeding efficiency. We hypothesized that inclusion of a collagen network as filling material within the open-pore architecture of polycaprolactone-tricalcium phosphate (PCL-TCP) scaffolds increases human bone marrow stromal cells (hBMSC) seeding efficiency under perfusion and in vivo osteogenic capacity of the resulting constructs. PCL-TCP scaffolds, rapid prototyped with a honeycomb-like architecture, were filled with a collagen gel and subsequently lyophilized, with or without final crosslinking. Collagen-free scaffolds were used as controls. The seeding efficiency was assessed after overnight perfusion of expanded hBMSC directly through the scaffold pores using a bioreactor system. By seeding and culturing freshly harvested hBMSC under perfusion for 3 weeks, the osteogenic capacity of generated constructs was tested by ectopic implantation in nude mice. The presence of the collagen network, independently of the crosslinking process, significantly increased the cell seeding efficiency (2.5-fold), and reduced the loss of clonogenic cells in the supernatant. Although no implant generated frank bone tissue, possibly due to the mineral distribution within the scaffold polymer phase, the presence of a non crosslinked collagen phase led to in vivo formation of scattered structures of dense osteoids. Our findings verify that the inclusion of a collagen network within open morphology porous scaffolds improves cell retention under perfusion seeding. In the context of cell-based therapies, collagen-filled porous scaffolds are expected to yield superior cell utilization, and could be combined with perfusion-based bioreactor devices to streamline graft manufacture.