1,721,078 research outputs found
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Electroanalytical techniques for probing neurochemical mechanisms
Get PDFBehavioral, as with all, functions of the brain are governed by biochemical processes in and between brain cells. Neurotransmitters are particularly important in these processes because they are responsible for relaying information between the brain cells. The neurotransmitter dopamine is believed to be directly involved in the neuronal circuitry of pleasure and reward, and hence is an important contributor to behavior. Extensive research into understanding mechanisms involved in dopamine release and regulation may allow us to develop potential pharmacological solutions to eliminate negative behaviors such as drug addiction. Detection of dopamine was performed electrochemically, placing microelectrodes into regions of the brain that were abundant in dopamine release and receptor sites. With a stimulating electrode, artificial action potentials were generated that mimicked biological conditions conducive to dopamine release. The dopamine release was then analyzed under various pharmacological conditions to determine which biological mechanisms were responsible for regulating the amount of dopamine released per stimulation. Additional experiments were performed to determine which other neurotransmitters might be involved in reward mechanisms. A microsensor capable of selectively detecting the neuromodulator nitric oxide was developed to measure changes of nitric oxide in vitro. My thesis has focused primarily on developing detection methods that allow for an understanding of the factors and mechanisms that regulate extracellular concentrations of neurotransmitters
Borosilicate and Fused-Silica Capillary Microelectrode Electrochemical Comparison and Micromanipulator Adaptor for Fused-Silica Capillary Microelectrodes
Get PDFBorosilicate glass is the material predominately used for encasing carbon fiber (CF) microelectrodes for fast-scan cyclic voltammetry (FSCV) experiments. These capillaries insulate unexposed CF from the surrounding environment, while leaving a 150-200 um exposed CF tip in vivo that allows for electrochemical measurements of neurotransmitters. However, borosilicate glass commonly breaks during implantation; this can leave debris in the rat brain and prevent lowering of subsequent electrodes, reducing experimental yield. Additionally, borosilicate glass electrodes often contain cracks at the seal, resulting in undesirable electrochemical characteristics. This can be alleviated with the use of epoxy resin to fill cracks and solidify the seal. Recently, a new design of CF microelectrodes using fused-silica (FS) was announced, which was more flexible and resilient to strain1. This study compares non-epoxied borosilicate, epoxied borosilicate capillary microelectrodes, and FS microelectrodes. The electrochemical characteristics (RMS noise, background amplitude, and background peak locations) between these three designs were compared.
Additionally, the larger diameter of the glass electrodes (600 um compared to 90 um FS) allow them to be used in micromanipulators, which allow for precise control of electrode depth along the dorsal-ventral axis and new electrodes to be used for each experiment. The FS electrodes lack any such device, so an adaptor prototype was developed to match the smaller diameter; the FS electrode construction process was also adjusted to fit the design. These two experiments make FS a more acceptable and useful electrode for in vivo testing.Bachelor of Scienc
Advancing Electroanalytical Methods for Monitoring Chemical Messenger Release
Get PDFChemical communication between cells within the body has been long recognized as an integral part of understanding how the human body functions, especially within the central and peripheral nervous system. Electrochemistry at microelectrodes in tissue slices extracted from the body has become a method of choice for probing the release, reuptake, and overall function of chemical messengers. Here, fast-scan cyclic voltammetry (FSCV) and constant potential amperometry at carbon-fiber microelectrodes is used in various different types of tissues such as the adrenal gland and the spinal cord in order to detect and quantify the release of various chemical messengers including epinephrine, norepinephrine, and adenosine. Pharmacological manipulation of these signals allows for further analysis of the origins of these signals as well as their overall function in integral bodily functions. Work performed on the adrenal gland gave new insight into the exocytotic release characteristics of the hormones epinephrine and norepinephrine which had never been carried out in intact tissue. Also, extensive studies on the methods used to probe the release of these hormones were performed, and allowed for the distinction of electrical stimulation of two different cell types, both the neurons which innervate the tissue, and the chromaffin cells which release the neuromodulators detected with this technology. Moving the detection of adenosine by FSCV into lamina II of the spinal cord allowed for determination of enzymatic activity of ectonucleotidases. Furthermore, it provided new proof of adenosine as an antinociceptive neurotransmitter, and gave evidence for an adenosinergic tone in these circuits with the first ever detection of adenosine transientsanywhere in the nervous system. Also, total tissue analysis of various brain regions by high performance liquid chromatography (HPLC) is used to determine fundamental difference or similarities in different strains of animals, animals which have been genetically altered, or animals which have been treated with drugs. Finally, the coating of Ag/AgCl reference electrodes with Nafion for chronic implantation is discussed. These studies use fundamental analytical tools to advance our understanding of the nervous system, and shows new methods of answering long standing biological questions
Investigation of membrane protein dynamics of gamma-glutamyl carboxylase using liquid chromatography and mass spectrometry
Get PDFMembrane proteins are involved in numerous biological processes, including transport, signal transduction, and a variety of metabolic pathways. Despite their abundance, however, membrane proteins largely remain resistant to biophysical characterization due to complexities in sample preparation and limited knowledge regarding structural elucidation. The overlying objective of the work reported in this dissertation is focused upon developing proteomics based approaches for the structural investigation of integral membrane proteins involved in the vitamin K cycle. Vitamin K is an essential micronutrient that functions as a coenzyme in the carboxylation of vitamin K-dependent (VKD) proteins by the integral membrane protein Gamma-Glutamyl Carboxylase (GGCX). Concomitant with VKD modification, vitamin K is regenerated by a mechanism involving the enzyme Vitamin K Epoxide Reductase (VKOR) and the cycle continues. Structural analysis of GGCX and VKOR is of particular interest in understanding their functional involvement in blood coagulation, calcification, and cell growth control. Although the mechanisms for carboxylation and epoxidation have been investigated for over thirty years, biological recognition involving structural conformations and protein associations are not yet completely understood. As an alternative approach to classical biochemical experimentation, methods were developed to investigate GGCX protein dynamics by ultra-performance liquid chromatography (UPLC) coupled to mass spectrometry. Following a brief overview of the vitamin K cycle (Chapter 1), Chapters 2-4 aim to develop analytical approaches for identifying the catalytic active site in GGCX using covalent cross-linking mass spectrometry. A comprehensive bottom-up proteomics methodology (Chapter 2) was applied to identify site-specific covalent attachment of synthetically modified VKD cross-linker substrates in Chapters 3 and 4. In Chapter 5, a new class of model membrane, Nanodiscs, are introduced providing a controlled, native phospholipid structure in which membrane proteins can be isolated in a water-soluble environment. Incorporation of GGCX embedded Nanodiscs are further investigated by hydrogen exchange mass spectrometry (HX MS) in Chapter 6. This novel system demonstrates the first reported application for investigation of membrane protein dynamics in a near-native environment by HX MS. The work outlined in this dissertation not only offers significant advancements in the structural investigation of GGCX, but provides unique platforms in which to investigate other complex membrane protein systems
SOL-GEL-DERIVED MATERIALS FOR ANTIMICROBIAL COATINGS AND ELECTROCHEMICAL NITRIC OXIDE ANALYSIS
Get PDFSol-gel-derived coatings modified to release nitric oxide (NO), an endogenous broad-spectrum antimicrobial, have been described as highly promising antimicrobial biomaterials. As part of my thesis work, I extended the study of the antimicrobial properties of NO-releasing xerogels against the adhesion, viability, and biofilm formation of the pathogenic fungus, Candida albicans. Nitric oxide fluxes as low as 14 pmol cm-2 s-1 proved sufficient to reduce fungal adhesion by ~49% over controls (non-NO-releasing substrates) after 90 min of exposure. By utilizing a fluorescence live/dead assay and replicate plating, the NO flux was determined to reduce fungal viability in a dose dependent manner. Likewise, the formation of C. albicans biofilms on NO-releasing xerogel-coated silicon rubber (SiR) coupons was impeded when compared to control and bare SiR surfaces. To begin an examination of the likelihood of exogenous NO fostering NO resistance, bacteria were exposed to NO in long- and short-term mutagenesis assays. Even after 20 d of continuous sub-therapeutic exposure, resistance to NO was not observed for gram-positive and -negative species. The next phase of my research thus focused on the synthesis of superhydrophobic xerogel coatings from a mixture of nanostructured fluorinated silica colloids, fluoroalkoxysilane, and a backbone silane. Quantitative bacterial adhesion studies performed using a parallel plate flow cell demonstrated that the adhesion of Staphylococcus aureus and Pseudomonas aeruginosa were reduced by 2.08 ± 0.25 and 1.76 ± 0.12 log over controls, respectively. The straightforward and mild synthesis of this chemistry enables its application to any surface regardless of geometry making such interfaces ideal as biopassivation strategies. Along with NO's ability to serve a potent exogenous antimicrobial, endogenous NO serves many important physiological roles (e.g., immune response, vasodilatation, neurotransmission). The final phase of my dissertation research focused on the development of microfluidic NO sensors capable of selectively measuring NO in small volumes (<1 mL). The final device enabled sensitive NO detection in PBS, blood, and simulated wound fluid at concentrations as low as 0.7-2.0 nM. Future studies using this device may prove useful clinically.Doctor of Philosoph
Studies of exocytosis at single cells
Get PDFIntercellular communication via chemical signaling is vital to the healthy functioning of multicellular organisms. In exocytosis, intracellular vesicles undergo Ca2+-triggered fusion with the cell plasma membrane, releasing their chemical messengers into the extracellular space. As exocytosis serves as the primary mechanism of communication at neuronal synapses, great emphasis has been placed on understanding the complex cellular regulation of this process. This dissertation focused on the use of amperometry and fast scan cyclic voltammetry at carbon-fiber microelectrodes to monitor exocytosis in real-time at both isolated neurons and chromaffin cells, well-characterized model cells for neuronal exocytosis. These techniques provide the necessary temporal resolution and sensitivity required to detect the chemical signals resulting from individual vesicular release events. Amperometric recordings at midbrain dopamine neurons showed that somatodendritic dopamine release is exocytotic, with a bimodal distribution of vesicular events. A combinatorial approach was used to demonstrate alterations in biogenic amine exocytosis in mice lacking the mitochondrial uncoupling protein UCP2 or the hormone leptin. Conversely, a mouse model of fragile X syndrome revealed no deficiencies in vesicular release mechanisms. Electrochemical methodologies were developed to distinguish catecholamine transmitters from the L-tyrosine-derived trace amines. Application of these methods revealed poor vesicular accumulation of trace amines precludes their function as false transmitters. Finally, vesicular quantal size in chromaffin cells was shown to be resistant to exogenous application of catecholamine precursors
Electrochemical investigation of dopamine neurotransmission involving iontophoresis
Get PDFIdentification of the role of transient dopamine release in behavior has proved difficult. In order to interpret the complex processes involved in dopamine neurotransmission, dopamine release and cell firing rates must be recorded and the signals correlated through pharmacological manipulation. For this purpose, the carbon-fiber electrodes employed for electrochemical dopamine detection and extracellular unit recordings were combined with the local drug delivery technique, iontophoresis. The application of glutamate, dopamine, and the glutamate receptor antagonist, CNQX, by iontophoresis strongly modulated cell firing rates in anesthetized rats with short (20 s) ejections. The ejection of glutamate and the dopamine receptor agonist, quinpirole HCl, strongly decreased stimulated dopamine release though required ejections several minutes long. The stimulated dopamine release studies are believed to require greater distances the ejected compound must diffuse in order to generate a detectable effect. This increased distance may explain the drastic difference in ejection durations. Glutamate was found to increase firing in a minority of neurons and no observed effect from dopamine in freely-moving animals. However, long ejections of the D1 antogonist, SCH23390, inhibited cell firing during ICSS behavior. The amount of basal dopamine present in the extracellular fluid was also studied. Dopamine levels were monitored during microinjection of saline and lidocaine in the ventral tegmental area and systemic cocaine administration. With both impulse-dependent dopamine release and dopamine reverse transport blocked, minimal decreases in dopamine were observed (less than 25 nM) that are similar to levels obtained in recent microdialysis estimates. A novel action of cocaine was also investigated using genetically-modified mice. The effect of cocaine upon stimulated dopamine release in mice lacking all 3 isoforms of the protein, synapsin, was investigated using fast-scan cyclic voltammetry and amperommetry. Cocaine was found to increase dopamine release through not only decreasing uptake but through increasing the amount of dopamine released per stimulus event. During depleting (15 second) electrical stimulations and after synthesis inhibition via a-methyl-para-tyrosine administration, the synapsin TKO mice showed a decreased response to cocaine with respect to wild-type mice. This suggests an interaction between a synapsin-dependent pool of dopamine vesicles and cocaine
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