This study focuses on the mechanism of flow-acoustic coupling in a T-junction, which has a transitional section entailing an expansion of pipe diameter. Six cases with varying length of the transitional section have been tested. It is shown that T-junctions with transitional sections can be a strong source of acoustic excitation and therefore can cause powerful acoustic resonance in the associated piping systems. Elimination of the transitional section reduces the excitation level drastically. The length of the transitional section with enlarged diameter is found to be the fundamental length scale determining the Strouhal number of flow excitation in the T-junction. To investigate the mechanism further, a series of experiments were conducted in a rectangular pipe system, which was considered as the 2D case of the T-junction. The results showed that the vortices formed in the outer and inner sides of the T-junction are both acoustic sources. They appear to form independently without interaction with each other, but are synchronized by the acoustic field. Flow visualization was carried out to gain images of the flow pattern when the resonance occurs. The images from flow visualization provided support to the measurements of the rectangular pipe system. Numerical simulations of the mean flow and acoustic field in the rectangular T-junction were performed. A simplied analysis of acoustic energy generation was also carried out. / Thesis / Master of Applied Science (MASc)

The results of an experimental investigation of the separation phenomena in dividing two-phase flow is presented. This work involved the commissioning of a steam-water loop to obtain detailed data on the characteristics of steam-water flow in a horizontal tee junction. Measurements included the pressure and void fraction distributions as well as the total flow rate and quality along the inlet and branching legs. A detailed set of experiments were performed enabling the effects of flow split, inlet quality and inlet mass flux on the separation and pressure characteristics to be determined. For the annular inlet flow conditions considered herein, total separation was approached when more than 40% of the inlet flow was removed through the branch. At lower branch flow rates, the degree of phase separation was strongly dependent on the branch flow split and the inlet quality. The pressure change from the inlet through the run of the tee was modelled from an axial momentum balance at the junction for both homogeneous and separated flow assumptions. The separated flow momentum correction factor was distributed about a value of unity indicating that the branching flow carriers little or no axial component of momentum. The pressure change from the inlet through the branch was considered in terms of reversible an'd irreversible components for separated and homogeneous flow assumptions. Both models yielded loss coefficients that were strongly dependent on the branch flow split and inlet quality. / Thesis / Master of Engineering (ME)

Sudden cardiac death occurs due to aberrations in the multifactorial process that is cardiac conduction. Conduction velocity (CV) and its modulation by several determinants, like cellular excitability, tissue structure and electrical coupling by gap junctions (GJ), have been extensively studied. However, there are several discrepancies in cardiac electrophysiology research that have extended over decades, suggesting elements that are still not completely understood about this complex phenomenon. This dissertation will focus on one such mechanism, ephaptic coupling (EpC). The purpose of this work is twofold, 1) to identify ionic determinants of EpC, and its interactions with gap junctional coupling (GJC) and, 2) to investigate the possible role of serum ion modulation in cardiac arrhythmia therapy. First, the effects of altering extracellular ion concentration – sodium, potassium and calcium at varying GJ protein expression were studied. Briefly, reducing sodium was related to CV slowing under conditions of reduced EpC (wide intercalated disc nanodomains – perinexi) and GJC (reduced GJ protein – Connexin43). On the other hand, increasing potassium slowed CV in hearts with wide perinexi independent of GJC. Elevating calcium, reduced perinexal width and was associated with fast CV during physiologic sodium concentration. However, under conditions associated with disease, like hyponatremia, elevating calcium still reduced perinexal width but slowed CV. These findings are the first to suggest that ionic modulators of EpC could modulate CV during health and disease. Next, the potential of perfusate ion modulation in cardiac arrhythmia therapy was investigated. Briefly, in a model of myocardial inflammation, TNFα, a pro-inflammatory cytokine, slowed CV relative to control conditions and this was associated with widening of the perinexus (reduced EpC). Increasing extracellular calcium restored CV to control values by improving not only EpC but also GJC. Finally, in a model of metabolic ischemia in the heart, CV response due to solutions with varying sodium and calcium concentrations were tested. The solutions that were associated with wider perinexi and elevated sodium performed best during ischemia by attenuating CV slowing, reducing arrhythmias and increasing time to asystole. Taken together, these findings provide evidence for the possibility of ionic determinants of EpC in cardiac arrhythmia therapy. / Ph. D.

Blast neurotrauma represents a significant mode of traumatic injury to the brain. The incidence of blast neurotrauma is particularly high amongst military combat personnel and can be debilitating and endure clinically for years after injury is sustained. Mechanically, blast represents a unique and complex loading paradigm associated with compressive shock waves that propagate out from an explosive event and interact with the head and other organs through high-rate loading. When subjected to such insult, brain cells undergo characteristic injury responses which include neuroinflammation, oxidative stress, edema and persistent glial activation. These features of the injury have emerged as important mediators of the chronic brain damage that results from blast. Astrocytes have emerged as a potential therapeutic target because of their ubiquitous roles in brain homeostasis, tissue integrity and cognitive function. This glial subtype has a characteristic reactive response to mechanical trauma of various modes. In this work, custom in vitro injury devices were used to characterize functional models of astrocyte reactivity to high-rate insult to study mechano-stimulation mechanisms associated with the reactive phenotype. The working hypothesis was that high-rate overpressure exposure would cause metabolic aberrations, cell junction changes, and adhesion signal transduction activation, all of which would contribute to astrocyte response and reactivity. Astrocyte cultures were exposed to a 20 psi high-rate overpressure scheme using an underwater explosion-driven device. Astrocytes experienced dynamic energetic fluctuations in response to overpressure which were followed by the assumption of a classically defined reactive phenotype. Results indicated specific roles for cationic transduction, cell junction dynamics (gap junction and anchoring junctions) and downstream signal transduction mechanisms associated with adhesion alterations in onset of the astrocyte reactive phenotype. Investigation into adhesion signaling regulation by focal adhesion kinase in 2D and 3D cultures was also explored to better understand cellular reactivity as a function of extracellular environment. Additionally, another underwater in vitro device was built to study combination effects from overpressure and fluid shear associated with insult. Overall, the combined studies offer multiple mechanisms by which to explore molecular targets for harnessing astrocytes' potential for repair after traumatic injury to the brain. / PHD / Blast neurotrauma represents a significant mode of traumatic injury to the brain. The incidence of blast neurotrauma is particularly high amongst military combat personnel in which close to 80% of the injuries sustained in combat are attributed to explosive mechanisms. This injury, like other traumatic brain injuries, can be debilitating and result in altered quality of life for years after injury is sustained. There is a critical need to understand how brain cells are injured by and respond to blast loading in order to develop effective therapeutic strategies. The following work approaches this problem through the use of cellular models of blast-type insult. Custom injury devices were used to develop models of brain cell reactive response to high-rate insult based on experimental simulations of blast neurotrauma. In particular, a sub-type of brain cells called astrocytes were studied. Astrocytes have emerged as a potential therapeutic target because of their ubiquitous roles in brain homeostasis, tissue integrity and cognitive function. The working hypothesis was that high-rate overpressure exposure would cause metabolic aberrations, cell junction changes, and adhesion signal transduction activation, all of which would contribute to astrocyte response and reactivity. Astrocytes experienced dynamic energetic fluctuations in response to overpressure which were followed by the assumption of a classically defined reactive phenotype. Results indicated specific roles for cationic transduction, cell anchorage and downstream signaling mechanisms associated with adhesion alterations in onset of the astrocyte reactive phenotype. Investigation into adhesion signaling regulation by focal adhesion kinase in 2D and 3D cultures was also explored to better understand cellular reactivity as a function of extracellular environment. Additionally, another underwater cell injury device was built to study combination effects from overpressure and fluid shear associated with insult. Overall, the combined studies offer multiple mechanisms by which to explore molecular targets for harnessing astrocytes’ potential for repair after traumatic injury to the brain.

Connexins are transmembrane proteins that form hemichannels allowing the exchange of molecules between the extracellular space and the cell interior. Two hemichannels from adjacent cells dock and form a continuous gap junction pore, thereby permitting direct intercellular communication. Connexin 36 (Cx36), expressed primarily in neurons, is involved in the synchronous activity of neurons and may play a role in aberrant synchronous firing, as seen in seizures. To understand the reciprocal interactions between Cx36 and seizure-like neural activity, we examined three questions: a) does Cx36 deficiency affect seizure susceptibility, b) does seizure-like activity affect Cx36 expression patterns, and c) does acute blockade of Cx36 conductance increase seizure susceptibility. We utilize the zebrafish pentylenetetrazol (PTZ; a GABA(A) receptor antagonist) induced seizure model, taking advantage of the compact size and optical translucency of the larval zebrafish brain to assess how PTZ affects brain-wide neuronal activity and Cx36 protein expression. We exposed wild-type and genetic Cx36-deficient (cx35.5-/-) zebrafish larvae to PTZ and subsequently mapped neuronal activity across the whole brain, using phosphorylated extracellular-signal-regulated kinase (pERK) as a proxy for neuronal activity. We found that cx35.5-/- fish exhibited region-specific susceptibility and resistance to PTZ-induced hyperactivity compared to wild-type controls, suggesting that genetic Cx36 deficiency may affect seizure susceptibility in a region-specific manner. Regions that showed increased PTZ sensitivity include the dorsal telencephalon, which is implicated in human epilepsy, and the lateral hypothalamus, which has been underexplored. We also found that PTZ-induced neuronal hyperactivity resulted in a rapid reduction of Cx36 protein levels within. 30 minutes and one-hour exposure to 20 mM PTZ significantly reduced the expression of Cx36. This Cx36 reduction persists after one-hour of recovery but recovered after 3-6 hours. This acute downregulation of Cx36 by PTZ is likely maladaptive, as acute pharmacological blockade of Cx36 by mefloquine results in increased susceptibility to PTZ-induced neuronal hyperactivity. Together, these results demonstrate a reciprocal relationship between Cx36 and seizure-associated neuronal hyperactivity: Cx36 deficiency contributes region-specific susceptibility to neuronal hyperactivity, while neuronal hyperactivity-induced downregulation of Cx36 may increase the risk of future epileptic events. / Doctor of Philosophy / Within the brain, cells (neurons) communicate with each other to pass along information. This communication is important for normal functions of the brain such as learning and memory, muscle movement, etc. Epilepsy is a disease of the brain that is caused by rapid over synchronized communication between cells. This leads to seizures which can include convulsions, loss of attention, and much more. Currently, 30% of patients suffering from epilepsy do not have a treatment option that works for them, it is, therefore, imperative to investigate new targets for treatment in this disease. Connexin36 is a protein in the brain that directly connects cells so they can pass information quickly between them. Connexin36, therefore, might make a good target for treatment. Previous work has aimed to understand this relationship but has been limited in their ability to look at the entire brain at any one time. The goal of this study was to understand the relationship between connexin 36 and brain hyperactivity across the whole brain simultaneously. To understand this relationship, we first determined what happened to brain activity if the protein was missing entirely after exposure to a seizure causing drug. We were asking: How does connexin 36 affect hyperactivity. We found that different regions of the brain responded differently without the connexin 36 protein. This suggests that one size does not fit all, and one must look at the whole brain to understand the effects of the connexin 36 protein. Next, we asked a similar question, but in the opposite direction, how does hyperactivity affect connexin 36? We found, in the short-term, hyperactivity reduced the amount of connexin 36 present in certain regions of the brain. This continued until 3 hours following exposure to the seizure causing drug Pentylenetetrazol (PTZ). Lastly, to determine if this short-term reduction in connexin 36 meant that an individual was more likely to experience hyperactivity. To do this, we used a connexin 36 blocking drug, then introduced the seizure causing drug at different concentrations. We found, at all concentrations, the connexin 36 blocking drug caused significant changes in neuronal activity, depending on the brain regions. Overall, our results showed that connexin 36 plays an important role in hyperactivity and that a short-term reduction in connexin 36 is detrimental, and may contribute to an increase in the possibility of subsequent hyperactivity.

Electronic devices, for logical and memory applications, are constructed based on bistable electronic units that can store binary information. Molecular electronics proposes the use of single molecules—with two distinctive states of conductance—as bistable units that can be used to create more complex electronic devices. The conductance of a molecule is strongly influenced by the contacts used to address it. The purpose of this work is to determine the electrical characteristics of several candidate molecular junctions, which are composed of a molecule and contacts. Specifically, we are interested in determining whether binary information, “0” or “1,” can be encoded in the low- and high-conductance states of the molecular junctions. First, we calculate quantum-mechanically the electronic structure of the molecular junction. Second, the continuous electronic states of the contacts, originated from their infinite nature, are obtained by solving the Schrödinger equation with periodic boundary conditions. Last, the electron transport through the molecular junctions is calculated based on a chemical interpretation of the Landauer formalism for coherent transport, which involves the information obtained from the molecule and the contacts. Metal-molecule-metal and metal-molecule-semiconductor junctions are considered. The molecule used is an olygo(phenylene ethynylene) composed of three benzene rings and a nitro group in the middle ring; this molecule is referred hereafter as the nitroOPE molecule. Gold, silicon, and metallic carbon nanotubes are used as contacts to the molecule. Results from the calculations show that the molecular junctions have distinctive states of conductance for different conformational and charge states. High conductance is found in the conformation in which all the benzene rings of the nitroOPE are coplanar. If the middle benzene ring is made perpendicular to the others, low conductance is found. Also, the negatively charged junctions (anion, dianion) show low conductance. Whenever a semiconducting contact is used, a flat region of zero current is found at low bias voltages. The results indicate that the use of Si contacts is possible; however, because of the flat region, the operating point of the devices needs to be moved to higher voltages.

Hintergrund: Connexine (Cx) spielen eine wichtige Rolle bei Wachstum und Differenzierung. Die Angiogenese ist an vielen physiologischen und pathologischen Prozessen beteiligt. Jedoch ist noch unklar, ob Connexine einen Einfluss auf die Angiogenese haben. Fragestellung; In dieser Arbeit sollte die Rolle endothelialer Connexine an der Angiogenese untersucht werden. Methoden: Venöse Zellen der menschlichen Nabelschnur (HUVEC) wurden kultiviert bis sie subkonfluent waren. Vor der 3D Kultur im Matrigel wurden die Zellen entweder mit Nicotin, dem Gap Junction Inhibitor Palmitoleinsäure (PA), einem siRNA-Knockdown von entweder Cx37, Cx40 oder Cx43 oder mit Isoprenalin behandelt. Die Zellen wurden dann auf ein in vitro Angiogenese-Assay (3D-Kultur, Matrigel) gegeben und nach 18h wurden unterschiedliche Angiogeneseparameter erhoben, um die Komplexität der Angiogenese zu beurteilen. Änderungen der Expression der Cx mRNA- und Proteinexpression sowie der Kommunikation über Gap Junctions wurden mittels PCR, Immunfluoreszenzmikroskopie, Western Blot und Lucifer Yellow Dye Transfer untersucht. Bei Gewebeproben der A. mammaria von Rauchern und Nichtrauchern wurden mittels Immunofluoreszenzmikroskopie die Expression der Cx beurteilt. Ergebnisse: Die Behandlung mit spezifischer siRNA führte zu einer signifikanten Abnahme der Expression des jeweiligen Connexins in den HUVECs. Sowohl der Knockdown als auch die Behandlung mit PA verringerte die Kommunikation über Gap Junctions signifikant und reduzierte die Anzahl der Abzweigungen im Angiogenese-Assay. Der Knockdown des Cx43 und Cx40 sowie die Behandlung mit PA reduzierten ebenfalls die Komplexität des Musters im Matrigel-Assay. Nicotin führte zu einer Reduktion der Cx43- und Cx37-Proteinexpression, wohingegen Cx40 durch transskriptionelle Gegenregulation konstant gehalten wurde, sowie zu einer Abnahme der Länge der Kapillar-ähnlichen-Strukturen, der Anzahl der Abzweigungen und des Musters im Matrigelassay, während die Anzahl der Zellen zunahm. Die mRNA-Expression der Connexine war hingegen erhöht. In Gewebeproben von Rauchern konnte analog eine verminderte Expression von Cx43 und Cx37, aber nicht von Cx40, in der Intima gezeigt werden. Isoprenalin erhöhte die Proteinexpression der endothelialen Connexine und verbesserte sowohl die interzelluläre Kommunikation als auch das Muster im Matrigel-Assay. Ebenso waren die Kapillar-ähnlichen Strukturen im Vergleich zur Kontrolle länger. Schlussfolgerung: Aus den erhobenen Ergebnissen lässt sich der Schluss ziehen, dass Connexine bei der Angiogenese involviert sind, vor allem beim Vorgang der Verzweigung. Dies kann teilweise die Veränderung erklären, die eine Nicotinbehandlung auf die Angiogenese hat.

Since the discovery of peripheral nerve regeneration nearly a century ago, the mechanisms that guide this regeneration have been elusive. This project aimed to describe how an axon is able to traverse the environment of the body and precisely reinnervate its target cell. Using a novel technique of combining light and electron microscopy, I observed reinnervating axons in transgenic mice to answer the questions of whether Schwann cells are an important guidance cue for the motor neuron and whether the outgrowing axon is fully developed or the process is a step-wise process of activation. The data suggests that Schwann cell contact is important for the tip of the regenerating axon to guide the axon back to its synapse on the muscle fiber. Further, it seems that the tip of the axon is not capable of synaptic transmission as it lacks active zones, suggesting that reinnervation is a step-wise process. / Department of Physiology and Health Science

Nerve signals in the form of action potentials are relayed between neurons through specialized connections called synapses via neurotransmitter released from synaptic vesicles. The release process is Ca2+ dependent, and relies on fusion of neurotransmitter filled synaptic vesicle with the presynaptic membrane. During high frequency stimulation, the amount of vesicle release increases at some synapses (e.g., frog neuromuscular junction (NMJ)), a process known as short-term plasticity. Due to the micron scale size of the presynaptic active zone where vesicle fusion takes place, experimentally study is often difficult. Thus, computational modeling can provide important insight into the mechanism of synaptic vesicle release at active zones. In the first part of my thesis, I used the frog NMJ as a model synapse for computer simulation studies aimed as testing various mechanistic hypotheses proposed to underlie short-term plasticity. Building off a recently reported excess-bindingsite model of synaptic vesicle release at the frog NMJ (Dittrich et al., 2013), I have investigated several mechanisms of short-term facilitation at the frog NMJ. My studies placed constraints on previously proposed mechanistic models, and concluded that the presence of a second calcium sensor protein on synaptic vesicles distinct from synaptotagmin, can explain known properties of facilitation observed at the frog NMJ. In addition, I was able to identify a second facilitation mechanism, which relied on the persistent binding of calcium bound synaptotagmin molecules to lipids of the presynaptic membrane. In the second part of my thesis, I investigated the structure function relationship at active zones, with the hypothesis that active zones are organized from the same basic synaptic building block consisting of a docked vesicle and a small number of closely associated voltage-gated-calcium-channels (VGCCs). To test this hypothesis, I constructed a vesicle release model of the mouse NMJ by reassembling frog NMJ model building blocks based on electron-microscopy imaging data. These two models successfully predicted the functional divergence between frog and mouse NMJ in terms of average vesicle release and short-term plasticity. In the meanwhile, I found that frog NMJ loses facilitation when VGCCs were systematically removed from active zone. By tracking Ca2+ ions from each individual VGCCs, I further show how the difference in short-term plasticity between frog and mouse NMJ may rise from their distinct release building block assemblies. In summary, I have developed a stochastic computer model of synaptic transmission, which not only shed light on the underlying mechanisms of short-term plasticity, but was also proved powerful in understanding structural and functional relationships at synaptic active zones.

Gap Junction Kanäle sind wesentlich für die interzelluläre Kommunikation im Herzen. Die wichtigsten kardialen Gap Junction Proteine sind Connexin 43, Connexin 40 und in frühen Entwicklungsstadien Connexin 45. Da Katecholamine eine wichtige Rolle in der kardialen Physiologie bzw. Pathophysiologie spielen, sollte geklärt werden, ob Katecholamine die Expression von Connexin 40 und Connexin 43 beeinflussen. Es wurden neonatale Rattenkardiomyozyten in Zellkultur 24 Stunden mit aufsteigenden Noradrenalinkonzentrationen (1-10000 nM) (physiologischer Agonist für α-und β-Adrenozeptoren) inkubiert. Hier zeigte sich ein signifikanter Anstieg der Connexin 43 Expression, während die Connexin 40 Expression nicht beeinflußt wurde. Um zu zeigen über welchen Adrenozeptor (α oder β) die Hochregulation von Cx 43 durch Noradrenalin erfolgt, wurden die Zellkulturen in zwei weiteren Versuchsreihen zusätzlich mit einem β-Blocker (Propranolol 100µM) bzw. einem α- Blocker (Prazosin 10 µM) über 24 Std. inkubiert. Hier zeigte sich eine deutliche Inhibierung des Noradrenalineffektes durch die Blockade der α-Rezeptoren durch Prazosin. Eine Blockade der β-Rezeptoren hatte keinen inhibitorischen Effekt auf die Hochregulation der Expression von Cx 43 durch Noradrenalin. Es kam hier sogar zu einer verstärkten Expression verglichen mit der Cx 43 Expression unter Noradrenalin ohne Rezeptorblockade. Desweiteren wurde überprüft, ob es auch unter der Behandlung von Sprague– Dawley-Ratten mit einem β-Mimetikum (Isoprenalin) bzw. einem α-Mimetikum (Phenylephrin) zu einer Veränderung der Connexinexpression kommt. Die Western Blot Analysen ergaben hierbei wieder einen α-Rezeptoren vermittelten Effekt, indem es zu einem wesentlichen Konzentrationsanstieg von Connexin 43 unter der Behandlung mit Phenylephrin kam.