Cardiac tissue slices, a rarely used pseudo two-dimensional preparation, have gained increasing popularity for applications such as drug testing over the last ten years as they combine ease of handling with patho-physiologically relevant cell-type representation, distribution and inter-connection. The most well-established methods to measure electrophysiology in cardiac tissue are sharp electrodes and multi-electrode-arrays, techniques which are limited in spatial resolution or signal content. In this work, we have applied dual voltage Ca²⁺ optical mapping on cardiac slices, allowing us to record these two key parameters simultaneously at high spatio-temporal resolution, yielding better visualisation of conduction waves, spatial dispersion in action potential (AP) characteristics, and intracellular Ca²⁺ transient (CaT). The slice preparation method and the measurement protocols were refined to yield good reproducibility. Data analysis routines were developed to extract relevant parameters reliably. Despite being a promising candidate for drug testing, little is known about how slice and intact whole-heart AP properties are interrelated, and how to scale-up from observations in two dimensions (2D) to the three dimensional (3D) heart. In this thesis, we present a method to compare directly AP properties of intact whole-heart and tissue slices, and show the extent to which slices preserve AP characteristics. We have explored the suitability of tissue slices as an experimental model to study stretch induced changes in AP and CaT. During axial stretch, a dynamic profile of both AP and CaT was observed with an initial shortening of both AP and CaT duration, followed by a gradual recovery/prolongation. We have also used tissue slices to study spatial heterogeneity of AP and CaT properties in the rabbit left ventricular free wall. A transmural gradient can be captured in CaT and AP (with the longest APD and CaT durations being captured in the subendocardium). No large AP prolongation was found in the mid-myocardium. We conclude that the cardiac tissue slice preparation preserves some key functional parameters of the whole heart and is a promising model to study cardiac electrophysiology.

Osteocytes are widely regarded as mechanosensors, capable of detecting changes in the mechanical environment of the bone tissue and modifying cellular responses accordingly. Indeed, an intact osteocyte network is required for bone changes in response to unloading, and studies have shown that loading/unloading influences osteocyte expression of proteins that modulate bone turnover, such as sclerostin and receptor activator of nuclear factor kappa B ligand (RANKL). However, mechanisms underlying osteocyte mechanotransduction remain unclear. For instance, one of the earliest responses of bone cells to mechanical stimuli is a rise in intracellular, or cytosolic, calcium (Ca2+cyt), but the mechanisms by which osteocytes generate or utilize Ca2+ signals to direct bone adaptation are largely unknown. In this thesis, I explored the mechanisms underlying the sustainment of Ca2+cyt oscillations in osteocytes as well as downstream consequences of these patterns. I discovered that Ca2+cyt oscillations are generated in osteocytes by Ca2+ release from the endoplasmic reticulum and that the predominant expression of T-Type voltage sensitive Ca2+ channels in these cells facilitates this behavior. I also explored the role of the actin cytoskeleton – another prominent feature in osteocytes – and found that actin dynamics are important for the generation of Ca2+cyt signals. Furthermore, I confirmed that Ca2+cyt transients subsequently activate actomyosin contractions in osteocytes by monitoring interactions of osteocytes exposed to Ca2+ agonists on micropillar substrates. With this information, I sought to relate Ca2+cyt signaling and actomyosin contractility in osteocytes to their roles as coordinators of bone adaptation. Ca2+-dependent contractions have been shown to facilitate the release of extracellular vesicles, small membrane-enclosed packages of proteins that cells use for communication, in other cell types. I found that mechanical stimulation increased the production and release of extracellular vesicles in osteocytes, and this was dependent on Ca2+ signaling. These extracellular vesicles contained key bone regulatory proteins and were small enough to plausibly transport through the lacunocanalicular system. Thus, I uncovered a novel mechanotransduction pathway by which osteocytes may coordinate tissue-level adaptation. As an extension of this work, I also characterized these behaviors in new osteocyte cell lines which may better reflect native cell physiology. The work in this thesis anchors Ca2+ signaling as a critical osteocyte response to mechanical loading and adds to the body of work exploring how and why these signals are generated. The results of these studies add new information to the still limited knowledge of this important bone cell and extend Ca2+ signaling research by connecting early mechanosensation events to subsequent protein responses to mechanical loading. Understanding the mechanisms behind the robust Ca2+cyt oscillations in osteocytes and how they relate to their roles as coordinators of bone adaptation may improve our ability to prevent or treat bone degeneration in diseases like osteoporosis where mechanosensitivity is impaired.

Phospholipase C enzymes are a class of enzymes that catalyse the cleavage of the membrane phospholipid, phosphatidylinositol bisphosphate (PtdIns(4,5)P₂) into the second messengers, inositol trisphosphate (Ins(4,5)P₃) and diacylglycerol (DAG). Six classes of PLC enzymes have been identified based on their structure and mechanism of activation. PLCηs are the most recently identified family and consist of two isozymes, PLCη1 and PLCη2. The aim of this thesis is to further understand the mechanisms of PLCη activation, the role of PLCη2 in relation to neuritogenesis and their roles in certain disease states. Both isoforms were found to be activated by physiological concentrations of intracellular Ca²⁺. Activation of PLCη2 by Gß₁γ₂ was confirmed using a bacterial 2A co-expression system to allow expression of PLCη2, Gß₁ and Gγ₂ with a single plasmid. Localisation studies show a nuclear distribution for PLCη2, but a cytoplasmic distribution for PLCη1 in a neuroblastoma cells line (Neuro2A). PLCη2 has been implicated in brain development and neurite formation. Building on this, a neuronal differentiation model using RA-treated Neuro2A cells stably expressing mutant forms of PLCη2 was utilised, revealing that PLCη2 activity is essential for neuritogenesis but that this process is independent of the enzymes high sensitivity towards Ca²⁺. Furthermore, the direct interaction of PLCη2 and LIMK-1, a previously identified PLCη2 associated protein, is confirmed in the aforementioned neuronal model. Due to the high sensitivity of PLCη enzymes to Ca²⁺ and because of their presence within neurons, they may be involved in Ca²⁺ dysregulation that occurs in certain diseases such as Alzheimer's disease (AD). The role of PLCη2 was assessed in amyloid-ß (Aß) treated differentiated Neuro2A cells, a cellular model for AD pathogenesis. Also a developmental role for PLCη1 was investigated due to a recently identified PLCη1 polymorphism in patients with holoprosencephaly, an embryonic midline defect.

Gap junctions provide direct electrical and biochemical communication between cardiomyocytes in the heart. Connexin40 (Cx40) is the major connexin in the atria of the heart and little is known regarding its regulation. Thus, the goal was to investigate the regulation of Cx40 in both physiological and pathophysiological conditions. The first objective of this thesis was to determine whether Cx40 gap junctions were regulated by â-adrenergic receptor activation. Cx40 has previously been shown to be acutely activated by cAMP, this cAMP-induced increase in Cx40-mediated cell-to-cell dye transfer has been shown to be effected through the â-adrenergic receptor-adenylyl cyclase- Protein Kinase A (PKA) pathway in Cx40-transfected HeLa cells. The second objective of this thesis was to determine whether Cx40 gap junctions were regulated by intracellular Ca2+ concentration ([Ca2+]i ). [Ca2+]i was increased by addition of the ionophore ionomycin and elevating extracellular calcium [Ca2+]o from 1.8 mM to 21.8 mM. This resulted in an elevation of [Ca2+]i and effected an inhibition of Cx40-mediated cell-to-cell dye transfer (IC50 of 500 ± 0.72 nM) which was Calmodulin-dependent. The third objective of this thesis was to determine whether Cx40 gap junctions were regulated by ischemia. Inducing ischemia chemically by inhibiting the electron transport chain with sodium cyanide and glycolysis with iodoacetate and 2-deoxyglucose effected an inhibition of Cx40-mediated cell-to-cell dye transfer that was shown to be Calmodulin dependent. The main conclusions of this thesis were: (1) â-adrenergic receptor activation increases Cx40-mediated cell-to-cell dye transfer which requires the activation of PKA; (2) A sustained elevation in [Ca2+]i causes a partial inhibition of Cx40 gap junction-mediated cell-to-cell dye transfer which was Ca2+-and Calmodulin dependent; (3) Chemical ischemia causes a partial inhibition of Cx40 gap junction-mediated cell-to-cell dye transfer which was shown to be Calmodulin-dependent.

Stroke is the fifth leading cause of death in the United States and a major cause of long-term disability in industrialized countries. The core region of an ischemic stroke dies within minutes due to activation of necrotic pathways. Outside of this core region is the penumbral zone, where some perfusion is maintained via collateral arteries. Delayed cell death occurs in this area due to the triggering of apoptotic mechanisms, which expands the ischemic injury over time. The cellular and molecular events that produce the expansion of the ischemic core continue to be poorly understood. The increases in the amyloid precursor protein and pathogenic secretases lead to the increase in amyloid-β (Aβ) production. The relatively small amount of research in this area has hampered development of stroke therapy designed to prevent neuronal and glial cell degeneration in the penumbra. Currently, there is a significant lack of therapeutic options for acute ischemic stroke, and no drug has been approved for treating patients at delayed time points (≥ 4.5 hr post-stroke). Afobazole, an anxiolytic currently used clinically in Russia, has been shown to reduce neuronal and glial cell injury in vitro following ischemia, both of which have been shown to play important roles following an ischemic stroke. Treatment with afobazole decreased microglial activation in response to ATP and Aβ, as indicated by reduced membrane ruffling and cell migration. Prolonged exposure of microglia to ischemia or Aβ conditions resulted in glial cell death that was associated with increased expression of the pro-apoptotic protein, Bax, the death protease, caspase-3 and a reduced expression in Bcl-2. Co-application of afobazole decreased the number of cells expressing both Bax and caspase-3, while increasing the cells expressing Bcl-2 resulting in a concomitant enhancement in cell survival. While afobazole inhibited activation of microglia cells by Aβ25-35, it preserved normal functional responses in these cells following exposure to the amyloid peptide. Intracellular calcium increases induced by ATP were depressed in microglia after 24 hr exposure to Aβ25-35. However, co-incubation with afobazole returned these responses to near control levels. Therefore, stimulation of sigma-1 and sigma-2 receptors by afobazole prevents Aβ25-35 activation of microglia and inhibits Aβ25-35-associated cytotoxicity. Examining the molecular mechanisms involved in the increased neuronal survival demonstrates that ischemia or Aβ results in an increased expression of the pro-apoptotic protein Bax and the death protease caspase-3, while at the same time decreasing expression of the anti-apoptotic protein, Bcl-2. However, unlike observations made with microglia, afobazole was unable to modulate this ischemia-induced expression, but was able to modulate Aβ-induced expression of apoptotic proteins while still rescuing neurons from death. Additional experiments were carried out to understand this disparity between the failures of afobazole to prevent the up-regulation of pro-apoptotic genes while retaining the ability to mitigate neuronal death. Although the neurons were still alive they were in a senescent state and were unresponsive to depolarization by high K+. However, these findings are still positive due to the ability of afobazole to delay neuron death, thus minimalizing the toxic environment of the penumbra. These comorbidities of ischemia and Aβ toxicity may lead to potentiated responses and increase the risk for various vascular dementias. It was of particular interest to study how the convergence of ischemia, acidosis and Aβ influence cellular activity and survival within core and penumbral regions. Application of Aβ increased the [Ca2+]i overload produced by concurrent ischemia + acidosis application in isolated cortical neurons. We found that the acid-sensing ion channels 1a (ASIC1a) are involved in the potentiation of [Ca2+]i overload induced by Aβ. Furthermore, afobazole (100 uM) abolished Aβ potentiation of ischemia + acidosis evoked [Ca2+]i overload, which may represent a therapeutic strategy for mitigating injury produced by Aβ and stroke.

Cystic fibrosis (CF) is an inborn, hereditary disease, due to mutations in the gene for a cAMP-activated chloride (Cl-) channel, the cystic fibrosis transmembrane conductance regulator (CFTR). As a result of impaired ion and water transport,the airway mucus is abnormally viscous, which leads to bacterial colonization.Recurrent infections and inflammation result in obstructive pulmonary disease.Similar changes in the pancreas lead to pancreatic insufficiency.Several compounds have been tested to improve transepithelial ion transport in CF patients, either via activation of the mutant CFTR, or via stimulation of alternative chloride channels. The main purpose of this thesis was to find substances that might correct the defective ion transport in epithelial cells in CFand could be useful for the pharmacological treatment of CF patients. Long-term treatment with the macrolide antibiotic azithromycin (AZM)improved clinical parameters and lung function in CF patients and increased Cl- transport in CF bronchial epithelial cells (CFBE) (Paper I); although mRNA expression of the CFTR gene remained unchanged.In contrast, pre-exposure to the mucolytic antioxidant N-acetylcysteine (NAC) increased CFTR protein expression and was associated with increased Cl- efflux from CFBE cells (Paper II). Clinical trials of this substance might be warranted. Duramycin has been the subject of clinical trials that finished in June2009. Up till now, no results from this study are available. The effect of this substance on Cl- efflux from three CF and three non-CF cell lines (Paper III) was disappointing. An effect was found only in CFBE cells, the effect was minimal, occurred in a narrow concentration range, and was not associated with an increase in the intracellular calcium concentration [Ca2+]i. The fact that NO-donors stimulated Cl- efflux from CFBE cells (but did notchange [Ca2+]i) after several hours of preincubation suggests that these substances may be a potentially interesting group of compounds for the treatment of CF (Paper IV). A model for the effect of NO-donors on Cl- efflux is presented. / Cystisk fibros (CF) är en medfödd, ärftlig, sjukdom, som förorsakas av en mutation i en gen som innehåller koden för en kloridkanal som aktiveras av cykliskt AMP (cystic fibrosis transmembrane conductance regulator, CFTR). Som en följd av otillräcklig transport av joner och vatten är slemmet i luftvägarna onormalt segt, vilket leder till att det koloniseras av bakterier. Upprepade infektioneroch inflammation av luftvägarna leder slutligen till obstruktiv lungsjukdom.Liknande förändringar i bukspottkörteln leder till att också detta organ inte fungerar. Flera kemiska ämnen har testats för sin förmåga att förbättra jontransporten över epitelet hos CF-patienter. Detta skulle kunna göras antingen genom aktivering av det muterade CFTR-proteinet, eller genom stimulering av alternativa kloridkanaler. Huvudsyftet med den forskning som beskrivs i denna avhandling var att hitta kemiska substanser som skulle kunna korrigera den defekta jontransporten i epitelceller hos CF-patienter, och därför vara nyttiga för behandlingen av patienterna. Behandling under längre tid med azithromycin (AZM), ett makrolidantibiotikum,förbättrade CF-patienternas kliniska status och lungfunktion,samt ökade kloridutflödet från CF bronkialepitelceller (CFBE-celler) (Arbete I).Däremot ändrades inte uttrycket av mRNA för CFTR-genen. I kontrast till detta ökade uttrycket av CFTR-proteinet om CFBE-cellerna utsattes för den slemlösande anti-oxidanten N-acetylcystein (NAC), vilket ledde till ökat kloridutflöde från denna cellinje (Arbete II). Det vore rimligt att utföra kliniska prövningar av detta ämne. Duramycin har testats i kliniska prov som slutade i juni 2009, men några resultatfrån dessa prov har inte offentliggjorts än. Effekten av detta ämne på kloridutflödet från tre CF-cellinjer och tre icke-CF cellinjer (Arbete III) var en besvikelse. Duramycin hade endast effekt på CFBE-celler, effekten var mycket liten, förekom endast i ett litet koncentrationsområde av duramycin, och var inte kopplad till en ökning av den intracellulära kalciumkoncentrationen [Ca2+]i. Att ämnen som avger kväveoxid (NO) stimulerade kloridutflödet från CFceller (men inte påverkade [Ca2+]i) efter några timmar, visar att denna grupp av ämnen kan vara potentiellt intressant för behandlingen av CF (arbete IV). En modell för effekten av NO på kloridtransporten i CF-celler presenteras.

Sudden cardiac death (SCD) is a leading cause of fatalities. Several methods have been developed to predict instability in myocytes which could lead to SCD. The focus of this study was on altering memory in myocytes, i.e. hysteresis in restitution of action potential duration (APD), by differing levels of calcium. Determination of alteration was implemented by using a diastolic interval (DI) control program that implements a sinusoidal change in DI. Plotting APD versus previous DI, i.e. restitution, produces a hysteresis loop. From these hysteresis loops, five parameters were used to determine measures of memory: area, thickness, overall tilt, max delay and min delay. Calcium levels were then altered with either verapamil or BAPTA-AM. Statistically significant effects were found for the verapamil study, but not for the BAPTA-AM study. Simulations were used to explain significant results. The verapamil findings support clinical studies that have shown verapamil to not have anti-arrhythmic effects. Theory predicts that a decrease in memory would decrease the stability of a system, and perhaps verapamil may not increase stability as hypothesized previously. The results of the BAPTA-AM study were inconclusive, and further investigation is needed before it can be determined that BAPTA-AM has no significant effect on memory.

abstract: Euendolithic cyanobacteria have the remarkable ability to actively excavate and grow within certain minerals. Their activity leads to increased erosion of marine and terrestrial carbonates, negatively affecting coral reef and bivalve ecology. Despite their environmental relevance, the boring mechanism has remained elusive and paradoxical, in that cyanobacteria alkalinize their surroundings, typically leading to carbonate precipitation, not dissolution. Thus, euendoliths must rely on unique adaptations to bore. Recent work using the filamentous model euendolith Mastigocoleus testarum strain BC008 indicated that excavation relied on transcellular calcium transport mediated by P-type ATPases, but the phenomenon remained unclear. Here I present evidence that excavation in M. testarum involves an unprecedented set of adaptations. Long-range calcium transport is achieved through the coordinated pumping of multiple cells, orchestrated by the localization of calcium ATPases in a repeating annular pattern, positioned at a single cell pole, adjacent to each cell septum along the filament. Additionally, specialized chlorotic cells that I named calcicytes, differentiate and accumulate calcium at concentrations more than 500 fold those of canonical cells, likely allowing for fast calcium flow at non-toxic concentrations through undifferentiated cells. I also show, using 13C stable isotope tracers and NanoSIMS imaging, that endolithic M. testarum derives most of its carbon from the mineral carbonates it dissolves, the first autotroph ever shown to fix mineral carbon, confirming the existence of a direct link between oxidized solid carbon pools and reduced organic pools in the biosphere. Finally, using genomic and transcriptomic approaches, I analyze gene expression searching for additional adaptations related to the endolithic lifestyle. A large and diverse set of genes (24% of 6917 genes) were significantly differentially regulated while boring, including several master regulators and genes expectedly needed under this condition (such as transport, nutrient scavenging, oxidative stress, and calcium-binding protein genes). However, I also discovered the up-regulation of several puzzling gene sets involved in alternative carbon fixation pathways, anaerobic metabolism, and some related to photosynthesis and respiration. This transcriptomic data provides us with several new, readily testable hypotheses regarding adaptations to the endolithic lifestyle. In all, my data clearly show that boring organisms show extraordinarily interesting adaptations. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2016

Intracellular calcium is a major determinant of a wide variety of cell functions and thus of organ function. In order to get a clear picture of the intracellular calcium status it is preferable to assess the content of the various intracellular calcium pools as well as the characteristics of the transmembrane calcium movements, Le., the magnitude of the transmembrane Ca2+ flux upon stimulation and the rate of the subsequent return to baseline levels. The first aim of this study was to establish and evaluate the methods in the laboratory. The methods investigated include atomic absorption spectrometry, graphite furnace atomic absorption spectrometry and inductively coupled plasma mass spectrometry for the determination of the total cell calcium content, fluorescence spectrophotometry for the determinations of intracellular free Ca2+ and transmembrane Ca2+ movements and transmission electron microscopy for the localisation of intracellular calcium. The methods eventually identified as feasible included fluorescence spectrophotometry for the determination of intracellular free Ca2+ and transmembrane Ca2+ movements and transmission electron microscopy for the localisation of intracellular calcium. The newly developed fluorescent calcium indicator, fura-PE3, was presently shown to be the most reliable fluorescent indicator for the intracellular free Ca2+ determinations. The best method for the calcium localisation by transmission electron microscopy was an adaptation of the antimonate precipitation technique. The following objectives were set in order to contribute to the knowledge in chronic renal failure; examination of the intracellular free Ca2+ content in the neutrophils of end stage renal failure patients on maintenance haemodialysis treatment, as the result of renal failure, dialysis treatment and medication combined; examination of the characteristics of the transmembrane Ca2+ movements; investigation of the intracellular calcium distribution in the neutrophils; exploration of a possible link between the alterations in intracellular calcium status and factors known to influence the calcium status, including the lipid composition of the membrane, the oxidative status as reflected by anti-oxidant vitamin levels, as well as the levels of parathyroid hormone, and ionised serum calcium. This study involved 14 chronic renal failure patients on maintenance haemodialysis. An increase in intracellular free Ca2+, the magnitude of the transmembrane Ca2+ flux upon fMLP stimulation and an increase in the rate of the subsequent decrease in intracellular free calcium were found. In separating the patients into those receiving rHuEPO and those not receiving rHuEPO, it was seen that the significance in the increase in intracellular free Ca2+ could be ascribed to the values obtained in those patients receiving rHuEPO - despite the fact that they were the only patients receiving calcium channel blockers. No overt indications of oxidative stress could be detected by anti-oxidant vitamin levels. Nevertheless, a decrease in the content of specific membrane fatty acids occurred, supporting the previous suggestions of the presence of a mild chronic inflammatory condition in the chronic renal failure patient on maintenance haemodialysis treatment. These results suggest that factors other than those associated with uraemia, such as rHuEPO administration, might result in an increase in intracellular free Ca2+ in cells of CRF/MHT patients. The magnitude of the rHuEPD-induced increase in intracellular free Ca2+ and the effects of the various calcium channel blockers need urgent further investigation as ineffective counteraction of the rHuEPO effect, as indicated by the relative ineffectivity of Norvasc, may have serious side-effects. / Dissertation (MSc)--University of Pretoria, 2000. / Physiology / unrestricted

Proteinase-activated receptor-2 (PAR-2) is a member of a family of G-protein-coupled, seven-transmembrane domain receptors that are activated by proteolytic cleavage. The receptor is expressed in a number of different tissues and potential physiological activators identified thus far include trypsin and mast cell tryptase. Acrosin, a trypsin-like serine proteinase found in spermatozoa of all mammals, was found to cleave a model peptide fluorescent quenched substrate representing the cleavage site of PAR-2. This substrate was cleaved with kinetics similar to those of the known PAR-2 activators, trypsin and mast cell tryptase. Acrosin was also shown to induce significant intracellular calcium responses in Chinese hamster ovary cells stably expressing intact human PAR-2, most probably due to activation of the receptor. Immunohistochemical studies using PAR-2 specific antibodies indicated that the receptor is expressed by mouse oocytes, which suggests that acrosin may play additional role(s) in the fertilization process via the activation of PAR-2 on oocytes.