A Quantum Degenerate Mixture of 87^{87}Rb and 133^{133}Cs

This thesis reports the formation of a dual-species Bose-Einstein condensate of $^{87}$Rb and $^{133}$Cs in the same trapping potential. Quantum degenerate mixtures exhibit rich physics inaccessible to single species experiments and provide an ideal starting point for the creation of ultracold dipolar molecules. These molecules offer a wealth of new research avenues including precision metrology, quantum simulation and computation. The experimental method exploits the efficient sympathetic cooling of $^{133}$Cs via elastic collisions with $^{87}$Rb, initially in a magnetic quadrupole trap and subsequently in a levitated optical trap. Evaporative cooling in the dipole trap must compete against a high interspecies three-body inelastic collision rate \mbox{$\sim10^{-25}-10^{-26}$~cm$^{6}/$s}. The two condensates each contain up to \mbox{$2\times10^{4}$} atoms and exhibit a striking phase separation, revealing the mixture to be immiscible due to strong repulsive interspecies interactions. Sacrificing all the $^{87}$Rb during the cooling leads to the creation of single-species $^{133}$Cs condensates of up to \mbox{$6\times10^{4}$} atoms. In addition this thesis reports the observation of an interspecies Feshbach resonance at 181.7(5)~G and the creation of a pure sample of Cs$_{2}$ molecules via magneto-association on the 4(g)4 resonance at 19.8~G. These results represent important steps towards the creation of ultracold polar RbCs molecules

The transition of smooth muscle cells from a contractile to a migratory, phagocytic phenotype : direct demonstration of phenotypic modulation

Atherosclerotic plaques are populated with smooth muscle cells (SMCs) and macrophages. SMCs are thought to accumulate in plaques because fully-differentiated, contractile SMCs reprogram into a ‘synthetic’ migratory phenotype, so-called phenotypic modulation, whilst plaque macrophages are thought to derive from blood-borne myeloid cells. Recently, these views have been challenged, with reports that SMC phenotypic modulation may not occur during vascular remodelling and that plaque macrophages may not be of haematopoietic origin. Following the fate of SMCs is complicated by the lack of specific markers for the migratory phenotype and direct demonstrations of phenotypic modulation are lacking. Therefore, we employed long-term, high-resolution, time-lapse microscopy to track the fate of unambiguously identified, fully-differentiated, contractile SMCs in response to the growth factors present in serum. Phenotypic modulation was clearly observed. The highly-elongated, contractile SMCs initially rounded up, for 1-3 days, before spreading outwards. Once spread, the SMCs became motile and displayed dynamic cell-cell communication behaviours. Significantly, they also displayed clear evidence of phagocytic activity. This macrophage-like behaviour was confirmed by their internalisation of 1µm fluorescent latex beads. However, migratory SMCs did not uptake acetylated low-density lipoprotein or express the classic macrophage marker CD68. These results directly demonstrate that SMCs may rapidly undergo phenotypic modulation and develop phagocytic capabilities. Resident SMCs may provide a potential source of macrophages in vascular remodelling

FK506 regulates IP3 evoked Ca2+ release independently of FKBP in endothelial cells

Background and Purpose FK506 and rapamycin are modulators of FK-binding proteins (FKBP) that are used to suppress immune function after organ and hematopoietic stem cell transplantations. The drugs share the unwanted side-effect of evoking hypertension that is associated with reduced endothelial function and nitric oxide production. The underlying mechanisms are not understood. FKBP may regulate IP3 and ryanodine receptors to alter Ca2+ signalling in endothelial cells. Experimental Approach We investigated the effects of FK506 and rapamycin on Ca2+ release via IP3 and ryanodine receptors in large numbers of endothelial cells in intact arteries. Key Results While confirmed to be present, FKBP modulation with rapamycin did not alter IP3-evoked Ca2+ release. Conversely, FK506, which modulates FKBP and additionally blocks calcineurin, increased IP3-evoked Ca2+ release. Inhibition of calcineurin (using okadiac acid or cypermethrin) also increased IP3-evoked Ca2+ release and blocked FK506 effects. Indeed, when calcineurin was inhibited with okadiac acid, FK506 reduced IP3-evoked Ca2+ release. These findings suggest that FKBP does not modulate IP3-evoked Ca2+ release and FK506 increased IP3-evoked Ca2+ release by calcineurin inhibition. FK506 and rapamycin are also unlikely to mediate their effects via RyR. The RyR activator caffeine and ryanodine itself failed to evoke Ca2+ changes suggesting that RyR is not functional in native endothelium. Conclusion and Implications The hypertensive effects of the immunosuppressant drugs FK506 and rapamycin, while mediated by endothelial cells, do not appear to be exerted at documented cellular targets of the drugs on Ca2+ release and altered FKBP binding to IP3 and RyR

The endothelium solves problems that endothelial cells do not know exist

The endothelium is the single layer of cells that lines the entire cardiovascular system and that regulates vascular tone and blood-tissue exchange, recruits blood cells, modulates blood clotting and determines the formation of new blood vessels. To control each function, the endothelium uses a remarkable sensory capability to continuously monitor vanishingly small changes in the concentration of many simultaneously arriving extracellular activators that each provide cues to physiological state. Here, we suggest that the extraordinary sensory capabilities of the endothelium does not come from single cells but from the combined activity of a large number of endothelial cells. Each cell has a limited, but distinctive, sensory capacity and shares information with neighbours so that sensing is distributed among cells. Communication of information among connected cells provides a system-level sensing substantially greater than the capabilities of any single cell and, as a collective, the endothelium solves sensory problems too complex for any single cell

Pressure-dependent regulation of Ca2+ signaling in the vascular endothelium

The endothelium is an interconnected network upon which hemodynamic mechanical forces act to control vascular tone and remodeling in disease. Ca2+ signaling is central to the endothelium's mechanotransduction and networked activity. However, challenges in imaging Ca2+ in large numbers of endothelial cells under conditions that preserve the intact physical configuration of pressurized arteries have limited progress in understanding how pressure-dependent mechanical forces alter networked Ca2+ signaling. We developed a miniature wide-field, gradient-index (GRIN) optical probe designed to fit inside an intact pressurized artery which permitted Ca2+ signals to be imaged with subcellular resolution in a large number (∼200) of naturally-connected endothelial cells at various pressures. Chemical (acetylcholine) activation triggered spatiotemporally-complex, propagating IP3-mediated Ca2+ waves that originated in clusters of cells and progressed from there across the endothelium. Mechanical stimulation of the artery, by increased intraluminal pressure, flattened the endothelial cells and suppressed IP3-mediated Ca2+ signals in all activated cells. By computationally modeling Ca2+ release, endothelial shape changes were shown to alter the geometry of the Ca2+ diffusive environment near IP3 receptor microdomains to limit IP3-mediated Ca2+ signals as pressure increased. Changes in cell shape produce a geometric, microdomain-regulation of IP3-mediated Ca2+ signaling to explain macroscopic pressure-dependent, endothelial-mechanosensing without the need for a conventional mechanoreceptor. The suppression of IP3-mediated Ca2+ signaling may explain the decrease in endothelial activity as pressure increases. GRIN imaging provides a convenient method that provides access to hundreds of endothelial cells in intact arteries in physiological configuration

Flicker-assisted localization microscopy reveals altered mitochondrial architecture in hypertension

Mitochondrial morphology is central to normal physiology and disease development. However, in many live cells and tissues, complex mitochondrial structures exist and morphology has been difficult to quantify. We have measured the shape of electrically-discrete mitochondria, imaging them individually to restore detail hidden in clusters and demarcate functional boundaries. Stochastic “flickers” of mitochondrial membrane potential were visualized with a rapidly-partitioning fluorophore and the pixel-by-pixel covariance of spatio-temporal fluorescence changes analyzed. This Flicker-assisted Localization Microscopy (FaLM) requires only an epifluorescence microscope and sensitive camera. In vascular myocytes, the apparent variation in mitochondrial size was partly explained by densely-packed small mitochondria. In normotensive animals, mitochondria were small spheres or rods. In hypertension, mitochondria were larger, occupied more of the cell volume and were more densely clustered. FaLM provides a convenient tool for increased discrimination of mitochondrial architecture and has revealed mitochondrial alterations that may contribute to hypertension

Chapter 9 Mitochondria Structure and Position in the Local Control of Calcium Signals in Smooth Muscle Cells

Features of Ca2+ signals including the amplitude, duration, frequency and location are encoded by various physiological stimuli. These features of the signals are decoded by cells to selectively activate smooth muscle functions that include contraction and proliferation [1–3]. Central, therefore, to an appreciation of how smooth muscle is controlled is an understanding of the regulation of Ca2+

Heterogeneity in the proliferative capacity of smooth muscle cells (SMCs)

In cardiovascular disease, artery walls remodel through an increase in SMC numbers. The predominant hypothesis for this is that SMCs in the tunica media undergo phenotypic modulation into a proliferative cell type. However, direct evidence for SMC phenotypic modulation is scant. We therefore exploited time-lapse microscopy methods to track the fate of freshly isolated, contractile SMCs. As SMCs isolated from different smooth muscle (SM) tissues are heterogeneous in nature, we have used time lapse microscopy in combination with immunocytochemistry to investigate the proliferative capacity of SMCs from portal vein (PV), carotid artery (CA) and distal colon. The adventia/endothelium and mucosa/serosa were mechanically removed before isolating cells by enzymatic digestion and trituration. Highly elongated, contractile SMCs that stained for both SM α-actin (SMA) and SM myosin heavy chain (SM-MHC) were obtained from all tissues. Significantly, both colon and PV tissues also contained large numbers of spherical cells that did not stain for SMA or SM-MHC (nonSMCs). In standard culture conditions, the SMCs showed limited proliferative capacity: with the exception of one SMC that divided once (out of 15 tracked cells), colon SMCs did not proliferate. Of 11 PV SMCs tracked, 7 SMCs did divide, though none progressed beyond the 3rd generation (daughter of daughter) by confluency. Conversely, the nonSMCs proliferated rapidly (reaching the 5th generation in 5 days) and dominated the resulting cultures. In contrast, all CA cells stained for both SMA and SM-MHC i.e. no nonSMCs were present. Whilst most CA SMCs underwent apoptosis early in culture (27 of 31 cells), those that survived went on to proliferate at varying rates (up to the 6th generation in 5 days). These results illustrate the complexities involved in creating models of SMC proliferation

Elevations of intracellular calcium reflect normal voltage-dependent behavior, and not constitutive activity, of voltage-dependent calcium channels in gastrointestinal and vascular smooth muscle

In smooth muscle, the gating of dihydropyridine-sensitive Ca2+ channels may either be stochastic and voltage dependent or coordinated among channels and constitutively active. Each form of gating has been proposed to be largely responsible for Ca2+ influx and determining the bulk average cytoplasmic Ca2+ concentration. Here, the contribution of voltage-dependent and constitutively active channel behavior to Ca2+ signaling has been studied in voltage-clamped single vascular and gastrointestinal smooth muscle cells using wide-field epifluorescence with near simultaneous total internal reflection fluorescence microscopy. Depolarization (−70 to +10 mV) activated a dihydropyridine-sensitive voltage-dependent Ca2+ current (ICa) and evoked a rise in [Ca2+] in each of the subplasma membrane space and bulk cytoplasm. In various regions of the bulk cytoplasm the [Ca2+] increase ([Ca2+]c) was approximately uniform, whereas that of the subplasma membrane space ([Ca2+]PM) had a wide range of amplitudes and time courses. The variations that occurred in the subplasma membrane space presumably reflected an uneven distribution of active Ca2+ channels (clusters) across the sarcolemma, and their activation appeared consistent with normal voltage-dependent behavior. Indeed, in the present study, dihydropyridine-sensitive Ca2+ channels were not normally constitutively active. The repetitive localized [Ca2+]PM rises (“persistent Ca2+ sparklets”) that characterize constitutively active channels were observed rarely (2 of 306 cells). Neither did dihydropyridine-sensitive constitutively active Ca2+ channels regulate the bulk average [Ca2+]c. A dihydropyridine blocker of Ca2+ channels, nimodipine, which blocked ICa and accompanying [Ca2+]c rise, reduced neither the resting bulk average [Ca2+]c (at −70 mV) nor the rise in [Ca2+]c, which accompanied an increased electrochemical driving force on the ion by hyperpolarization (−130 mV). Activation of protein kinase C with indolactam-V did not induce constitutive channel activity. Thus, although voltage-dependent Ca2+ channels appear clustered in certain regions of the plasma membrane, constitutive activity is unlikely to play a major role in [Ca2+]c regulation. The stochastic, voltage-dependent activity of the channel provides the major mechanism to generate rises in [Ca2+]