Altered cerebral hemodynamics contributes to mechanisms of unexplained syncope. Wecompared dynamic interaction between respiration and cerebral autoregulation in two groups ofsubjects from 28 healthy adults. Based on development of tilt-induced presyncope, subjects wereclassified as Non-Presyncopals (n=23) and Presyncopals (n=5). Airflow, CO2, Doppler cerebralblood flow velocity (CBF), ECG and blood pressure (BP) were recorded. To determine whetherinfluences of mean BP (MBP) and systolic BP (SBP) on CBF were altered in Presyncopals, thecoherencies and transfer functions between these variables and mean and peak CBF (CBFm andCBFp) were estimated. To determine influence of end-tidal CO2 (ETCO2) on CBF, relative CO2reactivity was calculated. The two primary findings were, during tilt in Presyncopals: (1) Inrespiratory frequency region, coherence between SBP and CBFp (p=0.02) and transfer functiongain between BP and CBFm was higher (MBP, p=0.01, and SBP, p=0.01) than in Non-Presyncopals. (2) In the last 3 minutes prior to presyncope, Presyncopals had a reduced relativeCO2 reactivity (p=0.005). Thus the relationship of CBF with systemic BP was more pronouncedor cerebral autoregulation was less effective preceding presyncope. This decreasedautoregulation, secondary to decreased ETCO2, may contribute in the cascade of events leadingto unexplained syncope.

Under normotensive conditions, the progressive loss of renal function in diabetes mellitus is very slow. Since hypertension accelerates many forms of renal disease, we assessed the progression of nephropathy in Streptozotocin-induced type 1 diabetes mellitus under renin-mediated hypertension condition. We investigated the diabetic “salt paradox” as a modifiable susceptibility factor for renal damage. Since hyperfiltration occurs in early diabetes, the reduction of glomerular filtration rate due to an increased salt intake could be mediated by increased tubuloglomerular feedback sensitivity. We compared intact-hypertensive versus diabetic-hypertensive Long-Evans rats under normal and increased salt intake, 1 and 2.5% by weight of food eaten, respectively. Weekly 24-h blood pressure records were acquired by telemetry during the six months of the experiment. Target mean blood glucose of ~ 25 mmol/L was maintained by suboptimal insulin implants. Systolic blood pressure increased after induction of hypertension but was not affected by diabetes or increased salt intake, either alone or together. Autoregulation was highly efficient in both intact and diabetic rats. Nephropathy was scored by histology in the clipped and non-clipped kidneys at the end of the protocol. The non-clipped kidney, which was exposed to hypertension, showed a linear pressure-dependent glomerular injury in both intact and diabetic rats. The best fit line describing the linear relationship between pressure load and injury was shifted toward lower blood pressure in diabetic rats. Over the time course of our experiments, injury was entirely pressure dependent in intact and diabetic rats. Diabetes mellitus increased the susceptibility of the kidney to injury, but independent of blood pressure. Increased salt intake affected neither blood pressure nor renal susceptibility to hypertensive injury. / Graduate

<p>Abstract</p><p> Hfq is a pleiotropic posttranscriptional regulator and RNA chaperone that facilitates annealing of trans-encoded sRNA/mRNA pairs. It regulates many different cellular pathways including environmental stress responses, quorum sensing, virulence and maintenance of membrane integrity. Hfq is a member of the Sm/LSm family and forms a homohexamer that has two faces, termed proximal and distal. Hfq preferentially binds A/U rich regions that are near stem loop structures. Crystal structures have shown that poly-A sequences tend to bind the distal face while poly-U sequences bind the proximal face. Currently crystal structures reveal the binding mechanisms for short RNA sequences however; physiologically relevant RNA sequences are typically longer and more structured. To study how these more complex RNA sequences interact with Hfq, a tryptophan fluorescence quenching (TFQ) assay has been developed. Here it is presented that TFQ can correctly identify the binding face for two control sequences, A15 and U6, using the E. coli, S. aureus and L. monocytogenes Hfq homologues. Using fluorescence anisotropy and crystallography it is observed that Trp mutants necessary for TFQ may affect binding to some degree but do not affect the overall structure or RNA binding function of Hfq. TFQ is then used to examine the distal face binding motifs for both Gram-negative (E. coli) and Gram-positive (S. aureus/L. monocytogenes) Hfq, (A-R-N)n and (R-L)n respectively. Using sequences that either fulfilled just (A-R-N)n or both (A-R-N)n and (A-A-N)n motifs it is shown that the distal face motif for Gram-negative Hfq is the more specific (A-A-N)n motif. Using sequences that either fulfilled just (R-L)n or both (R-L)n and (A-L)n motifs it is shown that the Gram-positive distal face motif can be redefined to the (A-L)n motif. Finally TFQ is used to explore autoregulation of E. coli hfq. Two identified binding sites located in the 5'UTR of hfq mRNA, site A and site B, were used for TFQ, along with a longer RNA sequence that contains both sites and their native linker, 5' UTR. TFQ illustrates that the individual sites and the 5' UTR are capable of binding both faces. Each site appears to prefer binding to one face over the other, suggesting a model for hfq 5' UTR mRNA binding to Hfq where either one or two hfq mRNA bind a single Hfq hexamer. In conclusion, TFQ is a straightforward method for analyzing how RNA sequences interact with Hfq that can be utilized to study how longer, physiologically relevant RNA sequences bind Hfq.</p> / Dissertation

Introduction Cerebral autoregulation (CA) refers to the physiological mechanisms in the brain to maintain constant blood flow despite changes in cerebral perfusion pressure (CPP). It plays an important protective role against the danger of ischaemia or oedema of the brain. Over the years, various methods for CA assessment have been proposed, while most commonly used parameters include the autoregulation index (ARI), which grades CA into ten levels; transfer function (TF) analysis, describing CA as a high pass filter; the mean flow index (Mx), that estimates CA through the correlation coefficient between slow waves of mean cerebral blood flow velocity (CBFV) and CPP; and pressure reactivity index (PRx), calculated as a moving correlation coefficient between mean arterial blood pressure (ABP) and intracranial pressure (ICP). However, until now, how these parameters are related with each other is still not clear. A comprehensive investigation of the relationship between all these parameters is therefore needed. In addition, the methods mentioned above mostly assume the system being analysed is linear and the signals are stationary, with the announcement of non-stationary characteristic of CA, a more robust method, in particular suitable for non-stationary signal analysis, needs to be explored. Objectives and Methods This thesis addresses three primary questions: 1. What are the relationships between currently widely used CA parameters, i.e. Mx, ARI, TF parameters, from theoretical and practical point of view? 2. It there an effective method that can be introduced to assess CA, which is suitable for analyses of non-stationary signals? 3. How can bedside monitoring of cerebral autoregulation be improved in traumatic brain injury patients? These general aims have been translated into a series of experiments, retrospective analyses and background studies that are presented in different chapters of this thesis. Results and Conclusions This PhD project carefully scrutinised currently used CA assessment methodologies in TBI patients, demonstrating significant relationships between ARI, Mx and TF phase. A new introduced wavelet-transform-based method, wPRx was validated and showed more stable result for CA assessment than the well-established parameter, PRx. A multi-window approach with weighting system for optimal CPP estimation was described. The result showed a significant improvement in the continuity and stability of CPPopt estimation, which made it possible to be applied in the future clinical management of TBI patients.

The human brain acts as an intelligent sensor by helping in effective signal communication and execution of logical functions and instructions, thus, coordinating all functions of the human body. More importantly, it shows the potential to combine prior knowledge with adaptive learning, thus ensuring constant improvement. These qualities help the brain to interact efficiently with both, the body (brain-body) as well as the environment (brain-environment). This dissertation attempts to apply the brain-body-environment interactions (BBEI) to elevate human existence and enhance our day-to-day experiences. For instance, when one stepped out of the house in the past, one had to carry keys (for unlocking), money (for purchasing), and a phone (for communication). With the advent of smartphones, this scenario changed completely and today, it is often enough to carry just one's smartphone because all the above activities can be performed with a single device. In the future, with advanced research and progress in BBEI interactions, one will be able to perform many activities by dictating it in one's mind without any physical involvement. This dissertation aims to shift the paradigm of existing brain-computer-interfaces from just ‘control' to ‘monitor, control, enhance, and restore' in three main areas - healthcare, transportation safety, and cryptography. In healthcare, measures were developed for understanding brain-body interactions by correlating cerebral autoregulation with brain signals. The variation in estimated blood flow of brain (obtained through EEG) was detected with evoked change in blood pressure, thus, enabling EEG metrics to be used as a first hand screening tool to check impaired cerebral autoregulation. To enhance road safety, distracted drivers' behavior in various multitasking scenarios while driving was identified by significant changes in the time-frequency spectrum of the EEG signals. A distraction metric was calculated to rank the severity of a distraction task that can be used as an intuitive measure for distraction in people - analogous to the Richter scale for earthquakes. In cryptography, brain-environment interactions (BBEI) were qualitatively and quantitatively modeled to obtain cancelable biometrics and cryptographic keys using brain signals. Two different datasets were used to analyze the key generation process and it was observed that neurokeys established for every subject-task combination were unique, consistent, and can be revoked and re-issued in case of a breach. This dissertation envisions a future where humans and technology are intuitively connected by a seamless flow of information through ‘the most intelligent sensor', the brain.

No description available.

A mathematical model of blood flow in infants with hypoplastic left heart syndrome (HLHS) was improved by adding cerebral autoregulation. This is the process by which blood vessels constrict or dilate to keep blood flow steady in certain organs during pressure changes. The original lumped parameter model transformed the fluid flow into an electrical circuit. Its behavior is described using a system of thirty-three coupled differential equations that are solved numerically using a fourth-order Runge-Kutta method implemented in MATLAB. A literature review that includes a discussion of autoregulation mechanisms and approaches to modeling them is followed by a description of the model created for this paper. The model is based on the baroreceptor or neurogenic theory of autoregulation. According to this theory, nerves in certain places within the cardiovascular system detect changes in blood pressure. The brain then compensates by sending a signal to blood vessels to constrict or dilate. The model of the control system responded fairly well to a pressure drop with a steady state error of about two percent. Running the model with or without the control system activated had little effect on other parameters, notably cardiac output. A more complete model of blood flow control would include autonomic regulation. This would vary more parameters than local autoregulation, including heart rate and contractility. This is suggested as a topic of further research.

The brain accounts for around 2 % of human adult bodyweight but consumes 20 % of the resting oxygen available to the whole body. The brain is dependent on a constant supply of oxygen to tissue, transported from the heart via the vasculature and carried in blood. An interruption to flow can lead to ischaemia (a reduced oxygen supply) and prolonged interruption may result in tissue death, and permanent brain damage. The cerebral vasculature consists of many, densely packed, micro-vessels with a very large total surface area. Oxygen dissolved in blood enters tissue by passive diffusion through the micro-vessel walls. Imaging shows bursts of metabolic activity and flow in localised brain areas coordinated with brain activity (such as raising a hand). An appropriate level of oxygenation, according to physiological demand, is maintained via autoregulation; a set of response pathways in the brain which cause upstream or downstream vessels to expand or contract in diameter as necessary to provide sufficient oxygen to every region of the brain. Further, autoregulation is also evident in the response to pressure changes in the vasculature: the perfusing pressure can vary over a wide range from the basal-state with only a small effect on flow due to the constriction or dilation of vessels. Presented here is a new vasculature model where diameter and length are calculated in order to match the data available for flow velocity and blood pressure in different sized vessels. These vessels are arranged in a network of 6 generations each of bifurcating arterioles and venules, and a set of capillary beds. The input pressure and number of generations are the only specifications required to describe the network. The number of vessels, and therefore vessel geometry, is governed by how many generations are chosen and this can be altered in order to create more simple or complex networks. The flow, geometry and oxygen concentrations are calculated based on the vessel resistance due to flow from geometry based on Kirchoff circuit laws. The passive and active length-tension characteristics of the vasculature are established using an approximation of the network at upper and lower autoregulation limits. An activation model is described with an activation factor which governs the contributions of elastic andmuscle tension to the total vessel tension. This tension balances with the circumferential tension due to pressure and diameter and the change in activation sets the vessel diameter. The mass transport equation for oxygen is used to calculate the concentration of oxygen at every point in the network using data for oxygen saturation to establish a relationship between the permeability of the vessel wall to oxygen and the geometry and flow in individual vessels. A tissue compartment is introduced which enables the modelling of metabolic control. There is evidence for a coordinated response by surrounding vessels to local changes. A signal is proposed based on oxygen demand which can be conducted upstream. This signal decays exponentially with vessel length but also accumulates with the signal added from other vessels. The activation factor is therefore set by weighted signals proportional to changes in tissue concentration, circumferential tension, shear stress and conducted oxygen demand. The model is able to reproduce the autoregulation curve whereby a change in pressure has only a small effect on flow. The model is also able to replicate experimental results of diameter and tissue concentration following an increase in oxygen demand.

In normal tissues, blood supply is closely matched to tissue demand for wide ranges of oxygen demand and arterial pressure. This suggests that multiple mechanisms regulate blood flow. Theoretical models can be used to analyze these interacting mechanisms. One proposed mechanism for metabolic flow regulation involves the saturation-dependent release of ATP by red blood cells, which triggers an upstream conducted response signal and arteriolar vasodilation. To analyze this mechanism, oxygen and ATP levels are calculated along a flow pathway of seven representative segments, including two vasoactive arteriolar segments. The conducted response signal is dependent on ATP concentration. Arteriolar tone depends on the conducted response signal, local wall shear stress and wall tension. Arteriolar diameters are calculated based on vascular smooth muscle mechanics. The model can account for increases in perfusion consistent with experimental findings at low and moderate oxygen consumption rates despite the opposing effects of the myogenic and shear-dependent responses. Autoregulation, the maintenance of nearly constant blood flow as arterial pressure varies, is assessed in the presence or absence of the myogenic, shear-dependent and/or metabolic responses. The model results indicate that the combined effects of myogenic and metabolic regulation overcome the vasodilatory effect of the shear-dependent response to generate autoregulatory behavior. Capillary recruitment has been shown to increase the capacity for oxygen delivery during exercise. In the model, capillary density is assumed to depend on small arteriole diameter. The model predicts a significant increase in the range over which perfusion can be regulated when recruitment is included. Oscillations in diameter and tone are predicted under certain conditions, suggesting a novel mechanism for vasomotion. The conditions that give rise to oscillations are analyzed. It is shown that the appearance of oscillations depends in a complex way on a number of system parameters. In summary, the theoretical model provides a quantitative assessment of the myogenic, shear-dependent and metabolic responses that affect blood flow regulation and identifies a role for capillary recruitment and vasomotion in the control of blood flow.

ABSTRACT Phosphorylation is a post-translational modification important for regulating protein activity and protein binding capacity. It is used in many different signaling pathways within the cell. Src Family Kinases and Protein Kinase A (PKA) are two prototyptical non-receptor tyrosine and serine/ threonine kinases, respectively, which are found in canonical signaling pathways. These two kinases are critical for signaling in essentially every cell of a multicellular organism, and are particularly important in development, cell migration and proliferation. Although both proteins have been intensely studied for many decades, an understanding of the molecular mechanisms which govern their regulation and the regulation that they effect on other proteins are still being elucidated. Fyn, like its related Src Family Kinase members, has previously been shown to be regulated by two tyrosine phosphorylation events at residues Y420 and Y531. Y420 is located in the kinase (Src Homology 1(SH1)) domain and it is a highly-characterized intermolecular autophosphorylation site that increases the activity of the kinase. Y531 is located near the C-terminus and is phosphorylated by C-terminal Src kinase (Csk). Phosphorylation of Y531 allows it to bind to R176 in the SH2 domain in an intramolecular fashion. In this conformation Fyn has only basal activity. Since these sites are essential for regulating the activity of the kinase, we hypothesized that four novel sites of tyrosine phosphorylation in Fyn could also importantly regulate the protein. Three of the novel sites lie in the SH2 domain, and one is located in the kinase domain. Mass spectrometry, in vitro kinase assays, as well as western blot analysis aided in uncovering that these novel Fyn phosphorylation sites fine tune the activity and substrate binding of the protein. PKA has been implicated in a multitude of signaling pathways and is particularly important in cell growth, proliferation, and migration. Fyn and PKA have classically been considered to be in separate signaling pathways. However, research over the past several decades has provided evidence that there is crosstalk that exists between the two pathways. The SFK Fyn and PKA can phosphorylate each other, thereby regulating each other's activity. Based on these data, we hypothesized the existence of downstream effectors of this relatively uncharacterized pathway. It was hypothesized that the presence of Fyn could lead to PKA activation and to differences in PKA binding partners. Through the use of co-immunoprecipitations, Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) and quantitative mass spectrometry, many proteins were found to increase their binding to PKA in the presence of Fyn. Several proteins were selected and further biochemically validated. These data suggest that the presence of Fyn could allow for PKA to more importantly interact with discrete pools of proteins within the cell to effectuate its signal transduction. Together these studies provide understanding on critical and fundamental processes by which all cells function.