Renal autoregulation is unique and critically important in maintaining homeostasis in the body via control of renal blood flow and filtration. The myogenic reflex responds directly to pressure variation and is present throughout the vasculature in varying degrees, while the tubuloglomerular feedback (TGF) mechanism adjusts microvascular resistance and glomerular filtration rate (GFR) to maintain distal tubular NaCl delivery. No simple models are available which allow the independent contributions of the myogenic and TGF responses to be compared and which include control over multiple metabolic and physiological parameters. Independently developed mathematical models of myogenic autoregulation and TGF control of GFR have been combined to produce a comprehensive model for the rat kidney which is responsive to multiple small step changes in mean arterial pressure. The system encompasses every level of the renal vasculature and the tubular system of the nephrons while simultaneously incorporating the modulatory effects of changes in viscosity and shear stress-induced nitric oxide (NO) production. The vasculature of the rat kidney has previously been divided via a Strahler ordering scheme using morphological data derived from micro-CT imaging. This data, combined with an extensive literature review of the relevant experimental data, led to the development of order-specific parameter sets for each of the eleven vascular levels. The model of the myogenic response depends primarily on circumferential wall tension, corresponding to a distally dominant resistance distribution with the highest contributions localized to the afferent arterioles and interlobular arteries. The constrictive response is tempered by the vasodilatory influence of flow-induced NO. Experimental comparison with data from groups that inhibited the TGF mechanism showed that the model was able to accurately reproduce the characteristics of renal myogenic autoregulation. This myogenic model was coupled with a system of equations that represented both spatial and temporal changes in concentration of the filtrate in the tubular system of the nephrons and the corresponding resistance changes of the afferent arteriole via the TGF mechanism. Computer simulation results of the system response to pressure perturbations were examined, as well as the interaction between mechanisms and the modulatory influences of metabolic and hemodynamic factors on the steady state and transient characteristics of whole-organ renal autoregulation. The responses of the model were consistent with experimental observations and showed that the frequency of the myogenic reflex was approximately 0.4 Hz while that of TGF was 0.06 Hz, corresponding to a 2-3 sec response time for myogenic contraction and 16.7 sec for TGF. Within the autoregulatory range step increases in pressure induced damped oscillations in tubular flow, macula densa NaCl concentration, arteriolar diameter, and renal blood flow. The model demonstrated that these oscillations were triggered by TGF and confined to vessels less than 100 micrometer in diameter. The pressure response in larger vessels remained important in characterizing total autoregulatory efficacy. Examination of the steady-state and transient characteristics of the model results demonstrates the necessity of considering the whole organ response in studies of renal autoregulation. A comprehensive model of autoregulation also allows for the examination of pathological states, such as the altered NO production in hypertension or the excess tubular reabsorption of water seen in diabetes. The model was able to reproduce experimental results when simulating diseased states, enabling the analysis of impaired autoregulation as well as the identification of key factors affecting the autoregulatory response.

Indiana University-Purdue University Indianapolis (IUPUI) / Calcium/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a multimeric holoenzyme composed of 8–14 subunits from four closely related isoforms (α, β, γ, δ). CaMKII plays a strategic, multifunctional role in coupling the universal second messenger calcium with diverse cellular processes including metabolism, cell cycle control, and synaptic plasticity. CaMKII exhibits broad substrate specificity, targeting numerous substrates with diverse phosphorylation motifs. Binding of the calcium sensor CaM to the autoregulatory domain (ARD) of CaMKII functions to couple kinase activation with calcium signaling. Important sites of autophosphorylation, namely T287 and T306/7 (δ isoform numbering), reside within the ARD and control either CaM dependence or ability to bind to CaMKII respectively, thus determining various activation states of the kinase. Because autophosphorylation is critical to the function of CaMKII in vivo, we sought to determine the relationship between the activation state of the kinase and substrate selectivity. We show that the ARD of activated CaMKII tunes substrate selectivity by competing for substrate binding to the catalytic domain, thus functioning as a selectivity filter. Specifically, in the absence of T287 autophosphorylation, substrate phosphorylation is limited to high-affinity, consensus substrates. T287 autophosphorylation restores maximal kinase activation and broad substrate selectivity by disengaging ARD filtering. The unique multimeric architecture of CaMKII is an ideal sensor which encodes calcium-spike frequency into graded levels of subunit activation/autophosphorylation within the holoenzyme. We find that differential activation states of the holoenzyme produce distinct substrate phosphorylation profiles. Maximal holoenzyme activation/autophosphorylation leads to further broadening of substrate specificity beyond the effect of autophosphorylation alone, which is consistent with multivalent avidity. Thus, the ability of calcium-spike frequency to regulate T287 autophosphorylation and holoenzyme activation permits cellular activity to dictate switch-like behavior in substrate selectivity that is required for diverse cellular responses by CaMKII.

Clostridium difficile is a major nosocomial pathogen responsible for causing pseudomembranous colitis. It is estimated that 25% of antibiotic-associated diarrhea is due to C. difficile. These diseases result from intestinal tissue damage caused by two of the largest known bacterial toxins, A and B. Molecular studies of the C. difficile toxins have identified a 19.6 kb toxigenic element that contains both toxin genes flanked by three small open reading frames (ORFs). The focus of this study is to elucidate the function of the ORF, designated txeR, which is located at the beginning of the toxigenic element. The deduced amino acid sequence of txeR predicts a 22-kDa protein that contains a helix-turn-helix motif characteristic of DNA binding regulatory proteins. To determine if the protein TxeR regulates expression from the toxA, toxB, and txeR promoters, gene fusions were constructed that contained the various promoter regions and a reporter gene. The immunodominant region of toxin A located at the carboxy-terminus, termed the repeating units (ARU), was selected as the reporter gene. Expression studies were performed in Escherichia coli host strains. Levels of ARU expression were measured by enzyme-linked immunosorbent assay using an ARU-specific monoclonal antibody. Expression levels of ARU from the toxin B promoter region with TxeR supplied on the same plasmid (in cis) or on a different plasmid (in trans) were determined. In cis, ARU levels were 50-fold higher than strains without txeR. In trans, expression of ARU from the toxin B promoter region increased over 800-fold. When TxeR was supplied in trans to a toxin A promoter region-ARU fusion, expression levels of ARU increased over 500-fold. To test for autoregulation, TxeR was supplied in trans to the txeR promoter region fused to ARU. The effect was an increase of ARU expression up to 20-fold over background. These results suggest that TxeR is a trans-acting regulator that stimulates expression of the C. difficile toxins and is subjected to autoregulation. / Master of Science

Plant embryogenesis is an intriguing developmental process that is controlled by many genes. AGAMOUS Like 15 (AGL15) is a MADS-domain transcriptional regulator that accumulates preferentially during this stage. However, at the onset of this work it was unknown which genes are regulated by AGL15 or how AGL15 is regulated. This dissertation is part of the ongoing effort to understand the biological roles of AGL15. To decipher how AGL15 functions during plant development, a chromatin immunoprecipitation (ChIP) approach was adapted to obtain DNA fragments that are directly bound by AGL15 in vivo. Putative AGL15 targets were isolated, and binding and regulation was confirmed for one such target gene, ABF3. In addition, microarray experiments were performed to globally assess genes that are differentially expressed between wild type and agl15 young seeds. Among them, a gene, At5g23405, encoding an HMGB domain protein was identified and its response to AGL15 was confirmed. Preliminary results suggest that the loss-of-function of At5g23405 might have an effect on somatic embryogenesis, consistent with AGL15 repression of the expression of this gene. Lastly, to address the question about how the regulator is regulated, the cis elements controlling the expression of AGL15 must be identified. Deletion analysis of the AGL15 promoter indicated the presence of putative positive and negative cis elements contributing to the expression of AGL15. Further analysis suggested that AGL15 regulates the expression of its own gene and this regulation may partially be explained by the direct binding of the protein to the AGL15 promoter. The data presented in this dissertation demonstrate that ChIP can be used to identify previously unsuspected targets of AGL15. Based on ChIP, a ChIP-chip technique is being developed in the lab to allow a more global analysis of in vivo binding sites. The identification of target genes and cis elements in AGL15 promoter is a step towards characterization of the biological roles of AGL15.

An important protective feature of the cerebral circulation is its ability to maintain sufficient cerebral blood flow and oxygen supply in accordance with the energy demands of the brain despite variations in a number of external factors such as arterial blood pressure, heart rate and respiration rate. If cerebral autoregulation is impaired, abnormally low or high CBF can lead to cerebral ischemia, intracranial hypertension or even capillary damage, thus contributing to the onset of cerebrovascular events. The control and regulation of cerebral blood flow is a dynamic, multivariate phenomenon. Sensitive techniques are required to monitor and process experimental data concerning cerebral blood flow and metabolic rate in a clinical setting. This thesis presents a model simulation study and 4 related signal processing studies concerned with CBF regulation. The first study models the regulation of the cerebral vasculature to systemic changes in blood pressure, dissolved blood gas concentration and neural activation in a integrated haemodynamic system. The model simulations show that the three pathways which are generally thought to be independent (pressure, CO₂ and activation) greatly influence each other, it is vital to consider parallel changes of unmeasured variability when performing a single pathway study. The second study shows how simultaneously measured blood gas concentration fluctuations can improve the accuracy of an existing frequency domain technique for recovering cerebral autoregulation dynamics from spontaneous fluctuations in blood pressure and cerebral blood flow velocity. The third study shows how the continuous wavelet transform can recover both time and frequency information about dynamic autoregulation, including the contribution of blood gas concentration. The fourth study shows how the discrete wavelet transform can be used to investigate frequency-dependent coupling between cerebral and systemic cardiovascular dynamics. The final study then uses these techniques to investigate the systemic effects on resting BOLD variability. The general approach taken in this thesis is a combined analysis of both modelling and data analysis. Physiologically-based models encapsulate hypotheses about features of CBF regulation, particularly those features that may be difficult to recover using existing analysis methods, and thus provide the motivation for developing both new analysis methods and criteria to evaluate these methods. On the other hand, the statistical features extracted directly from experimental data can be used to validate and improve the model.

The ability of the brain to keep a stable and adequate cerebral blood flow (CBF) independently of fluctuations in systemic blood pressure is referred to as cerebral pressure autoregulation (CPA). When the brain is injured by trauma or hemorrhage, this ability may be impaired, leaving the brain vulnerable to events of high or low blood pressure. The aims of this thesis were to study CPA in patients with severe traumatic brain injury (TBI) or subarachnoid hemorrhage (SAH), the relation between CPA and other physiological parameters, and the influence of CPA on outcome. Four retrospective studies are included in the thesis. All patients were treated at the neurointensive care unit, Uppsala University hospital. In paper I, 58 TBI patients were studied. In patients with impaired CPA, cerebral perfusion pressure between 50-60 mm Hg was associated with favorable outcome while CPP > 70 and >80 mm Hg was associated with unfavorable outcome. In patients with intact CPA there was no association between CPP and outcome. In paper II, 107 TBI patients were studied. High CPP was associated with unfavorable outcome in patients with focal injuries. In patients with diffuse injury and impaired CPA, CPP > 70 mm Hg was associated with favorable outcome. In paper III, 47 SAH patients were studied. CBF was measured bedside with Xenon-enhance CT (Xe-CT). Patients with impaired CPA had lower CBF, both in the early (day 0-3) and late (day 4-14) acute phase of the disease. In paper IV, 64 SAH patients were studied. Optimal CPP (CPPopt) was calculated automatically as the level of CPP where CPA works best for the patient, i.e., where PRx is lowest. Patients with actual CPP below their calculated optimum had higher amounts of low-flow regions (CBF < 10 ml/100g/min). The findings in this thesis emphasize the importance of taking CPA into account in the management of TBI and SAH patients, and suggest that treatment should be individualized depending on status of autoregulation. PRx and CPPopt may be used bedside to guide management according to status of autoregulation. In the future CPA-guided management should be tested in prospective studies

"Renal autoregulation stabilizes renal blood flow to protect the glomerular capillaries and maintain glomerular filtration rates through two mechanisms: tubuloglomerular feedback (TGF) and the myogenic response (MR). It is considered that the feedback mechanisms operate independently in each nephron (the functional unit of the kidney) within a kidney, but renal autoregulation dynamics can be coupled between vascular connected nephrons. It has also been shown that the mechanisms are time-varying and interact with each other. Understanding of the significance of such complex behavior has been limited by absence of techniques capable of monitoring renal flow signals among more than 2 or 3 nephrons simultaneously. The purpose of this thesis was to develop approaches to allow the identification and characterization of spatial and temporal properties of renal autoregulation dynamics. We present evidence that laser speckle perfusion imaging (LSPI) effectively captures renal autoregulation dynamics in perfusion signals across the renal cortex of anaesthetized rats and that spatial heterogeneity of the dynamics is present and can be investigated using LSPI. Next, we present a novel approach to segment LSPI of the renal surface into phase synchronized clusters representing areas with coupled renal autoregulation dynamics. Results are shown for the MR and demonstrate that when a signal is present phase synchronized regions can be identified. We then describe an approach to identify quadratic phase coupling between the TGF and MR mechanisms in time and space. Using this approach we can identify locations across the renal surface where both mechanisms are operating cooperatively. Finally, we show how synchronization between nephrons can be investigated in relation to renal autoregulation effectiveness by comparing phase synchronization estimates from LSPI with renal autoregulation system properties estimated from renal blood flow and blood pressure measurements. Overall, we have developed approaches to 1) capture renal autoregulation dynamics across the renal surface, 2) identify regions with phase synchronized renal autoregulation dynamics, 3) quantify the presence of the TGF-MR interaction across the renal surface, and 4) determine how the above vary over time. The described tools allow for investigations of the significance and mechanisms behind the complex spatial interactions and time-varying properties of renal autoregulation dynamics. "

Objective: Ischemic spinal cord injury is a serious complication of aortic surgery. The mechanism underlying ischemic preconditioning (IPC) protection against spinal cord ischemia/reperfusion (I/R) injury is unclear. We investigated the role of spinal cord autoregulation in tolerance to spinal cord I/R injury induced by IPC. Although the extracellular signal-regulated kinases 1 and 2 (ERK1/2) are generally regarded as related to cell survival and proliferation, increasing evidence suggests that the role of the ERK1/2 pathway in I/R injury is contributory to inflammation. We investigated the effect of blocking ERK1/2 pathway to inhibit inflammation reaction in tolerance to spinal cord I/R injury. Methods: In the part 1 study, Sprague-Dawley rats were randomly assigned to 4 groups. IPC (P) group animals received IPC by temporary thoracic aortic occlusion (AO) with a 2-F Fogarty arterial embolectomy catheter for 3 min. I/R injury (I/R) group animals were treated with blood withdrawal and temporary AO for 12 min, and shed blood reinfusion at the end of the procedures. (P+I/R) group animals received IPC, followed by 5 min reperfusion, and then I/R procedures for 12 min. Sham (S) group animals received anesthesia and underwent surgical preparation only. Neurological functions were evaluated, and lumbar segments were harvested for histopathological examination. To evaluate the role of autoregulation in IPC, spinal cord blood flow and tissue oxygenation were continuously monitored throughout the procedure duration. In the part 2 study, spinal cord ischemia rats was induced by occluding the thoracic descending aorta with a balloon catheter introduced through a femoral artery, accompanied by concomitant exsanguinations. Rats in the control group were given dimethyl sulfoxide (vehicle) before undergoing spinal cord ischemia/reperfusion injury. In the U0126-treated group, rats were pretreated with an inhibitor of ERK1/2, U0126, to inhibit ERK1/2 phosphorylation. The sham rats underwent aortic catheterization without occlusion. Parameters, including neurologic status, neuronal survival, inflammatory cell infiltration, and interleukin-1£] production in the spinal cords, were compared between groups. Results: The Tarlov scores in the (I/R) group were significantly lower than those in the (S), (P), and (P+I/R) groups on days 1, 3, 5, and 7. The numbers of surviving motor neurons in the (S), (P), and (P+I/R) groups were significantly higher than those in the (I/R) group. The (P) group exhibited higher spinal cord blood flow and tissue oxygenation after reperfusion than the (S) group. The (P+I/R) group exhibited higher spinal cord blood flow and tissue oxygenation within the first 60 min after reperfusion than the (I/R) groups. In the part 2 study, early ERK1/2 phosphorylation was observed after injury in the control group, followed by abundant microglial accumulation in the infarct area and increased interleukin-1£] expression. In the U0126 group, U0126 treatment completely blocked ERK1/2 phosphorylation. Microglial activation and spinal cord interleukin-1£] levels were significantly reduced. Neuronal survival and functional performance were improved. Conclusions: IPC ameliorates spinal cord I/R injury in rats, probably mediated by triggering spinal cord autoregulation and improving local spinal cord blood flow and tissue oxygenation. The ERK1/2 pathway may play a noxious role in spinal cord ischemia/reperfusion injury by participating in inflammatory reactions and cytokine production. According to our findings, these concepts may be the new therapeutic targets in patients requiring aortic surgery.

A hypothetical causal link between ventilatory regulation of carbon dioxide anddevelopment of syncope during orthostatic challenges is reduction in arterial partialpressure of carbon dioxide and resultant reduction in cerebral blood flow. We performedtwo experiments to investigate the ventilatory sensitivity to carbon dioxide and factorsaffecting cerebral autoregulation (CA). We also studied the nonlinear phase couplingbetween cardio-respiratory parameters before syncope.For experiment one, in 30 healthy adults, we stimulated chemo and baro reflexesby breathing either room-air or room-air with 5 percent carbon dioxide in a pseudorandom binary sequence during supine and 70 degree head up tilt (HUT). Six subjectsdeveloped presyncope during tilt.To determine whether changes in ventilatory control contribute to the observeddecrease in PaCO2 during HUT, we assessed ventilatory dynamic sensitivity to changesin PaCO2 during supine and 70 degrees HUT. The sensitivity of the ventilatory controlsystem to perturbations in end tidal carbon dioxide increased during tilt.To investigate nonlinear phase coupling between cardio-respiratory parametersbefore syncope, bispectra were estimated and compared between presyncopal andnon-presyncopal subjects. Our results indicate that preceding presyncope, nonlinearphase coupling is altered by perturbations to baro and chemo reflexes.To investigate the effects of gender in CA, we selected 10 men and 10age-matched women and used spectral analysis to compare differences in CA betweenmen and women. Our results showed that gender-related differences in CA did exist andgender may need to be considered as a factor in investigating CA.To investigate the influence of induced hypocapnia on CA in absence ofventilatory variability, we performed experiment two in which subjects were randomlyassigned to a Control (under normocapnia) or Treatment (under hypocapnia) group. Bothgroups voluntarily controlled their breathing pattern yet two groups breathed in air withdifferent levels of carbon dioxide. Our results show that changes in mean blood pressureat middle cerebral artery level were less transferred into mean cerebral blood flow in theTreatment group than in the Control group, suggesting better CA under hypocapniarelative to under normocapnia.

The Circle of Willis (CoW) is a ring-like arterial structure forming the major anastomotic connection between arterial supply systems in the brain, and is responsible for the distribution of oxygenated blood throughout the cerebral mass. Among the general population, only approximately 50% have a complete CoW, where absent or hypoplastic vessels are common among a multitude of possible anatomical variations, reducing the degree to which blood may be rerouted. While an individual with one of these variations may under normal circumstances suffer no ill effects, there are certain pathological conditions which can present a risk to the person's health and increase the possibility of suffering an ischaemic stroke when compounded with an anatomical variation. This body of work presents techniques for generating 3D models of the cerebral vasculature using magnetic resonance imaging (MRI) and performing computational fluid dynamics (CFD) simulations in order to simulate the flow patterns throughout a circle of Willis. Incorporated with the simulations is a mathematical model of the cerebral autoregulation mechanism, simulating the ability of the smaller arteries and arterioles in the brain to either constrict or dilate in response to alterations in cerebral blood flow, thereby altering the cerebrovascular resistance of each major brain territory and regulating the amount of blood flow within a physiological range of cerebral perfusion pressure. The CFD simulations have the ability to predict the amount of collateral flow rerouted via the communicating arteries in response to a stenosis or occlusion, and the major objective of this study has been the investigation of how anatomical variations of the circle of Willis affect the capacity to provide this collateral flow. Initial work began with the development of three idealized models of common anatomical variations, created using computer aided design software (CAD) and based on the results of MRI scans. The research then shifted to developing a technique whereby patient specific models of the circle of Willis could be directly segmented from the MRI data. As a result of this shift, an interactive GUI-based tool was developed for the processing of the MRI datasets, allowing for rapid data enhancement and creation of a surface topology representing the arterial wall of the circle of Willis, suitable for a CFD simulation. The results of both sets of simulations illustrate that there exist a number of variables associated with a patients circle of Willis geometry, such as cerebral blood flow and combinations and degrees of stenosis, implying that the initial goal of drawing generalized conclusions was perhaps flawed. Instead, a crucial outcome of this body of work is that the future research should be directed toward extending the physiological complexity of both the geometry and the autoregulation model, with the intention of a patient specific application rather than producing large datasets with which to make broad generalizations.