The brain is protected by the blood brain barrier (BBB); areas lacking the BBB are termed circumventricular organs (CVOs). The SFO, a CVO is capable of detecting and responding to satiety signals that regulate energy balance. Insulin, a satiety signal, plays a role in energy balance and its actions at the SFO are unknown. The goal was to determine if cultured SFO neurons are electrophysiologically sensitive to insulin. Of 27 neurons tested 33% neurons hyperpolarized (-8.7 ± 1.7 mV), 37% neurons depolarized (10.5 ±2.8 mV) and 30% neurons (8 out of 27) showed no change in membrane potential. Input resistance changes indicated the modulation of two ion channels. Pharmacological data suggests hyperpolarization arises from the opening of KATP channels and depolarization results from the opening of non-selective cationic channels. Thus insulin modulates the electrical activity of SFO neurons and supports that the SFO is a sensor for maintaining energy homeostasis.

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The brain is protected by the blood brain barrier (BBB); areas lacking the BBB are termed circumventricular organs (CVOs). The SFO, a CVO is capable of detecting and responding to satiety signals that regulate energy balance. Insulin, a satiety signal, plays a role in energy balance and its actions at the SFO are unknown. The goal was to determine if cultured SFO neurons are electrophysiologically sensitive to insulin. Of 27 neurons tested 33% neurons hyperpolarized (-8.7 ± 1.7 mV), 37% neurons depolarized (10.5 ±2.8 mV) and 30% neurons (8 out of 27) showed no change in membrane potential. Input resistance changes indicated the modulation of two ion channels. Pharmacological data suggests hyperpolarization arises from the opening of KATP channels and depolarization results from the opening of non-selective cationic channels. Thus insulin modulates the electrical activity of SFO neurons and supports that the SFO is a sensor for maintaining energy homeostasis.

Iron is essential in nearly all organisms. It is a cofactor in many proteins and enzymes. This transition metal can also be toxic because it participates in reactions which produce reactive oxygen species. To avoid these toxic effects while still being used for essential processes, the cell must regulate tightly iron import, metabolism, trafficking, and homeostasis. These processes were studied using biophysical methods centered on Mossbauer spectroscopy supplemented by electron paramagnetic resonance, electronic absorption spectroscopy, and inductively coupled plasma mass spectrometry. This integrated biophysical approach was applied to yeast cells, isolated yeast mitochondria, and mouse brains. We determined the concentration of Fe, and the proportion of that Fe present as iron-sulfur clusters, heme centers, mononuclear nonheme centers, and as Fe3+ oxyhydroxide (phosphate) nanoparticles for each system. In yeast, the dependence of metabolic mode of growth and iron in the growth medium on this distribution was studied. Approximately three-quarters of the iron in fermenting cells was located in vacuoles, where it was present as high-spin mononuclear Fe3+ species with rhombic symmetry. The remaining quarter was present in the mitochondria. In fermenting mitochondria 4 distinct species of iron were observed, including [Fe4S4]2+ clusters and low-spin Fe2+ hemes arising from respiratory complexes, non-heme high spin (NHHS) Fe2+ species, high spin nonheme Fe3+ species, and nanoparticles. These distributions (in both the cells and mitochondria) change when the cells are grown on iron deficient medium but remained relatively unaltered as iron in the growth medium was increased. Respiring cells had less Fe associated with vacuoles, and more Fe present as HS Fe2+. Respiring mitochondria contain more [Fe4S4]2+ clusters and low-spin Fe2+ hemes, more S = 1/2 [Fe2S2]1+ clusters, and less NHHS Fe2+, HS Fe3+ species and Fe3+ nanoparticles. These changes were rationalized by assuming that the NHHS Fe2+ and Fe3+ species, and the nanoparticles were in equilibrium within the matrix of the mitochondria, and that the Fe2+ species served as feedstock for the synthesis of iron-sulfur clusters and heme centers. The iron in the mouse brain consisted mostly of [Fe4S4]2+ clusters and Fe2+ hemes from mitochondria respiratory complexes, and of ferritin, an Fe storage protein complex. NHHS Fe2+ and Fe3+ species were also observed. The ratio of stored Fe to mitochondrial Fe was sensitive to age. The brains of prenatal animals were dominated by ferritin. Following birth up to the first 4 weeks of life, there was an increase in mitochondrial Fe and a decline of ferritin Fe. Beyond 4 weeks up to 58 weeks, levels of ferritin increased and mitochondrial Fe remained constant. The brains of mice fed an Fe-deficient diet were also studied; most of the Fe in these brains was present as mitochondrial Fe, with little stored as ferritin. A model was developed to explain these changes.

Passive and active heat loading causes cardiovascular strain, which can have diverse and substantial effects. Thus, cardiovascular function is integral to work and heat stress tolerance, but recent hyperthermia and exercise literature has not emphasised this relationship, instead focusing on the roles of upper �critical� core temperature or rate of heat storage as primary mechanisms of fatigue. Therefore, the aim of this thesis was to examine some potential causes and effects of cardiovascular strain under heat stress, including potential strategies for attenuating that strain. Body precooling before exercise increases heat storage capacity; the primary mechanism by which attenuations in thermal and cardiovascular strain, and improved work capacity is thought to occur. However, no precooling study has utilised realistic airflow in the laboratory, possibly inflating its purported benefits. Therefore, Study One examined the cardiovascular, thermal, psychophysical and ergogenic effects of precooling with and without airflow in the heat (30�C, 50% rh). Ten males completed four trials in balanced order, comprising 60- min immersion in thermoneutral (35�C) or cool (24�C) water before cycling at 95% ventilatory threshold with airflow (~4.8 m�s⁻�) or no airflow, until exhaustion. Heart rate and mean core, body, and skin temperatures were attenuated for 15 min into cycling after precooling. Endurance time was extended by 30 � 23 min with airflow, and 16 � 15 min with precooling relative to control (28 � 12 min) but not further extended with strategies combined (29 � 21 min). Precooling removed 784 � 223 kJ�m⁻� (calorimetrically); less than the effect of airflow alone (1323 � 1128 kJ�m⁻�). Competition for blood between tissues is pronounced during exercise in the heat: skin and gut have marked increases and decreases, respectively. Gut ischemia affects epithelial tight junction integrity, allowing lippopolysaccharide ingress and immune responses. Bovine colostrum may attenuate gut permeability. Study Two (double-blind, placebo controlled) investigated the effects of aerobic fitness (7 highly fit, 8 moderately fit) and bovine colostrum on physiological and perceived strain, and performance during mixed-mode exercise; cycle 15 min at 50% maximal heart rate range (HRR), run 30 min at 80% HRR then 30 min self-selected paced before another 15 min cycle at the same work-rate. Airflow was graded to running speed. During the last cycle, blood pressure, stroke volume and total peripheral resistance were lower, heart rate and skin blood flow increased, and skin temperature was unchanged compared to the first cycle. Indices of fever response (IL-1β, TNF-α) were not evident during exercise, nor were those of blood-brain barrier permeability (S100β) or cognitive impairment (Stroop test). Neither bovine colostrum, nor higher fitness modified these measures. Moving to upright posture is orthostatically stressful and can initially decrease cerebral perfusion. Compression garments are used to assist venous return; while their effectiveness is unknown, they could reduce heat or orthostatic-induced hypoperfusion. Study Three investigated the cardiovascular and cerebrovascular responses to orthostatic stress with and without passive heating (+0.5�C). Fifteen participants completed two trials (compression v placebo garments) in balanced order. Cerebral autoregulation was assessed via 3-min stand, and via thigh cuff inflation. All participants experienced initial orthostatic hypotension upon standing in one or more trials, with 4/15 individuals experiencing presyncopal symptoms, aborting the standing protocol. In those who "fainted", reductions in blood pressure and partial pressure of end-tidal CO₂ reduced middle cerebral artery velocity. Neither training status nor compression trousers modified the responses. Collectively, cardiovascular strain to heat stress is attenuated when realistic airflow is provided. Increased cardiovascular strain does not inevitably result in clinical outcomes to heat stress. Higher fitness does not necessarily attenuate cardiovascular responses or higher tolerance to heat stress.

Colon cancer is the third most common type of cancer, with high mortality throughout the world. During tumorigenesis, normal cells transform into tumour cells following changes in the expression of oncogenes and/or tumour suppressor genes, which are involved in many processes including the cell cycle, differentiation and apoptosis. An imbalance in the regulation of proliferation and differentiation in colon epithelial cells is usually associated with the development of colon cancers. Uncontrolled proliferation with a lack of differentiation is one of the major characteristic features of cancer cells and a remodelling of the Ca2+ signalling is linked to these pathways. Among the Ca2+ transporting proteins, P-type Ca2+-ATPases, the plasma membrane Ca2+ ATPase (PMCA) pump, has a high-affinity for Ca2+ and is involved in the efflux of Ca2+ against the electrochemical gradient from the cytosol across the extracellular space. Four PMCA isoforms have been identified. PMCA1 and 4 are expressed in most tissues. Changes in the expression of PMCA have been documented in breast cancer cells, whereas the expression profile of PMCA isoforms in colon cancer cells remains unknown. Up-regulation of another P-type Ca2+-ATPase, expressed in the endoplasmic reticulum, SERCA3, occurs during the differentiation of colon cancer cell lines and is down-regulated in colon cancers. Changes in PMCA expression have not been assessed during colon cancer cell differentiation. The first part of this thesis describes the analysis of the expression profile of PMCA during colon cancer cell differentiation. Both PMCA mRNA and protein levels were assessed in differentiated HT-29 cells by real time RT-PCR and western blotting analysis, respectively. The results showed changes in PMCA4 expression, whereas changes in the expression of PMCA1 were not associated with differentiation of HT-29 cells. PMCA mRNA levels were also reduced in some colon cancers suggesting a remodelling of PMCA-mediated Ca2+ efflux during colon carcinogenesis. The second part of this thesis involved exploring the functional role of PMCA4 in Ca2+-mediated signalling pathways such as differentiation, proliferation and apoptosis. PMCA4 expression was altered in HT-29 colon cancer cells via transient and stable over-expression of a PMCA4 expressing plasmid or siRNA-mediated silencing of PMCA4. An increase in the PMCA4 level did not alter or induce differentiation of HT-29 cells. Hence, up-regulation of PMCA4 expression may be a consequence rather than a cause of HT-29 colon cancer cell differentiation. PMCA4-mediated reduction in proliferation was observed in HT-29 colon cancer cells where PMCA4 was stably over-expressed. Stable PMCA4 over-expression was also associated with the down-regulation of the transcription of the early response gene, FOS. Despite the apparent augmentation of cytosolic Ca2+ responses to G-protein coupled receptor Ca2+ mobilizing agents, the sensitivity of cells to the apoptotic inducing agents such as TRAIL and/or CCCP was not affected following siRNA-mediated PMCA4 inhibition in HT-29 cells. Collectively this thesis describes PMCA isoform-specific changes during differentiation of HT-29 colon cancer cells and alterations in PMCA levels in some colon cancers.Evidence is also presented to suggest that alterations in PMCA expression in colon cancer cells may provide a growth advantage by promoting proliferation without increasing sensitivity to apoptotic stimuli.

Thesis (Ph. D.)--Purdue University, 2006. / Includes bibliographical references (leaves 89-113). Also available online (PDF file) by a subscription to the set or by purchasing the individual file.

Thesis (Ph. D.)--Purdue University, 2006. / Includes bibliographical references (leaves 89-113).

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.