Browsing by Subject "Biology, Physiology"
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Item Open Access A Discrete Monolayer Cardiac Tissue Model for Tissue Preparation Specific Modeling(2010) Kim, JongmyeongEngineered monolayers created by using microabrasion and micropatterning methods have provided a simplified in vitro system to study the effects of anisotropy and fiber direction on electrical propagation. Interpreting the behavior in these culture systems has often been performed using classical computer models with continuous properties. Such models, however, do not account for the effects of random cell shapes, cell orientations and cleft spaces inherent in these monolayers on the resulting wavefront conduction. Additionally when the continuous computer model is built to study impulse propagations, the intracellular conductivities of the model are commonly assigned to match impulse conduction velocity of the model to the experimental measurement. However this method can result in inaccurate intracellular conductivities considering the relationship among the conduction velocity, intracellular conductivities and ion channel properties. In this study, we present novel methods for modeling a monolayer cardiac tissue and for estimating intracellular conductivities from an optical mapping. First, in the proposed method for modeling a monolayer of cardiac tissue, the factors governing cell shape, cell-to-cell coupling and the degree of cleft space are not constant but rather are treated as spatially random with assigned distributions. This approach makes it possible to simulate wavefront propagation in a manner analogous to performing experiments on engineered monolayer tissues. Simulated results are compared to reported experimental data measured from monolayers used to investigate the role of cellular architecture on conduction velocities and anisotropy ratios. We also present an estimate for obtaining the electrical properties from these networks and demonstrate how variations in the discrete cellular architecture affect the macroscopic conductivities. The simulation results agree with the common assumption that under normal ranges of coupling strengths, tissues whose cell shapes and connectivity show relatively uniform distributions can be represented using continuous models with conductivities derived from random discrete cellular architecture using either estimates. The results also reveal that in the presence of abrupt changes in cell orientation, local estimates of tissue properties predict smoother changes in conductivities that may not adequately predict the discrete nature of propagation at the transition sites. Second, a novel approach is proposed to estimate intracellular conductivities from the optical mapping of the monolayer cardiac tissue under subthreshold stimulus. This method uses a simplified membrane model, which represents the membrane as a second order polynomial of the membrane potential. The simplified membrane model and the intracellular conductivities are estimated from the optical mapping of the monolayer tissue under the subthreshold stimulus. We showed that the proposed method provides more accurate intracellular conductivities compared to a method using a constant membrane resistance.
Item Open Access Bifurcations in the Echebarria-Karma Modulation Equation for Cardiac Alternans in One Dimension(2009) Dai, ShuWhile alternans in a single cardiac cell appears through a simple
period-doubling bifurcation, in extended tissue the exact nature
of the bifurcation is unclear. In particular, the phase of
alternans can exhibit wave-like spatial dependence, either
stationary or traveling, which is known as discordant
alternans. We study these phenomena in simple cardiac models
through a modulation equation proposed by Echebarria-Karma. In
this dissertation, we perform bifurcation analysis for their
modulation equation.
Suppose we have a cardiac fiber of length l, which is
stimulated periodically at its x=0 end. When the pacing period
(basic cycle length) B is below some critical value Bc,
alternans emerges along the cable. Let a(x,n) be the amplitude
of the alternans along the fiber corresponding to the n-th
stimulus. Echebarria and Karma suppose that a(x,n) varies
slowly in time and it can be regarded as a time-continuous
function a(x,t). They derive a weakly nonlinear modulation
equation for the evolution of a(x,t) under some approximation,
which after nondimensionization is as follows:
&partialt a = σ a + L a - g a 3,
where the linear operator
L a = &partialxxa - &partialx a -Λ-1 ∫ 0 x a(x',t)dx'.
In the equation, σ is dimensionless and proportional to
Bc - B, i.e. σ indicates how rapid the pacing is,
Λ-1 is related to the conduction velocity (CV) of the
propagation and the nonlinear term -ga3 limits growth after the
onset of linear instability. No flux boundary conditions are
imposed on both ends.
The zero solution of their equation may lose stability, as the
pacing rate is increased. To study the bifurcation, we calculate
the spectrum of operator L. We find that the
bifurcation may be Hopf or steady-state. Which bifurcation occurs
first depends on parameters in the equation, and for one critical
case both modes bifurcate together at a degenerate (codimension 2)
bifurcation.
For parameters close to the degenerate case, we investigate the
competition between modes, both numerically and analytically. We
find that at sufficiently rapid pacing (but assuming a 1:1
response is maintained), steady patterns always emerge as the only
stable solution. However, in the parameter range where Hopf
bifurcation occurs first, the evolution from periodic solution
(just after the bifurcation) to the eventual standing wave
solution occurs through an interesting series of secondary
bifurcations.
We also find that for some extreme range of parameters, the
modulation equation also includes chaotic solutions. Chaotic waves
in recent years has been regarded to be closely related with
dreadful cardiac arrhythmia. Proceeding work illustrated some
chaotic phenomena in two- or three-dimensional space, for instance
spiral and scroll waves. We show the existence of chaotic waves in
one dimension by the Echebarria-Karma modulation equation for
cardiac alternans. This new discovery may provide a different
mechanism accounting for the instabilities in cardiac dynamics.
Item Open Access Characterization and Modeling of Fluctuating Hypoxia in Breast Cancer(2008-08-08) Cardenas-Navia, Laura IsabelTumor hypoxia is an enduring problem for traditional cancer therapies such as radiation and chemotherapy. This obstacle has traditionally been attributed to the widespread presence of chronic, diffusion-limited hypoxia in solid tumors; recent data suggests that tumor hypoxia may also be spatially and temporally variable. In this work we characterize the presence of spatial and temporal fluctuations in hypoxia, as well as use mathematical modeling to predict the impact of fluctuations on the hypoxic cytotoxin, tirapazamine, and examine potential mechanisms of fluctuations in tumor oxygenation. Using phosphorescence lifetime imaging on preclinical tumors, we show that instabilities in tumor oxygenation are a prevalent characteristic of three tumor lines and that previous characterization of tumor hypoxia as being primarily diffusion-limited does not accurately portray the tumor microenvironment. Then, using a one-dimensional theoretical model, we examine the effects of fluctuating hypoxia on metabolized tirapazamine concentration; we find that fluctuating oxygen reduces the concentration of metabolized tirapazamine at distances farther from the source, thereby limiting its ability to reach and kill the most hypoxic cells. Finally, we use a three-dimensional Green's function oxygen transport model to explore the effects of temporal fluctuations in hemoglobin saturation, blood flow, and overall perfusion on tumor tissue oxygenation. Results from the model predict that hemoglobin saturation has a dominant effect on tissue oxygenation. These studies collectively suggest that the pervasive temporal and spatial heterogeneity in tumor oxygenation are highly therapeutically relevant, and future clinical and preclinical studies are needed.
Item Open Access Defining Ankyrin-b Syndrome: Characterization of Ankyrin-b Variants in Mice and Men and the Discovery of a Role for Ankyrin-b in Parasympathetic Control of Insulin Release(2009) Healy, Jane AnneStudies in the ankyrin-B+/- mouse reveal that ankyrin-B deficiency is associated with both the benefits of enhanced cardiac contractility and the costs of arrhythmia, early senescence, reduced lifespan, and impaired glucose tolerance. This constellation of traits is known as ankyrin-B syndrome, which may have important implications for humans possessing functional ankyrin-B mutations. We found that ankyrin-B variants are surprisingly common, ranging from 2 percent of European individuals to 8 percent in individuals from West Africa. Furthermore, by studying of the metabolic phenotype associated with ankyrin-B mouse, we have uncovered a major new dimension to ankyrin-B syndrome, a link between ankyrin-B and parasympathetic control of insulin secretion. Stimulation of pancreatic beta cells by acetylcholine augments glucose-stimulated insulin secretion by inducing inositol-trisphosphate receptor (InsP3R)-mediated Ca2+ release. We report that ankyrin-B is also enriched in pancreatic beta cells. Ankyrin-B-deficient islets display impaired potentiation of insulin secretion by the muscarinic agonist carbachol, blunted carbachol-mediated intracellular Ca2+- release, and reduced InsP3R stability. Ankyrin-B(+/-) mice also display postprandial hyperglycemia, consistent with impaired parasympathetic potentiation of glucose-stimulated insulin secretion. R1788W mutation of ankyrin-B impairs its function in pancreatic islets and associates with type 2 diabetes in Caucasians and Hispanics. Finally, we have generated knockin mice corresponding to the R1788W and L1622I mutations. Functional characterization of these animals will allow us to better understand the relationship between human ankyrin-B variants and ankyrin-B syndrome.
Item Open Access Ecotoxicology of Natural and Anthropogenic Extreme Environments(2010) Osterberg, Joshua SamuelReactive oxygen species (ROS) are produced endogenously in all aerobes and are induced by environmental stressors. ROS oxidize and disable essential cellular components such as DNA, proteins, and lipid membranes. Exposure to metals, polycyclic aromatic hydrocarbons (PAHs), and some pesticides can induce oxidative stress in marine invertebrates. All aerobic organisms have a network of antioxidants and enzymes to quench ROS and prevent oxidative damage. This dissertation examines antioxidant and oxidative stress biomarkers in endemic molluscs and crabs from two natural extreme environments: deep-sea hydrothermal vents in the Lau and North Fiji Basin, and cold seeps in the Gulf of Mexico. In addition, the acute toxicity and sub-lethal effects of four insecticides and an herbicide are examined in the estuarine blue crab, Callinectes sapidus. Blue crabs are North Carolina's most important fishery species and are frequently found in agricultural drainage ditches, an example of an anthropogenic extreme environment.
Total glutathione, catalase, superoxide dismutase, and lipid peroxidation levels were of the same respective order of magnitude in the two vent gastropods, Alviniconcha sp. and Ifremeria nautilei, and vent mussel, Bathymodiolus brevior. These biomarkers activities were similar to those from previous reports on Mid-Atlantic Ridge mussels, except for ~100-fold higher lipid peroxidation levels among Lau molluscs. Principal component analysis (PCA) of mollusc tissue-specific biomarker levels grouped individuals by species rather than by site.
Biomarker levels in the seep mussels Bathymodiolus childressi, B. brooksi, and B. heckerae were similar across species except for elevated foot and gill cytosolic SOD in mussels from MC-640 compared to those from AC-645. PCA of seep mussel biomarker levels differentiated by species with B. childressi isolated from B. brooksi and B. heckerae. The addition of B. brevior biomarker data to the PCA showed them grouping around B. brooksi and B. heckerae. Bathymodiolus childressi is ancestral to the other species and contains only methanotrophic endosymbionts. Whether symbionts play a role in alleviating possible toxic conditions remains unknown.
Pesticides were acutely toxic to blue crabs in the order of Lambda-cyhalothrin > imidacloprid ≈ aldicarb > acephate ≈ Roundup® (glyphosate). Megalopae were almost always more sensitive to pesticides than early stage juveniles. Commercial formations of pesticides generally showed similar toxicity to active ingredients alone. Exposure to LC20 levels of acephate, aldicarb, imidacloprid and Roundup significantly increased the frequency of juvenile mortality after molting. There was no significant change in total glutathione or lipid peroxidation of exposed megalopae. Lambda-cyhalothrin-, imidacloprid-, and aldicarb-based products have the potential to cause acute toxicity and molting-related mortality in shallow creeks and ditches.
Item Open Access High Resolution X-ray Microscopy Using Digital Subtraction Angiography for Small Animal Functional Imaging(2008-08-04) Lin, Ming DeResearch using mice and rats has gained interest because they are robust test beds for clinical drug development and are used to elucidate disease etiologies. Blood vessel visualization and blood flow measurements are important anatomic and physiologic indicators to drug/disease stimuli or genetic modification. Cardio-pulmonary blood flow is an important indicator of heart and lung performance. Small animal functional imaging provides a way to measure physiologic changes minimally-invasively while the animal is alive, thereby allowing for multiple measurements in the same animal with little physiologic perturbation. Current methods of measuring cardio-pulmonary blood flow suffer from some or all of these limitations-they produce relative measurements, are limited to global or whole animal or organ regions, do not provide vasculature visualization, limited to a few or singular samples per animal, are not able to measure acute changes, or are very invasive or requires animal sacrifice. The focus of this work was the development of a small animal x-ray imaging system capable of minimally invasive real-time, high resolution vascular visualization, and cardio-pulmonary blood flow measurements in the live animal. The x-ray technique used was digital subtraction angiography (DSA). This technique is a particularly appealing approach because it is easy to use, can capture rapid physiological changes on a heart beat-to-beat basis, and provides anatomical and functional vasculature information. This DSA system is special because it was designed and implemented from the ground up to be optimized for small animal imaging and functional measurements. This system can perform: 1) minimally invasive in vivo blood flow measurements, 2) multiple measurements in the same animal in a rapid succession (every 30 seconds-a substantial improvement over singular measurements that require minutes to acquire by the Fick method), 3) very high resolution (up to 46 micron) vascular visualization, 4) quantitative blood flow measurements in absolute metrics (mL/min instead of arbitrary units or velocity) and relative blood volume dynamics from discrete ROIs, and 5) relative mean transit time dynamics on a pixel-by-pixel basis (100 µm x 100 µm). The end results are 1) anatomical vessel time course images showing the contrast agent flowing through the vasculature, 2) blood flow information of the live rat cardio-pulmonary system in absolute units and relative blood volume information at discrete ROIs of enhanced blood vessels, and 3) colormaps of relative transit time dynamics. This small animal optimized imaging system can be a useful tool in future studies to measure drug or disease modulated blood flow dynamics in the small animal.
Item Open Access Roles of Cytoplasmic Deacetylase Hdac6 in Oncogenic Tumorigenesis(2008-04-21) Lee, YishanReversible acetylation has emerged as an important post-translational modification that rivals phosphorylation in regulating chromatin structure and gene expression. Acetylation of histone is associated with transcriptional activation while deacetylation is linked to transcriptional repression. Moreover, histone deacetylase inhibitors induce growth arrest, differentiation and apoptosis of cancer cells and therefore appear to be promising anti-tumor agents. While transcriptional deregulation is thought to be the main mechanism underlying their therapeutic effects, the exact mechanisms and targets by which HDAC inhibitors, which are mostly non-specific, achieve their anti-tumor effects remain poorly understood. In other words, it is not known which and how each HDAC members are involved in supporting tumor growth.
In this thesis, I have showed that HDAC6, a cytoplasmic localized and cytoskeleton-associated deacetylase, is required for efficient oncogenic transformation and tumor formation. I have found that HDAC6 expression is induced upon oncogenic Ras-induced transformation in both human somatic cells and murine fibroblasts. Conversely, murine fibroblasts deficient in HDAC6 are more resistant to both oncogenic Ras and ErbB2-dependent transformation, indicating a critical role for HDAC6 in oncogene-induced transformation. Supporting this hypothesis, inactivation of HDAC6 in several human cancer cell lines effectively impairs anchorage-independent growth in vitro and their ability to form tumors in immunocompromised mice. I have demonstrated that the impairment of anchorage independent growth in HDAC6 deficient cells is associated with increased anoikis and mechanistically a defect in activation of the AKT and ERK kinase cascades. Additionally, HDAC6 null mice are more resistant to two-stage chemical carcinogen-induced skin tumors. Finally, I have demonstrated that the tumor-promoting effect of HDAC6 is probably mediated through the molecular chaperon Hsp90. While Hsp90 is known to be deacetylated by HDAC6 and has been implicated in stabilizing Raf-1 and ErbB2, I have found that suppression of HDAC6 impairs the stability of Raf-1 and the association between Hsp90 and ErbB2.
In conclusion, my work provides the first experimental evidence that of all the HDAC members, the cytoplasmic deacetylase HDAC6 is required for efficient oncogenic transformation, indicating that reversible acetylation plays a critical role in regulating cellular functions of non-histone non-nuclear cytoplasmic proteins that contributes to malignant transformation. Furthermore, this work identifies HDAC6 as an important component underlying the anti-tumor effects of HDAC inhibitors.
Item Open Access The role of hypoxia-inducible factor-1 in hyperthermia-induced tumor reoxygenation and therapy resistance(2010) Moon, Eui JungImbalance between oxygen consumption and supply often makes tumors hypoxic (Bristow and Hill 2008). Tumor hypoxia is significantly correlated with aggressive tumor growth, ineffective response to radiation and chemotherapy, and as a result, poor patient prognosis. Hyperthermia (HT) is a strong adjuvant treatment to overcome these challenges of tumor hypoxia because it causes tumor reoxygenation at temperatures lower than 43ºC (Song, Park, and Griffin 2001). However, the detailed molecular mechanisms of how HT enhances tumor oxygenation have not been elucidated. Here we determine that 1 hour HT activates hypoxia-inducible factor-1 (HIF-1) and its downstream targets, vascular endothelial growth factor (VEGF), lactate dehydrogenase A (LDHA), and pyruvate dehydrogenase kinase 1 (PDK1) in tumors. Consistent with HIF-1 activation and upregulation of its downstream genes, HT also enhances tumor perfusion/vascularization and decreases oxygen consumption rates. As a result, tumor hypoxia is reduced after HT suggesting that these physiological changes contribute to HT-induced tumor reoxygenation. Since HIF-1 is a potent regulator of tumor vascularization and metabolism, our findings suggest that HIF-1 plays a role in HT-induced tumor reoxygenation by transactivating its downstream targets. Mechanistically, we demonstrate that NADPH oxidase-mediated reactive oxygen species (ROS) production upregulates HIF-1 after HT. Further, we determine that this pathway is initiated by increased transcription of NADPH oxidase-1 (NOX1) through the ERK pathway.
A major research effort at Duke focuses on combinations of HT and doxorubicin in the treatment of locally advanced breast and other cancers. Thus, we investigated whether there are HIF-1 responses to doxorubicin treatment. We reveal that doxorubicin also activates HIF-1. Unlike HT, doxorubicin-induced HIF-1 promotes persistent tumor angiogenesis. We also reveal that the signal transducer and activator of transcription 1 (STAT1)/inducible nitric oxide synthase (iNOS) pathway causes HIF-1α accumulation in an oxygen-independent manner. We show that activated STAT1 upregulates iNOS expression and promotes nitric oxide (NO) production in tumor cells resulting in HIF-1α stabilization. We further determine that both iNOS inhibitor, 1400W and STAT1 inhibitor, epigallocatechin-3-gallate (EGCG) significantly decrease intracellular NO production and suppress doxorubicin-induced normoxic HIF-1α accumulation.
HIF-1 is often considered a promising therapeutic target because of its role in tumor progression (Semenza 2003) and therapy resistance (Moeller et al. 2004). However, our findings suggest that HIF-1 plays a pleiotropic role in response to HT and chemotherapy. Therefore, to preferentially take advantage of HT-induced HIF-1 activation and also to suppress its deleterious effects induced by chemotherapy or as we have previously reported, by radiation (Moeller et al. 2004), HIF-1 inhibition needs to be carefully regulated in a time-sensitive manner to achieve optimal therapeutic effects.
Item Open Access The Role of Threshold Size in Insect Metamorphosis and Body Size Regulation(2010) Preuss, Kevin MichaelThe initiation of metamorphosis causes the cessation of the larval growth period which determines the final body size of adult insects. Because larval growth is roughly exponential, differences in timing the initiation of metamorphosis can cause large differences body size. Although many of the processes involved in metamorphosis have been well characterized, little is known about how the timing of the initiation of metamorphosis is determined.
Using different strains from Tribolium castaneum, Tribolium freemani, and Manduca sexta and varied nutritional conditions, I was able to document the existence of a threshold size, which determines when the larva becomes competent to metamorphose. Threshold size, however, does not dictate the exact timing of initiation. The exact timing for the initiation of metamorphosis is determined by a pulse of the molting hormone, ecdysone, but only after threshold size has been reached. Ecdysone pulses before the larva attains threshold size only cause the larva to molt to another larval instar. These results indicate the timing of metamorphosis initiation is controlled by two factors: (1) attainment of threshold size, at which the larva becomes competent to initiate metamorphosis and (2) the timing of an ecdysone pulse after attaining threshold size.
I hypothesize the attainment of threshold size, and therefore competence to metamorphose, is mediated by the effect of changing juvenile hormone concentrations caused by the increase in size of the larva. While the larval body grows nearly exponentially, the corpora allata, which secretes juvenile hormone, grows very little if at all. The difference in relative growth causes juvenile hormone concentrations to gradually become diluted. When juvenile hormone concentrations fall below a threshold, changes in protein-protein binding occur that can cause changes in signaling networks and ultimately gene expression. These changes make the larva competent for metamorphosis.
I have demonstrated that only threshold size is consistently correlated with body size; other growth parameters such as growth rate, duration of instars, or number of instars do not consistently correlate with variation in body size. Using the black mutant strain of M. sexta I have shown that lower juvenile hormone titers correlate with lower threshold sizes. My hypothesis is consistent with the large body of literature indicating the involvement of juvenile hormone. I also hypothesize that the diversity of metamorphosis types in holometabolous insects can be explained by heterochronic shifts in the timing of threshold size and other developmental events related to metamorphosis. The heterochronic shifts affect not only the morphology of organs, but can also affect the overall phenotypic response of the larva to changes in the environment. The different phenotypic responses among species may make the more or less suited for certain types of niches.