Made available in DSpace on 2014-06-11T19:30:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-11-01Bitstream added on 2014-06-13T19:18:42Z : No. of bitstreams: 1 filogonio_r_me_rcla.pdf: 657959 bytes, checksum: 37b2e2fd5df4b378bb998638fbf738c2 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Em répteis não crocodilianos, o ventrículo parcialmente dividido permite que o sangue venoso pobre em oxigênio recircule no circuito sistêmico, configurando um desvio sanguíneo intracardíaco (“shunt”) da direita para a esquerda (DSIc D-E), ou que o sangue arterial rico em oxigênio retorne ao circuito pulmonar, configurando um desvio sanguíneo intracardíaco da esquerda para a direita (DSIc E-D). Teoricamente, se o DSIc apresentar alguma vantagem adaptativa, seria controlado, ao invés de uma mera consequência passiva do sistema circulatório às mudanças das resistências vasculares. O nervo vago inerva o tronco pulmonar e possui influência no controle da resistência do circuito pulmonar. Assim, o aumento ou diminuição desta resistência poderia definir a direção do desvio sanguíneo resultante. Neste estudo foi postulada a hipótese de que a ausência do tônus vagal causaria mudanças no controle do DSIc em animais submetidos a variações na temperatura e na atividade. Para tanto, foram comparados dois grupos de cascavéis, Crotalus durissus, com nervo vago intacto ou seccionado, submetidas às mesmas condições de temperatura e de atividade. Os parâmetros hemodinâmicos foram aferidos através de canulação oclusiva. De forma geral, o aumento da temperatura e da atividade aumentaram a frequência cardíaca (fH), débito cardíaco (DC), fluxo sanguíneo pulmonar (Qpul) e fluxo sanguíneo sistêmico (Qsis), embora apenas a atividade tenha feito com que as serpentes desenvolvessem um DSIc E-D como resposta ao aumento da demanda de O2 nos tecidos. A atividade também foi responsável pelo aumento das pressões arteriais média sistêmica e pulmonar. A vagotomia unilateral também causa aumento dos fluxos sanguíneos, embora não afete a fH. A ausência do controle vagal tem maior efeito no controle da resistência... / In non crocodilian reptiles, the ventricle is partially divided and allows venous blood, low in oxygen, to recirculate the systemic circuit, which is referred to as a right-to-left shunt (R-L shunt), or the oxygen-rich blood to return to the pulmonary circuit, referred to as a left-to-right shunt (L-R shunt). In theory, if intracardiac shunts provide any adaptive advantage, it would be controlled rather than be a passive consequence of changes in vascular resistances. The vagus nerve innervates the pulmonary trunk and has a role in controlling the resistance of the pulmonary circuit (Rpul). Thus, the control of Rpul could set the direction of the resulting shunt. In this study we hypothesized that the lack of vagal tone causes changes in control of intracardiac shunts in animals subjected to variations in temperature and activity. Therefore, we compared two groups of South American rattlesnakes, Crotalus durissus, with the vagus nerve intact or severed, subjected to the same conditions of temperature and activity. Hemodynamic parameters were measured by occlusive cannulation. Generally, increasing the temperature and the activity increased heart rate (fH), cardiac output (CO), pulmonary blood flow (Qpul) and systemic blood flow (Qsys), although only activity has caused to develop a R-L shunt in response to increased demand for O2 in the tissues. The activity was also responsible for increase in mean systemic and pulmonary arterial pressure. Unilateral vagotomy also increased blood flow, but did not affect fH. The absence of vagal control has more effect in controlling Rpul, causing changes in shunt patterns, especially when the animal was active. Under such condition, the organism seemed to offset the effects on hemodynamic shunt, resulting from changes in systemic resistance, with changes in CO. Thus, CO is elevated until Qpul is at an... (Complete abstract click electronic access below)

A produção de frangos de corte é um dos maiores segmentos em crescimento no mundo. É um importante fornecedor de proteínas para consumo humano, devido a sua facilidade e rapidez de produção. Devido a essa demanda, surgiram alguns problemas de manejo, que causam o aparecimento de problemas fisiológicos. Os problemas mais relevantes são relacionados com estresse, tanto físico como psicológico. No capítulo 1 estão descritas as alterações nas estruturas das miofibras e a distribuição de água na musculatura de frangos causada pelo estresse térmico em câmara climática. O estresse térmico pode causar alterações na fisiologia das aves, que levam a mudanças na cor, capacidade de retenção de água e maciez dos produtos cárneos, aspectos que influenciam diretamente na aquisição do produto pelo consumidor. Foram avaliados os seguintes parâmetros: hematócrito, creatina quinase plasmática, peso e rendimento de carcaça, vísceras, peito, pernas, asas e percentagem de água livre e ligada no peito. Conclui-se que a redução do hematócrito pode ser uma medida para prevenir uma possível hipovolemia devido à perda de água, principalmente pela hiperventilação. Embora exista efeito na estrutura da miofibra devido ao estresse térmico agudo, a drenagem de água de tecidos e órgãos nesta condição parece não envolver os músculos da ave, especialmente a musculatura do peito. No capítulo 2, estão descritas as modificações estruturais ao nível das proteínas musculares, que compõem a fibra muscular. O estresse térmico agudo causa alterações nas propriedades das miofibrilas, que afetam as características funcionais da carne, principalmente a capacidade de retenção de água. O presente experimento teve como objetivo identificar mudanças na proteólise miofibrilar e migração entre as frações miofibrilar e sarcoplasmática, decorrentes do estresse térmico pré-abate, através do índice de fragmentação miofibrilar (MFI), SDS-PAGE das frações miofibrilar e sarcoplasmática e imunodetecção de vinculina. Concluiu-se que a taxa de fragmentação miofibrilar pode ser prejudicada pelo estresse térmico agudo e que modificações na fração sarcoplasmática são observados em carne pálida, independente da condição ambiental pré-abate. / The production of broiler chickens is one of the biggest segments in growth in the world. It is an important protein supplier for human consumption, due to its easiness and velocity of production. Owed to this demand, some handling problems occurred and cause the appearance of physiological problems. The most important problems are related with stress, in such a way physicist as psychological. In chapter 1 the alterations in the structures of myofibril and the water distribution in the muscle of broilers caused by acute heat stress in climatic chamber are described. Acute heat stress can change birds physiology and leads to alterations in the colour, water holding capacity and tenderness of meat products, factors that affect consumers purchase decision. The following parameters had been evaluated: hematocrit, plasmatic creatine kinase, weight and yield of carcass, visceras, breast, legs, wings, back and percentage of free water in the breast. It is concluded that the hematocrit and creatine kinase can change due to acute heat stress. The weight of carcass and its parts is not altered. The percentage of free and bound water can change due to acute heat stress.In chapter 2, the structural modifications of the muscular proteins are described. The proteins are responsible for the structure of myofibrils and are related with the physical and biochemists processes which determine the characteristics of the meat product. Acute heat stress (AHS) causes alterations in the properties of myofibrils affecting functional characteristics of the meat, mainly the water holding capacity. The present experiment aimed to identify changes in myofibrillar proteolysis and migration between the myofibrillar and sarcoplasmatic fractions due to pre-slaughter heat stress. The myofibrillar fragmentation index (MFI), SDS-PAGE of the muscle protein fractions and western blot of vinculin were used. Its concluded that that AHS can be harmful in the process myofibrillar proteolysis. In the SDS-PAGE we observed modifications in sarcoplasmatic fraction, in pale meat, independent of the environmental condition.

Os eritrócitos dos mamíferos são anucleados e desprovidos de organelas citoplasmáticas, contando somente com o ciclo da glicólise, o ciclo das pentoses e algumas enzimas anexas, o que garante o fornecimento de energia calórica sob a forma de adenosina-5\'-trifosfato (ATP) e de energia redutora sob a forma de nicotinamida adenosina dinucleotídeo reduzida (NADH), nicotinamida adenosina dinucleotídeo fosfato reduzida (NADPH) e glutationa reduzida (GSH). A via glicolítica possui o desvio denominado de ciclo de Rapaport-Luebering, onde há a síntese de 2,3- difosfoglicerato (2,3-DPG), que é um importante metabólito e atua como modulador da afinidade da hemoglobina ao O2. Havendo poucos estudos comparativos sobre o metabolismo eritrócitário dos mamíferos propôs-se investigar as atividades das enzimas glicolíticas, anexas (2,3-difosfogliceratomutase, glicose-6-fosfato desidrogenase e 6-fosfogliconato desidrogenase) e a concentração dos compostos intermediários adenosina-5\' -trifosfato e 2,3-difosfoglicerato. Mamíferos das ordens Primates, Rodentia, Camivora, Lagomorpha, Artiodactyla, Didelphimorphia e Xenarthra oriundos da Fundação Parque Zoológico de São Paulo e Centro de Bioterismo da Faculdade de Medicina da USP foram investigados. O sangue foi colhido em ACD, os eritrócitos foram lavados em solução fisiológica a 4°C e hemolisados em solução hemolisante 1:20 por congelamento e descongelamento e as atividades das seguintes enzimas foram determinadas de acordo com Beutler (Red Cell Metabolism, a Manual of Biochemical Methods, Ed. Grune & Stratton, 3rd ed, 1984): hexoquinase, glicose fosfato isomerase , fosfofrutoquinase, aldolase, triose fosfato isomerase, gliceraldeído 3-fosfato desidrogenase, fosfoglicerato quinase, monofosfogliceromutase, enolase, piruvato quinase, lactato desidrogenase, bem como a 2,3-difosfoglicerato mutase, glicose-6-fosfato desidrogenase, 6-fosfogluconato desidrogenase, os metabólitos intermediários 2,3-difosfoglicerato e adenosina-5\'-trifosfato. As enzimas e os compostos intermediários estudados apresentaram grande variabilidade entre as espécies de mamíferos estudadas. Foi observada correlação positiva entre a atividade da triose fosfato isomerase e a 2,3-difosfoglicerato mutase e os teores de adenosina-5\'-trifosfato das espécies, bem como correlação positiva entre a 2,3-difosfoglicerato mutase em relação ao 2,3-difosfoglicerato. Os teores de adenosina-5\'-trifosfato mantiveram-se dentro de um patamar estável, ao redor de 4.000 a 6.000 nmoles / gHb, com as exceções das espécies das ordens Carnivora (Panthera leo, Leopardus pardalis, Canis lupus and Chrysocyon brachyurus) e Artiodactyla (Cervus elaphus), que exibiram teores ao redor de 2.000 a 3.000 nmoles / g Hb. Já os valores da concentração de 2,3-difosfoglicerato apresentaram variação considerável entre as espécies e ordens estudadas. / Mammalia red cells are non-nucleated and do not have cytoplasm organeles as well, and present the glycolytc pathway, the pentose shunt to attend the requirements in caloric energy as adenosine-5-triphosphate (ATP) and reducing power as reduced nicotinamide adenosine dinucleotide (NADH) and reduced nicotinamide adenosine dinucleotide phosphate (NADPH) and reduced glutathione (GSH). The glycolytic pathway exhibit besides the Luebering-Rappaport shunt, in which the 2,3-diphosphoglycerate is formed, which regulates the hemoglobin affinity to the molecular oxygen. As there are not so many studies on comparative about mammalian red cell metabolism, it was decided to study the glycolytic enzyme activities, the glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, 2,3-diphosphoglycerate mutase and the metabolites adenosine-5-triphosphate and 2,3-diphosphoglycerate (2,3-DPG). Mammalia representatives from Primates, Rodentia, Carnívora, Lagomorpha, Artyodactyla, Didelphimorphia and Xenarthra orders, obtained from Fundação Parque Zoológico de São Paulo and Centro de Bioterismo da Faculdade de Medicina da USP, were studied. The blood was collected in ACD, the red cells were washed in saline at 4° C, lysed 1:20 in hemolysing solution by freeze-and-thaw, and the following enzymes were assayed according to Beutler (Red Cell Metabolism, a Manual of Biochemical Methods, Ed. Grune & Stratton, 3rd ed, 1984): hexokinase, glucose-6-phosphate isomerase, phosphofructo kinase, aldolase, triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, monophosphoglycerate mutase, enolase, pyruvate kinase, lactate dehydrogenase activities, as well as 2,3-diphosphoglycerate mutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase activities, and adenosine-5-triphosphate, 2,3-diphophoglycerate concentrations. A remarkable variation among the studied species was observed. However, it was detected a significant positive correlation between the adenosine-5-triphosphate concentrations and triosephosphate isomerase and 2,3-diphosphoglycerate mutase activities, as well as significant positive correlation between 2,3-diphosphoglycerate concentration and 2,3-diphosphoglycerate mutase activity in all studied species as a whole. Most of studied species exhibited a steady ATP concentration range between 4,000 and 6,000 nmoles.g Hb -1 but the Artiodactyla (Cervus elaphus) and Carnivora (Panthera leo, Leopardus pardalis, Canis Lupus and Chrysocyon brachyurus,) which presented values between 2,000 and 3,000 nmoles Hb - 1. However, the 2,3-DPG concentration showed remarkable variation among the studied species and orders.

Doctor of Philosophy / Department of Animal Sciences and Industry / J. Ernest Minton / Toll-like receptors (TLR) are instrumental in discriminating between pathogenic and commensal bacteria and act as mediators, along with downstream chemokines, of subsequent innate and adaptive immune responses. However, little is known about the expression and regulation of TLR or chemokines in swine. The objectives of the experiments described herein were to characterize the expression of porcine TLR and to identify regulatory patterns in these receptors in the presence of live Salmonella enterica serovar Typhimurium (ST) or Choleraesuis (SC). The first two experiments evaluated the in vivo and in vitro expression of TLR2, 4, 5 and 9. Our results indicate that TLR2, 4, 5 and 9 are constitutively expressed in vitro in a porcine jejunal epithelial cell line (IPEC-J2), porcine mononuclear phagocytes (pMPs) and in vivo in the distal ileum. In IPEC-J2 cells, ST elicited an increase in TLR2 mRNA (P < 0.05), and both ST and SC increased TLR2 mRNA in pMPs (P < 0.05). In vivo, oral challenge with ST increased (P < 0.05) both TLR2 and TLR4 mRNA in the distal ileum. In addition, the second experiment evaluated interleukin 8 (IL8) and CC chemokine ligand 20 (CCL20) expression in IPEC-J2 cells in response to ST or purified bacterial flagellin (Flag). TLR5 was constitutively expressed in the ileum and in IPEC-J2 and pMP cells. Interestingly, IL8 and CCL20 mRNA and protein were increased (P < 0.05) by ST and Flag, even in the absence of changes in TLR5. In the third experiment, the expression of TLR and chemoattractive mediators were evaluated in a panel of tissues obtained from pigs challenged with ST and SC. All genes of interest were constitutively expressed; however, the effects of treatment were limited to isolated tissues and genes. Taken together, the data indicate that TLR and chemoattractive mediators are expressed in porcine tissues and cells and that the observations described represent novel evidence that pig pathogens may regulate TLR expression and activate chemokine secretion.

Master of Science / Department of Animal Sciences and Industry / J. Ernest Minton / Enteric pathogens are responsible for major economic losses in the swine industry. In the U.S., Salmonella enterica subspecies enterica serovar Typhimurium (ST) and serovar Choleraesuis (SC) account for essentially all cases of salmonellosis in swine. Previous studies documented that oral ST eroded growth and produced unmistakable changes in the endocrine stress and somatotropic axis of young growing pigs. However, these effects occurred in the absence of elevated systemic inflammatory cytokines that were previously thought to accompany disease-associated growth retardation. In the current study, it was hypothesized that SC would produce very different systemic inflammatory cytokine responses compared to ST given the likelihood of SC to produce systemic disease in pigs. Weaned pigs were housed two per pen with free access to feed and water during a 14 d experiment. On d 0, pigs were fed either 108 CFU SC or 108 ST, and bacteria were re-fed twice weekly through the course of the experiment. Control pigs were fed dough without bacteria. Serum was collected on d 0, 7, and 14 for determination of tumor necrosis factor alpha (TNFα), interleukin-1beta (IL-1β), and insulin-like growth factor-I (IGF-I) were determined. Rectal temperatures (RT) were monitored daily beginning 2 d prior to challenge with bacteria and until 7 d following the first bacterial feeding. Pigs were weighed initially, and at the conclusion of the study. Daily body weight gain was reduced by 25.4% in pigs fed SC (P<.0001) compared to control, while growth was similar between control pigs and those fed ST. Pigs fed SC had increased RT beginning on d 2 and continuing though d 7 (P < 0.05) with the greatest elevation spike on d 3 (P < 0.001) when compared to controls. On d 7, pigs fed SC had reduced IGF-I when compared to both control (P < 0.01) and ST pigs (P = 0.01). Despite the obvious febrile response, and the reductions in body weight gain and serum IGF-I, circulating TNFα and IL-1β were not affected by treatment. It was concluded that elevated TNFα and IL-1β are not obligatory correlates of SC-induced pathology and growth retardation in weaned pigs.

Doctor of Philosophy / Department of Anatomy and Physiology / Thomas J. Barstow / The overall aim of this dissertation is to better understand the dynamic matching between O2 delivery and uptake following the onset of exercise. The first study of this dissertation (Chapter 2) revealed that: i) the dynamics of muscle oxygenation were determined primarily by the QO2–VO2 interaction during the initial phase of QO2 response (first 15-20 s); and ii) absolute values in the steady state used to calculate blood flow from VO2 and O2 extraction did not affect the dynamics of blood flow response. Consistent with these predictions, using pulmonary gas exchange and near-infrared spectroscopy in humans (Chapter 3) we observed that the estimated kinetics of capillary blood flow (moderate exercise 25.4 ± 9.1 s and heavy exercise 25.7 ± 7.7 s) were not significantly different from the kinetics of muscle VO2 (moderate exercise 25.5 ± 8.8 s and heavy exercise 25.6 ± 7.2 s). In Chapter 4 we observed that nitric oxide (NO) is essential to maintain microvascular O2 pressure (PO2mv ~ QO2/VO2) of contracting rat muscles. Blockade of NO synthase with L-NAME accelerated the kinetics [ΔMean response time(L-NAME–CONTROL) = -6.5 ± 6.6 s, P< 0.05] and reduced the contracting steady-state PO2mv [ΔPO2mv(L-NAME–CONTROL) = -5.0 ± 1.0 mmHg; P < 0.05] compared to control. In Chapter 5 we focused on the kinetics of bulk limb blood flow (LBF) to show that a low-pass filter (LPFILTER) developed for LBF data improved the confidence of kinetic analysis by decreasing the standard error of the estimate (SEE ~ 95% confidence interval) for all kinetics parameters compared to the Beat-by-Beat method (e.g., time-constant phase 2: Beat-by-Beat = 16 ± 5 s; LPFILTER = 1.1 ± 0.5 s). In conclusion, the early increase in QO2 is the main determinant of muscle oxygenation dynamics and NO is essential to maintain the tight coupling of QO2 and VO2 kinetics during exercise. In this context, application of a LPFILTER to LBF data provides the best confidence for kinetic analysis of bulk QO2 that should facilitate investigations integrating bulk and microvascular QO2/VO2 matching in a variety of settings in health and disease.

Doctor of Philosophy / Department of Anatomy and Physiology / Thomas J. Barstow / The interaction between dynamic muscle contractions and the associated muscle blood flow is very intriguing leading to questions regarding the net effect of these contractions on oxygen delivery and utilization by the working muscle. Study 1 examined the impact of contractions on muscle blood flow at the level of the femoral artery. We demonstrated that muscle contractions had either a facilitory, neutral, or net impedance effect during upright knee extension exercise as intensity increased from very light to ~70% peak work rate. This led to the question of what impact a change in contraction frequency might have on the coupling of blood flow to metabolic rate during cycling exercise. The blood flow/VO2 relationship has been shown to be linear and robust at both the central (i.e., cardiac output/pulmonary VO2) and peripheral (leg blood flow/leg VO2) levels. However, an increase in contraction frequency has been reported to either decrease, have no effect, or increase the blood flow response during exercise. Study 2 determined if the steady state coupling between muscle blood flow and metabolic rate (centrally and/or peripherally) would be altered by varying contraction frequency. Our results indicate that both central and peripheral blood flow/VO2 relationships are robust and remain tightly coupled regardless of changes in contraction frequency. Study 3 examined muscle microvascular hemoglobin concentration and oxygenation within the contraction/relaxation cycle to determine if microvascular RBC volume was preserved and if oxygen extraction occurred during contractions. We concluded that microvascular RBC volume was preserved during muscle contractions (i.e., RBCs remained in the capillaries), which could facilitate continued oxygen delivery. Further, there was a cyclic pattern of deoxygenation/oxygenation that corresponded with the contraction/relaxation phases of the contraction cycle, with deoxyhemoglobin increasing significantly during the contractile phase. These data suggest that oxygen extraction continues to occur during muscle contractions. Significant insight has been gained on the impact of muscle contractions on oxygen delivery to and exchange in active skeletal muscle. This series of studies forms a base of knowledge that furthers our understanding of the mechanisms which govern the control of skeletal muscle blood flow and its coupling to muscle metabolic rate.

Doctor of Philosophy / Department of Anatomy and Physiology / Thomas J. Barstow / The overall aims of this dissertation are to determine the incidence and magnitude of insulin resistance (IR) in a cohort of normoglycemic college-age subjects with a family history of type 2 diabetes, and to ascertain if there is early macro- and microvascular dysfunction relative to IR. Study 1 (Chapter 2) revealed a 7-fold range in IR in healthy college subjects concomitant with measures of insulin, both fasted and during an oral glucose tolerance test, but not related with any measure of plasma glucose. These results emphasize that early in the etiology of carbohydrate dysregulation, abnormalities first occur with regard to insulin sensitivity. Using brachial artery blood flow (BABF, Doppler fluxometry) and near-infrared spectroscopy (NIRS) (Chapter 3) we extended the understanding of the use of these non-invasive tools to assess forearm resting metabolic rate and to compare the parameters of both the NIRS oxy-hemoglobin signal, as a index of perfusion in the microcirculation, and BABF, as an independent measure of microvascular reactivity during post occlusive reactive hyperemia (PORH). Resting metabolic rate ranged ~ 2 fold (2.83-5.15 [Mu]MO[subscript2]/min/100g) similar to direct measures. Amplitude, but not kinetic parameters for NIRS variables correlated with comparable parameters for BABF, providing evidence for the possible utility of NIRS in examining microvascular reactivity. In study 3 (Chapter 4), utilizing our extended understanding of hemodynamics garnered from the results of study 2, we assessed the influence of IR on macro- and microvascular reactivity. We observed that i) the magnitude of IR was significantly correlated with attenuation of endothelium-dependent vasodilation of the brachial artery (P< .01) indicating the possibility of a reduced nitric oxide bioavailability and an enhanced atherogenic milieu. Additionally we found ii) BABF at rest and during reactive hyperemia to be strongly correlated with conductance (reduced downstream resistance—an indicator of microvascular control abnormalities) independent of forearm metabolic rate, and iii) parameters of BABF (microvascular response) were also strongly correlated with brachial artery vasoreactivity (macrovascular response). In conclusion, this body of work furthers our insight into the need for earlier identification of "disease" earlier in the progression to type 2 diabetes, and provides direction for future investigations into prevention / intervention to improve microvessel functionality and to slow the atherosclerotic process in larger vessels.

Doctor of Philosophy / Department of Anatomy and Physiology / Deryl L. Troyer / Umbilical cord matrix stem (UCMS) cells are derived from Wharton's jelly and have been shown to express genes characteristic of primitive stem cells. They can be isolated in large numbers in a short time and thus potentially represent an abundant source of cells for therapeutic use. We investigated the migratory nature of human UCMS cells towards MDA 231 human breast carcinoma cells in an in vitro model of cell migration; UCMS cells cultured with or without MDA 231 cells for 24 hours. Next, we evaluated the effect of chemokines, stromal derived factor 1 (SDF-1) and vascular endothelial growth factor (VEGF) on human UCMS cells by treating with increasing doses of SDF-1 and VEGF. UCMS cells were found to migrate towards MDA 231 cells in a dose dependent manner. Both SDF-1 and VEGF induced migration of UCMS cells in a dose dependent manner. These results suggest that MDA 231 cells might be releasing chemokine factors, such as SDF-1 and VEGF, which promote UCMS cell migration towards the tumor cells in vitro. Stem cells that migrate to tumors may allow targeted delivery of therapeutic agents that otherwise may have severe side effects. To evaluate the selective engraftment and therapeutic efficiency of human UCMS cells that were engineered to express interferon beta (UCMS-IFN-beta) MDA 231 cells (2,000,000) were intravenously injected into severe combined immune deficient (SCID) mice, followed by three weekly intravenous injections of fluorescently labeled UCMS-IFN-beta cells (500,000). To evaluate the synergistic effect of 5-Fluorouracil (5-FU) and IFN-beta, MDA 231 cells were intravenously injected into SCID mice, followed by three weekly intravenous injections of fluorescently labeled UCMS-IFN-beta cells and three weekly intra peritoneal injections of 5-FU. In both of the above experiments, mice were euthanized one week after the last UCMS cell transplant and lung weights were compared to the controls to determine the differences in tumor burden. After transplantation of UCMS-IFN-beta cells into MDA 231 tumor-bearing mice, UCMS cells were found near or within metastatic lung tumors but not in other tissues, and in these animals, the lung weight was significantly less than MDA 231 tumor-bearing animals that received saline injections. Histologically, there was significant reduction in the tumor area in MDA 231 tumor bearing lungs after UCMS-IFN-beta treatment. When 5-FU was given along with UCMS-IFN-beta cells, there was further reduction in tumor area. These results indicate that UCMS cells can potentially be used for targeted delivery of cancer therapeutics.

Doctor of Philosophy / Department of Biochemistry / Bruce D. Schultz / John M. Tomich / NC-1059 is a synthetic channel-forming peptide that provides for ion transport across, and transiently reduces barrier integrity of, cultured epithelial monolayers derived from canine kidney (MDCK cells; Broughman, J. R. et al; Am J Physiol Cell Physiol 286: C1312-23). In this first study experiments were conducted to determine whether epithelial cells derived from other sources were similarly affected. Human (T84, Calu-3) and non-human (IPEC-J2, PVD9902) epithelial cells derived from intestinal (T84, IPEC-J2), airway (Calu-3), and genitourinary (PVD9902) tissues were grown on permeable supports. Ion transport and barrier function were assessed electrically in a modified Ussing chamber. Basal short circuit current (I[subscript sc]) was typically less than 3 [Mu]A cm[superscript-2]. Apical NC-1059 exposure caused, in all cell types, an increase in I[subscript sc] to >15 [Mu]A cm[superscript-2], indicative of net anion secretion or cation absorption that was followed by an increase in transepithelial conductance (g[subscript te] in mS cm[superscript-2]; T-84, 1.6 to 62; PVD9902, 0.2 to 51; IPEC-J2, 0.3 to 26; Calu-3, 2.2 to 13). NC-1059 induces a concentration dependent change in the I[subscript sc] and g[subscript te] across these epithelia. The results in all cases were consistent with both a transcellular and a paracellular effect of the peptide. NC-1059 enhanced permeation of dextrans ranging from 10 kDa to 70 kDa across all epithelia tested. These results document an effect of NC-1059 on the paracellular route of epithelial barriers. Immunolabeling, confocal microscopy and immunoblotting methods were used in a second study to assess the molecular changes associated with increased paracellular permeability. NC-1059 induced a substantial reorganization of actin within 60 minutes of exposure. Confocal microscopy revealed that the changes in actin organization were accompanied by a pronounced change in the abundance and distribution of tight junction proteins occludin and ZO-1. Immunoblotting results suggest a time and concentration dependent effect on cellular abundance of these tight junction proteins. The effects on g[subscript te] and junctional proteins are transient with > 85% of recovery in 24 hours post exposure and full recovery within 48 hours. The reversible modulation of the epithelial tight junctions has therapeutic potential to increase the efficiency of drug delivery across barrier membranes.