<p>A review of the available evidence relating herpes simplex type 2 (HSV-2) to cervical cancer in humans showed reasonable circumstantial evidence to suspect the participation of HSV-2 in the pathogenesis of the tumors. The best evidence was provided by seroepidemiological data, however, variations were observed among the data obtained from different populations. A mathematical model based on causal considerations and developed as an extension of a model used in chemical carcinogenesis was found to account for a large part of these variations. This model was found to reasonably describe the relation between the incidence rate of cervical cancer and the fraction of women with previous experience with the virus in different human populations. The fitting to the data suggested the existence of two kinds of etiologic factors involved in the pathogenesis of cervical cancer, one related to HSV-2 and another not related to the virus. The model described for average population values was expanded to age-specific data. This allowed the estimation of the age distribution of primary infection with HSV-2 (ϕ(v)), the distribution of the age of developing cancer unrelated to HSV-2 (Yᵤ(t)), the distribution of the age of developing cancer associated with HSV-2 (Yᵥ(t)), and the distribution of the time elapsing from the primary infection with HSV-2 up to the development of cervical cancer associated to HSV-2 (ψ(u)). The different shapes of Yᵤ(t) and Yᵥ(t) supported the existence of the two kinds of etiologic factors in the pathogenesis of the tumors. In addition, ψ(u) was found to correspond to relatively long times suggesting an important role for viral latency or viral reinfections in HSV-2 carcinogenesis. In view of the fact that some cervical cancers may not be related to HSV-2 the direct detection of HSV-2 genetic information in the cancer cells was considered fundamental for the correct assessment of the viral participation in the pathogenesis of the tumor. Large inconsistencies were found among reports of studies designed to detect HSV-2 genes in cancer cells. As a consequence a new approach was explored. The direct retrieval of viral endogenous genes was attempted from cells biochemically transformed by the virus. Contrary to reports from other systems, rescue was not detected. The frequency of rescue products was estimated as a value below 7x10^-17. These results suggested that rescue experiments which may be suitable for the detection of latent HSV, are not sensitive enough for the exposure of subgenomic viral sequence present in transformed cells. As a requirement for the rescue experiments, a new technique was developed to quantitate and clone HSV expressing the viral thymidine kinase in stocks predominantly lacking virus able to express this enzymatic activity.</p> / Doctor of Philosophy (PhD)

<p>Pulmonary structure and function were compared in healthy adult horses and cows to examine the hypothesis that differences in pulmonary function between two species of similar size were related to differences in pulmonary structure. The functional residual capacity and tidal volume were higher in the horses than in the cows while the respiratory rate was higher in the cows. The pattern of air flow differed between the two species, and flow rates were higher in the cows. The dynamic compliance was higher in the horses while the work of breathing was higher in the cows. Separation of the lower pulmonary system (trachea to pleural cavity) from the total pulmonary system (nares to pleural cavity) indicated that most of the resistance and work of breathing were in the upper airways (nares to trachea), and there were no differences in the mechanical indices of pulmonary function in the lower pulmonary system between the two species. To more adequately characterize the lungs, horses and cows were anaesthetized and pressure-volume manoeuvres were performed to determine the lung capacities and quasistatic compliance. The lung capacities were significantly greater in the horses, but the lung compliance did not differ significantly between the two species. Quantitative and qualitative observations revealed few significant differences between the structure of the lung parenchyma in horses and cows. The obvious structural differences were in the size and shape of the lungs, the branching pattern of the airways, and the amount and distribution of the interlobular septa. During the measurements of pulmonary mechanics in the standing animals there were several anomalous observations and examination of the pressure recordings revealed phase lags between the total and lower pulmonary systems. It would appear that it is invalid to assume that inertia is a negligible factor in pulmonary function in large mammals such as the horse and the cow, due to the large abdomen in these species. The size and shape of the abdomen has probably been a significant factor in the evolution of lung structure in horses and cows, and abdominal movements are important in determining the breathing patterns in these two species.</p> / Doctor of Philosophy (PhD)

<p>Increased platelet aggregation and Telease of granule contents in response to ADP,. epinephrine, and collagen, increased platelet factor 3-availability induced by ADP or collagen, increased platelet turnover and shortened template bleeding times in patients with type II- and type IV-hyperlipidemia have been reported from several laboratories. Furthermore, studies in experimental animals and in man have indicated that diet-induced elevation of blood Iipid levels may be associated with increased platelet adhesiveness, increased platelet factor 3-availability in response to ADP, and increased platelet turnover. Recently, evidence has been reported that in vitro exposure of pIatelets to increased levels of cholesterol in the medium may be associated with an increase in platelet cholesterol/phospholipid ratio and platelet aggregability. It has also been demonstrated that changes in phospholipid-fatty acid composition of the platelets may occur as a result of diet-induced changes in phospholipid-fatty acid composition of the plasma. Although long chain saturated fatty acids are known to induce or enhance platelet aggregation in vitro, uptake of free fatty acids appears to occur only when the concentration of free fatty acids exceeds the plasma albumin free fatty acid binding capacity. Phospholipids constitute a major proportion of the platelet membranes. Phosphatidic acid and the phosphoinositides appear to be involved in platelet shape change, aggregation, and the platelet release reaction. Certain phospholipids in platelets, when exposed, are powerful accelerators of blood coagulation reactions in vitro.</p> <p>The primary aims of this study were to investigate a) the possibility of a direct exchange of phospholipids between plasma and platelets in vitro; and b) the possibility that phospholipid exchange could result in changes in platelet function.</p> <p>In preliminary studies evidence was obtained to indicate that sodium pentobarbital used as anesthetizing agent in the blood collection procedure in rabbits inhibited platelet adherence to collagen-coated surfaces and the platelet release reaction when the compound was added to platelets in vitro. This effect of SPB appeared not to be related to SPB-induced suppression of platelet energy production but rather due to platelet membrane stabilization. The inhibitory effect of SPB on platelet function was readily reversible and SPB-induced anesthesia in rabbits was not found to cause measurable inhibition of pIatelet function.</p> <p>A sensitive and practical assay for collagen-induced platelet factor 3-availability; using a modified Stypven-clotting time method, was elaborated. During these methodological studies, new evidence with respect to the mechanism of platelet factor 3-availability was obtained. The findings indicated that with platelets from rabbits, pigs and humans platelet factor 3 did not become available in association with platelet shape change or platelet aggregation, but was rather closely linked to the platelet release reaction. However, a small amount of lysis was consistently observed in association with the platelet release reaction and it could not be clearly differentiated whether increased platelet factor 3 availability was truly a result of fusion of granule membranes with the external plate let membrane during the release reaction or a result of release-associated platelet damage.</p> <p>When hyperlipidemia was induced in rabbits by feeding them a diet enriched with egg yolk for a minimum period of 16 weeks, platelets isolated from the blood of these rabbits and resuspended in Tyrode-albumin solution showed a significant increase in collagen- or thrombin-induced aggregation and serotonin release, but not in aggregation induced by ADP. Collagen-induced platelet factor 3-availability was also greater with platelets from hyperlipidemic rabbits compared with platelets from control rabbits. There were no significant differences between the diet and control groups in platelet aggregation induced by ADP, collagen, or thrombin or serotonin release induced by collagen or thrombin in citrated platelet rich plasma. With washed platelets, there was no correlation between the percentage increase in aggregation induced by collagen or thrombin and the plasma cholesterol or triglyceride levels in hyperIipidemic or control rabbits. When washed normal rabbit platelets were resuspended in citrated plasma from a hyperlipidemic rabbit, their response to aggregating or release-inducing stimuli was not significantly different from that observed when platelets from normal rabbits were resuspended in normal plasma. When washed normal were incubated for 30 minutes in hyperlipidemic plasma at 37°C, isolated from the plasma and resuspended in Tyrode-albumin solution, their response to aggregating or release-inducing stimuli was not significantly different from that observed with normal platelets incubated in normal plasma. These findings, thus, support previous reports that diet-induced hyperlipidemia in rabbits may induce an intrinsic functional alteration of the platelets which cannot be readily reproduced by short-term in vitro exposure of platelets to hyperlipidemic plasma.</p> <p>The possibility of a direct exchange of preformed phospholipids between pIatelets and plasma was explored by adaptation of a model previously used in the study of phospholipid exchange in erythrocytes. Washed rabbit platelets were resuspended in plasma in which all of the major phospholipids had been isotopically labeled by injection of ³²PO₄ into rabbits. At certain intervals during a 6-hour incubation at 37°C, aliquots were removed from the incubation mixture and the platelets were isolated and subjected to lipid extraction and phosphilipid analysis. A continuous rise in platelet non-lipid-bound and lipid-bound radioactivity was observed-throughout the incubation period. Two platelet phospholipids, lecithin and lysolecithin, were significantly labeled, whereas little or no labeling of the other phospholipids was found. There was no detectable change in total or individual platelet phospholipid content. At 6 hours, 4% of total platelet phospholipids, 43% of platelet lysolecithin, and 7% of platelet lecithin were labeled. Platelets incubated in plasma from rabbits with diet-induced hyperlipidemia took up and incorporated significantly more label into their phospholipids than did platelets in normal plasma but the pattern of labeling (PC + LPC) was similar to that observed normal plasma. Labeling of both platelet lysolecithin and lecithin could be explained by uptake and metabolism of plasma lysolecithin by platelets. However, labeling of platelet lecithin could at least in part be the result of direct exchange of this phospholipid with the plasma. This is the first demonstration that platelets take up and metabolize endogenous plasma lysolecithin and, possibly, directly exchange lecithin and that these processes may be significantly accelerrated in hyperlipidemic plasma. Uptake and incorporation of endogenous plasma lysolecithin by platelets and, possibly, direct exchange of platelet lecithin appear to be important for platelet membrane phospholipid renewal and could be an important mechanism by which plasma lipids could modify platelet membrane phospholipid-fatty acid composition and, possibly, platelet function.</p> <p>In view of the possibility that lysolecithin might be an important link in the interaction between platelets and plasma lipo-proteins and apparent contradictions in the data reported from other laboratories, the effects of lysolecithin on aggregation, serotonin release, shape and lysis of rabbit, pig, or human platelets in platelet-rich plasma or Tyrode-albumin solution were examined during prolonged incubation. Lysolecithin added to citrated or heparinized PRP from humans or rabbits at a final concentration above 100 μM, caused instantaneous inhibition of platelet aggregation induced by ADP, epinephrine (human PRP only), collagen or thrombin. The inhibitory effect of lysolecithin was found to be partially reversible over a period of 60-90 minutes. Lysolecithin at final concentrations above 30 mM also caused inhibition of ADP-, collagen- and thrombin-induced aggregation and collagen and thrombin-induced release of serotonin in suspensions of rabbit, pig, or human platelets. With washed platelets, the inhibitory effect not only rapidly disappeared, but was followed by transient potentiation of aggregation and serotonin release. This potentiating effect of lysolecithin was most pronounced when thrombin was used as stimulus. Both inhibition and potentiation were observed at concentrations of lysolecithin that did not cause a significant change in platelet shape or loss from platelets of lactic dehydrogenase. Inhibition and potentiation were also observed when plafelets were added to suspending medium containing lysolecithin, although considerably higher concentrations of lysolecithin were required under these conditions. Potentiation was not observed when lysolecithin was added to citrated or heparinized rabbit or human PRP or to washed rabbit platelets suspended in a medium containing 4% bovine serum albumin. It seems Iikely that some or all of the observed effects of lysolecithin on platelet function are due to structural modification of the platelet membrane insufficient to result in gross membrane damage of platelet lysis. In addition, the results of experiments using ¹⁴C-Iysolecithin seemed to indicate that the reversal of inhibition and the potentiating effect of lysolecithin on platelet function may at least in part be related to its rapid uptake and metabolism by the platelets. Whether plasma lysolecithin plays a role in the regulation of platelet function under normal conditions and/or in hyperlipidemia and whether acute changes in plasma lysolecithin concentration (e.g. in response to heparin administration) can acutely modify platelet function remains speculative.</p> <p>Lysophosphatatidylethanolamine and Iysophosphatidylserine also caused inhibition of platelet aggregation and serotonin release in suspensions of washed rabbit and human platelets induced by ADP, collagen or thrombin. With washed pig platelets lysophosphatidylethanolamine inhibited platelet aggregation and the platelet release reaction whereas lysophosphatidylserine induced platelet aggregation and serotonin release. All of these effects on platelets were observed at concentrations of the lysophospholipids which did not cause gross platelet membrane damage or platelet lysis. The differences in the effects of these lysophospholipids on platelet function could be related to marked differences in their fatty acid composition.</p> <p>The results of this study contribute to our knowledge of the mechanisms of platelet factor 3-availability, platelet membrane phospholipid renewal and the role of plasma lipids in the modification of platelet phospholipid and phospholipid-fatty acid composition and platelet function.</p> / Doctor of Philosophy (PhD)

<p>Macrophages obtained from animals infected with intracellular parasites are activated with respect to their ability to inhibit the replication of the parasite. While normal rabbit macrophages support the replication of vaccinia virus at levels of 2 to 3 logs, macrophages obtained from the peritoneal cavity of rabbits infected 9 to 12 days earlier with vaccinia virus are activated and will not support virus replication. The fate of vaccinia virus in activated rabbit macrophages was studied in order to characterize the abortive infection of the virus within the activated macrophage. Vaccinia virus adsorption was measured using radioactively labelled virus and was similar with both normal and activated macrophages but was lower than, the amount of virus which adsorbed to Vero cells. Maximum adsorption took place during the first 10 minutes of incubation. A significant amount of the vaccinia virus which had adsorbed to the cells eluted from the cells during further incubation. However, the virus elution curves for activated and normal macrophages were similar. Virus uncoating was measured by infecting with ³H thymidine labelled vaccinia virus and then detecting DNase sensitive, TCA soluble counts. Vaccinia virus was able to uncoat to a similar degree in both activated and normal macrophages. Maximum virus uncoating took place one to four hours after adsorption and was approximately 55% of the virus which was adsorbed to the cells. DNA synthesis in vaccinia virus infected cells was detected by pulse labelling with ³H thymidine. A burst of DNA synthesis at 3 to 6 hours after infection took place in both activated and normal macrophages infected with vaccinia virus as well as infected Vero cells. The pattern of vaccinia virus antigen production in activated and normal macrophages was identical as detected by immunofluorescence and immunodiffusion. Autoradiographs of SDS-PAGE gels of lysates of infected cells pulsed with ³H amino acids demonstrated that most of the polypeptides formed within the infected macrophages were identical. However, at least three polypeptides present in the activated macrophages infected with vaccinia virus were absent in the infected normal macrophages and at least one polypeptide present in the virus infected normal macrophages was absent in the virus infected activated macrophages. Pulse chase experiments failed to demonstrate that the differences in polypeptide synthesis in activated and normal macrophages infected with vaccinia virus were due to differences in posttranslational cleavage. Lack of virus particle production in activated rabbit macrophages infected with vaccinia virus was the major detectable defect in the viral replicative cycle. Virus particles were defected by centrifuging on a continuous sucrose gradient cell lysates of virus infected cells labelled with radioactive thymidine or amino acids. No virus particles with the size and density of vaccinia virions were detected in lysates of activated macrophages infected with vaccinia virus. Virus particles were present in normal macrophages and Vero cells after vaccinia virus infection.</p> <p>It appears that the inhibition of production of infectious virus in activated macrophages is mediated by mechanisms other than those induced by interferon. Vaccinia virus DNA and protein were synthesized in activated macrophages. This is in contrast to numerous previous studies which have shown that no vaccinia viral DNA or protein is synthesized in interferon treated cells. Retreatment of normal rabbit macrophages with tissue culture interferon (type I) did not block the replication of vaccinia virus. Pretreatment of normal macrophages with serum from a poly (I)-poly (C) injected rabbit reduced the replication of vaccinia virus but the characteristics of the abortive infection appeared different than in activated macrophages. Therefore, it appears that interferon is not involved in the inhibition of viral replication by activated macrophages.</p> / Doctor of Philosophy (PhD)

<p>Congenital hip disease (CHD), a condition in which the head of femur may be partially or completely separated from the acetabulum, is detected in one to 4 infants per 1,000 live births. At birth the hip joint is cartilaginous and underdeveloped. In cases of CHD the acetabulum is shallow, the femoral head is small and aspherical, ligament of the head of femur (LHF) is elongated, and the femoral angles are abnormal. These are measureable characteristics. However, no previous studies have given quantitative data for several dimensions of this joint in a sample of fetuses, at regular intervals of age, throughout the fetal period.</p> <p>The pattern of normal growth and development was studied in 280 hip joints from 140 fetuses between 12 weeks and term. Measurements were taken of acetabular depth, diameter, femoral head diameter, the length and width of the LHF, and the femoral torsion and neckshaft angles. Histological studies of ossification and labrum structure were undertaken on a number of acetabula at intervals throughout the fetal period.</p> <p>Acetabular depth was shown to be the slowest growing variable at the hip joint. The study confirmed previous reports that the acetabulum tends to become shallower towards term but suggested that the greatest amount of change in shape may occur early in the fetal period. In a sample of femoral heads a tendency for the head to become less spherical with age was revealed. With the exception of the neckshaft angle all variables demonstrated a moderate to high positive correlation with age. Variables were best fitted by a regression model which included a polynomial on age. Only for left LHF length and right LHF width was growth a simple interest function. The velocity of growth for ail dimensions, except the neck-shaft angle, was highest between 12 and 18 weeks. After this period a deceleration in the growth rate was noted, most noticeable in acetabular depth.</p> <p>No significant differences were detected between males and females, or between the right and left sides. This indicates that the higher incidence of CHD in females, and the greater involvement of the left hip must be explained by factors other than the growth of cartilage and bone.</p> <p>The LHF was shown to be variable in shape throughout the fetal period. It was not a distinctly linear structure in normal or abnormal hip joints. A strong correlation with acetabular dimensions was not evident.</p> <p>Booth femoral angles demonstrated variability throughout the fetal period and values reported are lower than those currently accepted for the newborn. Since the angles demonstrated poor to moderate correlation with the other hip dimensions, neither angle alone appears to provide a useful indicator of normal development. A change in the orientation of the lesser trochanter with age, correlated with the increase in torsion was observed. This is considered pertinent to the reading of femoral angles on radiographs.</p> <p>In 65 hip joints from 46 fetuses variation from descriptions of normal hip joint morphology was detected. Measurements of these joints did not differ significantly from those of normal hip joints. It is suggested that a number of these variants are microforms of CHD. An unexpected number of variants and abnormal socket features were localized to the anterosuperior quadrant and anterior socket wall.</p> <p>Comparison of data from abnormal joints (18) with data from the normal growth study permitted a more precise evaluation of the abnormality. From this comparison underdevelopment of apparently "normal" joints was detected.</p> / Doctor of Philosophy (PhD)

<p>To test the error catastrophe theory of cell senescence, we have developed a cell-free protein synthetic system from human diploid fibroblasts capable of translating both the synthetic mRNA, poly (u), and endogenous cellular mRNA in vitro. We have measured the fidelity of poly(U)-directed protein synthesis in extracts from a variety of cells. The results contradict the error catastrophe in several ways: - cells from subjects suffering maladies of premature aging, progeria and Werner Syndrome, exhibited error frequencies within the normal range. - cells from an old donor did not have elevated error frequency of protein synthesis. - early-passage (young) normal cells had error frequencies higher than those of late-passage (old) cells. The error frequency in one normal strain of fibroblasts declined throughout its tissue culture lifespan. Cells maintained in the post-mitotic (terminal) state, in which there was no cell selection, exhibited a constant error frequency over 16 weeks. In a series of experiments using clonal populations of cells, it became evident that the decline in error frequency as a function of passage was dependent on the clonal heterogeneity of the mass culture. In no experiment, however, could any correlation between the error frequency of protein synthesis and cellular growth parameters be made. Overall, the senescence characteristic of human diploid fibroblasts was independent of the measured fidelity of protein synthesis in vitro.</p> / Doctor of Philosophy (PhD)

<p>The changes in the physiological properties of the sinoatrial node which follow a programme of exercise training have been examined in the rat. The influence on the intrinsic sinoatrial frequency (ISF) of altered levels of autonomic nervous activity accompanying the physical training has been studied in two series of experiments combining daily pharmacological agents with ten weeks of treadmill exercise. The first series (Study I) examined the influence on the ISF of the increased parasympathetic activity reported in the trained state by blocking with atropine or stimulating with carbachol the receptors of the parasympathetic system in the heart. The second series (Study 2) examined the influence on the ISF of the increased sympathetic activity which occurs during exercise by stimulating with noradrenaline or isoprenaline, or blocking with propranolol the receptors of the sympathetic system in the heart. The content of potassium (K) and sodium (Na) in the plasma and myocardium were measured in all animals at the completion of the ten week period, and an estimate of electrophysiological changes was derived from the plasma to myocardial K⁺ ratio (K⁺o/K⁺i). In addition, the chronotropic response of the sinoatrial node to acetylcholine and noradrenaline was examined in vitro for all animals in Studies 1 and 2. The chronotropic responses of the sinoatrial node to exercise and noradrenaline were further explored in the in vivo experiments of Study 3.</p> <p>In Study 1, ISF was reduced with training. Blockade of the parasympathetic receptors with atropine also reduced ISF, and the effects of exercise and atropine appeared to be additive. Stimulation of the parasympathetic receptors with carbachol had little effect on ISF. In Study 2, stimulation of the sympathetic receptors with either noradrenaline or isoprenaline caused a reduction in ISF, but when combined with exercise, no reduction occurred. Sympathetic receptor blockade with propranolol reduced ISF, and propranolol plus exercise also reduced ISF. It appears that the exercise-induced reduction of ISF was not a consequence of increased parasympathetic activity in the trained state, or of increased sympathetic activity during exercise. The K⁺o/K⁺i was reduced with training. This was consistent with a more negative maximum diastolic repolarization, and a reduced ISF. The K⁺o/K⁺i of the drug treated groups indicated that ISF was probably reduced through factors other than a more negative maximum diastolic repolarization. No difference was found in the sensitivity of the atria of any group to acetylcholine in vitro. Increased chronotropic sensitivity to noradrenaline was found in vitro in the two chronically noradrenaline treated groups. The maximum response to noradrenaline was reduced in vitro. The maximum cardiac frequency in response to either exercise or noradrenaline infusion in vivo was also reduced. This reduction of maximum frequency in both the atria and the intact heart was observed to be positively correlated with the ISF, thus those hearts with a low ISF obtained a low maximum frequency. This observation led to the proposal that electrophysiological properties of the sinoatrial node which establish the ISF are also involved in imposing a limit on the maximum sinoatrial frequency.</p> / Doctor of Philosophy (PhD)

<p>A number of cellular products present during fetal development, but absent from normal adult tissues, have been shown to be re-expressed in cancer cells. One example of these oncofetal substances is carcinoembryonic antigen (CEA). Studies were undertaken to examine the expression of CEA at the surface of human colon carcinoma cells grown in vitro and to develop a radioimmunoassay for quantitation of CEA and antibodies to CEA in the serum of cancer patients.</p> <p>Antibodies specific for CEA were prepared in goats and these antibodies were found to induce polar redistribution or capping of the antigen. As with other systems in which polar redistribution of surface molecules have been studied, the capping was temperature-dependent and required an intact microfilament system. Fluorescent-labeled antibodies were utilized to demonstrate that while CEA would undergo capping, blood group antigen A did not, hence these antigens exist as separate molecules at the cell surface. The capping process was further characterized using ¹²⁵I-labeled antibodies and it was demonstrated that upon capping the majority of cell surface CEA underwent endocytosis. The ability to specifically remove CEA from the cell surface with antibody was used to demonstrate a rapid reappearance of CEA on the tumor cell surface, and this reappearance appeared to require protein synthesis.</p> <p>A precise quantitative radioimmunoassay for CEA was developed and used to determine the amount of CEA expressed on cell surfaces. Various strains of cells were established in vitro which differed in the amount of CEA they produced. Two strains which differed in the amount of CEA expressed at their cell surfaces were shown to be equally tumorigenic in nude mice, which suggested a lack of correlation between CEA production and tumorigenicity.</p> <p>The radioimmunoassay was also used to study the control of genetic expression of CEA. There was a direct correlation between the amount of cell surface CEA and the amount of CEA secreted into the culture medium. Control over the level of CEA expressed by various strains appeared genetically stable. Yet, a number of lines of evidence suggested that the parental population from which the strains were derived was heterogeneous with respect to CEA synthesis.</p> <p>The effects of various inducing agents on CEA expression by various cell strains was examined. One strain (HCT-8 Nu2), a very low CEA producing strain, could be induced to express high levels of CEA by inclusion of theophylline in the culture medium. This effect appeared after three days of incubation and reached a maximum after five days. Enhanced expression was dosedependent and time-dependent, requiring continual presence of the drug. The effect also appeared to require continual protein synthesis and did not cause marked alteration of cell morphology or growth. It was demonstrated that the effect was not density-dependent and did not appear to be due to selective proliferation of a high expressor population. Further, the effect could not be mimicked with dibutyryl cyclic adenosine monophosphate. Similarly, another strain (HCT-8R) could be induced to produce higher levels of CEA with bromodeoxyuridine (BrdU). This effect was not as dramatic as the theophylline effect and only appeared transiently. The response to BrdU was dose-dependent.</p> <p>The specific inhibition of binding of ¹²⁵I-Iabeled anti-CEA antibodies by unlabeled anti-CEA antibodies, was used to demonstrate that no antibodies to CEA could be detected in control or cancer patient sera. The radioimmunoassay was also examined to determine its ability to quantitate the amount of CEA in serum from cancer patients and controls. It was determined that this test could measure comparable ranges of standard reference CEA, obtained from international or marketed sources. The results obtained from tests of patient sera closely correlated with results obtained using a marketed assay kit. A limited number of sera from patients was examined for CEA using the assay. Comparable percentages of patients with CEA-related cancers were found positive by my assay as reported in studies using standard assays. However, my assay appeared to have greater specificity than standard assays in that a lesser percent of patients without CEA-related cancers were positive.</p> / Doctor of Philosophy (PhD)

<p>The first step in hemostasis and thrombosis is the adherence of platelets to the damaged vessel wall. The factors involved in platelet adherence are not well understood, mainly because of the lack of a suitable method to measure quantitatively platelet adherence to surfaces. The aim of the present studies was to develop a quantitative method to measure platelet adherence to collagen containing surfaces. The following results were obtained with this method: (1) In this quantitative method for measuring platelet adherence to collagen-coated glass surfaces and to subendothelium the platelelets are labeled with ⁵¹Cr and adherence can be measured over a large area without sampling bias. Platelets are suspended at pH 7.35, at 37°C in an artificial medium which can be modified as required. The medium contains physiological concentrations of calcium and magnesium and platelet aggregation is prevented by the inclusion of apyrase to degrade any ADP released. There is no plasma present and thus no thrombin generation or fibrin formation occurs. Adherence is measured using a rotating probe device under controlled flow conditions and various surfaces can be tested (collagen-coated glass, everted aorta damaged to expose the subendothelium). (2) Platelet adherence to collagen-coated glass or to subendothelium is greatly reduced in the absence of divalent cations. Thus, methods which measure platelet adherence in the presence of chelating agents are difficult to interpret. (3) Increasing the albumin concentration of the medium decreases platelet adherence, but there is less variation among replicates. (4) Increasing the hematocrit increases the number of platelets adhering to the surface. (5) Platelets adhere tightly to collagen or to subendothelium and are not dislodged by agents which readily deaggregate platelets such as EDTA, EGTA, or PGE₁. (6) Exposure of undamaged endothelium to thrombin increases the number of platelets adherent to the surface and this is blocked by heparin. (7) This method of measuring platelet adherence to collagen can be used to screen drugs which may be useful as antithrombotic agents. The best system is to test the effect of a drug in the presence of 4% albumin and 40% hematocrit, however, for initial screening a simpler system can be used (0.35% albumin, zero hematocrit). (9) Several treatments which modify the platelet surface have been tested for their effect on adherence to collagen. Removal of sialic acid with neuraminidase do not affect platelet adherence whereas treatment with sodium periodate and the proteolytic enzymes thrombin, plasmin and chymotrypsin decrease adherence. (10) UDP and UDPG do not have an effect on platelet adherence to collagen and this observation does not support the collagen: glucosyltransferase theory of platelet adhesion to collagen. (11) Clq, a subcomponent of the first component of complement specifically inhibits platelet adhesion to collagen and may compete with the collagen receptor on the platelet membrane. (12) A wide variety of agents inhibits platelet adherence to collagen-coated surfaces and to subendothelium. Among the inhibitors which decrease platelet adherence in a system containing 4% albumin and 40% hematocrit are agents which chelate divalent cations (EGTA, EDTA, citrate), indomethacin, agents which increase cAMP levels (prostaglandin E₁, dipyridamole, RA 433), methylprednisolone, penicillin G and cephalothin. (13) Aspirin inhibits platelet adherence to collagen-coated surface or to subendothelium, but its inhibitory effect is not evident in the presence of 40% hematocrit or citrated plasma indicating that the conditions of the experiments are important in determining the effect of drugs. (14) Modifications of platelelets by treatment with thrombin, penicillin G or cephalothin which inhibit platelet adherence to collagen or subendothelium do not affect platelet survival. Modification of the platelet surface sialic acid by neuraminidase or periodate is followed by rapid clearance of platelets from the circulation. Thus there is no correlation between platelet adherence and platelet survival. (15) Treatments which decrease platelet to collagen and to the subendothelium also reduce the platelets in hemostasis.</p> / Doctor of Philosophy (PhD)

<p>The mechanism by which physiological stimuli increase cyclic GMP formation in platelets or in other cells is unknown. Agents that promote the formation of cyclic GMP in intact cells have in general not been found to stimulate the activity of guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2.] in broken cell preparations. Therefore, possible mechanisms for the activation and control of guanylate cyclase activity in platelets were investigated in this thesis.</p> <p>It was found that over 90% of the total guanylate cyclase activity is present in supernatant fractions of hypotonically lysed platelets. Platetet particulate fractions contained no guanylate cyclase activity that could not be accounted for by contaminating soluble enzyme, suggesting that physiological aggregating agents may increase cyclic GMP levels in intact platelets through the effects of intermediary factors. Because of the possibility that soluble as well as particulate factors may be involved in the control of enzyme activity, whole platelet lysate was used in studies of the properties and activation of guanylate cyclase.</p> <p>Under optimal ionic conditions (4.0 mM-MnCl₂), the specific activity of guanylate cyclase in fresh platelet lysates was about 10 nmol of cyclic GMP formed/20 min per mg of protein at 30°C, which is higher than that of any other mammalian cells or tissues studied. Activity was 15% of optimum with 10.0 mM-MgCl₂ and negligible with 4.0 mM-CaCl₂. Synergism between MnCl₂ and MgCl₂ or CaCl₂ was observed when [MnCI₂] ≤ [GTP]; under more physiological ionic conditions (Mg²⁺ present), micromolar concentrations of Ca²⁺ stimulated enzyme activity by about 50%.</p> <p>Lower than optimal specific activities were obtained in assays containing large volumes of platelet lysate, owing to the presence of inhibitory factors that could be removed by ultrafiltration. Adenine nucleotides and glutathione accounted for less than 50% of the inhibitory activity. The combined effects of inhibitory factors and of suboptimal ionic conditions are likely to lower the guanylate cyclase activity in intact platelets to almost negligible values in the absence of activating factors.</p> <p>Dithiothreitol (5.0 mM) and N-ethylmaleimide (0.1 mM) inhibited the activity of platelet lysate by about 70 and 50%, respectively. Preincubation of lysate for 60 min at 37°C increased guanylate cyclase activity on average by 225%. This effect could be blocked with dithiothreitol or N-ethylmaleimide, but dithiothreitol could not fully reverse activation once it had occurred. Oxidants such as 4,4'-dithiodipyridine (0.04 mM), diamide (0.4 mM) and tert-butylhydroperoxide (1.0 mM) increased enzyme activity on average, by 40, 87 and 165% respectively. Neither diamide nor tert-butylhydroperoxide had an effect on enzyme that had been preincubated or treated with N-ethylmaleimide.</p> <p>Sodium azide (10.0 mM) increased guanylate cyclase activity by an average of 335%; this effect was both time- and temperature-dependent. Activation by sodium azide was not prevented by dithiothreitol. Sodium nitroprusside (1.0 mM) increased enzyme activity by about 1000%; this effect could be blocked by preincubation or by tert-butylhydroperoxide, but not by either dithiothreitol or N-ethylmaleimide.</p> / Doctor of Philosophy (PhD)