Airway smooth muscle (ASM) shortening is the key event leading to broncho-constriction. The degree of airway narrowing which occurs with ASM shortening is a function both of the mechanical properties of the airway wall as well as the angle of orientation of ASM. If ASM is oriented very obliquely, ASM shortening would in part be transduced to a change in airway length rather than airway narrowing. Previous reports have suggested that the angle of ASM orientation may be as high as 30$ sp circ.$ To measure ASM orientation we have developed a technique based on en-face dissection. The lungs from 4 cats and one human were fixed with 10% buffered formalin at 25 cmH$ sb2$O for 48 hrs. The airway generations 4 to 17 were dissected out from the left lower lobes. Each airway generation was individually embedded in paraffin from which 5$ mu$m thick serial sections were cut parallel to the airway long axis ("en-face") and stained with haematoxylin-phloxine-saffron. Each block yielded 3-5 sections in which the orientation of ASM nuclei relative to the airway long axis ($ theta$) was measured as an index of ASM orientation. $ theta$ was measured clockwise and counterclockwise to the short axis by using a digitizing tablet and a light microscope (X250) equipped with a drawing tube attachment. Inspection of the sections revealed extensive ASM crisscrossing without a homogeneous orientation. Between 29 and 102 nuclei were measured per generation. Although there was considerable variation within airway generations, $ theta$ clustered between $-$20$ sp circ$ and 10$ sp circ$ in all generations and did not vary significantly between generations in any of the subjects. When $ theta$ was converted to an acute angle without regard to sign($ Theta$), the mean angle was 12-13$ sp circ$ both in cat and the human lung. (Abstract shortened by UMI.)

By techniques of immunohistochemical staining, flow cytometry, and image analysis, the present study demonstrated the following: (1) The ST3 antigen is expressed on the surface of thymocytes of young rats, and decreases in both percentage and intensity with increasing age (after 12 months). (2) The ST3 antigen also is expressed on the surface of thymic nurse cells. Although the majority of lymphoid cells within nurse cells also bear the ST3 antigen, a subpopulation contains lymphocytes that do not. These results suggest that the ST3 antigen may be a useful new marker for cortical lymphocytes, and imply that its expression may play a role in intra-thymic T-cell development, and in senescence of the organ.

Cell adhesion molecules are thought to have an important role in neoplastic progression as they are likely to be involved in the multiple steps of the metastatic cascade. We have focused on two adhesion molecules, E-cadherin and CD44, looking for changes in their expression in human colon cancer. The intercellular adhesion mediated by E-cadherin has been shown to be altered in cancer. We have investigated the expression of E-cadherin in normal and tumorigenic colorectal mucosa by immunohistochemistry. Tumor samples showed a down-regulation of expression correlating with the degree of tumor dedifferentiation. The second adhesion molecule, CD44, has been previously associated with metastasis in animal models. We have shown by Northern blotting that mRNA splice variants with domains IV + V are specifically over expressed in carcinomas. Immunohistochemistry showed redistribution of CD44 to the cellular basal membrane. Thus, the aberrant expression of E-cadherin and CD44 could be associated with malignant progression in colorectal cancer.

No description available.

Cells are constantly challenged by acute and chronic stresses that must be counteracted by upregulation of protective pathways. The premise of this thesis is that motor neurons have an impaired ability to trigger these protective mechanisms, which may contribute to their preferential vulnerability in the neurodegenerative disease, amyotrophic lateral sclerosis (ALS). The first objective was to study the involvement of metallochaperones in motor neuronal stress response, including their potential for rescuing motor neurons from toxicity conferred by a mutant Cu/Zn-superoxide dismutase (SOD1G93A ) that causes a form of familial ALS. Motor neurons in dissociated spinal cord cultures failed to induce the metallochaperone, metallothionein (MT), in response to classical MT inducers, although overexpression of MT in motor neurons failed to protect them from SOD1G93A. A second response system, involving protein chaperones called heat shock proteins (Hsp), was more therapeutically promising, but was also impaired in motor neurons due to an inability to activate the regulatory protein heat shock transcription factor 1 (Hsf1). The remaining objectives were to examine if activation of Hsf1 in motor neurons would protect against SOD1G93A and to understand the mechanisms responsible for its impaired activation. A constitutively active form of Hsf1 induced multiple Hsps in motor neurons and nearly eliminated SOD1G93A toxicity and aggregation. Experiments also demonstrated that failure of stressed motor neurons to activate endogenous Hsf1 is not a result of inappropriate or insufficient activity of kinases that phosphorylate key residues of Hsf1 in nonneuronal cell lines with a competent heat shock response. Disruption of inhibitory Hsp90/multichaperone complexes is another important step in Hsf1 activation. Four different pharmacological inhibitors of Hsp90 induced multiple Hsps in motor neurons, although failure to observe the same response by targeting inhibitory complexes with activator of Hsp90 ATPase 1 (Aha1) or Daxx suggested other mechanisms were involved. A constitutively active form of calcium/calmodulin-dependent kinase N induced Hsp70 in motor neurons, but not in fibroblasts and likely through an Hsf1-independent mechanism. These results provide further evidence for disparity between the stress response of motor neurons and other cells and suggest the possibility of a unique Hsp regulatory system in neurons.

The normal lung with its air-filled alveoli is designed primarily to function in gas-exchange. Pulmonary edema, particularly in its alveolar flooding phase, is life-threatening because it interferes with this gas-exchange function. An important safety factor against alveolar flooding is the pulmonary interstitium that has a high compliance to accommodate edema fluid. We tested the hypothesis that if we decreased the compliance of the interstitium by experimental fibrosis, lung edema would be redistributed from the interstitium and predominantly flood alveoli. To study this, severe left lung fibrosis was produced in six dogs with radiation and bleomycin. Twenty-four months later, hydrostatic edema was induced by fluid infusion and lungs were studied pre- and post-edema with computed tomography (CT) scanning. Gravimetry and morphology were used to assess the amounts and distribution of edema between fibrotic left and control right lungs. Fibrosis, pre-edema, produced a 11-fold decrease in lung volume and a 2.2-fold increase in tissue density. We found, by CT and gravimetry, that similar amounts of water accumulated per unit volume in control and fibrotic lungs. Morphometry and semi-quantitative light microscopic grading showed a two-fold rise in the volume fractions of connective tissue and alveolar edema, and a 50% reduction of air and of interstitial edema in the fibrotic lobes. By electron microscopy, the interstitial edema in the fibrotic lungs was randomly distributed, whereas in the controls it was found primarily around extra-alveolar vessels and airways, not in the alveolo-capillary septa. We conclude that fibrosis profoundly affects the distribution of edema in the lung.

The pathophysiological origins of airway hyperresponsiveness (AHR) in asthma are unknown. The objectives of this thesis were to establish an association between AHR in an animal model of asthma and altered contractility of airway smooth muscle (ASM) and to elucidate changes in contractile signalling that could account for any observed differences in ASM contractility. The Fisher strain of rat is spontaneously hyperresponsive to methacholine inhalation challenge relative to Lewis rats. These inbred rat strains provide a model with which to study genetically-determined variations in airway smooth muscle that may underlie AHR. Fisher rats were found to be also hyperresponsive to serotonin (5-HT) in vivo compared to Lewis rats, indicating that their AHR is not agonist specific the narrowing capacity and velocity of contraction of Fisher intraparenchymal airways in cultured explants were also greater than explanted. Lewis intraparenchymal airways in response to 5-HT. In addition, 5-HT stimulated higher Ca2+ transients in Fisher than Lewis ASM, in parallel with their rank order of intraparenchymal airway responsiveness. These results suggest that ASM contractility may be determined by the extent of Ca2+ mobilization in airway myocytes. To examine the mechanism of enhanced intracellular Ca2+ mobilization in Fisher ASM, the role of the inositol (1,4,5)trisphospbate (Ins (1,4,5)P 3) pathway in determining interstrain differences in ASM Ca2+ was studied. 5-HT produced higher levels of Ins (1,4,5)P3 in Fisher than Lewis ASM. This appeared to be caused by a lower expression and activity in Fisher ASM of the type I and II 5-phosphatases which inactivate Ins (1,4,5)P3. Inhibition of 5-phosphatase activity increased Ins (1,4,5)P3-mediated Ca2+ release in ASM from both strains of rat, equalizing Ca2+ signals between Lewis and Fisher ASM. Since Ins (1,4,5)P3 receptor sensitivity to Ins (1,4,5)P 3 was found to be similar between the two rat strains, the differences in 5-phosphatas

The injury-induced alterations in sulfated proteoglycans (PG) were studied, in neointima, developed in response to a selective deendothelialization of the aorta, of normocholesterolemic rabbits. Light microscopic radioautography and size exclusion chromatography revealed differences in PG between neointima not covered by regenerated endothelium, and reendothelialized neointima or normal aorta. These differences included radioautographic reaction, concentration, size distribution and composition. Further studies were conducted to examine the putative role of the altered PG in lipoprotein (LP) sequestration and lipid accumulation during atherogenesis. LP-PG complexes were isolated by anti apo-B affinity column, from intima-medial tissues from normal and injured aortas. These complexes contained low density and very low density LP, chondroitin sulfate PG and hyaluronic acid. It appeared that endothelial injury enhanced the formation of LP-PG complexes, particularly in areas covered by regenerated endothelium. The uptake and degradation of LP-PG complexes, derived from normal (LP-NPG) or injured aortas (LP-IPG), by arterial smooth muscle cells (SMC) and blood monocyte-derived macrophages (BMDM) were also examined. LP-PG complexes stimulated LP binding, internalization and degradation by SMC and BMDM. Both cell types showed a higher affinity for LP-IPG than LP-NPG. The uptake of LP-PG complexes was mediated mainly by the LDL receptor pathway and phagocytosis. The scavenger receptor played a minor part in the uptake of LP-PG complexes. Data from this study provide evidence that endothelial injury could trigger alterations in neointimal PG, which in turn, facilitate LP accumulation both extracellularly and intracellularly during atherogenesis.

Secondary amyloidosis was induced in mice with daily consecutive injections of casein or in an accelerated form with the injection of the "Amyloid-Enhancing Factor" (AEF). Innate susceptibility to the disease operated at the level of the production of AEF. AEF activity was purified to one protein component with an apparent molecular weight of 55,100Da. Increased disappearance of the amyloid A (AA) fibril precursor apo-SAA2 did not correlate exactly with amyloid deposition. Multiple splenic macrophage phenotypes and functions were modulated during the two induction protocols. Splenic macrophages from normal or casein-injected mice could not degrade in vitro HDL$ sb3$-SAA2. The catabolism was induced by the exogenous addition of soluble AEF and/or Serum Amyloid P. Finally, AA and amyloid PO proteins were simultaneously deposited in the spleen with respect to time and tissue localization during the two induction protocols. In conclusion, the appearance of AEF in organs during the development of secondary amyloidosis was accompanied by the modulation of the splenic macrophage phenotype and function.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in developed countries. Aggressive research is underway to elucidate putative molecular targets for therapy for both the atrophic and neovascular forms of this disease. Current pharmacotherapy is effective in some patients but not sufficient to halt disease progression or repair damage that has already occurred. Drug intervention and retinal cell replacement represent the two most promising potential treatment avenues. The purpose of this thesis was to investigate the role of a recently identified regulator of neural development, SIRT1, in retinogenesis and to further investigate whether pharmacological inhibition of this protein represents a possible treatment option in neovascular AMD. Via immunohistochemistry and immunocytochemistry we evaluated the expression and subcellular localization of SIRT1 and its innate inhibitor, DBC1, in mouse and human fetal and adult retinas. We further studied SIRT1 in mouse- and human-derived retinal progenitor cells, the former being using in small interfering RNA studies. We found SIRT1 to be widely expressed in developing and adult retinas and to be a regulator of key retinal development genes, namely PAX6, Nestin and CRX. Moreover, we found that photoreceptor precursor cells were among the smallest cells in the heterogeneous population of mouse retinal progenitors. Collectively, these results provide the foundation for manipulating SIRT1 expression in small retinal progenitors as a means of increasing the yield of photoreceptors for transplantation in models of retinal degeneration.We further found SIRT1 to be highly expressed in human-derived choroidal neovascular membranes and sought to pharmacologically inhibit its activity via the drug Nicotinamide. We found Nicotinamide to be a potent regulator of angiogenic and hypoxic signaling in a human retinal pigment epithelial cell line at both the protein level using angiogenesis arrays and at the RNA level using whole genome microarrays. These results point to the SIRT1 inhibitor, Nicotinamide, as a possible agent for treatment of neovascular AMD. Further studies of Nicotinamide are warranted in animal models of AMD. To the best of our knowledge, this is the first time that a detailed analysis of SIRT1 as a regulator of both retinal development and choroidal neovascularization has been reported. / La dégénérescence maculaire liée à l'âge (DMLA) est la principale cause de cécité chez les personnes âgées dans les pays développés. Une recherche dynamique est en cours pour élucider des cibles moléculaires potentiels pour le traitement de la dégénérescence à la fois pour la forme atrophique et la forme néovasculaire de cette maladie. L'actuelle pharmacothérapie est efficace chez certains patients mais pas suffisante pour arrêter la progression de la maladie ou la réparation des dommages qui ont déjà eu lieu. La decouverte de nouveaux medicaments et le remplacement de cellules rétiniennes représentent les deux avenues les plus prometteuses de traitements possibles. Le but de cette thèse est d'étudier le rôle d'un régulateur récemment identifié du développement neuronal, SIRT1, dans le developpement de la retine et de rechercher si l'inhibition pharmacologique de cette protéine représente une option de traitement possible dans la DMLA. Via l'immunohistochimie et l'immunocytochimie, nous avons évalué l'expression et la localisation subcellulaire de SIRT1 et de son inhibiteur inné, DBC1, chez la souris et les humains dans les retines fœtales et adultes. Nous avons également étudié SIRT1 dans les cellules souches de la rétine chez la souris et l'homme. Nous avons trouvé SIRT1 largement exprimé dans la rétine en développement et des adultes et à un régulateur de gènes clés du développement de la rétine, à savoir PAX6, Nestin et CRX. En outre, nous avons constaté que les cellules précurseurs des photorécepteurs ont été parmi les plus petites cellules dans la population hétérogène de cellules progénitrices. Collectivement, ces résultats fournissent la base pour la manipulation de l'expression SIRT1 dans les petits progéniteurs rétiniens comme un moyen d'augmenter le rendement des photorécepteurs à la transplantation dans les modèles de dégénérescence rétinienne. Nous avons en outre constaté que SIRT1 est fortement exprimé dans les membranes néovasculaires humaines et avons cherché à inhiber son activité pharmacologique par le nicotinamide. Nous avons trouvé que Nicotinamide est un puissant régulateur de l'hypoxie et de l'angiogenèse au niveau de la protéine et de l'ARN. Ces résultats indiquent que l'inhibiteur de SIRT1, Nicotinamide est un agent possible pour le traitement de la DMLA néovasculaire. D'autres études de la nicotinamide devraient être poursuivit dans des modèles animaux de la DMLA. Au meilleur de notre connaissance, c'est la première fois qu'une analyse détaillée de SIRT1 l'identifie comme un régulateur du développement tant de la rétine et de la néovascularisation choroïdienne.