Purpose: To model the time evolution of active caspase-3 protein expression in a healthy lens, and in a lens exposed to UVR-300 nm (UVR-B). To develop an automated method to classify the fluorescent signal of biomarkers in the lens epithelial cells. Methods: Six-week old Sprague-Dawley rats were used. Firstly, expression of active caspase-3 was studied in the lens epithelium of healthy rats. Secondly, rats were unilaterally exposed in vivo to 1 kJ/m2 UVR-B for 15 minutes. At 0.5, 8, 16, and 24 hours after the UVR-B exposure, the exposed and the contralateral non-exposed lenses were removed. Immunohistochemistry was done on three mid-sagittal sections from each lens. The florescent labelling for active caspase-3 in each lens section was counted three times. The time evolution of active caspase-3 expression in response to UVR-B exposure was modelled as a function of cell position in the lens epithelium. An automated objective method was developed to quantify the lens epithelial cells and to classify the fluorescent signal of active caspase-3. Active caspase-3 was selected as a model signal. Results: Active caspase-3 was abundant in the anterior pole of the normal lenses. Spatial distribution of active caspase-3 labelling in the lens epithelium was fitted to a logistic model. The probability of active caspase-3 expression was higher in the UVR-B exposed lenses (95% CI = 0.12 ± 0.01). There was no difference in the expression of active caspase-3 between the 0.5 and the 24 hours groups or between the 8 and the 16 hours groups. A difference was noted, when comparing the 0.5 and 24 hours groups with the 8 and 16 hours groups (Test statistic 7.01, F1;36;0.95= 4.11). Exposure to UVR-B has an impact on the average probability of labelling for active caspase-3 as a function of cell position. The probability of labelling as a function of cell number also varied as a function of time after UVR-B exposure. The automated method counted the lens epithelial cells and estimated the proportion of active caspase-3 labelling in the lens epithelium. Conclusions: Active caspase-3 is present in the healthy lens epithelial cells. Active caspase-3 exhibits higher expression at the anterior pole of the lens and the expression decreases towards the periphery. After UVR-B exposure, the expression of active caspase-3 in the lens epithelium increases with a peak of expression occurring around 16 hours after exposure. The average probability of labelling in the lens epithelium is dependent on both the UVR-B exposure and the time period elapsed after the exposure. The automated method enables objective and fast quantification of lens epithelial cells and the expression of fluorescent signal in the lens cells.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Also available online. Includes bibliographical references (leaves 93-111).

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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Apart from CAD, the transient liberation of AIF during day 6 of TF-1 differentiation could pose another threat to the genomic DNA in cells. We have demonstrated the absence of AIF in the nucleus of TF-1 cells despite its release from the mitochondria by using confocal studies. Moreover, the expression of heat shock protein 70 kDa (Hsp70), a well-known antagonist of AIF, was found to be temporarily increased at day 6. Taken together, our results implied a plausible retention of AIF in the cytoplasm by Hsp70. Although Hsp70 is commonly utilized by many cancer cells to counteract AIF and avoid DNA fragmentation, we are the first to demonstrate its role in suppressing AIF during normal erythroid maturation. / As a whole, we have illustrated that the activated caspase-3, mediated most likely by the mitochondrial pathway, is an essential component in the differentiation of TF-1 cells. Its activation was nevertheless not coupled with DNA fragmentation due to some protective mechanisms such as CAD downregulation, Hsp70 upregulation and overexpression of Bcl-XL. Our study therefore provides some insights in the understanding of the relationship between human erythropoiesis and apoptosis and a better understanding in this regard will undoubtedly facilitate the development of new drugs in the treatment of different hematopoietic diseases. / Caspases play a central role in apoptosis. Their activations during the process are accounted for different biochemical and morphological changes in apoptotic cells. Yet in recent years, increasing studies had shown that caspases were also involved in some non-apoptotic cellular events, including T and B-lymphocytes activation, as well as the terminal differentiation of lens cells, megakaryocytes and erythrocytes. / In order to find out other unknown cellular mechanisms in erythropoiesis, mRNA differential display was employed to compare the gene expression pattern of TF-1 cells at different stages of differentiation. Several differentially expressed genes were identified and subsequently confirmed by RT PCR. These genes include formin binding protein 3, destrin and T-complex protein-1 (TCP-1). Their involvement in erythroid differentiation was still not clear at the moment but would be investigated in the near future. Furthermore, aiming at identifying the interacting proteins or inhibitors of caspase-3 in the system, a pull down assay was developed by means of the bacterial expression of a recombinant human caspase-3 mutant protein. With the mutation in the active site, the binding of our recombinant caspase-3 mutant with two known partners ICAD and BIRII (Baculovirus Inhibitor of apoptosis protein Repeat II) domain has been demonstrated. We hope in the near future that it can be employed to fish out some novel caspase-3 substrates from the differentiating TF-1 cell lysate. / In the present study, the participation of caspase in in vitro erythropoiesis was investigated using a human erythroleukemia cell line TF-1. Erythropoietin (EPO) induced erythroid maturation of TF-1 as indicated by the expression of erythroid-lineage markers like glycophorin A (GPA), transferrin receptors (CD71) and synthesis of hemoglobin (Hb). Activation of caspase-3 was observed from day 6 to day 12 during TF-1 differentiation after EPO treatment. With the administration of caspase-3 specific inhibitor, expressions of GPA and CD71 were partially blocked, suggesting that caspase-3 activation is essential in erythropoiesis in our TF-1 model. / Possible involvement of the intrinsic and extrinsic apoptotic pathways was studied by investigating respectively the activation of pro-caspase-9 and -8. It was found that caspase-9, but not -8, was activated at the corresponding time point when caspase-3 was activated. Besides, a transient mitochondrial depolarization coupled with the release of cytochrome c and apoptosis inducing factor (AIF) were detected on day 6, strongly implying a role of mitochondria in triggering the activation of executioner caspase-3. On the other hand, GPA and CD71 expressions were blocked by the application of mitochondrial depolarization inhibitor cyclosporin A (CyA). Also, the recovery of mitochondrial membrane potential was found to be correlated with an overexpression of Bcl-XL at a late stage of TF-1 differentiation, and the role of Bcl-XL was subsequently manifested further by a significant retardation of erythroid differentiation in the siRNA Bcl-XL knocked down TF-1 cells. / The exact role of caspase-3 in erythroid differentiation is far from clear at this moment. Yet, its regulation in the process is equally intriguing. On the course of TF-1 maturation, activated caspase-3 was able to cleave and de-localize the Inhibitor of Caspase-activated DNase (ICAD) from the nucleus, but at the same time DNA fragmentation was not detected by TUNEL assay nor agarose electrophoresis. Furthermore, protection against DNA fragmentation was observed in the EPO-treated TF-1 cells when challenged with a potent apoptotic inducer staurosporine (STS). These observations are in contrast to our understanding that DNA is fragmented by CAD (Caspase-activated DNase) when ICAD in the ICAD-CAD complex is cleaved by caspase-3. For these apparently contradictory observations, we demonstrated that downregulation of CAD occurred at the mRNA and protein levels during the erythroid differentiation in TF-1. This provides a cell rescuing mechanism in non-apoptotic cells with activated caspases. / Lui Chun Kin Julian. / "September 2006." / Adviser: Siu Kai Kong. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1620. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 239-253). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

<p>The nasal passages are constantly exposed to both air- and bloodborne foreign compounds. In particular, the olfactory mucosa is demonstrated to be susceptible to a variety of drugs and chemicals. In this thesis, mechanisms involved in tissue-selective toxicity in the olfactory mucosa of rodents have been investigated using the olfactory toxicant 2,6-dichlorophenyl methylsulphone (2,6-diClPh-MeSO₂) as a model compound. Comparative studies were performed with the non-toxic 2,5-dichlorophenyl methylsulphone (2,5-diClPh-MeSO₂) and the reasons for the strikingly different toxicity were investigated. </p><p>A strong bioactivation and protein adduction of 2,6-diClPh-MeSO₂ in olfactory microsomes and S9-fractions of rodents was demonstrated. In contrast, no significant metabolic activation of 2,5-diClPh-MeSO₂ was observed and the bioactivation in the liver for both chlorinated isomers was negligible. <i>In vitro</i> studies with recombinant yeast cell microsomes expressing mouse cytochrome P450 2A5 (CYP2A5) demonstrated a metabolic activation of 2,6-diClPh-MeSO₂. The 2,6-diClPh-MeSO₂-induced lesions and CYP2A5 expression preferentially occurred in Bowman’s glands and sustentacular cells of the olfactory mucosa. A significant depletion of glutathione (GSH) in the olfactory mucosa was demonstrated <i>in vivo</i>, while no changes were observed in the liver. There was a rapid induction of the endoplasmic reticulum (ER)-specific chaperone Grp78, activation of the ER-specific caspase-12 and the downstream caspase-3 in the Bowman’s glands. Electron microscopy revealed swelling of ER and mitochondria and a lost integrity of the Bowman’s glands. </p><p>Based on these results, the proposed mechanism for 2,6-diClPh-MeSO₂-induced toxicity in the olfactory mucosa is bioactivation by CYP2A5 into a reactive intermediate causing protein adduction and GSH-depletion. This is initiating a sequence of downstream events of ER-stress, changes in ion homeostasis, ultrastructural organelle disruption and apoptotic signalling. In spite of the initial apoptotic signals, the terminal phase of apoptosis seemed to be blocked and necrotic features occurred. The predominant expression of CYP2A5 in the olfactory mucosa is proposed to play a key role for the tissue- and cell-specific toxicity induced by 2,6-diClPh-MeSO₂.</p>

The nasal passages are constantly exposed to both air- and bloodborne foreign compounds. In particular, the olfactory mucosa is demonstrated to be susceptible to a variety of drugs and chemicals. In this thesis, mechanisms involved in tissue-selective toxicity in the olfactory mucosa of rodents have been investigated using the olfactory toxicant 2,6-dichlorophenyl methylsulphone (2,6-diClPh-MeSO2) as a model compound. Comparative studies were performed with the non-toxic 2,5-dichlorophenyl methylsulphone (2,5-diClPh-MeSO2) and the reasons for the strikingly different toxicity were investigated. A strong bioactivation and protein adduction of 2,6-diClPh-MeSO2 in olfactory microsomes and S9-fractions of rodents was demonstrated. In contrast, no significant metabolic activation of 2,5-diClPh-MeSO2 was observed and the bioactivation in the liver for both chlorinated isomers was negligible. In vitro studies with recombinant yeast cell microsomes expressing mouse cytochrome P450 2A5 (CYP2A5) demonstrated a metabolic activation of 2,6-diClPh-MeSO2. The 2,6-diClPh-MeSO2-induced lesions and CYP2A5 expression preferentially occurred in Bowman’s glands and sustentacular cells of the olfactory mucosa. A significant depletion of glutathione (GSH) in the olfactory mucosa was demonstrated in vivo, while no changes were observed in the liver. There was a rapid induction of the endoplasmic reticulum (ER)-specific chaperone Grp78, activation of the ER-specific caspase-12 and the downstream caspase-3 in the Bowman’s glands. Electron microscopy revealed swelling of ER and mitochondria and a lost integrity of the Bowman’s glands. Based on these results, the proposed mechanism for 2,6-diClPh-MeSO2-induced toxicity in the olfactory mucosa is bioactivation by CYP2A5 into a reactive intermediate causing protein adduction and GSH-depletion. This is initiating a sequence of downstream events of ER-stress, changes in ion homeostasis, ultrastructural organelle disruption and apoptotic signalling. In spite of the initial apoptotic signals, the terminal phase of apoptosis seemed to be blocked and necrotic features occurred. The predominant expression of CYP2A5 in the olfactory mucosa is proposed to play a key role for the tissue- and cell-specific toxicity induced by 2,6-diClPh-MeSO2.

Exposure cigarette smoke (CS) during prenatal life is the leading cause of preventable premature death. In this study, we explored the hypothesis that in vitro exposure of fetal lung cells to cigarette smoke extract (CSE) may result in the alteration of apoptosis through activation of caspase-3. Alongside we compared the responses of fetal lung cells with A549 cells and rat periodontal ligament (PDL) fibroblasts exposed to CSE in a dose dependent manner. Caspase-3 activity and inhibition was measured using a fluorometric assay. Cell viability in smoke exposed cells was measured using MTT formazan assay. Caspase-3 expression and cellular localization was detected by western blot analysis and immunofluorescence. Our results indicate that caspase-3 activity was significantly (p < 0.05) elevated and cell viability was significantly inhibited in fetal rat lung cells exposed to 10% or 15 % (v/v) CSE. No significant differences were observed in the caspase-3 activity or cellular viability in A549 cells and rat PDL fibroblasts exposed to 5%, 10% or 15% (v/v) CSE. Activation of caspase-3 in fetal lung connective tissue and alveolar epithelial cells may be one of the reasons for the developmental pulmonary toxicity induced by CSE.

Exposure cigarette smoke (CS) during prenatal life is the leading cause of preventable premature death. In this study, we explored the hypothesis that in vitro exposure of fetal lung cells to cigarette smoke extract (CSE) may result in the alteration of apoptosis through activation of caspase-3. Alongside we compared the responses of fetal lung cells with A549 cells and rat periodontal ligament (PDL) fibroblasts exposed to CSE in a dose dependent manner. Caspase-3 activity and inhibition was measured using a fluorometric assay. Cell viability in smoke exposed cells was measured using MTT formazan assay. Caspase-3 expression and cellular localization was detected by western blot analysis and immunofluorescence. Our results indicate that caspase-3 activity was significantly (p < 0.05) elevated and cell viability was significantly inhibited in fetal rat lung cells exposed to 10% or 15 % (v/v) CSE. No significant differences were observed in the caspase-3 activity or cellular viability in A549 cells and rat PDL fibroblasts exposed to 5%, 10% or 15% (v/v) CSE. Activation of caspase-3 in fetal lung connective tissue and alveolar epithelial cells may be one of the reasons for the developmental pulmonary toxicity induced by CSE.

Dissertation (Ph.D.)--University of South Florida, 2008. / Title from PDF of title page. Document formatted into pages; contains 111 pages. Includes vita. Includes bibliographical references.