Doctor of Philosophy (PhD) / GABAA receptors are implicated in the pathology of psychiatric disorders such as schizophrenia and depression. They are rapidly affected by stress in a sex-dependent fashion, suggesting that GABAA receptors may be relevant to understanding the association between stress and psychiatric disorders. Thus, this thesis examined how GABAA receptors are affected in both male and female mice exposed to stress in adulthood (Chapter 2), early-life (Chapter 3-5) and a combination of both early-life and adulthood stress (Chapter 6). 2. The effects of acute adulthood stress (3 minute warm swim stress) on GABAA receptor binding in the brains of male and female mice were examined using quantitative receptor autoradiography. The total number of GABAA receptor [3H]GABA binding sites was increased following swim stress in specific forebrain cortical regions of female mice swum individually or in a group, but decreased in male mice when swum in a group only. These findings confirm and extend previous studies, identifying the cortical regions involved in rapid stress-induced changes in GABAA receptors. 3. Post-natal handling models in rodents comparing control (brief handling sessions; EH) with no intervention stress conditions (NH), indicate that the NH condition results in an anxious adulthood phenotype and this was confirmed in the present thesis using the elevated plus-maze behavioural test. Using this model the effects of early-life stress on adulthood GABAA receptors were then examined. 4. Regional densities of GABAA receptor α1 and α2 subunit proteins were observed in the adult brain of male and female mice using immunoperoxidase histochemistry. NH males showed a loss of the α2 subunit from the thalamus and the lower layers (IV-VI) of the primary somatosensory cortex, whilst NH females showed a reduction of α2 but an increase in α1 protein in the lower layers of the primary somatosensory cortex only. These regionally specific alterations in the α1:α2 subunit ratio suggest that early-life stress disrupts the developmental α subunit switch, which occurs in a regionally-dependent fashion over the first two weeks of rodent life. 5. Double-labelling immunofluorescence and confocal microscopy were used to examine the effects of sex and early-life stress on GABAA receptor synaptic clustering. Regardless of sex, mice exposed to early-life stress (NH) showed reduced colocalisation of the GABAA receptor α2 subunit with the synaptic marker protein gephyrin relative to the control condition (EH). This suggests that early-life stress impairs adulthood inhibitory synaptic strength and is consistent with the increased anxiety of the stressed relative to control mice. 6. Finally, the effects of early-life stress on adulthood swim stress-induced changes in GABAA receptor binding were examined using quantitative receptor autoradiography in forebrain cortical regions. Findings showed that the effect of adulthood stress on the total number of GABAA receptor binding sites for [3H]GABA in forebrain cortical regions was altered by early-life stress in both male and female mice, suggesting that the rapid adulthood stress response of GABAA receptors is affected by early-life experience. 7. Together these results show that GABAA receptors are sensitive to subtle changes in the environment in both early-life and adulthood and that these neurochemical responses to stress in adulthood are sex-dependent. The short and long-term stress-sensitivity of the GABAergic system implicates GABAA receptors in the non-genetic aetiology of psychiatric illnesses in which sex and stress are important factors.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Includes bibliographical references (p. 196-235).

Made available in DSpace on 2014-06-11T19:23:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-07-20Bitstream added on 2014-06-13T20:11:29Z : No. of bitstreams: 1 vitaliano_sn_me_jabo.pdf: 2132952 bytes, checksum: f71337d8a4f675ed29371324f60f293f (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O presente trabalho objetivou estudar a susceptibilidade do carcará (Caracara plancus) frente à infecção experimental pela amostra ME49 de Toxoplasma gondii pela observação da sintomatologia clínica, análise dos parâmetros hematológicos, estudo da cinética de anticorpos IgG anti-T. gondii, e constatação da presença do parasito nos diversos tecidos utilizando a imunoistoquímica, imunofluorescência direta em tecidos, bioensaio em camundongos e PCR. Sete carcarás soronegativos para anticorpos anti-T. gondii foram separados em dois grupos: G1 (grupo infectado - 5 aves) e G2 (grupo controle - 2 aves). As aves do grupo G1 foram alimentadas durante três dias consecutivos com roedores Calomys callosus previamente infectados com T. gondii e as aves do grupo G2 foram alimentadas com roedores não infectados. As aves infectadas não apresentaram sintomas clínicos de toxoplasmose, tampouco alterações nos parâmetros hematológicos. Todas as aves infectadas soroconverteram. A soroconversão das aves experimentalmente infectadas ocorreu a partir do sétimo dia pós-infecção, os picos iniciais na produção de anticorpos IgG anti-T. gondii ocorreram entre 15 e 30 dias pós-infecção sendo que em seguida houve uma tendência de queda dos títulos de anticorpos. No momento da necropsia não foram encontradas lesões sugestivas da infecção por T. gondii. Não foi observado no exame histopatológico a presença do parasito nos diversos tecidos amostrados das aves infectadas, entretanto em uma das aves do grupo não infectado e sorologicamente negativa para anticorpos anti-T. gondii, foi encontrado um cisto tecidual na musculatura. Pelas técnicas de imunoistoquímica e imunofluorescência direta em tecidos o parasito foi encontrado em pequeno número em diversos tecidos, porém tanto nas aves do grupo infectado quanto nas aves do grupo controle. Utilizando o bioensaio em camundongos... / The present study aimed to evaluate the susceptibility of crested caracara (Caracara plancus) to experimental infection with ME49 strain of Toxoplasma gondii, through clinical symptoms, hematological parameter analysis, kinetics of specific IgG antibodies to T. gondii, and parasite detection by immunohistochemistry, direct immunofluorescence, bioassay, and PCR. Seven crested caracara, seronegative to T. gondii antibodies, were separated into two groups: G1 (infected group - 5 birds), and G2 (control group - 2 birds). G1 birds were fed during three successive days with Calomys callosus previously infected with T. gondii, and G2 birds were fed with non-infected rodents. The infected birds did not present clinical symptoms, nor significant changes in hematological parameters. All infected birds had antibody titers to T. gondii. IgG antibodies to T. gondii were first detected at 7 dpi, with production peaks between 15 and 30 dpi, which were followed by a sharp decrease in detectable antibodies. No gross lesions sugestive of T. gondii infection were observed. By histopatological examination, it was not possible to observe the presence of the parasite in the infected birds' organs, however, in one bird of the control group, a T. gondii tissue cyst was visualized in muscle. By immunohistochemistry and direct immunofluorescence, the parasite was found in small number in different organs, although in animals from both groups. The presence of T. gondii was confirmed in heart samples of two bioassayed birds, both from the infected group. In this study, the experimentally infected crested caracaras did not show clinical symptoms, alteration on hematological parameters and the parasite was found in small numbers in the organs of the birds, for these reasons the crested caracara were resistant to T. gondii infection.

Non Melanoma Skin Cancer (NMSC) affects 3.3 million Americans each year and results from Ultra Violet Radiation (UVR) damage to DNA in the form of pyrimidine dimers and photoproducts [1]–[5]. Cells directly detect the damage and initiate apoptosis, cell cycle arrest, or DNA repair by modulating p53 and p21 levels [6]–[9]. Current methods of photoprotection include sunscreen, but controversy over safety of some active ingredients necessitates research into more natural alternatives [10]–[12]. In particular, 24 hour incubation with bovine milk sphingomyelin (BSM) has demonstrated photoprotective potential by reducing p21 and p53 levels in keratinocytes (KRTs) after UV radiation [13], [14]. This thesis aims to expand on past BSM research by exploring the mechanism for photoprotection. Normally, sphingomyelin (SM) is metabolically degraded to ceramide which then leads to cell apoptosis [6]. The goals of this thesis were to characterize a fluorescent SM (FSM) to assess changes in intracellular fluorescence distribution after various incubation and post-UV exposure times. FSM was deemed functionally equivalent to BSM by reducing levels of p21 after UV. Furthermore, quantification demonstrated that FSM trafficking and intracellular fluorescence were independent of continuous incubation time, warranting further investigation into shorter timepoints like 1 hour. Across several post-UV timepoints, the 1 hour incubation had a consistently higher average cytoplasmic mean gray value compared to 24 hour incubation. In addition, the no UV control was significantly lower compared to the 24 hour and 12 hour post-UV timepoints. No post-UV differences were observed for the 24 hour incubation, suggesting future work is necessary for the 1 hour incubation, which potentially streamlines future experiments. Two immunofluorescence stains for endogenous SM (lysenin) and ceramide were also optimized for preliminary fluorescence distribution studies and colocalization with FSM. Finally, a 3T3 fibroblast spheroid model was utilized as proof-of-concept for future 3D KRT cultures and depth of dye penetration quantification methods. These findings suggest FSM is an appropriate model for BSM trafficking, a shorter FSM incubation time could potentially be adopted in future studies, dual immunofluorescence staining for SM and ceramide is viable, and spheroids provide a promising model for future 3D KRT studies.

Non-melanoma skin cancer (NMSC) is the most common malignant tumor, representing more than a third of all malignant tumors combined and the incidence is increasing every year. Ultraviolet (UV) radiation from the sun is the most dominant factor contributing to tumor initiation and progression. The condition is most prevalent in populations with lighter skin and older age. Current pharmaceutical molecular research targets the inhibition of the Epidermal Growth Factor Receptor (EGFR), a receptor which is commonly over-expressed or dysregulated in skin malignancies. This study evaluates the content and location of the damage marker p21 within keratinocytes that were incubated in sphingomyelin (SM) and later exposed to UV. Confocal microscopy and automated image processing provided the tools to assess large populations of keratinocytes in the effort to accurately identify the photoprotective qualities of sphingomyelin. Classification of individual cells into subpopulations yielded results suggesting SM may be involved in the inhibition of EGFR, and could potentially be a more naturally derived treatment.

No description available.

Peptidoglycan not only protects bacterial cells against intracellular pressure but also provides the cells with a defined morphology. Penicillin-binding proteins (PBPs) catalyze the polymerization of the peptidoglycan in Bacillus subtilis. PBP2a and PBP2b are class B PBPs which have been known to have transpeptidase activities and they localize at different positions on the cell membrane. PBP2a spreads around the cylindrical wall as well as some at the septum, and PPB2b localizes exclusively to the septum and some at the cell poles. Both PBP2a and PBP2b are composed of four domains: S, N, P, and C domains from the N- to C- terminus. A FLAG epitope was tagged to the C-terminal ends of PBP2a and PBP2b. Cells with FLAG tagged PBP2a or PBP2b grow as well as wild type strain. Expression of PBP2a-FLAG and PBP2b-FLAG can be detected by western blotting using anti FLAG antibody. The expression of wild type PBP2a/PBP2b in these strains was tightly controlled by a xylose promoter. The FLAG fusion didn't influence the normal membrane localizations of PBP2a or PBP2b. PBP2a/2b mutant strains with the S and/or N domains switched between PBP2a and PBP2b were constructed. All these domain-switch proteins were tagged with a FLAG at the C-terminus. The expression of these recombinant proteins can be detected by western blotting. None of these domain-switch proteins was able to complement the wild type PBP2a and PBP2b and cells with only these recombinant proteins but no wild type proteins were non-viable. Cellular localization of these domain switch proteins were visualized using immunofluorescence microscopy. Proteins containing the PBP2a S domain had the same localization patterns as wild type PBP2a. Proteins that have the PBP2b S domain localized specifically at the septum and cell poles, which is similar to the wild type PBP2b. These results indicate that the S domain is the determinant to direct PBP2a and PBP2b to their cellular destinations. / Master of Science

The developing brain is sensitive to chemical exposures, however, currently there is a lack of good, regulatory accepted methods to study developmental neurotoxicity (DNT). The cell line C17.2 can be used as a model for DNT studies since it has the capacity to differentiate into neuronal and neuroglial populations. However, in a recent experiment, single cell RNA sequencing (scRNAseq) indicated that the cell line might contain a good proportion of fibroblasts. The aim of this project was to complement the scRNAseq with staining for protein markers for specific cell types in order to elucidate the cellular composition of differentiated and undifferentiated C17.2 cultures. The markers Hyaluronan-Mediated Motility Receptor (HMMR), vimentin and Platelet Derived Growth Factor Receptor A (PDGFRA) were identified as promising markers for radial glial cells (RGC) and subsequently fibroblast and validated by immunofluorescence. HMMR by itself was used as a marker for RGC and was, as expected, decreasing during the differentiation process. Co-localization of vimentin and PDGFRA was used as markers for fibroblast. However, the conditions for the PDGFRA-antibody could not be optimized due to time restraints. Thus no specific staining could be obtained and no conclusions could be drawn regarding the presence or absence of fibroblasts in the culture. The results emphasize the need for more optimisation or the selection of more specific markers, e.g. Collagen type I alpha 1 (Col1a1). Further, these findings highlight the complexity of the cellular composition and the need for other methods to characterize C17.2.

Le cancer de la prostate (CaP) est le plus diagnostiqué chez les hommes au Canada et représente le troisième cancer le plus meurtrier au sein de cette population. Malgré l’efficacité des traitements de première ligne, de nombreux patients finiront par développer une résistance et, le cas échéant, verront leur CaP progresser vers une forme plus agressive. Plusieurs paramètres, essentiellement cliniques, permettent de prédire la progression du CaP mais leur sensibilité, encore limitée, implique la nécessité de nouveaux biomarqueurs afin de combler cette lacune. Dans cette optique nous nous intéressons au facteur de transcription NF-κB. Des études réalisées au laboratoire et ailleurs, associent RelA(p65) à un potentiel clinique dans le CaP, soulignant ainsi l’importance de la voie classique NF-κB. L’implication de la voie alternative NF-κB dans la progression du CaP a aussi été suggérée dans une de nos études illustrant la corrélation entre la distribution nucléaire de RelB et le score de Gleason. Alors que la voie classique est largement documentée et son implication dans la progression du CaP établie, la voie alternative, elle, reste à explorer. La présente thèse vise à clarifier l’implication de la voie alternative NF-κB dans le CaP et répond à deux objectifs fixés dans ce but. Le premier objectif fut d’évaluer l’impact de l'activation de la voie alternative NF-κB sur la biologie des cellules cancéreuses prostatiques. L’étude de la surexpression de RelB a souligné les effets de la voie alternative NF-κB sur la prolifération et l'autophagie. Étant ainsi impliquée tant dans la croissance tumorale que dans un processus de plus en plus associée à la progression tumorale, quoique potentiellement létal pour les cellules cancéreuses, son impact sur la tumorigénèse du CaP reste encore difficile à définir. Il n'existe, à ce jour, aucune étude permettant de comparer le potentiel clinique des voies classique et alternative NF-κB. Le second objectif de ce projet fut donc l'analyse conjointe de RelA(p65) et RelB au sein de mêmes tissus de patients atteints de CaP afin de déterminer l'importance clinique des deux signalisations NF-κB, l'une par rapport à l'autre. Le marquage immunofluorescent de RelA(p65) et RelB en a permis l'analyse quantitative et objective par un logiciel d'imagerie. Nos travaux ont confirmé le potentiel clinique associé à RelA(p65). La variable RelA(p65)/RelB s’est, elle, avérée moins informative que RelA(p65). Par contre, aucune corrélation entre RelB et les paramètres cliniques inclus dans l'étude n’est ressortie. En définitive, mon projet de thèse aura permis de préciser l'implication de la voie alternative NF-κB sur la biologie du CaP. Son impact sur la croissance des cellules cancéreuses prostatiques ainsi que sur l'autophagie, dénote l’ambivalence de la voie alternative NF-κB face à la tumorigénèse du CaP. L’étude exhaustive de la signalisation NF-κB souligne davantage l'importance de la voie classique dont l’intérêt clinique est principalement associé au statut de RelA(p65). Ainsi, bien que RelB n’affiche aucun potentiel en tant que biomarqueur exploitable en clinique, l’analyse de l’intervention de la voie alternative NF-κB sur la biologie des cellules cancéreuses prostatiques reste d’intérêt pour la compréhension de son rôle exact dans la progression du CaP. / Prostate cancer (PCa) is the most frequently diagnosed cancer and represents the third cause of cancer-death in Canadian men. Despite effective first-line therapies, many patients experience disease recurrence where PCa progresses toward a more aggressive form. Several parameters, largely clinical, have been used to predict the progression of PCa but their accuracy is still limited and implies the need for new biomarkers to fill this gap. Previous research from the laboratory has demonstrated that the transcription factor NF-B, and its nuclear localization, could be such a prognostic biomarker. Studies in our laboratory and elsewhere have correlated RelA(p65) with a clinical progression in PCa, underlining the importance of the classical NF-B pathway. The involvement of the alternative NF-B pathway in the progression of PCa was also suggested in one of our studies showing the correlation between the nuclear distribution of RelB and Gleason score. While the classical pathway is well documented and its involvement in the PCa progression established, the alternative NF-B pathway remains largely unexplored. This thesis describes two research objectives that aims to clarify the involvement of the alternative NF-B pathway in PCa. The first objective assessed the impact of the alternative NF-B pathway activation on the biology of PCa cells. RelB overexpression in 22RV1 PCa cells highlighted an effect of the alternative NF-B pathway on cell proliferation and autophagy. Its dual role in cell growth and a form of cell death requires further study to understand the balance of these in PCa tumorigenesis. To date no study has addressed the comparative prognostic potential of both the classical and alternative NF-B pathways simultaneously. Therefore the second objective of this research project was to analyze both RelA(p65) and RelB at a cellular level in the same tissue of patients with PCa to determine their unique and combined contribution to predicting biochemical recurrence in patients. This analysis was possible through immunofluorescent labeling of RelA (p65) and RelB, and was followed by a quantitative and objective analysis using an appropriate software. Our work confirmed the predictive value of RelA(p65) for biochemical recurrance. Combining RelA(p65) with RelB weakened the association, and RelB on its own was not found to predict biochemical recurrance. Ultimately, the research presented here has clarified the involvement of the alternative NF-B pathway on the biology of PCa. Its impact on the growth of PCa cells as well as autophagy reveals the dual role of the alternative NF-B pathway in PCa tumorigenesis. This exhaustive study of NF-B in PCa tissues further underscores the importance of the classical pathway whose clinical interest is mainly associated with RelA(p65) status. Thus, although RelB shows no potential as a clinically exploitable biomarker, further studies are needed to determine whether RelB contributes, either positively or negatively, and in a temporal fashion, to PCa progression.

Le cancer de la prostate (CaP) est le plus diagnostiqué chez les hommes au Canada et représente le troisième cancer le plus meurtrier au sein de cette population. Malgré l’efficacité des traitements de première ligne, de nombreux patients finiront par développer une résistance et, le cas échéant, verront leur CaP progresser vers une forme plus agressive. Plusieurs paramètres, essentiellement cliniques, permettent de prédire la progression du CaP mais leur sensibilité, encore limitée, implique la nécessité de nouveaux biomarqueurs afin de combler cette lacune. Dans cette optique nous nous intéressons au facteur de transcription NF-κB. Des études réalisées au laboratoire et ailleurs, associent RelA(p65) à un potentiel clinique dans le CaP, soulignant ainsi l’importance de la voie classique NF-κB. L’implication de la voie alternative NF-κB dans la progression du CaP a aussi été suggérée dans une de nos études illustrant la corrélation entre la distribution nucléaire de RelB et le score de Gleason. Alors que la voie classique est largement documentée et son implication dans la progression du CaP établie, la voie alternative, elle, reste à explorer. La présente thèse vise à clarifier l’implication de la voie alternative NF-κB dans le CaP et répond à deux objectifs fixés dans ce but. Le premier objectif fut d’évaluer l’impact de l'activation de la voie alternative NF-κB sur la biologie des cellules cancéreuses prostatiques. L’étude de la surexpression de RelB a souligné les effets de la voie alternative NF-κB sur la prolifération et l'autophagie. Étant ainsi impliquée tant dans la croissance tumorale que dans un processus de plus en plus associée à la progression tumorale, quoique potentiellement létal pour les cellules cancéreuses, son impact sur la tumorigénèse du CaP reste encore difficile à définir. Il n'existe, à ce jour, aucune étude permettant de comparer le potentiel clinique des voies classique et alternative NF-κB. Le second objectif de ce projet fut donc l'analyse conjointe de RelA(p65) et RelB au sein de mêmes tissus de patients atteints de CaP afin de déterminer l'importance clinique des deux signalisations NF-κB, l'une par rapport à l'autre. Le marquage immunofluorescent de RelA(p65) et RelB en a permis l'analyse quantitative et objective par un logiciel d'imagerie. Nos travaux ont confirmé le potentiel clinique associé à RelA(p65). La variable RelA(p65)/RelB s’est, elle, avérée moins informative que RelA(p65). Par contre, aucune corrélation entre RelB et les paramètres cliniques inclus dans l'étude n’est ressortie. En définitive, mon projet de thèse aura permis de préciser l'implication de la voie alternative NF-κB sur la biologie du CaP. Son impact sur la croissance des cellules cancéreuses prostatiques ainsi que sur l'autophagie, dénote l’ambivalence de la voie alternative NF-κB face à la tumorigénèse du CaP. L’étude exhaustive de la signalisation NF-κB souligne davantage l'importance de la voie classique dont l’intérêt clinique est principalement associé au statut de RelA(p65). Ainsi, bien que RelB n’affiche aucun potentiel en tant que biomarqueur exploitable en clinique, l’analyse de l’intervention de la voie alternative NF-κB sur la biologie des cellules cancéreuses prostatiques reste d’intérêt pour la compréhension de son rôle exact dans la progression du CaP. / Prostate cancer (PCa) is the most frequently diagnosed cancer and represents the third cause of cancer-death in Canadian men. Despite effective first-line therapies, many patients experience disease recurrence where PCa progresses toward a more aggressive form. Several parameters, largely clinical, have been used to predict the progression of PCa but their accuracy is still limited and implies the need for new biomarkers to fill this gap. Previous research from the laboratory has demonstrated that the transcription factor NF-B, and its nuclear localization, could be such a prognostic biomarker. Studies in our laboratory and elsewhere have correlated RelA(p65) with a clinical progression in PCa, underlining the importance of the classical NF-B pathway. The involvement of the alternative NF-B pathway in the progression of PCa was also suggested in one of our studies showing the correlation between the nuclear distribution of RelB and Gleason score. While the classical pathway is well documented and its involvement in the PCa progression established, the alternative NF-B pathway remains largely unexplored. This thesis describes two research objectives that aims to clarify the involvement of the alternative NF-B pathway in PCa. The first objective assessed the impact of the alternative NF-B pathway activation on the biology of PCa cells. RelB overexpression in 22RV1 PCa cells highlighted an effect of the alternative NF-B pathway on cell proliferation and autophagy. Its dual role in cell growth and a form of cell death requires further study to understand the balance of these in PCa tumorigenesis. To date no study has addressed the comparative prognostic potential of both the classical and alternative NF-B pathways simultaneously. Therefore the second objective of this research project was to analyze both RelA(p65) and RelB at a cellular level in the same tissue of patients with PCa to determine their unique and combined contribution to predicting biochemical recurrence in patients. This analysis was possible through immunofluorescent labeling of RelA (p65) and RelB, and was followed by a quantitative and objective analysis using an appropriate software. Our work confirmed the predictive value of RelA(p65) for biochemical recurrance. Combining RelA(p65) with RelB weakened the association, and RelB on its own was not found to predict biochemical recurrance. Ultimately, the research presented here has clarified the involvement of the alternative NF-B pathway on the biology of PCa. Its impact on the growth of PCa cells as well as autophagy reveals the dual role of the alternative NF-B pathway in PCa tumorigenesis. This exhaustive study of NF-B in PCa tissues further underscores the importance of the classical pathway whose clinical interest is mainly associated with RelA(p65) status. Thus, although RelB shows no potential as a clinically exploitable biomarker, further studies are needed to determine whether RelB contributes, either positively or negatively, and in a temporal fashion, to PCa progression.