Accessible in vitro models recapitulating the human airway that are amenable to study whole cannabis smoke exposure are needed for immunological and toxicological studies that inform public health policy as well as medicinal and recreational cannabis use. In the present study, we developed and validated a novel three-dimensional (3D)-printed in vitro exposure system (IVES) that can be directly applied to study the effect of cannabis smoke exposure on primary human bronchial epithelial cells (HBECs). Using commercially available design software and a 3D printer, we designed a four-chamber Transwell insert holder for exposures to whole smoke. COMSOL Multiphysics software was used to model gas distribution, concentration gradients, velocity profile, and shear stress within IVES. Following simulations, primary HBECs cultured at the air–liquid interface on Transwell inserts were exposed to whole cannabis smoke using a modified version of the Foltin puff procedure. Following 24 h, outcome measurements included cell morphology, epithelial barrier function, lactate dehydrogenase (LDH) levels, cytokine expression and gene expression. HBECs exposed to cannabis smoke using IVES showed changes in cell morphology and disruption of barrier function without significant cytotoxicity. Cannabis smoke elevated interleukin-1 (IL-1) family cytokines and elevated CYP1A1 and CYP1B1 expression relative to control. These findings validate IVES to have an effect in HBECs at a molecular level following cannabis smoke exposure. In addition, HBECs stimulated with a viral mimetic, Poly I:C, challenge following cannabis smoke exposure showed a suppression of key antiviral cytokines. The growing legalization of cannabis on a global scale must be paired with research related to potential health impacts on lung exposures. IVES represents an accessible, open-source, exposure system that can be used to model varying types of cannabis smoke exposures with HBECs grown under air–liquid interface culture conditions. / Thesis / Master of Science (MSc) / Despite its recent legalization in Canada, cannabis smoke has been understudied and a lack of evidence exists to inform legislative policies, medicinal and recreational usage. Due to a lack of relevant ways to study cannabis smoke in a lab setting, it is difficult to accumulate literature around its impacts in the lungs. Here, we addressed this gap by engineering and validating a novel model to expose lung cultures to cannabis smoke. In addition, we investigated its impact on the immune response. Our findings suggest exposure to cannabis smoke alters the immune functions of these cells. We also found that in response to a viral mimetic stimulus, cell cultures pre-exposed to cannabis smoke exhibited impaired immune responses. Our novel model to expose cell cultures to cannabis smoke creates a foundation for future researchers to investigate environmental insults, such as cannabis smoke, in the context of respiratory health and infectious disease.

The FHIT gene is located on the most fragile site in the human genome. FHIT gene deletions are among the earliest and most frequent events in carcinogenesis, particularly in carcinogen-exposed tissue. Previous work in mouse and cell culture models established FHIT to be an authentic tumor suppressor. Re-expression of FHIT in cell culture causes cell death via initiation of apoptosis, but the precise mechanism underlying this process is unclear. It is well established that cellular transition from normal to transformed occurs in multiple steps and requires the accumulation of several genetic changes. Relying on the compelling phenotype of tumor development in FHIT knockout mice, this project aimed to elucidate a mechanism through which FHIT-deficient cells are primed to survive multiple genetic and environmental stresses, and promote progression of cancer. My work indicates that FHIT expression is required for the normal cellular response to oxidative stress, and presents evidence that in the absence of FHIT, an oxidative stress response pathway is superinduced. When FHIT is depleted from cells exposed to cigarette smoke, the expression of a subset of oxidative stress response genes is enhanced. Enhanced activation of these genes can occur as an adapative response to stress induced by reactive oxygen species production, and is frequently detected in cancer. Investigation into the mechanism underlying the enhanced gene expression determined that FHIT loss is associated with decreased levels of the transcriptional repressor Bach1. In this manner, we propose that loss of Fhit supports an antioxidant program that is pivotal in establishing and maintaining carcinogenic transformation.

No description available.

<p>Epithelial damage and shedding of the epithelium are common observations in many airway diseases such as asthma, Sjögren’s syndrome, chronic obstructive pulmonary disease and cystic fibrosis. The ability of the cells to attach to each other and/or to the matrix seems to be altered. In the present study, cultured normal human bronchial epithelial cells were used as a model system. The desmosomes and also the focal adhesions were investigated to see if changes in these structural components as well as metabolic alterations could explain the observed shedding of the epithelium.</p><p>Inflammatory mediators such as tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-13 (IL-13), hypochlorous acid (HOCl) and nitric oxide (NO) are present in increased amounts in inflammation. The Th-1 cytokines, IFN-γ and TNF-α, as well as HOCl and NO affected the number of desmosomes and their ability to attach to each other. Interestingly, the Th-2 cytokines IL-4, IL-5 and IL-13 did not affect the cell-cell adhesion. HOCl and NO also affected the focal adhesions of the cells. </p><p>Both morphological and functional studies indicated that TNF-α, IFN-γ, HOCl and NO affect the mitochondria. A decreased glucose oxidation rate could result in a decreased production of ATP, which in turn could lead to inhibition of many cellular activities including an impaired ability of the ciliary activity in bronchial epithelial cells and mucus transport. The antioxidant N-acetyl-L-cysteine and the nitric oxide synthase inhibitor N-propyl-L-arginine inhibited these effects of HOCl. This indicates that HOCl can induce damage both by induction of free radicals and also through an increased production of NO. TNF-α and IFN-γ also induced an increased production of NO. N^ω-monomethyl-L-arginine reduced the cytokine-induced production of NO. The NO donor DETA NONOate reduced the total protein biosynthesis as well as the DNA content. NO can react with superoxide anions generated by inflammatory cells in the airways to form peroxynitrite ions, which in turn could generate hydroxyl radicals. These toxic ions may contribute to damage of the airway epithelial cells. </p><p>In conclusion, pro-inflammatory cytokines such as TNF-α, IFN-γ and also the reactive oxygen species HOCl and NO could contribute to airway epithelial shedding by affecting the adhesion properties of the epithelial cells. More generalized morphological and metabolic changes could be other contributing factors, together with the increased production of NO.</p>

Epithelial damage and shedding of the epithelium are common observations in many airway diseases such as asthma, Sjögren’s syndrome, chronic obstructive pulmonary disease and cystic fibrosis. The ability of the cells to attach to each other and/or to the matrix seems to be altered. In the present study, cultured normal human bronchial epithelial cells were used as a model system. The desmosomes and also the focal adhesions were investigated to see if changes in these structural components as well as metabolic alterations could explain the observed shedding of the epithelium. Inflammatory mediators such as tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-13 (IL-13), hypochlorous acid (HOCl) and nitric oxide (NO) are present in increased amounts in inflammation. The Th-1 cytokines, IFN-γ and TNF-α, as well as HOCl and NO affected the number of desmosomes and their ability to attach to each other. Interestingly, the Th-2 cytokines IL-4, IL-5 and IL-13 did not affect the cell-cell adhesion. HOCl and NO also affected the focal adhesions of the cells. Both morphological and functional studies indicated that TNF-α, IFN-γ, HOCl and NO affect the mitochondria. A decreased glucose oxidation rate could result in a decreased production of ATP, which in turn could lead to inhibition of many cellular activities including an impaired ability of the ciliary activity in bronchial epithelial cells and mucus transport. The antioxidant N-acetyl-L-cysteine and the nitric oxide synthase inhibitor N-propyl-L-arginine inhibited these effects of HOCl. This indicates that HOCl can induce damage both by induction of free radicals and also through an increased production of NO. TNF-α and IFN-γ also induced an increased production of NO. Nω-monomethyl-L-arginine reduced the cytokine-induced production of NO. The NO donor DETA NONOate reduced the total protein biosynthesis as well as the DNA content. NO can react with superoxide anions generated by inflammatory cells in the airways to form peroxynitrite ions, which in turn could generate hydroxyl radicals. These toxic ions may contribute to damage of the airway epithelial cells. In conclusion, pro-inflammatory cytokines such as TNF-α, IFN-γ and also the reactive oxygen species HOCl and NO could contribute to airway epithelial shedding by affecting the adhesion properties of the epithelial cells. More generalized morphological and metabolic changes could be other contributing factors, together with the increased production of NO.

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2015-07-27T14:08:11Z No. of bitstreams: 1 Vera Lucia Silva Rigoni.pdf: 1348554 bytes, checksum: 28c01d5b4e69fddd38792d76d20fcf02 (MD5) / Made available in DSpace on 2015-07-27T14:08:11Z (GMT). No. of bitstreams: 1 Vera Lucia Silva Rigoni.pdf: 1348554 bytes, checksum: 28c01d5b4e69fddd38792d76d20fcf02 (MD5) Previous issue date: 2013-12-13 / The scorpionism is considered a public health worldwide, with Tityus serrulatus the main cause of accidents in Brazil. Symptoms of envenomation by Tityus serrulatus ranging from local pain to severe systemic reactions such as cardiac dysfunction and pulmonary edema, which is the leading cause of death. The amount of venom injected the age, physical and genetic factors of the victims are related to the severity of the symptoms presented by patients. Endothelial cells under physiological conditions play a protective role in the cardiovascular system. In the case of dysfunction or injury of that tissue occurs repercussion in the vascular structure, surrounding tissue, and finally on the cardiovascular system. In bronchial epithelial cells after injury, occurs repair mechanism mediated by cytokines and growth factors also occur increase in cell proliferation and differentiation after epithelial damage. Thus, this study aims to evaluate the modulating effect of Tityus serrulatus scorpion venom on endothelial cells and bronchial epithelial cells analyzing the viability of these cells and the mechanism by which inflammation occurs. Therefore, the cells in this study are cultured and incubated in culture plates with Tityus serrulatus scorpion venom in different concentrations and periods of time. The inflammatory mechanism was assessed through the measurement of cytokines in the supernatant of these cells. The results showed that Tityus serrulatus venom changed the viability of monolayers of endothelial cells (tEnd), and bronchial epithelial cells (BEAS), and this effect was more marked in bronchial epithelial cells. The results also demonstrate that the venom induces the release of cytokines IL-1, IL-6 and IL-8. iii This study extend the knowledge of the actions of the venom of the scorpion Tityus serrulatus on bronchial epithelial and endothelial cells thus demonstrating changes in pulmonary physiology that may contribute to a better treatment strategy of scorpion envenomation. / O escorpionismo é considerado um problema mundial de saúde pública, sendo a espécie Tityus serrulatus a principal causadora de acidentes graves no Brasil. Os sintomas do envenenamento pelo escorpião Tityus serrulatus vão desde dor local até reações sistêmicas severas, como disfunção cardíaca e edema pulmonar agudo, sendo este a principal causa de morte. A quantidade de veneno inoculado, idade, condições físicas e fatores genéticos das vítimas relacionam-se com a gravidade dos sintomas apresentados pelos pacientes. As células endoteliais, em condições fisiológicas, desempenham papel protetor do sistema cardiovascular. Em caso de disfunção ou lesão desse tecido há repercussão sobre a estrutura vascular, tecido adjacente e, por fim sobre o sistema cardiovascular. Já, nas células epiteliais brônquicas após injúria, ocorre mecanismo de reparo mediado por citocinas e fatores de crescimento, ocorrendo também proliferação e diferenciação celular após o dano epitelial. Assim, o presente estudo tem como objetivo avaliar o efeito modulador do veneno do escorpião Tityus serrulatus em células endoteliais e células epiteliais brônquicas, analisando a viabilidade destas células e o mecanismo de inflamação pela qual são acometidas. Para tanto, as células em estudo foram cultivadas e incubadas em placas de cultura com o veneno do escorpião Tityus serrulatus, em diferentes concentrações e em diferentes períodos de tempo. O mecanismo inflamatório foi avaliado através da dosagem de citocinas no sobrenadante destas células. Os resultados demonstraram que o veneno de Tityus serrulatus alterou a viabilidade de i monocamadas das células epiteliais brônquicas e células endoteliais, sendo que esse efeito foi mais evidente nas células epiteliais brônquicas. Ainda, os resultados demonstraram que o veneno induz a liberação das citocinas IL-1, IL-6 e IL-8. Estes estudos ampliam o conhecimento das ações do veneno do escorpião Tityus serrulatus nas células endoteliais (tEnd) e epiteliais brônquicas (BEAS), comprovando assim alterações na fisiologia pulmonar que poderão contribuir para uma melhor estratégia de tratamento do envenenamento escorpiônico.

Les canaux TRP (Transient Receptor Potential) sont des acteurs clés de l'homéostasie calcique. Plusieurs de ces canaux interviennent dans l'influx calcique des cellules épithéliales bronchiques, notamment TRPC6, qui est impliqué dans un couplage fonctionnel avec le canal Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Les mutations du CFTR (F508del et G551D) sont à l'origine de la mucoviscidose (Cystic Fibrosis (CF)), qui aboutit à l'augmentation de l'influx calcique dans les cellules CF. L'objectif de ce travail a été d'étudier l'implication des canaux TRP dans la dérégulation de l'influx calcique des cellules épithéliales bronchiques CF. Nous avons mis en évidence que CFTR régulait négativement l'activité de TRPC6, tandis que l'influx calcique via TRPC6 permettait de potentialiser l'activité du canal muté CFTR-G551D, activé au préalable par le VX-770. Nous proposons donc une nouvelle stratégie thérapeutique, combinant un potentiateur de CFTR et un activateur spécifique de TRPC6. Nous nous sommes ensuite intéressés au rôle des canaux TRPV, en particulier TRPV5 et TRPV6, dans l'influx calcique des cellules épithéliales bronchiques. Nous avons observé que l'influx Ca2+ constitutif, attribuable à ces deux canaux, était doublé dans les cellules CF, dû à une augmentation de l'activité de TRPV6. En effet, l'expression de la PLC-δ1, une enzyme régulant négativement TRPV6, est dramatiquement réduite dans les cellules CF. La correction de l'adressage du F508del-CFTR a permis de normaliser l'activité de TRPV6 sans restaurer l'expression de la PLC-δ1 dans les cellules CF, suggérant un contrôle plus complexe de TRPV6 dans les cellules épithéliales bronchiques. / TRP (Transient Receptor Potential) channels are keys actors of Ca2+ homeostasis. Several of these channels are involved in the Ca2+ influx of bronchial epithelial cells, including TRPC6 which is implicated in a functional coupling with the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) channel. CFTR mutation leads Cystic Fibrosis (CF) disease and causes abnormal Ca2+ homeostasis trought an increased of Ca2+ influx in CF bronchial epithelial cells. Our objective is to investigate the implication of TRP channels in abnormal Ca2+ influx of CF bronchial epithelial cells.We showed that CFTR down regulates TRPC6 activity whereas Ca2+ influx through TRPC6 potentiates G551D-CFTR, activated by VX-770. We propose a new therapeutic strategy that combines a CFTR potentiator and a specific activator of TRPC6. Then, we focused on the role of TRPV channels, particularly TRPV5 and TRPV6, in Ca2+ influx of bronchial epithelial cells. We observed that constitutive Ca2+ influx, related to TRPV5/TRPV6 activity, was twice higher in CF cells due to the increase of TRPV6 activity. The expression of PLC-δ1, an enzyme that negatively regulates TRPV6 activity, is dramatically decreased in CF cells. The correction of F508del-CFTR trafficking allows TRPV6 activity normalization but do not restore PLC-δ1 expression level in CF cells, suggesting a more complex control of TRPV6 in bronchial epithelial cells.

This PhD research relates to the design, fabrication, characterization, and optimization of on-chip electrochemical microelectrode arrays (MEAs) for measurement of transient concentrations and gradients, focusing on fast transients and physiological applications. In particular, this work presents the determination of kinetic mechanisms taking place at an active interface (either physiological or non-physiological) in contact with a liquid phase using the MEA device to simultaneously estimate the concentration and gradient of the analyte of interest at the surface of the active interface. The design approach of the MEA device and the corresponding measurement methodology to acquire reliable concentration information is discussed. The ability of the MEA device to measure fast (i.e., in sub-second time scale) transient gradients is demonstrated experimentally using a controllable diffusion-reaction system which mimics the consumption of hydrogen peroxide by a 2D cell culture. The proposed MEA device and measurement methodology meet effectively most of the requirements for physiological applications and as a demonstration of this, two physiological applications are presented. In one application, the MEA device was tailored to measure the hydrogen peroxide uptake rate of human astrocytes and glioblastoma multiforme cells in 2D cell culture as a function of hydrogen peroxide concentration at the cell surface; the results allowed to quantitatively determine the uptake kinetics mechanisms which are well-described by linear and Michaelis-Menten expressions, in agreement with the literature. In the other application, further customization of the MEA device was realized to study the glucose uptake kinetics of human bronchial epithelial and small cell lung cancer cells, these latter with and without DDX5 gene knockdown; the results allowed to distinguish mechanistic differences in the glucose uptake kinetics among the three cell lines. These results were complemented with measurements of glycolytic and respiration rates to obtain a bigger picture of the glucose metabolism of the three cell lines. Finally, additional applications, both physiological and non-physiological, are proposed for the developed MEA device.