As EPEC típicas apresentam padrão de adesão localizada (LA) em células epiteliais, formando de microcolônias compactas. As EPEC atípicas aderem no padrão de adesão localizada-like (LAL), no qual não se observa microcolônias e que ocorre tardiamente em relação à LA. O objetivo deste estudo foi caracterizar a interação de EPEC atípica que apresenta o padrão LAL com a célula epitelial. A cinética da formação da lesão A/E, característica da patogênese de EPEC, demonstrou um atraso na adesão e na formação da lesão em EPEC atípica, que correspondeu à expressão tardia de Intimina, Tir e EspA. A complementação de EPEC atípica com o regulador perABC antecipou a expressão destes fatores e a formação da lesão A/E. Não foi observada relação entre outras adesinas descritas em DEC com o padrão LAL, bem como a mobilização dos receptores eucarióticos nucleolina e <font face=\"symbol\">b1-Integrina para o foco de adesão. Observou-se a possível interação da Intimina com um peptídeo da membrana da célula epitelial de 300 kDa, podendo tratar-se de um provável receptor para essa adesina. / Typical EPEC exhibit a localized adherence (LA) pattern in epithelial cells, where tight bacterial clusters are produced. Atypical EPEC produce a localized-like (LAL) adhesion pattern, in which the compact clusters are not formed and that occurs in a later stage of the infection. The aim of this study was to characterize the interaction of LAL-adherent atypical EPEC with the epithelial cell. The kinetics of the A/E lesion, the main feature of EPEC pathology, showed a clear delay in its formation by the atypical strains, probably a consequence of the delay observed in the expression of Intimin, Tir and EspA. The complementation of atypical EPEC with the perABC regulator revealed to be effective in not only advancing the expression of these factors, as well the formation of A/E lesion. No correlation was found between the presence of different adhesins already described in DEC and the LAL pattern. The analysis of the possible interaction of Intimin with epithelial cell receptors showed the presence of a 300 kDa peptide that it seems to interact with this adhesin.

Os gânglios da raiz dorsal (GRD) armazenam neurônios TrkA-positivos. A percepção e transmissão de estímulos por estes neurônios dependem de uma neuritogênese adequada. Miosina (MioVa) é expressa no tecido nervoso e está presente em neuritos, corpo celular e cone de crescimento. Caracterizamos o padrão de expressão de MioVa na neuritogênese de células TrkA-positivas do GRD de galinha in vivo e in vitro. In vivo, MioVa é expressa em células que não começaram a emitir neuritos em HH25, e sua expressão persiste por toda neuritogênese. In vitro, é recrutada para o processo de re-emissão de neuritos de neurônios TrkA positivos na presença de NGF, sendo expressa em neuritos em diferentes estádios de neo-neuritogênese. Nos ensaios funcionais, observamos que a superexpressão do domínio globular de MioVa em culturas de GRD com 10 ou 100ng/ml de NGF reduz a população de células com neuritos longos e aumenta a população de células com neuritos curtos ou sem neuritos. Em conjunto, estes dados sugerem que a MioVa é importante para o estabelecimento de neuritos nociceptores. / The dorsal root ganglia (DRG) harbor the TrkA-positive neurons. The stimuli perception and transmission by these neurons depend on a proper neuritogenesis. Myosin (MyoVa) is widely expressed in nervous tissue and is present in neurites, cell body and growth cone. Here, we characterized the MyoVa expression pattern in chicken DRG TrkA-positive cells neuritogenesis, in vivo and in vitro. In vivo, at stage HH25, MyoVa was present both in cells with and without neurites and its expression persists throughout neuritogenesis. In vitro, it is recruited for the regeneration process and TrkA-positive neurons neurites re-emission in the presence of NGF, being expressed in neurites at different stages of neo-neuritogenesis. In functional assays, we observed that MyoVa globular tail overexpression in GRD cultures maintained with 10 or 100ng/ml NGF reduces the number of neurons with long neurites and increased the number of neurons with short neurites or no neurites. Taken together, these results suggest that Myosin Va is important for the establishment of nociceptor neurites.

As junções comunicantes são canais protéicos formados entre células adjacentes que permitem a passagem de moléculas e íons menores do que 1kDa; conexinas são proteínas que formam estas junções. Vêm sendo demonstradas na literatura a diminuição da capacidade de comunicação celular e alterações na expressão e/ou localização das conexinas em neoplasias. Este estudo foi realizado com o intuito de se verificar a influência da deleção de um dos alelos da Cx43 na carcinogênese pulmonar. Para tanto, camundongos geneticamente manipulados heterozigotos (Cx43± ou selvagens (Cx43±) de ambos os sexos receberam 3g/kg de uretana aos 15 e 17 dias de idade, e foram sacrificados após 25 semanas. As quantificações macro e microscópicas das lesões revelaram que os camundongos Cx43± apresentaram maior multiplicidade de adenomas pulmonares. Estes apresentavam também maior taxa de proliferação celular, avaliada pela quantificação de núcleos positivos para o PCNA. As expressões das Cxs 26, 32, 43 e 46, presentes no epitélio pulmonar, foram avaliadas por PCR em tempo real (RT-PCR) e por imunoistoquímica. A expressão da Cx43 revelou-se cerca de 50% menor em camundongos Cx43± quando comparada à dos correspondentes Cx43+/+, como esperado. Estudos in vitro mostraram que os pneumócitos de tipo II (APTII) extraídos de camundongos Cx43±, apresentaram capacidade de comunicação menor do que os APTII de camundongos Cx43+/+. Quando submetidos ao tratamento com uretana, a expressão de Cx 43 aumentou em 100% no tecido pulmonar. As demais Cxs tiveram a expressão reduzida pelo tratamento e não foram evidenciadas no epitélio pulmonar livre de lesões após o tratamento com uretana. Não foi detectada a expressão da Cx43 e da Cx32 nos adenomas provenientes dos camundongos Cx43±. A expressão das Cxs 26 e 46 foi correlacionada com o fenótipo papilífero das lesões. Constatou-se que a Cx32 acumulava-se no citoplasma das células epiteliais pulmonares e teve sua expressão, juntamente com a da Cx43, associada ao sexo, provavelmente contribuindo para a menor susceptibilidade das fêmeas aos adenomas induzidos pela uretana. Em conclusão, a redução da expressão da Cx43 conferiu maior susceptibilidade ao desenvolvimento de adenomas pulmonares pela uretana. Este foi o primeiro estudo in vivo mostrando a influência da deleção de um único alelo da Cx43 na carcinogênese / Gap junctions are communicating protein channels formed between adjacent cells that allow the exchange of molecules and ions smaller than 1kDa; connexins are proteins that form these junctions. Studies in the literature have been showing the lower level of cell communication capacity and alterations in the expression and/or localization of connexins in neoplasia. This study was performed to verify the influence of the deletion of one allele of Cx43 on lung carcinogenesis. Genetically manipulated heterozygous (Cx43Gap junctions are communicating protein channels formed between adjacent cells that allow the exchange of molecules and ions smaller than 1kDa; connexins are proteins that form these junctions. Studies in the literature have been showing the lower level of cell communication capacity and alterations in the expression and/or localization of connexins in neoplasia. This study was performed to verify the influence of the deletion of one allele of Cx43 on lung carcinogenesis. Genetically manipulated heterozygous (Cx43± or wild type mice (Cx43+/+) were injected with 3g/kg of at the age of 15 and 17 days and were euthanized after 25-weeks. Macroscopic and microscopic quantification of pulmonary lesions revealed that Cx43± mice presented higher multiplicity of pulmonary adenomas. These presented also a higher cell proliferation index, as evaluated by counting PCNA positive nuclei. Cxs 26, 32, 43 and 46 expressions in the pulmonary epithelium were investigated by Real-Time PCR and by immunohistochemistry. Cx43 expression was about 50% lower in Cx43± mice, in comparison to Cx43+/+ mice, as expected. In vitro studies showed that the APTII cells extracted from Cx43± mice presented a reduced communication capacity. When treated with urethane, the expression of Cx43 was increased by 100%. Other Cxs were down-regulated after the treatment with urethane, and were not observed lung areas devoid of adenomas after the treatment with urethane. Cx43 and Cx32 were not detected in Cx43± mouse adenomas. However, Cx26 and Cx46 were correlated with papillary lesions. Cx32 was cumulated in the cytoplasm of the lung epithelial cells and its expression, together Cx43, were associated with the sex, maybe contributing to the lower susceptibility of the female mice to urethane. In conclusion, the reduced expression of Cx43 determines a higher susceptibility to the development of pulmonary adenomas by urethane. This study was the first in vivo showing the influence of the deletion of one allele of Cx43± or wild type mice (Cx43+/+) were injected with 3g/kg of at the age of 15 and 17 days and were euthanized after 25-weeks. Macroscopic and microscopic quantification of pulmonary lesions revealed that Cx43± mice presented higher multiplicity of pulmonary adenomas. These presented also a higher cell proliferation index, as evaluated by counting PCNA positive nuclei. Cxs 26, 32, 43 and 46 expressions in the pulmonary epithelium were investigated by Real-Time PCR and by immunohistochemistry. Cx43 expression was about 50% lower in Cx43± mice, in comparison to Cx43+/+ mice, as expected. In vitro studies showed that the APTII cells extracted from Cx43± mice presented a reduced communication capacity. When treated with urethane, the expression of Cx43 was increased by 100%. Other Cxs were down-regulated after the treatment with urethane, and were not observed lung areas devoid of adenomas after the treatment with urethane. Cx43 and Cx32 were not detected in Cx43± mouse adenomas. However, Cx26 and Cx46 were correlated with papillary lesions. Cx32 was cumulated in the cytoplasm of the lung epithelial cells and its expression, together Cx43, were associated with the sex, maybe contributing to the lower susceptibility of the female mice to urethane. In conclusion, the reduced expression of Cx43 determines a higher susceptibility to the development of pulmonary adenomas by urethane. This study was the first in vivo showing the influence of the deletion of one allele of Cx43 in carcinogenesis

O ciclo de vida de Dictyostelium discoideum é composto de duas fases independentes. Durante o crescimento vegetativo, as amebas crescem isoladas até que a fonte de nutrientes seja esgotada. A carência nutricional induz sua entrada num processo de desenvolvimento, que inclui a parada do crescimento, a agregação das células e a formação de um organismo multicelular onde as células se diferenciam em esporos que sobrevivem as condições desfavoráveis. A proteína quinase YakA é requerida para a transição entre o crescimento e o desenvolvimento. YakA regula os níveis da PKA. Mutantes yakA- apresentam o crescimento acelerado, são deficientes no processo de agregação e são hiper-sensíveis a estresse oxidativo e nitrosoativo. Uma mutação em um segundo sítio em keaA, suprime a morte induzida por SNP (um gerador de óxido nítrico) no mutante yakA-. O papel de keaA foi determinado em resposta a estresse oxidativo, nitrosoativo e carênica nutricional. O gene keaA é necessário para o crescimento e desenvolvimento. Uma mutação em keaA confere resistência a estresse nitrosoativoloxidativo confirmando que uma mutação em keaA confere resistência a estresse. Um segundo supressor da morte induzida por SNP no mutante yakA- foi isolado pela mesma técnica de REMI e identificado como pkaC um regulador da resposta a estresse. YakA e PKA integradam a resposta a vários estresse em Dictyostelium. Os resultados indicam que a yakA regula a parada do ciclo celular em resposta a estresses através da modulação de keaA. keaA regula, por sua vez, a expressão da pkaC, um regulador chave da produção de cAMP e do processo de desenvolvimento. A interação gênica entre estes elementos é complexa e deve ser ajustada para permitir que as células sobrevivam a mudanças ambientais encontradas durante o seu ciclo de vida. / Dictyostelium discoideum\'s life cycle is composed of two phases. During the vegetative phase, amoebae grow as single cells until the nutrients are depleted. Starvation induces a developmental program where isolated amoebae adopt a multicellular mode of living and differentiate into spores to survive the harsh conditions. The protein Kinase YakA is required for the growth to development transition. YakA regulates the leveis of PKA. yakA null cells have a faster cell cycle, are aggregation deficient and are hypersensitive to nitrosoative/oxidative stress. A second-site mutation in keaA, supresses the death induced by SNP, a generator of nitric oxide. The role for keaA has been determined in response to oxidative, nitrosoative and nutrient starvation stresses. keaA is required for proper growth and development. The keaA- cells are more resistant to nitrosoative and oxidative stress confirming that a mutation in keaA confers stress resistance. A second supressor of the death induced by SNP in the mutant yakA- was isolated with REMI and identified pkaC as a regulator of the nitrosoative stress response. YakA and PKA may integrate the responses to several stresses in Dictyostelium. The results indicate that yakA regulates the cell cycle arrest in response to stress through the modulation of keaA. KeaA regulates the expression of pkaC, a key regulator of cAMP prodution and development. Genetic interactions among these elements is complex and must be finely tuned in the many environmental changes cells endure during the life cycle.

The phosphatidylinositol-3-kinase/AKT (Protein Kinase B)/mammalian target of rapamycin (PI3K/AKT/mTOR) signalling pathway, downstream of tyrosine kinase receptors, is upregulated in human cancers including breast and colon cancers. Glycogen synthase kinase 3 (GSK 3) is a serine/threonine protein kinase plays important role in various cellular processes including glycogen synthesis mediated by insulin signalling pathway. Moreover, 5' adenosine monophosphate activated protein kinase (AMPK), a crucial cellular energy sensor, has regulatory role in cell growth and proliferation through mTOR pathway. Phosphatidylcholine (PtdCho) is the major phospholipid in the mammalian cell membranes and is mainly synthesized by the CDP-choline pathway. Malignant transformation has been reported to be associated with altered choline metabolism. Hyperactivation of the PI3K/AKT signalling pathway upregulates the key enzymes of phospholipid metabolism. The first line antidiabetic drug, metformin, modulates glucose and concomitant lipid metabolism through AMPK activation. Studies suggest phosphatidylcholine biosynthesis and breakdown through CDP-choline pathway are modulated by glucose metabolism and de novo fatty acid synthesis. Cancer cell growth inhibitory effect of PI3K/AKT/mTOR/GSK3 pathway inhibitors and metformin were investigated by cytotoxic assay, western blot and cell cycle analysis in breast and colon cancer cells. IC50 values of anticancer drugs and combination indices between drug combinations were determined. 31P-NMR was carried out on cell extracts after drug treatments. [14C (U)] glucose and [3H] choline incorporation into lipids were also determined. All inhibitors targeting PI3K/AKT/mTOR signaling pathway, GSK3 and metformin have cancer cell growth inhibition. By 31P-NMR, PI3K/AKT/mTOR pathway inhibition induced agent-specific changes in PCho intensity. Increased UDP-sugars observed in breast and colon cancer cell extracts treated with LY294002 and AZD8055, an effect abrogated by inclusion of a GSK3 inhibitor. A link between glycolytic intermediates and phosphatidylcholine biosynthesis was investigated by metformin and GSK3 inhibitor in breast and colon cancer cells.

As junções comunicantes são canais protéicos formados entre células adjacentes que permitem a passagem de moléculas e íons menores do que 1kDa; conexinas são proteínas que formam estas junções. Vêm sendo demonstradas na literatura a diminuição da capacidade de comunicação celular e alterações na expressão e/ou localização das conexinas em neoplasias. Este estudo foi realizado com o intuito de se verificar a influência da deleção de um dos alelos da Cx43 na carcinogênese pulmonar. Para tanto, camundongos geneticamente manipulados heterozigotos (Cx43± ou selvagens (Cx43±) de ambos os sexos receberam 3g/kg de uretana aos 15 e 17 dias de idade, e foram sacrificados após 25 semanas. As quantificações macro e microscópicas das lesões revelaram que os camundongos Cx43± apresentaram maior multiplicidade de adenomas pulmonares. Estes apresentavam também maior taxa de proliferação celular, avaliada pela quantificação de núcleos positivos para o PCNA. As expressões das Cxs 26, 32, 43 e 46, presentes no epitélio pulmonar, foram avaliadas por PCR em tempo real (RT-PCR) e por imunoistoquímica. A expressão da Cx43 revelou-se cerca de 50% menor em camundongos Cx43± quando comparada à dos correspondentes Cx43+/+, como esperado. Estudos in vitro mostraram que os pneumócitos de tipo II (APTII) extraídos de camundongos Cx43±, apresentaram capacidade de comunicação menor do que os APTII de camundongos Cx43+/+. Quando submetidos ao tratamento com uretana, a expressão de Cx 43 aumentou em 100% no tecido pulmonar. As demais Cxs tiveram a expressão reduzida pelo tratamento e não foram evidenciadas no epitélio pulmonar livre de lesões após o tratamento com uretana. Não foi detectada a expressão da Cx43 e da Cx32 nos adenomas provenientes dos camundongos Cx43±. A expressão das Cxs 26 e 46 foi correlacionada com o fenótipo papilífero das lesões. Constatou-se que a Cx32 acumulava-se no citoplasma das células epiteliais pulmonares e teve sua expressão, juntamente com a da Cx43, associada ao sexo, provavelmente contribuindo para a menor susceptibilidade das fêmeas aos adenomas induzidos pela uretana. Em conclusão, a redução da expressão da Cx43 conferiu maior susceptibilidade ao desenvolvimento de adenomas pulmonares pela uretana. Este foi o primeiro estudo in vivo mostrando a influência da deleção de um único alelo da Cx43 na carcinogênese / Gap junctions are communicating protein channels formed between adjacent cells that allow the exchange of molecules and ions smaller than 1kDa; connexins are proteins that form these junctions. Studies in the literature have been showing the lower level of cell communication capacity and alterations in the expression and/or localization of connexins in neoplasia. This study was performed to verify the influence of the deletion of one allele of Cx43 on lung carcinogenesis. Genetically manipulated heterozygous (Cx43Gap junctions are communicating protein channels formed between adjacent cells that allow the exchange of molecules and ions smaller than 1kDa; connexins are proteins that form these junctions. Studies in the literature have been showing the lower level of cell communication capacity and alterations in the expression and/or localization of connexins in neoplasia. This study was performed to verify the influence of the deletion of one allele of Cx43 on lung carcinogenesis. Genetically manipulated heterozygous (Cx43± or wild type mice (Cx43+/+) were injected with 3g/kg of at the age of 15 and 17 days and were euthanized after 25-weeks. Macroscopic and microscopic quantification of pulmonary lesions revealed that Cx43± mice presented higher multiplicity of pulmonary adenomas. These presented also a higher cell proliferation index, as evaluated by counting PCNA positive nuclei. Cxs 26, 32, 43 and 46 expressions in the pulmonary epithelium were investigated by Real-Time PCR and by immunohistochemistry. Cx43 expression was about 50% lower in Cx43± mice, in comparison to Cx43+/+ mice, as expected. In vitro studies showed that the APTII cells extracted from Cx43± mice presented a reduced communication capacity. When treated with urethane, the expression of Cx43 was increased by 100%. Other Cxs were down-regulated after the treatment with urethane, and were not observed lung areas devoid of adenomas after the treatment with urethane. Cx43 and Cx32 were not detected in Cx43± mouse adenomas. However, Cx26 and Cx46 were correlated with papillary lesions. Cx32 was cumulated in the cytoplasm of the lung epithelial cells and its expression, together Cx43, were associated with the sex, maybe contributing to the lower susceptibility of the female mice to urethane. In conclusion, the reduced expression of Cx43 determines a higher susceptibility to the development of pulmonary adenomas by urethane. This study was the first in vivo showing the influence of the deletion of one allele of Cx43± or wild type mice (Cx43+/+) were injected with 3g/kg of at the age of 15 and 17 days and were euthanized after 25-weeks. Macroscopic and microscopic quantification of pulmonary lesions revealed that Cx43± mice presented higher multiplicity of pulmonary adenomas. These presented also a higher cell proliferation index, as evaluated by counting PCNA positive nuclei. Cxs 26, 32, 43 and 46 expressions in the pulmonary epithelium were investigated by Real-Time PCR and by immunohistochemistry. Cx43 expression was about 50% lower in Cx43± mice, in comparison to Cx43+/+ mice, as expected. In vitro studies showed that the APTII cells extracted from Cx43± mice presented a reduced communication capacity. When treated with urethane, the expression of Cx43 was increased by 100%. Other Cxs were down-regulated after the treatment with urethane, and were not observed lung areas devoid of adenomas after the treatment with urethane. Cx43 and Cx32 were not detected in Cx43± mouse adenomas. However, Cx26 and Cx46 were correlated with papillary lesions. Cx32 was cumulated in the cytoplasm of the lung epithelial cells and its expression, together Cx43, were associated with the sex, maybe contributing to the lower susceptibility of the female mice to urethane. In conclusion, the reduced expression of Cx43 determines a higher susceptibility to the development of pulmonary adenomas by urethane. This study was the first in vivo showing the influence of the deletion of one allele of Cx43 in carcinogenesis

A doença granulomatosa inflamatória envolve interações coordenadas entre linfócitos, monócitos/macrófagos, células epitelióides, eosinófilos, neutrófilos e fibroblastos. A comunicação intercelular mediada por junções gap, constituídas por conexinas, é responsável pela homeostase tecidual. A Cx43 está presente em células linfóides, células mielóides, fibroblastos e outras. Assim, para compreendermos o possível envolvimento das conexinas no granuloma, nós analisamos o efeito da deleção heterozigótica de uma Gja1 (gene Cx43) na formação e desenvolvimento dos granulomas hepáticos, induzidos por ovos de Schistosoma mansoni. Para tanto, camundongos heterozigotos (Cx43+/-) e selvagens (Cx43+/+) foram infectados com cercárias de Schistosoma mansoni e avaliados nos tempos: 6, 8 e 12 semanas após a infecção. As células dos granulomas apresentaram expressão de conexina 43, notadamente após 12 semanas de infecção. As lesões dos camundongos Cx43+/- apresentaram índice de proliferação reduzido e aumento na deposição de colágeno em fases tardias da doença. Apesar desses achados, não se encontrou redução no tamanho e celularidade das lesões em comparação aos camundongos selvagens. Também não obtivemos diferenças em relação ao hemograma ou população de linfócitos esplênicos CD4, CD8 e CD19. As células peritoneais dos animais de ambos genótipos apresentaram produção de NO e H<SUB2O<SUB2 similares. Contudo, os neutrófilos e monócitos sanguíneos dos animais Cx43+/-, estimulados por PMA, apresentaram aumento significante do burst oxidativo. Concluindo, nossos resultados indicam que a deleção de um alelo do gene da Cx43 modifica claramente a evolução da doença granulomatosa, mostrando um papel desta conexina no desenvolvimento do granuloma. / Inflammatory granulomatous disease involves coordinated interactions among lymphocytes, monocytes/macrophages, epithelioid cells, eosinophils, neutrophils and fibroblasts. Intercellular communication mediated by gap junctions constituted of connexins, is responsible for tissue homeostasis. Cx43 is present in lymphoid cells, myelogenous cells, fibroblasts and others. In order to understand the possible involvement of connexins in granuloma, we analyzed the effect of the heterologous deletion of a Gja1 (Cx43 gene) on the formation and development of hepatic granulomas induced by Schistosoma mansoni eggs. Heterozigous (Cx43+/-) and wild-type (Cx43+/+) mice were infected with S. mansoni cercarie and evaluated after 6, 8 and 12 weeks. Granuloma cells express Cx43, with considerable reduction in Cx43+/- mice. Moreover, granuloma cells from Cx43v+/- mice displayed reduced proliferation and increased collagen deposition at late phases of the disease. Despite these findings, no reduction of size or cellularity of the lesions was found in comparison to wild-type mice. We didn?t find differences in relation to blood count and splenic lymphocyte population CD4, CD8 and CD19. Peritoneal cells from animals of both genotypes presented similar production of NO and H2O2. However, blood neutrophils and monocytes from Cx43+/- mice, stimulated by PMA, displayed significantly increased oxidative burst. In conclusion, our results indicate that the deletion of one allele of the Cx43 gene clearly modifies the evolution of a granulomatous disease, supporting a role for this connexin on granuloma development.

"December 2001." Includes bibliographical references (leaves 178-225) Focuses on the ex vivo growth of human haemopoietic progenitor cells with the objective of defining culture conditions for generating myeloid post-progenitor cells for therapy

"December 2001." / Includes bibliographical references (leaves 178-225) / xviii, 225 leaves : ill. (some col.), plates, charts ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Focuses on the ex vivo growth of human haemopoietic progenitor cells with the objective of defining culture conditions for generating myeloid post-progenitor cells for therapy / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2001

The following thesis research was undertaken to gain a better understanding of the mechanisms that regulate the cellular trafficking and signaling of the endothelial differentiation gene (EDG) family of G-protein coupled receptors, LPA1, LPA2, and LPA3. This thesis will specifically focus on the regulation of the trafficking of the LPA1 Lysophosphatidic acid receptor, which is the most widely expressed and has been shown to be a major regulator of migration of cells expressing it. The initial studies undertaken in this project were aimed at understanding the endocytic pathway followed by the LPA1 receptor. Lysophosphatidic acid (LPA), an abundant serum phospholipid, stimulates heterotrimeric G protein signaling by activating three closely related receptors, termed LPA1, LPA2 and LPA3. In the first part of the project we show that in addition to promoting LPA1 signaling, membrane cholesterol is essential for the association of LPA1 with β-arrestin, which leads to signal attenuation and clathrin dependent endocytosis of LPA1. The second phase of the project was aimed at elucidating the different structural motifs required for the trafficking and signaling of the LPA1 receptor and helping us gain a more mechanistic view of the processes involved in its regulation. In the second part of the project we show that agonist-independent internalization of the LPA1 receptor is clathrin adaptor, AP-2 dependent and PKC-dependent and that it requires a distal dileucine motif, whereas agonist-dependent internalization of the LPA1 receptor is β-arrestin and clathrin-dependent and requires a cluster of serine residues in the tail region, which is upstream of the dileucine motif. These studies collectively vastly enhance our understanding of mechanisms that regulate LPA1 trafficking and signaling. These studies can also be applied to other G-protein coupled receptors making the task easier for other scientists to understand this vast family of receptors.