Thesis (Ph.D.)--Uniformed Services University of the Health Sciences, 2006 / Typescript (photocopy)

Thesis (Ph.D.)--Uniformed Services University of the Health Sciences, 2006 / Typescript (photocopy)

Η Ανθεκτική στην Αναισθησία Μνήμη είναι ένας ιδιαίτερος τύπος μνήμης που προς το παρόν έχει χαρακτηριστεί μόνο στη Drosophila. Ελάχιστα είναι γνωστά για τον ρόλο της καθώς και για τα μόρια που παίζουν ρόλο στο σχηματισμό της. Στόχος της παρούσας διπλωματικής εργασίας είναι να διερευνήσει χρησιμοποιώντας τόσο συμπεριφορικά όσο και μοριακά εργαλία τον ρόλο της πρωτεΐνης προσαρμοστή DRK στην δημιουργία αυτού του ιδιαίτερου τύπου μνήμης. / Anesthesia Resistant Memory is a unique form of memory characterising Drosophila. Very few are known about its function and the molecules participating in its formation. The aim of this research is to examine the role of the adaptor protein DRK in the formation of Anesthesia Resitant Memory using both behavioural and molecular asseys.

Chlamydia trachomatis (C. trachomatis) is the most prevalent sexually transmitted bacteria with devastating reproductive consequences that lead to tubal factor infertility (TFI). Recent studies have implicated apoptosis – associated speck – like protein containing a caspase recruitment domain (ASC) as an adaptor of inflammasomes that stimulate IL – 1β and IL – 18 secretion, pro – inflammatory cytokines with critical functions in host defense against a variety of pathogens. Therefore, for the first time, we are reporting the use of ASC-/- mice in a mouse model of Chlamydia infection that might provide some information on the role of inflammasomes in the pathogenesis of Chlamydia infection. In this study, wild type (WT) and ASC-/- mice were infected with Chlamydia. Infectivity, pathology of the upper genital tract (UGT), and, fertility were evaluated. In addition, expression of ASC – dependent inflammasomes and the activation of immune cells within the genital tract (GT) were studied. Results showed that Chlamydia infectivity in ASC-/- mice was significantly higher (p-/- mice which, when compared to infected WT mice, was exhibited by decrease in average number of pups and percent pregnancy. There was also severe UGT damage in ASC-/- mice compared to WT mice, correlating with the higher number of hydrosalpinx observed on the UGT of Chlamydia infected ASC-/- mice. Furthermore, IL – 1β and IL – 18 production as well as immune cell activation were down regulated in the GT of Chlamydia infected ASC-/- mice. This finding indicates that in absence of ASC, host innate and adaptive immunity is impaired. Results imply that ASC plays a protective role in the mucosal immunity against GT Chlamydia infection.

Submitted by Renata Fontoura (comunicaicc@fiocruz.br) on 2014-11-26T15:13:12Z No. of bitstreams: 2 Dissertação Claudia Maria do Nascimento Moreira - 01.pdf: 4994656 bytes, checksum: c9acb588dbfcaa318394a1f54b594a32 (MD5) Dissertação Claudia Maria do Nascimento Moreira - 01.pdf: 4994656 bytes, checksum: c9acb588dbfcaa318394a1f54b594a32 (MD5) / Made available in DSpace on 2014-11-26T15:15:37Z (GMT). No. of bitstreams: 2 Dissertação Claudia Maria do Nascimento Moreira - 01.pdf: 4994656 bytes, checksum: c9acb588dbfcaa318394a1f54b594a32 (MD5) Dissertação Claudia Maria do Nascimento Moreira - 01.pdf: 4994656 bytes, checksum: c9acb588dbfcaa318394a1f54b594a32 (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / O complexo adaptador 1 (AP-1) atua na formação do revestimento de vesículas com clatrina na rede trans-Golgi em células eucariontes. O conhecimento sobre o complexo AP-1 em tripanosomatídeos é escasso, mas já foi demonstrada sua importância na infectividade de Leishmania mexicana em macrófagos, assim como na viabilidade de Trypanosoma brucei. O Trypanosoma cruzi é um protozoário flagelado pertencente à família Trypanosomatidae, sendo o agente etiológico da doença de Chagas, a qual afeta milhões de pessoas no mundo. Neste contexto, esta dissertação teve como objetivo identificar os genes que codificam as quatro subunidades do complexo AP-1 no genoma de T. cruzi, analisar sua expressão e localização subcelular nesse parasita. Busca em banco de dados genômicos permitiu identificar as sequências codificantes para todas as subunidades do complexo AP-1, sendo obtidos os seguintes números de acesso gênicos: AP1-γ: XP_818958.1; AP1-β: XP_820334.1; AP1-µ:XP_818899.1; AP1-σ: XP_804127.1. Foram produzidos anticorpos em camundongos contra as proteínas recombinantes de todas as subunidades do complexo AP-1. Análise da especificidade dos anticorpos foi realizada por western blot e a localização subcelular das proteínas foi feita por imunofluorescência em microscopia de epifluorescência e microscopia confocal a laser. Resultados negativos foram obtidos com o antisoro contra a subunidade AP1-σ. Anticorpos obtidos contra as subunidades AP1-µ (policlonal) e AP1-β (monoclonal) reconheceram polipetídeos de tamanho compatível ao peso molecular da proteína endógena em extratos de formas epimastigotas, porém não reconheceram as proteínas por imunofluorescência. O antisoro policlonal obtido contra a subunidade AP1-γ reconheceu em extratos de diferentes formas evolutivas de T. cruzi (epimastigotas, tripomastigotas e amastigotas) um polipeptídeo de peso molecular compatível com o predito em banco de dados (~90 kDa). Imunolocalização demonstrou reação positiva pontual em região compatível com a do complexo de Golgi deste protozoário: entre núcleo e cinetoplasto de formas tripomastigotas e entre cinetoplasto e bolsa flagelar de epimastigotas e amastigotas. Colocalização da AP1-γ com a GTPase Rab7 (marcador de complexo de Golgi em T. cruzi) confirmou a localização desta subunidade no complexo de Golgi desse parasita. Nossos resultados demonstram que as subunidades do complexo AP-1 são conservadas e expressas em T. cruzi e que pelo menos a subunidade AP1-γ possui localização celular em complexo de Golgi, similar ao que é descrito em outras células eucariontes. / The adaptor complex 1 (AP-1) acts in the formation of clathrin-coated vesicles at the transGolgi network of eukaryotic cells. Knowledge about the AP-1 complex in trypanosomatids is scarce, but it has been already demonstrated its importance in the infectivity of Leishmania mexicana in macrophages, as well as the viability of Trypanosoma brucei. Trypanosoma cruzi is a protozoan flagellate that belongs to the family Trypanosomatidae, being the etiologic agent of Chagas disease, which affects millions of people worldwide. In this context, this study aimed to identify the genes encoding the four subunits of the AP-1 complex in the genome of T. cruzi and analyze their expression and subcellular localization in this parasite. Search in genomic database identified gene sequences coding for all subunits of the AP-1 complex, the following accession numbers being obtained: AP1-γ: XP_818958.1; AP1-β: XP_820334.1; AP1-µ: XP_818899 .1; AP1-σ: XP_804127.1. Antibodies were produced in mice against recombinant proteins of all subunits of the AP-1 complex. Analysis of the specificity of the antibodies was performed by western blot and subcellular localization of the proteins by immunofluorescence was done by epifluorescence microscopy and confocal laser microscopy. Negative results were obtained with the antiserum against subunit AP1-σ. Antibodies raised against the AP1-µ (polyclonal) and AP1-β (monoclonal) subunits recognized polypeptides with size compatible to the molecular weight of the endogenous protein in extracts from epimastigotes, but no proteins could be localized by fluorescence microscopy. The antiserum polyclonal obtained against the AP1-γ subunit recognized a polypeptide in extracts of different evolutionary forms of T. cruzi (epimastigotes, trypomastigotes and amastigotes) of molecular weight consistent with that predicted in database (~90 kDa). Immunolocalization showed punctual positive reaction in the region compatible with the Golgi complex of this protozoan: between nucleus and kinetoplast of trypomastigotes and between kinetoplast and flagellar pocket of epimastigotes and amastigotes. Colocalization of AP1-γ with the GTPase Rab7 (a marker of the Golgi complex in T. cruzi) confirmed the location of this subunit in the Golgi apparatus of this parasite. Our results demonstrate that the subunits of the AP-1 complex are conserved and expressed in T. cruzi and that at least the AP1-γ subunit has cellular localization in the Golgi apparatus, similar to that described in other eukaryotic cells.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O cÃncer colorretal (CCR) Ã uma das neoplasias mais prevalentes em todo o mundo, sendo uma das principais causas de Ãbito por cÃncer. Dentre as drogas utilizadas como primeira linha no tratamento do CCR e do CCR metastÃtico hepÃtico, o irinotecano apresenta destaque pelo impacto sobre o aumento da sobrevida dos pacientes. Contudo, o surgimento de efeitos colaterais associados ao irinotecano (IRI), como a mucosite intestinal (MI), tem impactado negativamente no curso terapÃutico do paciente, observando-se atrasos nos ciclos subsequentes de quimioterapia, reduÃÃo de doses e, por vezes, interrupÃÃo do tratamento. A MI e a diarrÃia grave sÃo efeitos colaterais frequentes que pode atingir de 15-25% dos pacientes em quimioterapia. Objetivos: Estudar os parÃmetros funcionais da barreira intestinal e os mecanismos envolvidos na mucosite intestinal induzida pelo Irinotecano e seu metabÃlito ativo, SN-38. MÃtodos: Camundongos C57BL/6 machos (WT), 20-25g, foram divididos em grupos (n=6-8), administrados por 4 dias com salina (5 mL/Kg, i.p) ou com irinotecano (IRI, 75 mg/Kg, i.p). Os animais foram analisados no 5Â dia [D5] ou 7Â dia [D7] quanto ao peso corpÃreo, escores de diarreia, contagem de leucÃcitos. ApÃs sacrifÃcio, uma amostra de intestino foi coletada para dosagem de mieloperoxidase, anÃlise histopatolÃgica, morfomÃtrica, e imunohistoquÃmica para TLR4. Adicionalmente, realizou-se o teste de permeabilidade e perfusÃo intestinal in vivo. Avaliou-se tambÃm a bacteremia e a translocaÃÃo bacteriana em linfonodo mesentÃrico e fÃgado. Em adiÃÃo, a participaÃÃo de receptores Toll-like 2 (TLR2), 4 (TLR4) e 9 (TLR9) da proteÃna adaptadora MyD88 e NOD1 na mucosite intestinal foi verificada pelo uso de camundongos knockout com deleÃÃo gÃnica especÃfica para aqueles receptores e seus respectivos camundongos selvagens (WT). Adicionalmente, realizou-se a avaliaÃÃo dos efeitos in vivo e in vitro do SN-38. Os dados foram analisados com ANOVA/teste de Bonferroni ou Kruskal Wallis/teste de Dunn. P<0,05 foi aceito. (Protocolo CEPA 99/10). Resultados: A injeÃÃo de IRI causou uma significativa (P<0,05) perda ponderal, leucopenia e diarreia, associada a um aumento da infiltraÃÃo de neutrÃfilos no jejuno, Ãleo e pulmÃo, com alteraÃÃes morfomÃtricas e uma intensa destruiÃÃo da arquitetura dos vilos e criptas, apoptose celular em camundongos WT versus animais injetados com salina. AlÃm disso, o IRI induz uma alteraÃÃo da barreira intestinal evidenciada pela diminuiÃÃo da excreÃÃo de lactulose, aliado a um aumento significativo (P<0,05) da secreÃÃo intestinal de sÃdio, potÃssio e cloreto. Os camundongos injetados com Irinotecano apresentaram bacteremia e translocaÃÃo bacteriana (P<0,05) no linfonodo mesentÃrico e fÃgado, quando comparados ao grupo salina. A identificaÃÃo bioquÃmica das bactÃrias translocadas evidenciou a presenÃa de Escherichia coli (75%), Citrobacter sp. (17,2%), BactÃrias Gram-Negativas NÃo-Fermentadoras e Pseudomona aeruginosa (18%) no grupo injetado com Irinotecano, aliado a um significativo aumento (P<0,05) da imunomarcaÃÃo para TLR4 no intestino de animais injetados com IRI D5 (4[3-4]) e D7 (4[3-4]) versus o grupo salina (1,5[1-4]). Observamos que a deleÃÃo gÃnica para o receptor TLR2 e a proteÃna adaptadora MyD88, mas nÃo para TLR4 ou TLR9, preveniram a perda ponderal e o dano funcional, relacionado aos eventos de diarreia, bem como as alteraÃÃes morfomÃtricas, histopatolÃgicas, infiltraÃÃo de neutrÃfilos e bacteremia induzida pelo Irinotecano versus o grupo WT injetado com IRI (P<0,05). Entretanto, a deficiÃncia genÃtica de NOD1 conferiu uma reduzida diarreia, sem reverter o dano prÃ-inflamatÃrio induzido pelo IRI. Adicionalmente, o SN-38 causou um aumento da atividade de mieloperoxidase (P<0,05), sem alterar a secreÃÃo intestinal na alÃa isolada de camundongos (P>0.05) versus o grupo injetado com salina. O SN38 foi capaz de induzir alteraÃÃes morfolÃgicas nas cÃlulas intestinais de ratos (IEC-6). ConclusÃo: O IRI induziu alteraÃÃo dos parÃmetros funcionais, detectadas pelo teste de permeabilidade e de perfusÃo intestinal. O IRI induziu uma bacteremia e translocaÃÃo bacteriana para ÃrgÃos perifÃricos. AlÃm disso, a deficiÃncia do receptor Toll-like do tipo 2, e da proteÃna MyD88 previniu o dano intestinal e a diarreia induzida pelo IRI. Contudo, a deficiÃncia de receptores NOD1 somente melhorou a diarreia. Adicionalmente, o SN38 foi associado a um aumento da infiltraÃÃo de neutrÃfilo, sem alteraÃÃo da secreÃÃo intestinal no modelo de alÃa isolada. / The Colorectal Cancer (CRC) is one of the most prevalent neoplastic diseases in the world and is one leading cause of death. Irinotecan is a drug used as first line treatment for CRC and its liver metastases and has markedly improved the overall survival of patients. However, irinotecan-related side-effects, which include intestinal mucositis (IM), have impacted negatively on therapeutic outcome, leading to delayed chemotherapy cycles, dose reductions and treatment interruption. IM and life-threatening diarrhea may affect up to 15-25% of patients under irinotecan-based cancer chemotherapy regimens. Aims: To study the intestinal barrier function and the mechanisms involved in the IM induced by irinotecan and its active metabolite, SN-38. Methods: Male C57BL/6 mice (WT, n=6-8) were divided into groups and injected with saline (5 mL/kg, i.p.) or irinotecan (IRI, 75 mg/kg, i.p.) for 4 days. Body weight, diarrhea and blood leukocyte count were assessed on days 5 [D5] and 7 [D7]. Following euthanasia, intestinal samples were collected for histopathology, morphometry, mieloperoxidase and imunohistochemistry assays. In addition, in vivo intestinal permeability and perfusion tests were performed. Bacteremia and bacterial translocation to mesenteric lymph node and liver were further carried out. Additionally, the participation of toll-like receptors 2 (TLR2), 4 (TLR4) and 9 (TLR9), the adaptor protein MyD88 and NOD1 receptor in the pathogenesis of IM were investigated by the use of WT mice and knockout with target gene disruptions. Furthermore, the in vivo and in vitro effects of SN-38 were studied. Data analysis was performed with ANOVA/Bonferroniâs test or Kruskal Wallis/Dunnâs test. P<0,05 was accepted. (CEPA 99/10). Results. IRI-injected WT mice presented a marked (P<0.05) weight loss, leukopenia, diarrhea, increased neutrophil infiltration in lung, jejunum, ileum associated with villi and crypt morphologic alteration and apoptotic cell death versus saline-administered mice. Besides, reduced lactulose renal excretion, gut secretion of sodium, potassium and chloride evidenced intestinal barrier dysfunction in IRI-injected WT mice versus saline-administered control mice (P<0.05). Bacterermia and bacterial translocation to mesenteric lymph node and liver were also observed in the IRI group. Biochemical identification of translocating bacteria revealed the presence of Escherichia coli (75%), Citrobacter sp. (17.2%), non-fermenting gram-negative bactÃria and Pseudomona aeruginosa (18%) in blood samples of IRI-injected mice. In addition, an increased TLR4 imunoexpression was detected in that group (IRI D5: 4[3-4] and D7: 4[3-4]) when compared with saline control (1.5[1-4]). Gene deletion to TLR2 and MyD88, but not to TLR4 or TLR9, prevented weight loss, diarrhea, intestinal morphometric alterations, neutrophil infiltration in the gut and bacteremia development versus the IRI-injeted WT group (P<0.05). However, NOD1 deletion was protective only against IRI-induced diarrhea without affecting the inflammatory infiltration. Furthermore, SN-38 promoted a marked neutrophil infiltration in ileum loops (P<0.05) but did not induce intestinal secretion of liquids (P>0.05) versus saline injected mice. Besides, cultured intestinal cells (IEC-6) incubated with SN-38 presented morphological changes in comparison to DMEN-cultured cells. Conclusions: IRI induced functional alterations in the gut and also bacteremia and bacterial translocation to peripheral organs. TLR2 and MyD88 deficiency prevented IRI-related intestinal damage and the diarrhea. However, NOD1 deficiency was protective only against diarrhea development. In addition, SN-38 might be responsible for the intestinal inflammatory reaction without affecting gut secretion of liquids.

Nef, an accessory protein of HIV-1, is a critical determinant of viral pathogenicity. The pathogenic effects of Nef are in large part dependent on its ability to decrease the amount of CD4 on the surface of infected cells. Early studies suggested that Nef induces downregulation by linking the cytosolic tail of CD4 to components of the host-cell protein-trafficking machinery. However, the specific sorting pathway that Nef uses to modulate CD4 expression remained uncertain. According to one model, Nef was thought to interfere with the transport of newly synthesized CD4 from the TGN to the cell-surface. Another model claimed that Nef facilitated the removal of CD4 from the plasma membrane. The primary goal of this thesis was to determine which of these models was correct. To accomplish this objective, a novel Nef-CD4 system was developed in Drosophila S2 cells. Nef was not only able to downregulate human CD4 in S2 cells, but it did so in a manner that was phenotypically indistinguishable from its activity in human cells. An RNAi screen targeting protein-trafficking genes in S2 cells revealed a requirement for clathrin and the clathrin-associated, plasma membrane-localized AP-2 complex in the Nef-mediated downregulation of CD4. In contrast, depletion of the related AP-1 and AP-3 complexes, which direct transport from the TGN and endosomes, had no effect. The requirement for AP-2 was subsequently confirmed in a human cell line. Yeast three-hybrid and GST pull-down assays were then used to demonstrate a robust, direct interaction between Nef and AP-2. This interaction was found to depend on a [D/E]xxxL[L/I]-type dileucine motif, located in the C-terminal loop of Nef, that is essential for CD4 downregulation. While mapping the binding site of AP-2 on Nef, a second determinant of interaction in the C-terminal loop was identified. Mutation of this motif, which conforms to a consensus [D/E]D diacidic sequence, prevented Nef from binding to AP-2 and down-regulating CD4. However, the same mutations did not affect the ability of Nef to interact with either AP-1 or AP-3, providing further evidence that these complexes are not required for the modulation of CD4 expression. Additional experiments indicated that the Nef diacidic motif most likely binds to a basic patch on AP-2 α-adaptin that is not present in the homologous AP-1 γ and AP-3 δ subunits. As with the Nef diluecine and diacidic motifs, the α-adaptin basic patch was shown to be necessary for CD4 downregulation. Moreover, all three of these motifs were needed for the cooperative assembly of a CD4-Nef-AP-2 tripartite complex, which was observed here for the first time using a yeast four-hybrid system. The data in this thesis uniformly support an endocytic model of Nef-mediated CD4 downregulation. Indeed, there is now strong evidence that Nef simultaneously binds CD4 and AP-2, thereby connecting the receptor to the cellular endocytic machinery and promoting its rapid internalization from the plasma membrane. In addition, the identification of novel motifs required for this process has provided new insights on endocytosis, and may facilitate the development of pharmacological inhibitors of Nef function.

Clathrin coated vesicles were the first type of coated vesicle to be characterised. The coat consists of two components, clathrin and adaptor (or AP) complexes, the AP-1 complex is associated with the clathrin coated vesicles that bud from the TGN and the AP-2 complex is associated with the clathrin coated vesicles that bud from the plasma membrane. A new type of adaptor-like complex was discovered in our laboratory and was published in 1996. The complex has been shown to consist of two known proteins, beta3B and mu3B, and two unknown proteins of 160kD and 22kD. Unlike the conventional adaptor complexes this complex is not associated with clathrin. The aim of this thesis was to complete the characterisation of the adaptor-like complex and to establish its function. My studies have shown that, the adaptor-like complex consist of an alpha/gamma like subunit, delta, a beta subunit (beta3A/B), a mu subunit (mu3A/B) and a sigma subunit (sigma3A/B). We named the adaptor-like complexAP-3, by analogy with the AP-1 and AP-2 complexes. The AP-3 complex is localised to perinuclear and more peripheral membranes in non-neuronal cells, with little overlap with endocytic markers. The beta subunit of the AP-3 complex is the major target for phosphorylation. Analysis of mice with mutations in the beta3A subunit, and in the delta subunit of the AP-3 complex, have revealed that the beta subunit is required for the stability of the mu subunit and that the delta subunit is essential for the stability of the whole complex. Further analysis of the mutant mice indicated that the mice lack significant levels of functional AP-3 complex. Studies on fibroblasts generated from these mice revealed that the AP-3 complex plays a role in the trafficking of LAMPI to lysosomes.

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第22087号 / エネ博第395号 / 新制||エネ||76(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 森井 孝, 教授 木下 正弘, 教授 片平 正人 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

P130Cas (Crk-associated substrate, CAS) is a multiadaptor protein important in integrin signalling where it positively regulates cell motility, invasion, proliferation and survival. CAS lacks enzymatic activity, but its binding to other signalling proteins could lead to the change of phosphorylation status of its substrate domain, which is the main mode, through which CAS takes part in regulating cell behavior. Local tensions in focal adhesions lead to an extension of CAS substrate domain, leaving phosphorylation sites more accessible for kinases, which subsequently leads to an increased CAS substrate domain phosphorylation. The CAS anchorage in focal adhesions is mediated by its SH3 domain, probably through the interactions with FAK, and also by C-terminal domain, where interaction partners are not known. The aim of my project is to find out, which proteins mediate the CAS anchorage to the focal adhesions. The elucidation of CAS anchorage to focal adhesions will contribute to the understanding of mechanosensory function of CAS. Experimental data suggest that tyrosine phosphorylation of the CAS SH3 domain plays an important role in the regulation of its binding properties. Another goal of my diploma project was to analyze the significance of tyrosine phosphorylation within SH3 domain and other...