Bedrock geology of the Winchendon quadrangle is divided into two zones based on structures and lithology. The eastern zone consists of tightly folded Silurian Rangeley and Paxton metasediments and pegmatites. Planar, NNE-SSW striking, W dipping foliations are characterized by alternating phyllosilicate-rich and leucosome layers. Fold axes of tight to isoclinal upright and recumbent folding are parallel to SSW-NNE shallowly plunging sillimanite, quartz and muscovite lineations. In the western zone Rangeley schists were intruded by Devonian Hardwick and Coys Hill and Fitzwilliam plutons. Planar NNE-SSW striking foliations dip shallowly to steeply west. Fold axes of tight-to-isoclinal asymmetric to recumbent folds plunge shallowly to steeply SSW, parallel to elongate quartz and feldspar lineations. Fabrics in both portions developed in by greenschist facies mineral assemblages Asymmetric porphyroclasts and S-C/C’ fabrics from both areas display dextral asymmetry. Lateral extrusion as the result of pure-shear dominated E-W shortening and N-S extension is recorded by structures that exhibit strong contractional strains, accompanied by stretching fabrics. A 2-3 kilometer wide belt of deformation named the Bronson Hill-Central Maine Boundary Zone (BHCMBZ) correlates with the Conant Brook Shear Zone. This creates a zone of deformation that widens to the north and separates Ordovician plutons of the Bronson Hill zone from units of the Central Maine zone. North of Winchendon, retrograde deformation is absent and Acadian metamorphism and structures are preserved. This creates an inconsistency in along-strike deformation the length of the inferred terrane boundary. A larger terrane, composed of the Bronson Hill and Central Maine zones is proposed to account for the discontinuous deformation along strike.

Human immunodeficiency virus type 1 (HIV-1) infection causes general loss of immune response in humans. Presently, an estimated 34 million (31.4-35.9 million) people worldwide are HIV-1 positive and many more are being newly infected. In the absence of a definitive cure, anti-HIV-1 drug therapy helps to manage the infection by suppressing virus replication. However, extensive drug resistance against most of existing drugs demands alternative anti-HIV-1 strategies. The proper knowledge about HIV-1 replication is essential to guide the development of new anti-HIV-1 strategies. The research presented in this thesis aims to understand the role of HIV-1 Integrase (IN) and cellular co-factors interactions in the early stage virus replication. In the cytoplasm, HIV-1 cDNA exists as a high molecular weight nucleoprotein complex called pre-integration complex (PIC). The cDNA enters the nucleus as a part of PIC by active nuclear import and integrates into the host genome. HIV-1 Integrase (IN) protein has been recognized as a primary viral factor for HIV-1 nuclear import, but the key contributing cellular factor(s) is unknown. We have examined the requirement of different Importinα (Impα) isoforms for HIV-1 replication and identified the requirement of Impα3 for HIV-1 replication in HeLa cells, C8166T cells, and human macrophages. Further investigations showed the specific requirement of Impα3 for HIV-1 nuclear import. By analyzing the Impα3 interaction with HIV-1 proteins, we detected the IN interaction with Impα3 and C-terminal domain (CTD) of IN was essential for Impα3 interaction. These data led to the conclusion that Impα3 is required for HIV-1 nuclear import and interacts with IN. The IN-CTD consists of conserved basic amino acid rich motifs (211KELQKQITK, 236KGPAKLLWK, and 262RRKAK) that closely resemble the consensus classical nuclear localization signal (NLS) for Impα interaction. By substitution mutation and interaction analysis, 211KELQKQITK and 262RRKAK motifs in IN were identified as required for Impα3 interaction, IN nuclear localization, and HIV-1 nuclear import. Together, these data were useful in explaining the molecular mechanism of IN and Impα3 interaction and its requirement for HIV-1 nuclear import. Retrograde transportation of macromolecules in the cytoplasm is one of the prerequisites for their nuclear import. Although an earlier study implicated the dynein complex in retrograde transport of HIV-1, cellular and viral factors that are involved in this process are unknown. In this study, we have elucidated the HIV-1 IN interaction with the dynein light chain 1 (DYNLL1) in 293T cells, in vitro, and in HIV-1 infected cells. DYNLL1 is one of the adapter proteins that mediate the cargo recruitment to dynein complex. However, our data suggested that the IN and DYNLL1 interaction is essential for proper HIV-1 uncoating and cDNA synthesis but not for nuclear import. Surprisingly, DYNLL1 interaction of IN was dispensable for HIV-1 recruitment to dynein complex. These data led to the conclusion that the IN and DYNLL1 interaction is essential for proper HIV-1 uncoating and cDNA synthesis but not required for HIV-1 recruitment to the dynein complex or for retrograde transport. In summary, this study advances our knowledge on the role of IN and cellular factors interactions in different early steps of HIV-1 replication and offers potential contributions in the development of future anti-HIV-1 strategies.

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No description available.

Subtilase cytotoxin (SubAB) is the prototype of a novel class of AB₅ cytotoxins produced by Shiga-toxigenic Escherichia coli (STEC). The A subunit (SubA) is a serine protease that cleaves the ER chaperone BiP causing cell death by a previouslyundetermined mechanism. The B subunits of AB₅toxins typically recognise host cell glycan receptors and direct the subcellular transport of the A subunit. Although the function of SubA and its intracellular substrate have been elucidated, the B subunit (SubB) is relatively uncharacterised. The subcellular trafficking pathway of SubAB was initially examined. SubAB conjugated to Oregon Green 488 (SubAB-OG) was internalised by Vero cells by 5 min, and co-localised with its ER target BiP within 30 min. When Vero cells were incubated with SubAB-OG and either Alexa Fluor 594-conjugated Cholera toxin B subunit (CtxBAF594) or Texas Red-conjugated Shiga toxin B subunit (StxB-TR), individual cells exhibited differential toxin uptake. This was shown to be cell cycle-dependent, in which, SubAB-OG was preferentially internalised by cells migrating through G1 and early S phases. In contrast, CtxB-AF594 was taken up by cells in S through M phases and by a majority of cells in G1, while StxB-TR endocytosis occurred in cells traversing G1. Fluorescent SubAB co-localised with the clathrin marker transferrin, but not with Caveolin-1 (a marker for cholesterol-associated caveolae) and was subsequently trafficked via a retrograde pathway to the TGN, Golgi and ER. The clathrin inhibitor phenylarsine oxide prevented SubAB entry and BiP cleavage in SubAB-treated Vero, HeLa and N2A cells, while cholesterol depletion did not, demonstrating that, unlike either Stx or Ctx, SubAB internalisation is exclusively clathrin-dependent. Identification of the SubB receptor was initially approached using toxin overlay assays in which Vero cell glycolipid extracts were separated by thin-layer chromatography and overlaid with SubAB. SubAB exhibited a high affinity for particular acidic species in the ganglioside fraction. However, none co-migrated with commercial glycolipid standards. SubAB-OG also exhibited an affinity for the oligosaccharide structures of chimeric LPS from GM₂ and GM₃ bacterial receptor mimic constructs in an LPS toxin overlay assay. Glycan array analysis revealed that SubB possessed a unique affinity for carbohydrate receptors with a terminal Neu5Gcα(2→3)Galβ disaccharide. Monovalent receptor analogues with distal Neu5Gc or Neu5Gcα(2→3)Galβ and highly-sialylated α₁-AGP did not prevent endocytosis of SubAB-OG, BiP cleavage or cytotoxicity in Vero cells. This indicated that SubAB has a greater affinity for the host cell receptors than the receptor analogues and may engage multiple receptors displayed on a lipid bilayer. In addition to mediating toxin binding and subcellular trafficking, CtxB and StxB can also potentiate the immune response to co-administered antigen. Accordingly, the systemic immunomodulatory properties of SubB administered by the i.p. route were assessed in mice. Using SubAA₂₇₂ as a bystander antigen, SubB significantly increased mouse anti-SubAA₂₇₂ titres to levels that were comparable to those obtained using Alum adjuvant. However, when admixed with structurally-unrelated OVA, SubB did not significantly affect anti-OVA titres whereas Alum and CtxB did. This indicated that SubB may function as a systemic carrier protein (rather than an adjuvant) for particular antigens. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1363363 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2009

Previous research has shown that nitric oxide signalling in the lateral nucleus of the amygdala is required for the consolidation of Pavlovian conditioned fear. Given the evidence that nitric oxide can act as a retrograde signalling molecule in in vitro models of memory consolidation the question arises whether this is also occurring within behavioural memory models? Using auditory fear conditioning this research shows that nitric oxide does indeed act as retrograde signalling molecule in the fear system. Its synthesis in the lateral nucleus of the amygdala regulates conditioning induced expression of the immediate early gene early growth response gene 1 (EGR-1) in cells of the auditory thalamus that project to the lateral nucleus of the amygdala. The regulation of EGR-1 expression by the lateral nucleus of the amygdala was proven to be dependent on amygdala-based cellular excitation, nitric oxide synthesis and NR2B-NMDA receptor activation but not ERK/MAPK activity. Using an EGR-1 antisense oligonucleotide to prevent training induced EGR-1 expressions in the auditory thalamus it was shown that this gene upregulation is necessary for the consolidation of conditioned fear. Finally, inhibition of EGR-1 upregulation in the auditory thalamus was proven to impair conditioning induced increases in the presynaptic proteins synaptophysin, and synapsin II and II back in the lateral nucleus of the amygdala. Overall, the results of this dissertation have shown that nitric oxide acts as a retrograde messenger in a mammalian memory system by modulating gene expression in presynaptic cells. This modulation of gene expression serves to increase levels of presynaptic proteins back at the origin of nitric oxide synthesis. This supports the long standing doctrine that nitric oxide acts as a retrograde signalling molecule to coordinate presynaptic changes associated with memory formation.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / O objetivo deste trabalho consistiu na análise da infiltração apical em dentes retrobturados por três materiais: MTA, iROOT SP e Endo CPM Sealer. Para tal, foram utilizados 51 dentes humanos extraídos, incisivos centrais superiores, que foram instrumentados manualmente com limas tipo K, pela técnica Crown-down, obturados com compactação lateral e, após serem apicectomizados a 3mm aquém do ápice foram submetidos à retrobturação, com os três materiais propostos. As amostras foram divididas, randomicamente, em três grupos: GI MTA, GII iROOT SP e GIII Endo CPM Sealer, cada grupo com 15 amostras. Os dentes foram inseridos em tubos de eppendorfs, e feitos a impermeabilização do remanescente radicular utilizando duas camadas de cianocrilato, epóxi, e outra camada de esmalte. Em cada eppendorf foi adicionado caldo TSB estéril e uma suspensão de Enterococcos faecalis e adaptado ao frasco de vidro com meio de cultura enterococcosel. A infiltração bacteriana foi verificada pela turvação do meio de cultura. Após a análise no período de 60 dias, podemos concluir que durante esse tempo ocorreram infiltrações no Grupo I, 43,75 % das amostras apresentaram turvamento do meio de cultura demonstrando persistência da infecção. Já no Grupo II, 31,25 % das amostras tiveram crescimento bacteriano. Por fim no Grupo III, 25,00 % houve a infiltração. Grupos controle positivo e negativo para crescimento bacteriano foram realizados (n=3, cada). Os cimentos testados comportaram-se de maneira semelhante frente à infiltração bacteriana durante o período testado. / The objective of this study consisted in the analysis of apical leakage in retrofilled teeth by three materials: MTA, iROOT SP and Endo CPM Sealer. 51 extracted human central incisors were instrumented manually with K-type files by Crown-down technique, filled by lateral compactation and apicectomyzed to 3mm below the apex. Then, teeth were retrofilling with the three proposed materials. The samples were divided, randomly, in three groups: GI - MTA, GII - iROOT SP and GIII - Endo CPM Sealer, each group with 15 samples. Teeth were inserted into eppendorf tubes, and the remaining root were sealed using two layers of cyanoacrylate, epoxy, and another layer of nail varnish. In each eppendorf tube were added sterile TSB broth and a suspension of Enterococcos faecalis. The body proof was adapted to a glass bottle with culture medium Enterococcosel. The bacterial leakage was checked by turbidity of the culture medium. After analysis within 60 days the bacterial leakage was recorded: the Group I showed infiltration in 43.75% of body proof, the Group II in 31.25% and Group III 25%. Positive and negative control groups for bacterial growth were performed (n = 3, each). We can conclude that the tested cements behave similarly to bacterial infiltration front during the period tested.

Vaccinia virus (VACV), the prototypic poxvirus, undergoes a complex life cycle, with multiple stages that are not yet fully understood. This work studied two cellular proteins which had previously been identified by siRNA screens as playing proviral roles in the replication cycle of VACV: the dual specificity mitogen-activated protein kinase kinase 3 (MKK3) and vacuolar protein sorting 52 (Vps52). MKK3 is an upstream regulator in the p38 pathway which, along with MKK6, phosphorylates and therefore activates p38. In HeLa cell cultures, siRNA depletion experiments confirmed that MKK3 supported VACV replication. MKK3 knockdown reduced production of both early and late-class VACV proteins, suggesting that it facilitates viral gene expression. However, this difference did not translate to an in vivo model, as comparison between wild type and MKK3 knockout mice infected with VACV revealed no significant differences in virus replication or overall disease. The Golgi-associated retrograde protein complex (GARP) is composed of four large heteromeric proteins: Vps51, Vps52, Vps53 and Vps54, and plays a key role in retrograde transport from endosomes to the TGN. The effects of loss of GARP function were investigated using three techniques: mouse embryonic fibroblasts (MEFs) containing the hypomorphic Vps54 “wobbler” mutation, Vps52-targetting siRNA in HeLa cells and pharmacological inhibition of retrograde transport using the drug Retro-2. GARP loss resulted in a marked reduction in VACV spread due to a reduction specifically in “double wrapped” extracellular enveloped virion (EEV) production. Investigation of the mechanism by which GARP facilitates EEV production revealed a disruption of the VACV morphogenesis pathway prior to the double wrapping event, resulting in mislocalisation and aggregation of the viral membrane protein B5 within the cytoplasm. The effects of GARP loss translated to an in vivo model, as mice infected with VACV and treated with Retro-2 exhibited reduced viral replication and overall disease. These results identify GARP as a pro-viral host complex required for EEV production, and suggest that cellular retrograde transport pathways are required for double-wrapping of VACV virions. Overall, the study illustrates both the potential pitfalls of carrying out genetic screens in a transformed cell line and the power of such studies to nevertheless identify novel features of virus biology as well as druggable targets for antiviral intervention.

Propofol é amplamente utilizado para sedação consciente durante a colangiopancreatografia endoscópica retrógrada (CPER) e a dexmedetomidina tem potenciais propriedades sedativas de eficácia equivalente. O objetivo deste estudo foi examinar a hipótese de que dexmedetomidina é tão eficaz quanto propofol combinado com fentanil para promover sedação consciente durante a CPER. Trata-se de um ensaio clínico, randomizado, cego, double-dummy, que abrangeu 26 pacientes adultos, estado físico I-III de acordo com a Sociedade Americana de Anestesiologia. Os pacientes foram randomizados para receber propofol (n = 14) (concentração plasmática alvo de 2 a 4 μg. ml-1) associado a fentanil (1 μg. kg-1) ou dexmedetomidina (n = 12) (1 μg.kg-1 por 10 minutos), seguida de infusão de 0,2 a 0,5 μg.kg-1.min. Sedativos adicionais foram utilizados se não fosse alcançada sedação adequada com a dose máxima permitida. Os desfechos primários foram o nível de sedação mensurado através da Escala Richmond de Agitação-Sedação e a necessidade de sedativos adicionais. Os desfechos secundários foram as variações hemodinâmicas e respiratórias aferidas pela freqüência cardíaca, pressão arterial, saturação de oxigênio, pressão parcial exalada de dióxido de carbono e freqüência respiratória. Quanto à sedação, o risco relativo (RR) foi de 2,71 (IC 95%, 1,31 a 5,61) e o número de pacientes que foi necessário tratar (NNT) foi de 1,85 (IC95%, 1,19 a 4,21) para observar um paciente adicional apenas sonolento, 15 minutos após o início da sedação no grupo dexmedetomidina. Também o RR foi de 9,42 (IC95%, 1,41 a 62,80), e o NNT, de 1,42 (IC 95%, 1,0 a 2,29) para requerer analgesia adicional. Entretanto, no pós-operatório, o grupo dexmedetomidina apresentou maior redução na pressão arterial e na freqüência cardíaca e sobretudo, maior nível de sedação. Dexmedetomidina isolada não foi tão eficaz quanto propofol combinado a fentanil para promover sedação consciente durante a CPER. Além disso, foi associada com maior instabilidade hemodinâmica e tempo de recuperação prolongado. / Propofol is a wildly used therapeutic for conscious sedation during endoscopic retrograde cholangiopancreatography (ERCP), and dexmedetomidine has sedative properties of equivalent efficacy. The aim of this study was to examine the hypothesis that dexmedetomidine is as effective as propofol combined with fentanyl for providing conscious sedation during ERCP. A randomized clinical trial, blind and double-dummy, was conducted where twenty-six adults with physical status I-III according to the American Society of Anesthesiology were studied. Patients were randomized to receive propofol (n= 14) (target plasma concentration ranged from 2 to 4 μg/ mL) combined with fentanyl (1 μg/kg) or dexmedetomidine (n=12) (1 μg/kg over 10 min), followed by 0.2 to 0.5 μg/kg/min. Additional sedatives were used if adequate sedation was not achieved at the maximum dose allowed. The main outcomes were sedation level that was assessed by the Richmond Agitation-Sedation Scale and the demand for additional sedatives. The secondary outcomes were the effects on hemodynamic and respiratory parameters, which included heart rate, blood pressure, oxygen saturation, partial pressure of exhaled carbon dioxide and respiratory rate. In sedation, the relative risk (RR) was 2.71 (95% CI, 1.31 to 5.61) and number of patients needed to treat (NNT) was 1.85 (95% CI, 1.19 to 4.21) to observe one additional patient only drowsiness 15 minutes after beginning of sedation in the dexmedetomidine group. Also, the RR was 9.42 (95% CI, 1.41 to 62.80) and NNT 1.42 (95% CI, 1.0 to 2.29) to require additional analgesic. However, in the postoperative period, the dexmedetomidine group had a greater reduction in blood pressure and heart rate, and especially greater sedation level. Dexmedetomidine alone was not as effective as propofol combined with fentanyl for providing conscious sedation during ERCP. Furthermore, it was associated with more hemodynamic instability and prolonged recovery.