A sepse é a principal causa de morte em unidades de terapia intensiva (UTIs) no mundo. A reduzida disponibilidade do aminoácido mais abundante do organismo, a glutamina contribui para o complicado estado catabólico da sepse. No presente estudo investigamos os efeitos da suplementação oral com L-glutamina e L-alanina (GLN+ALA), ambos na norma livre e como dipeptídeo, L-alanil-L-glutamina (DIP), sobre o eixo glutamina-glutationa (GSH), sistema imune, inflamação, proteínas de choque térmico (HSPs) e expressão de genes envolvidos com vias de sinalização proteica em animais endotoxêmicos. Camundongos C57/B6 foram submetidos à endotoxemia (Escherichia coli LPS, 5 mg.kg-1, grupo LPS) e suplementados por 48 horas com L-glutamina (1 g.kg-1) e L-alanina (0,61 g.kg-1, grupo GLN+ALA-LPS) ou 1,49 g.kg-1 de DIP (grupo DIP-LPS). A endotoxemia promoveu depleção da concentração de glutamina no plasma (71%), músculo esquelético (50%) e fígado (49%), quando comparado ao grupo CTRL, sendo restauradas nos grupos DIP-LPS e GLN+ALA-LPS (P<0,05), fato que atenuou a redução da GSH e o estado redox (taxa GSSG/GSH) em eritrócitos circulantes, musculo e fígado (P<0,05). A suplementação em animais endotoxêmicos resultou em uma upregulation dos genes GSR, GPX1 e GCLC no músculo e fígado. A concentração das citocinas plasmáticasTNF-&#945;, IL-6, IL-1&#946; e IL-10 foi atenuada pelas suplementações, bem como a expressão de mRNAs envolvidos com a resposta inflamatória, ativadas pela via do NF-&#954;B(P<0,05). Concomitantemente, verificou-se aumento da capacidade proliferativa de linfócitos T e B circulantes nos grupos GLN+ALA-LPS e DIP-LPS. A expressão de mRNAs e a concentração de HSPs no tecido muscular foi restabelecida pelas suplementações, contudo, a expressão mRNAs relacionados às vias de síntese e degradação proteica foi somente estimulada no tecido hepático(P<0,05). Os resultados do presente estudo demonstram que a suplementação por via oral com GLN+ALA ou DIP podem ser utilizados clinicamente como métodos nutricionais em reverter o quadro de depressão da disponibilidade de glutamina corporal da sepse induzida por LPS, tendo impacto no eixo glutamina-glutationa, sistema imune e inflamatório. / Sepsis is the leading cause of death inintensive care units (ICUs) in the world.The availability ofthe most abundant amino acid in the body, glutamine, is reduced in this situation, fact that contribute to the complicated catabolic state of sepsis. In the present study, we investigated the effects of oral supplementation with L-glutamine and L-alanine (GLN+ALA), both in their free form and as a dipeptide, L-alanyl-L-glutamine (DIP) on glutamine-glutathione axis (GSH), immune and inflammatory system, heat shock proteins (HSPs) expression and gene expressions involved in protein signaling pathways during endotoxemia. C57/B6 mice were subjected to endotoxemia (Escherichia coli LPS, 5 mg.kg-1, LPS group) and supplemented for 48 hours with L-glutamine (1 g.kg-1) plus L-alanine(0.61 g.kg-1, GLN+ALA-LPS group) or 1.49 g.kg-1of DIP (DIP-LPS group). Endotoxemia promoted depletion glutamine concentration in plasma (71%), skeletal muscle (50%) and liver (49%), when compared to the CTRL group, and was restored in the DIP-LPS e GLN+ALA-LPS (P<0.05), fact that attenuate the reduction of GSH and the redox state (GSSG/GSH rate) in circulating erythrocytes, liver and muscle (P<0.05). Supplementations in endotoxemic mice resulted in upregulation of GSR, GCLC and GPX1 genes in muscle and liver. Plasma concentration of TNF-&#945;, IL-6, IL-1&#946; and IL-10 were attenuated by supplementation as well as the expression of mRNAs involved in the inflammatory response, activated by NF&#954;-B pathway (P <0.05). At the same time, high proliferative capacity of circulating T and B lymphocytes GLN+ALA-LPS e DIP-LPS were observed. HSPs (protein and mRNAs) and in muscle were restored by the supplements, however, the mRNAs expression related to the synthesis and degradation of protein pathways was only stimulated in the liver (P <0.05). Our results demonstrate that oral supplementation with GLN+ALA or DIP can be used as clinically nutritional methods to reverse the depression of body glutamine availability during sepsis induced by LPS, impacting on the glutamine-glutathione axis, immune and inflammatory system.

A vacina contra hepatite B tem sido implicada como um desencadeador de doenças auto-imunes, mas ainda não existem estudos prospectivos no lúpus. Assim, avaliamos prospectivamente a segurança e eficácia da imunização com a vacina recombinante contra hepatite B (Euvax B® - LG) em pacientes com diagnóstico de lúpus. Foram selecionadas 28 pacientes com a doença inativa (SLEDAI<4), com idade entre 18 e 50 anos e sorologia negativa para o vírus da hepatite B (VHB). Os critérios de exclusão foram o uso de prednisona >=20 mg/dia e drogas imunossupressoras, anti-dsDNA e anticardiolipina negativos. Os dados clínicos e laboratoriais foram coletados na entrada do estudo e um mês após cada dose da vacina. Além disso, obtivemos dados do ano anterior usando o prontuário eletrônico padronizado. A média de idade foi de 34 ± 7,7 anos e a média da duração da doença foi de 10,4 ± 6,7 anos. Soroconversão adequada foi atingida no final do estudo (93%), embora tenhamos observado uma baixa freqüência após a primeira dose (4%) e após a segunda dose (54%). Nenhuma alteração significativa na média de SLEDAI foi detectada após cada dose durante o estudo (0,14 ± 0,52 vs. 0 vs. 0,61 ± 1,66 vs. 0,36 ± 1,34, p=0,11). Reforçando estes achados, os 11% de atividade de doença durante o período de vacinação foi semelhante aos 21% observados no ano anterior (p=0,46). Além disso, a média da dose de prednisona na entrada foi comparável à dose do final do estudo (2,86 ± 3,06 vs. 4,64 ± 8,25 mg/d, p=0,32). A freqüência do uso de terapia imunossupressora no período da vacinação (11%) foi semelhante aos 14% observados no ano anterior (p=0,66). A vacinação contra hepatite B apresentou uma resposta de anticorpos protetores adequada e foi segura nos pacientes com lúpus inativo. / Hepatitis B vaccination has been implicated as a potential trigger for autoimmune diseases but there are no prospective studies in lupus. We therefore assessed prospectively the safety and efficacy of immunization with recombinant DNA hepatitis B vaccine (Euvax B® - LG) in SLE patients. Twenty-eight consecutive inactive SLE patients (SLEDAI<4), age between 18-50 years and negative serology for hepatitis B virus (HBV) were selected. Exclusion criteria were prednisone > 20mg/day and immunosuppressive drugs. Clinical and laboratorial assessments were obtained at study entry and one month after the three doses. In addition, a previous one year evaluation was performed using a standard electronic protocol. The mean age was 34 ± 7.7 years and disease duration was 10.4 ± 6.7 years. An adequate seroconversion was achieved at the end of the study (93%), although a lower frequency after the first (4%) and second dose (54%) was observed. No significant change in mean SLEDAI score was detected after each dose throughout the study (0.14 ± 0.52 vs. 0 vs. 0.61 ± 1.66 vs. 0.36 ± 1.34, p=0.11). Reinforcing these findings, the 11% flares during vaccination was similar to the 21% observed in the previous year (p=0.46). Furthermore, the mean prednisone dose at study entry was comparable to the end of the study (2.86 ± 3.06 vs. 4.64 ± 8.25 mg/d, p=0.32). In addition, the frequency of immunosuppressive therapy during the vaccination period (11%) was alike to the 14% observed in the previous year before entry (p=0.66). Hepatitis B vaccination was safe in inactive SLE patients with an adequate vaccine response rate.

Le myélome multiple (MM) est un cancer hématologique qui se caractérise par une prolifération et une accumulation de cellules plasmocytaires malignes au niveau de la moelle osseuse (MO). Il représente 10% des hémopathies malignes et 2% de la mortalité par cancer dans le monde occidental. La principale conséquence de l’expansion plasmocytaire clonale médullaire est la sécrétion excessive d’une immunoglobuline (Ig) unique qui va être à l’origine du caractère multi-symptomatique de cette pathologie. Ainsi, les manifestations du MM se caractérisent par des lésions osseuses, une atteinte rénale, une anémie, une hypercalcémie et une immunodéficience humorale conduisant à des infections récidivantes. Son pronostic est mauvais avec une médiane de survie qui se situe entre cinq et sept ans sous chimiothérapie qui vise à éliminer les cellules plasmocytaires malignes.A partir de la lignée U266 de myélome, nous avons dérivé des clones résistants au bortézomib (R6). Grâce à une analyse par biopuces à ADN, nous avons identifié 160 gènes significativement régulés dans les cellules R6 par rapport à la lignée parentale U266. Nous avons établi, par une approche fonctionnelle, que la surexpression de la protéine HspB8 conduit, via l’activation de la dégradation autophagique, à l’élimination des agrégats protéiques et à compenser l’effet de l’inhibition du protéasome conférant ainsi la résistance des cellules de myélome aux inhibiteurs du protéasome.Dans un second temps, nous nous sommes intéressés à l’implication de la protéine Bcl-B (BCL2L10) dans la pathogenèse du MM. Nous avons confirmé que Bcl-B est impliqué dans la pathogénèse du MM (patients et modèle murin). / Velcade is one of the inescapable drug to treat patient suffering from multiple myeloma (MM) and resistance to this drug represents a major drawback for patients. However, the mechanisms underlying velcade resistance remain incompletely understood. We derived several U266 MM cell clones that resist to velcade. We derived several U266 MM cell clones that resist to velcade. U266- resistant cells were resistant to velcade-induced cell death but exhibited a similar sensitivity to various proapoptotic stimuli. Careful analysis of proteosomal subunits and proteasome enzymatic activities showed that neither the composition nor the activity of the proteasome was affected in velcade-resistant cells.In addition, pangenomic profiling of velcade-sensitive and resistant cells showed that the small heat shock protein HSPB8 was overexpressed in resistant cells. Finally, gain and loss of function experiment demonstrated that HSPB8 is a key factor for velcade resistance. In conclusion, HSPB8 plays an important role for the elimination of aggregates in velcade-resistant cells that contributes to their enhanced survival. Multiple myeloma (MM) evolves from a premalignant condition known as monoclonal gammopathy of undetermined significance (MGUS). However, the factors underlying the malignant transformation of plasmocytes in MM are not fully characterized. We report an MM phenotype in transgenic mice with Eμ-directed expression of the Bcl-B protein. With age, Eμ-bcl-b transgenic mice develop the characteristic features of human MM. In addition, this MM-like disease is serially transplantable, underlying the tumoral origin of plasmocytes.

Orientador: James Venturini / Resumo: Devido ao crescente isolamento de espécimes de Klebsiella pneumoniae resistentes a praticamente todas as classes de drogas antimicrobianas, a busca por novas drogas que sejam alternativa terapêutica para o tratamento das infecções por eles causadas se torna relevante. Nesse contexto, a complexação de íons metálicos a drogas antimicrobianas é uma das abordagens empregadas, uma vez que é possível gerar novas drogas com atividade superior as drogas já existentes. No presente estudo, foi realizada a síntese de metalofármaco por reação de coordenação entre sulfato de polimixina B e cobre (II). O produto desta reação foi caracterizado e sua atividade antimicrobiana frente a espécimes de K. pneumoniae resistentes aos carbapenêmicos e à polimixina B foi avaliada. Os resultados demonstraram que as concentrações inibitórias mínimas (MIC) do complexo sintetizado foram menores em relação aos MICs de polimixina B para 44,44% dos espécimes avaliados; para 33,33% dos espécimes os MIC’s foram equivalentes, e para 22,23% dos espécimes os MIC’s do complexo foram superiores aos MIC’s da polimixina B. Esses resultados são promissores, uma vez que houve um incremento na atividade bacteriana da polimixina complexada ao metal para quase metade dos espécimes avaliados, mostrando que a síntese de novas drogas antimicrobianas através da complexação de íons metálicos é uma técnica que deve ser mais explorada. Além disso, nossos resultados devem conduzir a novos estudos que visem a melhor compreensão da... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Due to the increasing isolation of Klebsiella pneumoniae specimens resistant to virtually all classes of antimicrobial drugs, the research for new drugs that are alternative therapeutics for the treatment of infections caused by them becomes relevant. In this context, the complexation of metal ions to antimicrobial drugs is one of the approaches used, since it is possible to generate new drugs with higher activity than existing drugs. In the present study, metallodrugs synthesis was performed by a coordination reaction between polymyxin B sulfate and copper (II). The product of this reaction was characterized and its antimicrobial activity against specimens of K. pneumoniae resistant to carbapenems and polymyxin B was evaluated. The results showed that the minimum inhibitory concentrations (MIC) of the synthesized complex were lower than the polymyxin B MICs for 44.44% of the evaluated specimens; for 33.33% of the specimens the MICs were equivalent, and for 22.23% of the specimens the MICs of the complex were superior to the MICs of polymyxin B. These results are encouraging, since there was an increase in the bacterial activity of metal complexed polymyxin for almost half of the evaluated specimens, showing that the synthesis of new antimicrobial drugs through the complexation of metallic ions is a technique that should be further explored. In addition, our results should lead to further studies aiming to a better understanding of the structure, mechanisms of action, toxicit... (Complete abstract click electronic access below) / Mestre

Blood group glycolipids are important tools in the study of microbial receptor interactions and other biological phenomena. Presently blood group glycolipids of interest are isolated from biological samples. However, all glycolipids are not readily available due to the low frequency of some phenotypes in the general population. The ability to acquire the rare glycolipids from the degradation of common glycolipids would be a useful alternative to trying to obtain the molecules from biological sources.This research set out to establish the ability of blood group glycolipids to be degraded into useful glycolipids in a controlled manner by acid hydrolysis and possibly metal catalysis. The initial experiments investigated the degradation/hydrolysis of the more readily available glycolipid globoside with a range of salts and acids to establish degradation concepts such as; temperature, type of acid, acid concentration, and the role of metal ions in glycolipid degradation. These concepts then led to a series of degradation experiments with the blood group glycolipids Leb and ALeb. These glycolipids were incubated with a range of acid concentrations and varying temperatures. Thin layer chromatography separation and chemical and immunochemical staining were the main methods used to identify the products of degradation.It was established that metal ions were not directly involved in the catalysis of glycolipids in the short-term, however some metal ions were indirectly implicated in their degradation due to their ability to form acid solutions. Acid hydrolysis was established as the principle mechanism for glycan chain degradation. In general it was found that the glycan chain primarily lost its fucose groups (in no particular order) and was then followed by sequential degradation of the remaining glycan chain. The glycan chain also appeared to have a protective function on the ceramide moiety. Degradation of globoside established a simple sequential pathway of glycan chain reduction from the non-reducing end. Blood group glycolipids ALeb and Leb first lost their fucose side groups followed by sequential reduction of the glycan chain. Although not fully controllable, degradation of Leb was able to produce Lea, Led and Lec. In contrast degradation of ALeb did not produce any Lea or Led. Instead A-type 1 and two novel A-like structures, 'linear A' and 'GalNAc-Lea' were generated. Lec was only produced from ALeb in extremely acidic conditions. This research established the ability to generate, by acid hydrolysis, a range of rare and "unnatural" novel glycolipids from more commonly available structures. It is of interest that the so-called unnatural glycolipids obtained from the acid hydrolysis of ALeb may, in theory, occur naturally in the acid environment of the stomach, and as such could have the potential to be implicated in disease. It is probable that by applying the principles learned here, a range of novel and natural structures suitable for use in the study of biological interactions can be obtained.

Systemic lupus erythematosus (SLE) and Sj?gren?s syndrome (SS) are both heterogeneous autoimmune diseases with strong B cell aspects. A proportion of SLE and SS patients exhibit elevated serum BAFF (B cell activating factor of the TNF family); BAFF plays a key role in B cell homeostasis, survival and tolerance. BAFF transgenic (Tg) mice develop nephritis and salivary gland destruction that resemble aspects of SLE and SS respectively. Autoimmune disease development in BAFF Tg mice correlates with marginal zone (MZ) B cell expansion and the abnormal presence of MZ-like B cells outside of the spleen. The role of MZ B cells in BAFF induced autoimmune disease was analysed by crossing BAFF Tg mice with Lymphotoxin-β knockout mice (creating LTβ-BTg mice) which lack most peripheral lymph nodes, and also lack MZ B cells as a result of disrupted splenic architecture. LTβ-BTg mice were not protected against nephritis but exhibited reduced salivary gland infiltration and destruction. Indicating that the development of sialadenitis but not nephritis in BAFF Tg mice is MZ B cell dependent. Nephritis development in LTβ-BTg mice was associated with the detection of B-1 B cells in the inflamed kidneys. As B-1a B cell survival is dependent on the spleen, the contribution of B-1a B cells to nephritis development in BAFF Tg mice was assessed by crossing BAFF Tg mice to congenitally asplenic Hox11-/- mice (creating Hox11 -BTg mice). The absence of a spleen and B-1a B cells in Hox11-BTg mice delayed the nephritis development. In contrast, splenectomy of BAFF Tg mice at 12 weeks of age did not alter nephritis onset. In these mice B-1a B cells persisted in the peritoneal cavity and MZ-like B cells were detected in the periphery 8 months after surgery. In summary, nephritis development in BAFF Tg mice is unaltered by the absence of MZ B cells, but delayed in the absence of a spleen, MZ and B-1a B cells. Thus, B-1a and B-1b B cells may be potential targets for the treatment of nephritis in SLE patients with elevated BAFF.

Background: The most recent estimate for the number of Australians living with chronic hepatitis B virus (HBV) infection is 150,000, with over one million ever having been infected. One in four people with chronic infection will die as a result. Worldwide, the burden of chronic HBV infection is great. As many as 400 million people are chronically infected, and the World Health Organisation estimates that as a result HBV infection is the tenth leading cause of death. / Aim: The aim of the research presented in this thesis is to improve the accuracy and relevance of our understanding of the epidemiology and control of HBV infection in Australia, through the development of new methodological approaches to the collection and analysis of relevant epidemiological data. / Methods: Three novel approaches were adopted. First, a serosurvey of a randomised, age-structured convenience sample of over 3200 specimens was performed spanning the period from 1995 to 2005 to estimate the prevalence of markers of infection with, and immunity to HBV. Secondly, a comparative analysis of the serosurvey results with national surveillance notifications since 1971 and migration records since 1945 was undertaken. Finally, a complex deterministic mathematical model of HBV infection in Australia was constructed simulating the entire population between 1951 and 2050. / Results: The serosurvey indicates that chronic infection with HBV is more common in the Victorian population than existing national serosurvey estimates suggest, and the coverage of immunisation programs (particularly of adolescents) is far from universal. Significant geographic, age, and gender disparities in the prevalence of chronic HBV infection were identified in the serosurvey, which appear in part to relate to historical migration patterns and which could be used to develop a targeted and effective public health response. The comparative analysis of the serosurvey results with notifications and migration data demonstrates coherence of these disparate sources of information, and suggest that knowledge of migration patterns can lead to robust predictions of future notifications. The novel regression model developed implies that at least 50,000 people with chronic HBV infection are undiagnosed. The mathematical model of HBV infection in the Australian population is unique in many respects, and has been validated against external data to provide reassurance regarding the accuracy of the simulated outcomes. Some of these outcomes include an estimated 160,000 Australians living with chronic HBV infection in 2009, increasing by several thousand people every year, and that less than 5 per cent of chronic infections entering the population are able to be addressed by domestic vaccination or other prevention programs. / Conclusion: The new insights into the epidemiology of HBV infection in Australia provided by the approaches described all suggest a large and increasing burden of chronic HBV infection. New approaches are needed to provide essential policy outcomes to assist and empower Australians living with chronic HBV infection. If this does not occur, the economic and human costs to our community are likely to become great.

<p>Examensarbetet som beskrivs i denna rapport handlar om en ny teknik för att förbättra säkerheten för flygplan i luften och i närheten av flygplatser. Denna teknik benämns ADS-B (Automatic Dependent Surveillance Broadcast), och är tänkt att göra det möjligt för piloter att själva få information om trafik i närområdet. Nuvarande system baserar sig i huvudsak på visuella observationer från flygledare i kontrolltorn samt radarspaning omkring flygplatserna. Med det nuvarande systemet kommer det att bli både dyrt och svårt att upprätthålla en acceptabel nivå på flygsäkerheten när trafiken ökar. </p><p>Arbetet har bedrivits i AerotechTelubs regi i Linköping samt med hjälp ifrån företaget Sectra Wireless Technologies AB. Huvuddelen av arbetet inriktar sig på implementerandet av C-funktioner för att hantera kommunikationen och sammankopplandet av ett tidigare skapat system, för grafikvisning, med en transponder som hanterar ADS-B (tillverkad av Sectra). Målet med detta var att göra förberedande arbete åt AerotechTelub som de förhoppningsvis kommer att kunna använda i ett eventuellt kommande projekt. Rapporten tar upp några standarder som hör till konceptet GP&C (Global Positioning & Communication), samt beskriver de delar som ligger till grund för programmets funktion. </p><p>Examensarbetet har resulterat i ett demonstrationsprogram för att visa hur en lösning av problemet kan se ut. Det har tyvärr inte gått att säkerställa om programmet fungerar till fullo, men genom simuleringar och andra tester har huvuddelen av programmets funktioner gått att verifiera.</p>

ABA type tri-block amphiphilic polyelectrolyte consisting of poly(methyl methacrylate-block-methacrylic acid-block-methyl methacrylate) (P(MMA-b-MAA-b-MMA)) was synthesized by atom transfer radical polymerization technique (ATRP) and the self-assembly behavior of the polymers in aqueous solution was studied over the course of neutralization. Combination of potentiometric and conductometric titrations along with dynamic light scattering (DLS) techniques were used to investigate the size and shape of aggregates at various degrees of neutralization. The effect of hydrophobic-hydrophilic (MMA-MAA) ratio and polymer chain length on the aggregation behavior during neutralization was studied. P(MMA-b-MAA-b-MMA) with longer MMA segment self-assembles via the close association mechanism through stronger self-entanglement of MMA chains, whereas P(MMA-b-MAA-b-MMA) with shorter MMA chain self-assembles via the open association mechanism, as confirmed by transmission electron microscopy (TEM). Conductometric titration was used to determine the counterion condensation during the course of neutralization. When the charge density of micelle approaches a critical value as neutralization progresses, counterion condensation of Na+ ions on the polymer chains occurs. The effect of counterion condensation on the aggregation behavior during neutralization was elucidated. / Singapore-MIT Alliance (SMA)

Thesis (Ph.D.)--Memorial University of Newfoundland, 2005. / Bibliography: leaves 268-300.