Abstract Interruption of cerebral blood flow during hypothermic circulatory arrest (HCA) predisposes neurons to glutamate excitotoxicity. Reperfusion is followed by leukocyte infiltration, which results in an inflammatory reaction in the brain tissue. In the first study, the presynaptic glutamate release inhibitor lamotrigine (L) and the leukocyte-depleting filter (LF) were studied to determine if their combination could mitigate brain injury after HCA (I). The aim of the second study was to evaluate the possible neuroprotective effect of a 14-hour period of mild (32°C) hypothermia after HCA (II). Recent experimental research has demonstrated the neuroprotective properties of erythropoietin (EPO) and fructose-1,6-bisphosphate (FDP), whose effects during and after HCA were evaluated in the third and the fourth studies (III, IV). A chronic porcine model was used. The animals were randomly assigned to the study groups as follows: 8 animals in the L+LF group, 8 in the L group, and 8 in the control group (I); 10 animals in the hypothermia group and 10 in the normothermia group (II); 10 animals in the EPO group and 10 in the control group (III), and 12 animals in the FDP group and 12 in the control group (IV). Monitoring of hemodynamics, metabolism, temperature, electroencephalogram (EEG), brain microdialysis, intracranial pressure (II-IV), and brain tissue oxygen (II-IV) was carried out. A daily behavioral assessment was performed until death or until elective sacrifice on the seventh postoperative day, after which the brain was prepared for a histopathologic examination. The results of these studies indicate that lamotrigine has a neuroprotective effect during HCA. This is observed in terms of EEG burst recovery, behavioral and histopathologic outcome, and brain microdialytic findings. The combined use of lamotrigine and leukocyte filtration may further improve survival. A 14-hour period of mild hypothermia after HCA is associated with a poor outcome. However, it may preserve its efficacy when used for no longer than 4 hours. Administration of EPO before HCA proved ineffective in reducing mortality or brain histopathologic injury. Findings from brain microdialysis, brain tissue oxygen tension, and neuronal apoptosis, however, suggest that the drug has neuroprotective properties. Administration of FDP before and after HCA is associated with better survival, behavioral outcome, and brain histopathologic scores. The metabolic and brain microdialytic findings also suggest that this drug has supportive effects on myocardial and brain metabolism.

Insulin resistance, a key risk factor for type 2 diabetes, can be defined as when cells fail to respond effectively to insulin. In striated muscle and fat, this manifests as impaired insulin-stimulated glucose uptake due to reduced plasma membrane insertion of the glucose transporter GLUT4. In cell culture models, insulin resistance induced by chronic exposure to insulin, endothelin-1 or glucosamine, is correlated with reduced immunoreactivity of the lipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in plasma membrane sheets. However, the reason for this decrease, and whether other factors that induce insulin resistance affect PtdIns(4,5)P2 levels, is unknown. Using L6 skeletal muscle myotubes and 3T3-L1 adipocytes, this project has investigated whether PtdIns(4,5)P2 levels are perturbed in insulin resistance induced by several factors, including exposure to insulin, oxidative stress, and treatment with tumour necrosis factor α, endothelin-1 or angiotensin II (Ang II).All these pre-treatments were found to abolish insulin-stimulated 3H 2-deoxy-glucose uptake, and significantly decrease PtdIns(4,5)P2 levels, measured in cell extracts by quantitative blotting using a PtdIns(4,5)P2-specific probe, developed from the PH domain of phospholipase C (PLC) δ. Importantly the ability of insulin to stimulate glucose uptake can be restored by replenishing PtdIns(4,5)P2 in L6 myotubes treated with insulin and Ang II. PtdIns(4,5)P2 levels are regulated by three families of proteins; PIP kinases, which synthesise it, phosphatases, which remove phosphate groups from the inositol headgroup, and PLCs, which hydrolyse it. Membrane preparations from Ang II- and insulin-induced insulin resistant L6 myotubes showed no differences in PtdIns(4,5)P2 production or dephosphorylation. However a significant increase in PLC activity was detected in membranes from insulin resistant cells and membrane localisation of PLCβ family members was increased in insulin resistant cells. Furthermore, studies using PLC inhibitors show a restoration of PtdIns(4,5)P2 levels in insulin resistant cells, leading to partial reversal of insulin resistance.This study therefore shows a causal link between decreased PtdIns(4,5)P2 levels and insulin resistance in L6 myotubes, and that PLCs are the reason for the PtdIns(4,5)P2 decrease in Ang II- and insulin-induced insulin resistance. PLCs, or their activation pathways, may thus be a novel target for combating insulin resistance, and preventing type 2 diabetes.

Chloroplasts of many species of hornworts (Anthocerotae) have a structure that resembles the pyrenoid of green algae but whether these two structures are homologous has not been determined. We utilized immunogold labelling on thin sections to determine the distribution of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the major protein of algal pyrenoids, in sixteen hornwort species with and without pyrenoids. Several species (Phaeoceros laevis, Anthoceros punctatus, A. formosae, A. laminiferus, Folioceros fuciformis, Folioceros sp., Dendroceros tubercularis, D. japonicus, D. validus, Notothylas orbicularis, N. temperata, and Spaerosporoceros adscendens) have uniplastidic (or primarily uniplastidic) cells with large prominent multiple pyrenoids. In all of these species, the labelling is found exclusively in the pyrenoid and, with the exception of the Folioceros, Dendroceros, and Notothylas species, the labelling is randomly distributed throughout the pyrenoid. In the exceptional species, the pyrenoids have prominent pyrenoglobuli or other inclusions that are unlabelled. In Megaceros flagellaris and M. longispirus, the cells are multiplastidic (with the exception of the apical cell and some epidermal cells) and the chloroplasts lack pyrenoids. Anthoceros fusiformis and Phaeoceros coriaceus have primarily uniplastidic cells but the chloroplasts lack pyrenoids; only an area of stroma in the center of the plastid devoid of starch, reminiscent of a pyrenoid, is found. In all of the species lacking pyrenoids, RuBisCo is found throughout the stroma, including the stromal spaces made by the so-called channel thylakoids. No preferential accumulation of RuBisCo is found in the pyrenoid-like region in A. fusiformis and P. coriaceus. These data indicate that 1) the hornwort pyrenoid is homologous to algal pyrenoids in the presence of RuBisCo; 2) that at least some of the RuBisCo in the pyrenoid must represent an active form of the enzyme; and 3) that, in the absence of pyrenoids, the RuBisCo is distributed throughout the stroma, as in higher plants.

Glucose 1,6-bisphosphate is an important small molecule involved in the regulation of glycolysis. Four enzymes synthesize this compound. One enzyme is known to degrade it, glucose 1,6-bisphosphatase. Other groups have produced work that indicates that there are two isoforms of this enzyme, one predominant in the brain and one in the muscle. This thesis contains the work performed in attempts to isolate and characterize the muscle isoform of glucose 1,6-bisphosphatase. While this enzyme was not isolated, much was learned about it and the results from this work may help in the future identification of this enzyme.

No description available.

No description available.

Phospholipase Cβ (PLCβ) cleaves phosphatidylinositol-4,5-bisphosphate (PIP₂) into the second messengers inositol-1,4,5-triphosphate (IP₃) and diacylglycerol (DAG). IP₃ increases intracellular Ca²⁺, while DAG remains in the membrane, and together with increased Ca²⁺, activates protein kinase C (PKC). PLCβ has low basal activity but is activated following stimulation of G_i- and G_q-coupled receptors through direct interactions with Gα_q and Gβγ. PLCβ is essential for normal cardiomyocyte and vascular smooth muscle function and regulates cell proliferation, survival, migration, and differentiation. However, increased PLCβ activity and expression results in arrhythmias, hypertrophy, and heart failure. PLCβ must interact with the cell membrane for its activity. While heterotrimeric G proteins stimulate PLCβ, they are insufficient for full activation, suggesting the membrane itself contributes to increased lipid hydrolysis, potentially via interfacial activation. However, how the composition of the membrane and its resulting properties, such as surface charge, contribute to adsorption and interfacial activation is not well-established. Furthermore, whether or how interfacial activation also impacts other regulatory elements in PLCβ and Gα_q-dependent activation is unknown. Using an innovative combination of atomic force microscopy on compressed lipid monolayers and biochemical assays, we are beginning to understand how the membrane itself, PLCβ autoinhibitory elements and Gα_q regulate PLCβ activation. These studies provide the first structure-based approach to understanding how the cell membrane regulates the activity of this essential effector enzyme.

No description available.

A solução de preservação da Universidade de Wisconsin (UW) é considerada a solução padrão para preservação de fígados, rins e pâncreas. A frutose-1,6-bisfosfato (FBP) é uma substância que apresenta efeito protetor do fígado contra injúrias provocadas por agentes químicos e ocorridas durante o período de isquemia-reperfusão. O objetivo deste estudo foi avaliar os efeitos da FBP na composição de soluções para preservação de fígados para transplante. Neste estudo experimental, a perfusão e a preservação dos fígados foram realizadas em cada grupo com as soluções de UW, UW contendo 10 mmol/L de FBP (UWM) e FBP 10 mmol/L (FBPS), respectivamente. Os fígados foram armazenados em um recipiente plástico contendo solução de preservação a 4oC por 24 horas. As mensurações bioquímicas de AST, ALT, LDH e TBARS foram realizadas em amostras das soluções de preservação nos tempos de 0, 12, 18 e 24 horas. A análise histológica foi realizada em fígados preservados por 24 horas. O grau de preservação observado com as soluções de UW e FBPS foi similar até 18 horas de armazenamento. A adição de 10 mmol/L de FBP à solução de UW provocou um aumento das injúrias ocorridas e uma pior preservação quando comparado ao grupo armazenado em UW. A FBP protegeu os fígados contra danos causados pelos radicais livres por tempos de preservação inferiores a 18 horas. Não houve diferença significativa entre os grupos na análise histológica dos fígados preservados no tempo de 24 horas. A FBP utilizada em solução foi eficaz na preservação de fígados, podendo ser um importante constituinte para outras formulações. / University of Wisconsin (UW) solution is considered the standard preservation solution for livers, kidneys and pancreas. Fructose-1,6-bisphosphate (FBP) has been reported to have a protective effect in liver injury caused by chemical agents and ischemic-reperfusion damages. The aim of this study was to evaluate the effects of FBP in storage solution for cold liver preservation. In this experimental study, we divided the rats in three experimental groups. The perfusion and preservation of the liver were performed on each group with UW, UW containing 10 mmol/L of FBP (UWM) and FBP 10mmol/L (FBPS) solutions, respectively. The livers were stored in a plastic bag containing storage solution at 4oC for 24 hours. Biochemical measurements of AST, ALT, LDH e TBARS were realized in cold storage solution samples at 0, 12, 18 and 24 hours of preservation. Histological evaluation was performed in tissue samples obtained after 24 hors of cold storage. The preservation grade was similar in FBPS and UW solutions during 18 hours. Addition of 10 mmol/L of FBP in UW solution induced liver injury and a poor preservation when compared to group of livers preserved in UW solution. FBP protects the liver from injury caused by free radicals for preservation times bellow 18 hours. There were no significant histological differences between groups after 24 hours of preservation. FBP could be an important component of cold storage solutions for liver preservation.

O diabetes mellitus tipo 1 (DM1) é uma síndrome autoimune órgão-específica caracterizada pela destruição seletiva de células β que leva à morte celular. A DM1 é a causa de mais de 5% do total de mortes na população por ano e são poucas as estratégias de tratamento disponíveis. As linhagens celulares, são amplamente utilizadas nos estudos da DM1 como um modelo in vitro. A linhagem celular derivada de insulinoma murino, chamada de MIN6, assemelha-se as ilhotas de camundongo. Sendo assim, pesquisadores comprovaram que estas células apresentam características funcionais semelhantes às células β-pancreáticas em resposta a glicose e a outros secretagogos. Através de agregação espontânea e crescimento tridimensional, estas células formam as pseudoilhotas (PIs). A frutose-1,6-bisfosfato (FBP) é um açúcar bifosforilado, que apresenta duas estruturas anoméricas denominadas α e β furanose. Já são conhecidas algumas ações importantes da FBP, como: ação citoprotetora, antioxidante e anti-inflamatória. Com base no descrito, o objetivo deste estudo foi avaliar a possível ação citoprotetora da FBP sobre células MIN6, cultivadas em monocadas e como pseudoilhotas. Para tal, foram avaliados os efeitos do tratamento com diferentes concentrações (0,30 mM, 0,62 mM e 1,25 mM) de FBP sobre a viabilidade, proliferação e síntese de insulina em células MIN6. Também foram determinados os efeitos da FBP na formação, crescimento e viabilidade das PIs. MIN6 Os resultados mostraram, que em qualquer das doses testadas, a FBP aumentou a adesão das células MIN6. No entanto, os tratamentos com 0,62 e 1,25 mM de FBP provocaram uma inibição significativa da proliferação. A sintese de insulina foi detectada por imunocitoquímica e sua secreção basal estimulada por glicose determinada por ELISA. Como esperado, a formação e o crescimento de PIs é tempo dependente, tanto nas culturas controle como nas tratadas com FBP. Nas culturas tratadas com FBP, a formação de PIs maiores (150-300 μm) foi menor que nas culturas controle. A incorporação de iodeto de propídeo mostrou um aumento da porcentagem de células inviáveis nas culturas de PIs tratadas com 1,25 mM de FBP, sendo citotóxica nessa concentração. Embora ainda sejam necessários mais estudos para aprofundar o conhecimento da atividade citoprotetora da FBP em outros modelos celulares e em ilhotas pancreáticas, nossos resultados mostram que em monocamadas e PIs, da linhagem celular MIN6 utilizadas nos experimentos, que a FBP não apresentou atividade citoprotetora. / Diabetes mellitus type 1 (DM 1) is an organ-specific autoimmune syndrome characterized by the selective destruction of β cells that leads to cell death.The DM1 is the cause of more than 5% of all deaths in the population per year and there are few treatment strategies available. Cell lines are widely used in studies of DM1 in vitro model. The cell line derived from a mouse β insulinoma MIN6 is similar to the mouse islets. Studies of these cells exhibit functional characteristic similar to pancreatic β-cells in response to glucose and other secretagogues. Spontaneous aggregation and tridimensional growth those cells form the pseudoislets (PIs). Fructose-1,6-bisphosphate (FBP) is a biphosphorylated sugar with two anomeric forms designated α and β furanoses. Important actions of FBP as antioxidant, cytoprotective and anti-inflammatory. Based on the described, in this work, our objective is evaluate the cytoprotective effect of FBP on MIN6 cells in monolayer and pseudoislets agregate cells. The effects of FBP with different concentrations (0.30 mM, 0.62 mM and 1.25 mM) in MIN6 cells on the proliferation, viability and insulin synthesis. We also determined the effects of FBP in the formation, growth and viability of PIs. The results showed that all doses of FBP tested increased adhesion on MIN6 cells. However, the treatment with 0.62 and 1.25 mM of FBP causes a significant decrease in cell proliferation. Insulin synthesis detected by immunocytochemistry, basal and glucose-stimulated insulin secretion was determined by ELISA. As expected, PIs formation and growth is time dependent in the control cultures and treated with FBP. In cultures treated with FBP the formation of larger (150-300 μm) PIs was lower than in control cultures. Propidium iodide incorporation showed an increasing percentage of nonviable PIs cells in cultures treated with 1.25 mM of FBP, including cytotoxic. More studies are required to deepen understanding of the role of cytoprotective activity of FBP in other cell types and in pseudoislets, but our results indicate in cell line MIN6, monolayers and pseudoislets, that FBP did not show cytoprotective activity.