A participação do fígado na homeostasia da glicose materna ainda não está esclarecida. Este estudo avaliou a produção hepática de glicose em ratas prenhes e controle bem como algumas das principais vias metabólicas hepáticas envolvidas nas alterações metabólicas mediadas pelo fígado ao final do período de prenhez em ratas. Houve diminuição do conteúdo de glicogênio hepático e glicose circulante em ratas prenhes. A taxa de gliconeogênese diminuiu em prenhes assim como o conteúdo de IR e sua fosforilação em tirosina mediante estímulo com insulina, porém a fosforilação em serina da AKT e o conteúdo e expressão gênica da PEPCK, mantêm-se como em controles. Há aumento do conteúdo de PTP1B e de sua associação ao IR na presença de insulina. Houve diminuição do conteúdo de G6Pase, AMPK total e da fosforilação da AMPKa/1 e, conseqüentemente, da fosforilação da ACC em ratas prenhes. Há aumento de triglicérides circulantes e diminuição de triglicérides no tecido hepático. Aumenta a expressão gênica da FAS e diminuem as expressões de G-6-Pase, MEs, LXR, SREBP1 e PFK. Os resultados indicam não haver resistência à insulina no fígado de ratas prenhes e que a atividade metabólica hepática no período está voltada para a síntese de novo de ácidos graxos. / The involvement of the liver in maternal glucose homeostasis is still unclear. This study evaluated the hepatic glucose production in pregnant rats and some of the hepatic metabolic pathways involved in metabolic disorders mediated by the liver in late pregnancy.There was a decrease in liver glycogen content and plasma glucose in pregnant rats. The rate of gluconeogenesis decreased as well as the contents of IR tyrosine phosphorylation after insulin stimulus, but the pSer-AKT, PEPCK, SHP2 contents, SHP2-IR association and the expression of PEPCK remained unaltered. The content of PTP1B and its association with IR decreases in the presence of insulin. The content of G6Pase, AMPK and the phosphorylation of AMPK a/1 and consequently the phosphorylation of ACC decreases in liver from pregnant animals. Triglyceride levels and the expression of FAS was increased in pregnant rats whereas the triglycerides reduced in the liver. The expression of G-6-Pase, MEs, LXR, SREBP1 and PFK decreased in the liver of pregnant rats. The results indicate no insulin resistance in the liver of pregnant rats, and that hepatic metabolic activity in this period is focused on de novo fatty acids synthesis.

A análise de fluxos metabólicos para a produção de PHAMCL por Pseudomonas sp. LFM046 revelou que a baixa eficiência de conversão de carboidratos em PHA (60-70% do valor máximo teórico) deve estar associada à utilização principal da via das pentoses (VP) para o metabolismo de carboidratos. Eficiências significativamente maiores poderiam ser obtidas se uma parcela maior da glicose fosse metabolizada pela via Entner-Doudoroff (ED). Assim, neste trabalho foi realizada a análise de fluxos metabólicos utilizando glicose marcada (C13) e o melhoramento genético de Pseudomonas sp. LFM046 utilizando estratégia de engenharia metabólica. Experimentos com glicose marcada confirmaram um maior fluxo de carboidratos pela VP em relação a ED. A superexpressão de genes específicos de ED em Pseudomonas sp. LFM046 demonstrou apenas pequenos aumentos na eficiência de conversão de carboidratos em PHA pelas linhagens recombinantes. / The metabolic flux analysis for PHAMCL production by Pseudomonas sp. LFM046 revealed that the low efficiency of carbohydrates conversion into PHA (60-70% of the maximum theoretical value) should be associated with the main use of pentose phosphate pathway (PP) for the carbohydrate metabolism. Significantly higher efficiencies could be obtained if a larger portion of the glucose was metabolized via the Entner-Doudoroff (ED). Therefore, the metabolic fluxes analysis was performed in this work using labeled glucose (13C) and the genetic improvement of Pseudomonas sp. LFM046 by using metabolic engineering approach. Experiments with labeled glucose confirmed an increased flow of carbohydrates by PP compared to ED. The overexpression of specific genes from ED in Pseudomonas sp. LFM046 showed only small increases in the efficiency of carbohydrates conversion into PHA by recombinant strains.

Esse estudo teve como objetivo avaliar as alterações hemodinâmicas e do nível de glicemia decorrentes do uso do anestésico local articaína a 4% com epinefrina nas concentrações 1:100.000 (A100) e 1:200.000 (A200) em cirurgias periodontais na maxila, realizadas em diabéticos. Em relação aos anestésicos, foram avaliados: tempo de início de ação, duração da anestesia sobre os tecidos mole, analgesia pós-operatória, sangramento trans-operatório, qualidade da cicatrização, parâmetros hemodinâmicos e glicemia medidos durante as cirurgias. Para isso, 18 voluntários com idades entre 40 e 65 anos foram selecionados. Destes, 10 não apresentavam alterações sistêmicas (não diabéticos-não DM), enquanto 8 eram portadores de diabetes mellitus não insulinodependentes (DM), todos com condições periodontais semelhantes. Foram submetidos a cirurgias periodontais bilateralmente na região da maxila sob anestesia local com A100 e A200, de forma duplo-cega, randomizada e cruzada. O tempo cirúrgico foi semelhante para todos os grupos, e A100 e A200 mostraram-se igualmente eficazes para cirurgias periodontais. Foi utilizada quantidade idêntica de ambos anestésicos em todas as cirurgias (1 tubete; 1,8ml), o tempo cirúrgico foi semelhante em todos os procedimentos. O tempo de inicio de ação foi similar para todos, independentemente da concentração de epinefrina ou presença de diabetes. O tempo de duração da anestesia foi significativamente maior para os DM, sem haver correlação com a concentração de epinefrina. O sangramento trans-operatório foi significativamente maior nos pacientes diabéticos apenas na fase de incisão com A200. Nas demais fases, o sangramento foi muito semelhante entre DM e Não DM. A analgesia pós-operatória foi considerada excelente, refletindo na baixa ingestão de analgésicos (paracetamol), especialmente pelo grupo DM, independentemente da concentração de epinefrina. Quanto à cicatrização, não houve diferença entre os grupos. As mudanças transitórias nos parâmetros hemodinâmicos (frequência cardíaca-FC; pressão arterial-PA) tiveram pouco significado clínico, apesar de os diabéticos apresentarem certa tendência a elevação na PA nas fases de incisão e debridamento. Os diabéticos não apresentaram elevação da glicemia ao longo das fases cirúrgicas, independente da concentração de epinefrina presente na solução anestésica, ao passo que os não diabéticos mostraram que a maior concentração de epinefrina resulta num maior tempo para a normalização dos níveis glicêmicos. Concluindo, tais resultados mostram que A100 e A200 são equieficazes para a realização de cirurgias periodontais. Sendo assim, a utilização de anestésico com menor concentração de epinefrina (1:200.000) parece ser a melhor escolha para os indivíduos portadores de alterações sistêmicas como os diabéticos. / The present study compared the effect of articaine 4% associated with epinephrine in two different concentrations, 1:100.000(A100) and 1:200.000(A200), in periodontal surgeries performed in diabetic patients. We analyze hemodynamic parameters, blood glucose concentration, onset and duration of anesthetic action on soft tissues, intraoperative bleeding and wound healing. Eighteen volunteers, age range 40 to 65 years, with similar periodontal disease and conditions, were separate in two groups, type 2 diabetes mellitus (DM, 8 volunteers) or with no diabetes mellitus (Non DM, 10 volunteers). They´re submitted to a matched bilateral periodontal surgery in maxilla, under local anesthesia with either A100 or A200, in a double blind, randomized, crossed manner. The duration of surgery was the same for all groups, with A100 and A200 being equally effective for periodontal surgeries. Identical volumes of both anesthetic solutions were used (1 cartridge:1,8ml) in all surgeries. The anesthetic latency was similar in diabetics or non-diabetics for both epinephrine concentration. In diabetic patients the anesthetic duration was increased regardless the epinephrine concentration. Intraoperative bleeding only increased in diabetic patients with A200 during incision phase. The duration of postoperative analgesia was excellent, reflecting by a low intake of postoperative medications (paracetamol). Wound healing was relatively normal for all volunteers regardless the local anesthetic employed or presence of diabetes. The transient changes in blood pressure or hart hate were not clinically significant but the diabetic patients have some tendency to increase their blood pressure in some surgical phases. In diabetic subjects, blood glucose have no increase throughout surgical phases, regardless the epinephrine concentration present in the anesthetic solution, but the Non DM presents a prolonged time for normalize their blood glucose after A100. In conclusion, this study demonstrate that epinephrine concentration (1:100.000 or 1:200.000) in articaine 4% solution have the same efficacy for periodontal surgeries. Therefore, the formulation with a lower vasoconstrictor concentration (A200) seems to be the more adequate choice for patients with systemic diseases like diabetes.

Chan Chung Sing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 93-101). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ix / List of Abbreviations --- p.xvii / Chapter CHATPER ONE --- INTRODUCTION / Chapter 1.1 --- Glucose Homeostasis --- p.1 / Chapter 1.1.1 --- Function --- p.1 / Chapter 1.1.2 --- Origins and regulation of glucose --- p.2 / Chapter 1.1.3 --- Glucoregulatory factors --- p.4 / Chapter 1.1.4 --- Insulin --- p.6 / Chapter 1.1.4.1 --- Function of Insulin --- p.7 / Chapter 1.1.4.2 --- Discovery and Production of Insulin --- p.7 / Chapter 1.1.4.3 --- Insulin Signaling Pathway --- p.8 / Chapter 1.1.4.3.1 --- Insulin Receptor --- p.8 / Chapter 1.1.4.3.2 --- MAPK Pathway --- p.9 / Chapter 1.1.4.3.3 --- Phosphatidylinositol 3-kinase (PI3-K) Pathway --- p.10 / Chapter 1.1.5 --- Glucose Transporters --- p.11 / Chapter 1.1.6 --- Role of skeletal muscle in glucose homeostasis --- p.13 / Chapter 1.1.7 --- Insulin Resistance --- p.14 / Chapter 1.1.8 --- Glucose abnormality and its complications --- p.16 / Chapter 1.2 --- Actin --- p.19 / Chapter 1.2.1 --- Function of Actin --- p.20 / Chapter 1.2.2 --- Actin Accessory Protein --- p.22 / Chapter 1.2.3 --- Actin Polymerization --- p.23 / Chapter 1.3 --- "Interaction between Insulin, GLUT4 and Actin in Glucose Homeostasis" --- p.24 / Chapter 1.3.1 --- Insulin-Induced Actin Remodeling --- p.25 / Chapter 1.3.2 --- Actin Remodeling and Insulin-Induced GLUT4 Translocation --- p.26 / Chapter 1.3.3 --- Involvement of Insulin Signaling Molecules in Actin Remodeling --- p.27 / Chapter 1.3.4 --- Actin Remodeling and Insulin Resistance --- p.30 / Chapter 1.4 --- Hypothesis and Objective --- p.30 / Chapter 1.4.1 --- Rationale --- p.30 / Chapter 1.4.2 --- Hypothesis --- p.31 / Chapter 1.4.3 --- Objective --- p.31 / Chapter CHAPTER TWO --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.33 / Chapter 2.2 --- Cell Culture --- p.36 / Chapter 2.2.1 --- Cell Culture --- p.36 / Chapter 2.2.2 --- Reagents Preparation and Incubation --- p.39 / Chapter 2.3 --- 2-Deoxyglucose Uptake --- p.39 / Chapter 2.4 --- Immunofluorescence Microscopy --- p.41 / Chapter 2.4.1 --- Permeabilized cell staining --- p.41 / Chapter 2.4.2 --- Membrane-intact cell staining --- p.43 / Chapter 2.4.3 --- The analysis of actin remodeling reduction --- p.44 / Chapter 2.5 --- Live Image Microscopy --- p.44 / Chapter 2.6 --- Transmission Electron Microscope Study --- p.44 / Chapter 2.7 --- Statistical Analysis --- p.46 / Chapter CHAPTER THREE --- RESULTS / Chapter 3.1 --- Cell Growth --- p.48 / Chapter 3.2 --- Acute Effect of Insulin on L6 myotubes --- p.48 / Chapter 3.2.1 --- Immunofluorescence Microscopy --- p.49 / Chapter 3.2.1.1 --- The time profile of insulin on actin cytoskeletonin permeabilized L6 myotubes --- p.49 / Chapter 3.2.1.2 --- The concentration effect of insulin on actin cytoskeletonin permeabilized L6 myotubes --- p.50 / Chapter 3.2.1.3 --- Relationship between actin cytoskeleton and GLUT4mycin permeabilized L6 myotubes --- p.51 / Chapter 3.2.1.4 --- Translocation of GLUT4myc in membrane-intact L6 myotubes --- p.51 / Chapter 3.2.1.5 --- "Effect of methyl-β-cyclodextrins, MeOH or EtOHin permeabilized and membrane-intact L6 myotubes" --- p.52 / Chapter 3.2.2 --- 2-Deoxyglucose Uptake --- p.52 / Chapter 3.2.2.1 --- "Effects of insulin, methyl-β-cyclodextrins, MeOH and EtOH in L6 myotubes" --- p.52 / Chapter 3.2.3 --- TEM Study --- p.53 / Chapter 3.2.3.1 --- Effects of insulin on actin cytoskeleton and GLUT4myc in L6 myotubes --- p.53 / Chapter 3.3 --- Effect of high glucose and high insulin incubation in L6 myotubes --- p.54 / Chapter 3.3.1 --- Immunofluorescence Microscopy --- p.54 / Chapter 3.3.1.1 --- High insulin and high glucose preincubation in permeabilized L6 myotubes --- p.55 / Chapter 3.3.1.2 --- Effect of high insulin and high glucose incubationin membrane-intact L6 myotubes --- p.55 / Chapter 3.3.2 --- 2-Deoxyglucose Uptake --- p.56 / Chapter 3.3.2.1 --- Effect of high insulin and high glucose incubation in L6 myotubes --- p.56 / Chapter 3.3.3 --- TEM Study --- p.57 / Chapter 3.3.3.1 --- Effect of high insulin and high glucose incubation in L6 myotubes --- p.57 / Chapter 3.4 --- Effect of FFA incubation in L6 myotubes --- p.58 / Chapter 3.4.1 --- Immunofluorescence Microscopy --- p.58 / Chapter 3.4.1.1 --- FFA preincubation in permeabilized L6 myotubes --- p.58 / Chapter 3.4.1.2 --- FFA incubation in membrane-intact L6 myotubes --- p.59 / Chapter 3.4.2 --- 2-Deoxyglucose Uptake --- p.59 / Chapter 3.4.2.1 --- FFA incubation in L6 myotubes (24 hours) --- p.60 / Chapter 3.4.3 --- TEM Study --- p.62 / Chapter 3.4.3.1 --- FFA incubation in L6 myotubes --- p.62 / Chapter 3.5 --- Effect of CHO incubation in L6 myotubes --- p.62 / Chapter 3.5.1 --- Immunofluorescence Microscopy --- p.62 / Chapter 3.5.1.1 --- CHO preincubation in permeabilized L6 myotubes --- p.63 / Chapter 3.5.1.2 --- CHO incubation in membrane-intact L6 myotubes --- p.63 / Chapter 3.5.2 --- 2-Deoxyglucose Uptake --- p.64 / Chapter 3.5.2.1 --- CHO incubation in L6 myotubes (24 hours) --- p.64 / Chapter 3.5.3 --- TEM Study --- p.65 / Chapter 3.5.3.1 --- CHO incubation in L6 myotubes --- p.65 / Chapter 3.6 --- Overall changes in glucose uptake after preincubation experiment --- p.65 / Chapter CHAPTER FOUR --- DISCUSSION / Chapter 4.1 --- Effect of insulin on L6 myotubes --- p.69 / Chapter 4.2 --- "Effect of methyl-β-cyclodextrins, MeOH and EtOH on L6 myotube" --- p.75 / Chapter 4.3 --- Effect of pretreatment of cells in conditions of insulin resistance --- p.76 / Chapter 4.3.1 --- Effect of high glucose and high insulin preincubation on L6 myotubes --- p.76 / Chapter 4.3.2 --- Effect of FFA preincubation on L6 myotubes --- p.78 / Chapter 4.3.3 --- Effect of CHO preincubation on L6 myotubes --- p.82 / Chapter 4.3.4 --- Effect of cell preincubation in conditions of insulin resistance on L6 myotubes (TEM) --- p.83 / Chapter 4.4 --- Summary of the effects of cell preincubation in conditions of insulin resistance --- p.84 / Chapter 4.5 --- Possible mechanisms involved in insulin resistance induction --- p.86 / Chapter 4.5.1 --- Possible changes in GLUT expression and activities --- p.87 / Chapter 4.5.2 --- Possible changes in insulin signaling propagation --- p.88 / Chapter 4.5.3 --- Altered functioning of various actin accessory proteins --- p.89 / Chapter 4.6 --- Limitation of the study --- p.90 / Chapter 4.7 --- Conclusion --- p.90 / Chapter 4.8 --- Future study --- p.91 / REFERENCES --- p.93 / TABLES

Situações de estresse metabólico e térmico deflagram a expressão celular e liberação para o plasma, de proteínas de choque térmico da família de 70 kDa (HSP70) para desempenhar função de defesa-chave em um processo conhecido como resposta ao choque térmico ou ao estresse, que as caracterizam como proteína citoprotetora. Estudos indicam a ocorrência de HSP70 extracelular (eHSP70) na circulação por até 24 h após desafios como o exercício físico, cuja liberação dá-se por tecidos do território hepatoesplâncnico, em uma resposta mediada por receptores α-adrenérgicos. Esta liberação de HSP70 para o plasma é atenuada pela ingestão de glicose. Estas evidências levaram-nos a supor que a liberação de eHSP70 em resposta ao estresse possa ter um papel fisiológico na regulação da glicemia, a qual é afetada por estímulo simpático. Neste trabalho, os resultados dos experimentos in silico indicaram a possibilidade de ancoragem e interação estável entre a HSP70 e a molécula de insulina. Ensaios de imunoprecipitação de insulina com amostras de plasma de animais submetidos ao tratamento de insulina Regular e Detemir revelaram HSP70 coimunoprecipitada especialmente em situação de jejum severo. Parte destas amostras foi submetida à quantificação de glicemia, insulinemia e HSP72 circulante, cujos resultados evidenciam elevada concentração desta proteína de choque térmico por ocasião de hipoglicemia induzida por insulina. Parte dos estudos in vivo deste trabalho foram desenvolvidos com ratos submetidos ao choque térmico agudo (uma sessão de 15 minutos) com hipertermia induzida (41,5 °C) ou com injeção de HSP70 (HSPA1A) exógena, com grupos experimentais organizados em grupos Controle (C) e Choque Térmico (HS) e, em tempos experimentais: imediatamente (tempo zero), 6, 12 e 24 h pós-choque. Foi observada a ocorrência das formas constitutiva e induzível de eHSP70 no plasma de animais submetidos ao choque térmico com valor maior nos períodos de 12 e 24 h pós-choque. Nossos estudos mostram que, in vivo, a eHSP70 plasmática produz alteração na tolerância à glicose, com maior hiperglicemia, quando o teste é realizado 12 h após o choque térmico. Este efeito foi bloqueado pela administração (i.p.) de anticorpo anti-HSP70 e se mostrou independente da síntese de novo de HSP70. Ainda, a presença de eHSP70 plasmática (induzida por choque térmico ou administrada via endovenosa) causa menor captação de glicose pelo músculo esquelético. Ensaios com músculo isolado indicam que o efeito inibidor da eHSP70 sobre a captação de glicose é dependente de insulina. Apesar das evidências de que a ligação HSP70-insulina no plasma tenha implicação sobre a oferta de glicose, não se pode descartar também a possibilidade de que as ações da eHSP70 estejam ocorrendo pela sua ligação a receptores específicos, e/ou de que sejam parte de uma resposta ao estresse na qual estejam implicados os hormônios contrarregulatórios. Em suma, os resultados indicam que a eHSP70 plasmática é capaz de se ligar- à insulina, provocar tolerância alterada à glicose e diminuir a captação de glicose pelo músculo esquelético, aumentando a disponibilidade de glicose circulante. / Metabolically stressful situations trigger the expression and release of the 70 kDa (HSP70) as an essential protective response, in a process known as heat shock (HS) response or stress response. HSP70 is characterized as cytoprotector entities. Previous studies indicate the occurrence of extracellular HSP70 (eHSP70) in the circulation up to 24 h after stressful challenges, such as physical exercise, and that such eHSP70 liberation comes from hepatosplancnic tissues, in an 1-adrenergic dependent response, which is attenuated by glucose ingestion. The above findings led us to suppose that stress-elicited eHSP70 release could play a physiological role on glycemic control, which are affected by sympathetic stimuli. In this work, in silico experiments indicated the possibility of docking and stable interaction between HSP70 and the insulin molecule. Immunoprecipitation of plasma samples revealed that HSP70 co-immunoprecipitates with insulin, especially under severe fast. Part of these samples was subjected to quantification of glucose, insulin and circulating HSP72, whose results show high concentration of heat shock protein during severe hypoglycemia induced by insulin. Part of the in vivo studies from this work were also carried out in healthy rats submitted to an acute (15 min) HS (41.5 °C) session or in HSP70 (HSPA1A)-injected rats, assigned into control (C) and HS groups which were observed immediately and after 6, 12 and 24 h post-shock. Both the constitutive (predominantly) and the inducible forms of eHSP70 were observed in the plasma of heat shocked animals, being the times 12 and 24 h those in which the highest concentrations were noted. Our studies suggest that in vivo produces eHSP70 change in plasma glucose tolerance, greater hyperglycemia when glucose tolerance test is performed 12 h after heat shock, and this effect was abrogated by anti-HSP70 antibody administration (i.p.) and independent of de novo synthesis of HSP70. Moreover, the presence of eHSP70 (HS-induced or injected) in the plasma or in soleus muscle incubations elicits lower glucose uptake from the skeletal muscle. Isolated muscle studies indicate that eHSP70 inhibiting effect over glucose uptake is insulin-dependent. However, although evidence suggests a role for HSP70 binding to insulin over glucose offering, it cannot be discarded the possibility that eHSP70 actions are due to their binding to specific HSP70 membrane receptors within the muscle cell, and/or that are part of a stress response in which the counter-regulatory hormone are involved. In short, the results indicate that eHSP70 plasma is able to bind to insulin, causing impaired glucose tolerance and decrease glucose uptake by skeletal muscle, increasing blood glucose availability.

Glucose-6-phosphate dehydrogenase (G6PD) is a cytoplasmic enzyme with an important function in cell oxidative damage prevention. Erythrocytes have a predisposition towards oxidized environments due to their lack of mitochondria, giving G6PD a major role in its stability. G6PD deficiency (G6PDd) is the most common enzyme deficiency in humans; it affects approximately 400 million individuals worldwide. The overall G6PDd allele frequency across malaria endemic countries is estimated to be 8%, corresponding to approximately 220 million males and 133 million females. However, there are no reports on the prevalence of G6PDd in Andean communities where bartonellosis is prevalent.

Photocopy of typescript. / Digitized by Kansas Correctional Industries

The central nervous system (CNS) has a crucial role in the maintenance of energy homeostasis by orchestrating a plethora of signals from peripheral organs about the state of energy stores and the current energy intake needed to match energy expenditure. These signals converge into the hypothalamic regions and its complex local circuitry. CNS-derived cholecystokinin (CCK) is acting at central level to modulate energy balance by regulating the neuronal activity of hypothalamic neuronal populations that regulate food intake, energy storage and consumption. Moreover, our recent published work identifies CCK neurons as key integrators of the neuroendocrine negative feedback of glucocorticoids to the PVN. Glucose sensing neurons of the Ventromedial Hypothalamus (VMH) are integrating energy signals and are essential for mounting a counter-regulatory response and glucose homeostasis. VMH is also important in energy expenditure by regulating body weight and thermogenesis. CCK neurons are present in high density in the VMH.The source of endogenous CCK that acts on distinct neuronal components has not been elucidated. The research so far does not address the purpose of CCK neurons in the hypothalamus and their potential role in the network dynamics regarding energy homeostasis. In this study, we untangle the role of CCK neurons in the VMH nucleus by employing stereotactic intracranial delivery of adeno-associated viruses that result in cell-type specific chemogenetic inhibition or ablation of these neurons. Acute silencing of their neurotransmission with the cre-dependent AAV expression of the chemogenetic tool of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) increases their daily food intake due to increased meal numbers and eating frequency without meal size or meal duration being affected. CCK ablation by a newly generated double-recombinase-mediated Diphtheria Toxin Receptor (DTR) mouse line or AAV-DTA-mediated ablation resulted in hyperphagia, obesity and hyperglycaemia. We conclude that CCK^VMH neurons are implicated in the regulation of food intake, body weight and glucose homeostasis in the adult brain.

A 3D-printed microfluidic lab-on-a-disc (LOAD) device was designed and manufactured using a low cost ( ̃£1600) consumer grade fused deposition modelling (FDM) Ultimaker 2+ 3D printer with imbedded microfluidic channels 1 mm wide, 400 μm depth and with a volumetric capacity of approximate 23 μl. FDM printers are not typically used, or are capable, of producing the fine detailed structures required for microfluidic fabrication; in addition 3D-printed objects can suffer from poor optical transparency. However, in this work, imbedded microfluidic channels were produced and the optical transparency of the device was improved though manufacture optimisation to such a point that optical colourimetric assays can be performed in a microfluidic cuvette device with sample path length of 500 μm and volumetric capacity of 190 μl. When acetone vapour treatment was used, it was possible to improve transparency of plastic samples by up to a further 30%. The LOAD device is capable of being spun using an unmodified optical disc drive (ODD), demonstrating the centrifugation based separation of plasma from whole blood in a low-cost FDM 3D-printed microfluidic LOAD device. A cholesterol assay and glucose assay was developed and optimised using cholesterol oxidase (ChOx) or glucose oxidase (GlOx) respectively and horseradish peroxidase (HRP) for the oxidative coupling of chromotropic acid (CTA) and 4-aminoantipyrine (AAP). This produced a blue quinoneimine dye with a broad absorbance peaking at 590 nm for the quantification of cholesterol/glucose in solution. The colourimetric enzymatic cascade assays were developed for use within low-cost FDM 3D-printed microfluidic devices to demonstrate the capabilities and functionality of the devices. For comparison, the assay was run in standard 96 well plates with a commercial plate reader. The results demonstrated that the quantification of 0-10 mM glucose solution using a 3D-printed microfluidic optical device had a performance comparable to a plate reader assay; glucose assay in whole blood samples R2 = 0.96.

A glicose é um açúcar redutor importante na dieta humana, sendo abundante em diversos alimentos, como sucos de frutas, mel, iogurtes e refrigerantes e pode ser facilmente ingerido e metabolizado. Ela fornece a energia para o corpo humano, entretanto, diversos desequilíbrios metabólicos estão associados com variações no teor de glicose no sangue, saliva ou urina. Sendo assim é preciso desenvolver métodos de baixo custo, simples e rápidos que permitam o monitoramento deste metabólito. Portanto, no presente trabalho foi desenvolvido e caracterizado um eletrodo composto por uma superfície bifuncional de Pt/Au, onde a superfície de Au foi modificada com uma monocamada auto-organizada de cistamina na qual foi ancorada a enzima glicose oxidase e a superfície da Pt com ferroceno, para determinação de glicose em amostras alimentícias. O eletrodo foi construído utilizando-se um eletrodo de platina, sobre o qual foram eletrodepositado nanoestruturas de ouro, através de voltametria linear em uma solução contendo o ânion tetracloroáurico. As nanoestruturas de ouro foram modificadas com o alcanotiol cistamina, formando uma camada auto-organizada, para servir de plataforma para ancoragem da glicose oxidase. O peróxido de hidrogênio, que é um dos produtos da reação enzimática foi determinado na platina modificada com ferroceno. Sendo assim, as nanoestruturas de ouro serviriam de sítios específicos para as enzimas, e a platina modificada com ferroceno servirá para quantificar o produto da reação enzimática. Para a detecção da glicose foram construídos três eletrodos, o eletrodo embutido (EE) para detecção de glicose em refrigerantes, o eletrodo por evaporação de platina (EEP) para detecção de glicose em amostras de iogurte e o ultramicroeletrodo (UME) para futuras aplicações de detecção de glicose por métodos não invasivos. Encontrou-se um limite de detecção de 2,4 µmol L-1 para o eletrodo EE, de 2,2 µmol L-1 para o eletrodo EEP e 0,03 µmol L-1 para o UME. Foram realizados diversos tratamentos estatísticos como erro relativo, desvio padrão e incertezas para verificar a resposta do eletrodo. Por fim, os eletrodos desenvolvidos foram aplicados na detecção de glicose em amostras de refrigerantes, chás e iogurtes, e obteve-se uma resposta satisfatória para a detecção do analito nestas amostras. / Glucose is a reducing sugar very important in the human diet and is abundant in many foods, such as fruit juices, honey, yogurt and soft drinks and can be easily ingested and metabolized. It provides the energy for the human body perform its healthy functioning. However, many metabolic imbalances associated with variations in the level of glucose in the blood, urine or saliva. Therefore it is necessary to develop methods cheap, simple and quick to allow the monitoring of this metabolite. Therefore, in the present work was developed and characterized an electrode composed of a bifunctional surface Pt/Au, where Au surface was modified with a selforganized monolayer of cystamine which was anchored in the enzyme glucose oxidase and the surface of Pt with ferrocene for the determination of glucose in food samples. The electrode was constructed using a platinum electrode, which was electrodeposited gold nanostructures, using the technique of linear voltammetry in a solution containing the anion tetrachloroauric. The gold nanostructures were modified with cystamine alkanethiol forming a self-organized layer to serve as a platform for anchoring the glucose oxidase. Hydrogen peroxide, which is a product of the enzyme reaction, was determined in the platinum modified with ferrocene. Therefore, the gold nanostructures serve as sites for specific enzymes, and platinum modified with ferrocene serve to quantify the product of the enzymatic reaction. For detection of glucose were built three electrodes, the electrode embedded (EE) for detecting glucose in soft drinks, the electrode by evaporating platinum (EEP) for detection of glucose in samples of yogurt and ultramicroelectrode (UME) for future sensing applications glucose through non-evasive. We found a detection limit of 2.4 µmol L-1 to the electrode EE, 2.2 µmol L-1 to the electrode EEP and 0.03 µmol L-1 for UME. Various statistical treatments were performed as relative error, standard deviation and uncertainties to check the response of the electrode. Finally, the electrodes developed were applied to the detection of glucose in samples of soft drinks, teas and yogurts, which gave a satisfactory response to the detection of the analyte in food samples.