Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 1982. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND SCIENCE / Vita. / Bibliography: leaves 211-223. / by Douglas Elliott Koshland. / Ph.D.

Human and murine diabetes is characterized by pancreatic β-cell dedifferentiation, a process in which β-cells lose expression of markers of maturity and gain those of endocrine progenitors. Failing β-cells inappropriately metabolize lipids over carbohydrates and exhibit impaired mitochondrial oxidative phosphorylation. Therefore, pathways involved in mitochondrial fuel selection and catabolism may represent potential targets for the prevention or reversal of dedifferentiation. In chapter I of this dissertation, we isolated and functionally characterized failing β-cells from various experimental models of diabetes. We found a striking enrichment in the expression of aldehyde dehydrogenase 1 isoform A3 (Aldh1a3) as β-cells become dedifferentiated. Flow-sorted Aldh1a3-expressing (ALDH+) islet cells demonstrate impaired glucose-induced insulin secretion, are depleted of Foxo1 and MafA, and include a Neurogenin3-positive subset. RNA sequencing analysis demonstrated that ALDH+ cells are characterized by: (i) impaired oxidative phosphorylation and mitochondrial complex I, IV, and V; (ii) activated RICTOR; and (iii) progenitor cell markers. We propose that impaired mitochondrial function marks the progression from metabolic inflexibility to dedifferentiation in the natural history of β-cell failure. In chapter II of this dissertation, we report that cytochrome b5 reductase 3 (Cyb5r3) is a FoxO1-regulated mitochondrial oxidoreductase critical to β cell function. Expression of Cyb5r3 is greatly decreased in multiple murine models of diabetes, and in vitro Cyb5r3 knockdown leads to increased ROS generation and impairment of respiration, mitochondrial function, glucose-stimulated insulin secretion, and calcium mobilization. In vivo, mice with β-cell-specific ablation of Cyb5r3 (B-Cyb5r3) display impaired glucose tolerance with decreased insulin secretion, and their islets have significantly lower basal respiration and glucose-stimulated insulin secretion. B-Cyb5r3 β-cells lose expression of Glut2, MafA, and Pdx1 expression despite a compensatory increase in FoxO1 expression. Our data suggest that Cyb5r3 is a critical mediator of FoxO1’s protective response in β-cells, and that loss of Cyb5r3 expression is an early event in β-cell failure.

Made available in DSpace on 2018-08-01T23:27:08Z (GMT). No. of bitstreams: 1 tese_10622_73 - Karlo Gregorio Guidoni Martins20180117-83910.pdf: 1263773 bytes, checksum: 9e4a68c6b84a54ea3ee7487d5b735aa0 (MD5) Previous issue date: 2017-02-22 / A diversidade beta tem sido utilizada para testar se comunidades são estruturadas por pro-cessos determinísticos (relacionados às respostas das espécies ao ambiente e às interações entre elas) ou estocásticos (relacionados à dispersão das espécies no espaço). No entanto, esses estudos não têm levado em conta que comunidades são estruturadas por espécies dominantes e subordinadas que podem ter contribuições distintas para a diversidade beta. Aqui, nós abordamos as seguintes questões: 1) qual é a contribuição de espécies dominantes e subordinadas para a diversidade beta de uma comunidade?; 2) qual é a importância relativa dos processos determinísticos e estocásticos para a diversidade beta desta comunidade?; 3) como a importância relativa desses processos muda para espécies dominantes e subordinadas?; e 4) esses padrões são consistentes em diferentes escalas espaciais? Nós utilizamos uma comunidade tropical de sub-bosque ao longo de um gradiente edáfi-co para responder estas questões. Foram amostradas 50 parcelas de 100 m² cobrindo uma extensão espacial de 750 m. Todos os indivíduos com o diâmetro à altura do peito (1,30 cm a partir do solo) entre 1 e 10 cm foram registrados. Amostras de solo foram coletadas em 21 parcelas ao longo do gradiente amostral. As espécies dominantes e subordinadas foram classificadas a partir do índice de valor de importância logaritmizado. A diversidade beta total da comunidade de sub-bosque foi quantificada pelo índice de similaridade de Jaccard para múltiplas unidades amostrais. A partir des-te índice, a diversidade beta foi decomposta em valores relativos ao aninhamento e à substituição das espécies entre unidades amostrais. O mesmo critério de quantificação e decomposição da diver-sidade beta foi aplicado às espécies dominantes e subordinadas. As análises foram conduzidas em dois esquemas: 1) utilizando os dados da vegetação e dos solos coletados em 21 parcelas e 2) usan-do os dados da vegetação coletados em 50 parcelas e os dados de solos interpolados para as 29 par-celas que não tiveram solos amostrados. Os dois esquemas têm a mesma extensão espacial, mas a distância entre algumas parcelas foi menor no esquema com 50 parcelas, acarretando em uma reso-lução espacial mais fina. A importância relativa do ambiente e do espaço foi quantificada por orde-nações canônicas de redundância seguidas pela análise de partição da variação. Nossos resultados revelaram que as substituições entre parcelas, tanto de espécies dominantes quanto de subordinadas, contribuem para a diversidade beta desta comunidade. Padrões de aninhamento foram evidentes apenas para as espécies dominantes. Somente a importância relativa do espaço foi significativa para a comunidade de sub-bosque, independentemente da resolução espacial. Processos determinísticos se mostraram preponderantes em resoluções amostrais mais grosseiras, enquanto a importância relativa do espaço (relacionado aos processos estocásticos) foi preponderante em resolução mais fina tanto para espécies dominantes quanto para subordinadas. Nosso estudo mostrou que padrões em comunidades podem ser confundidos quando as diferenças entre espécies dominantes e subordi-nadas não são levadas em consideração. Diferenças nos balanços entre os processos emergiram somente entre diferentes resoluções espaciais, sendo percebidos, no entanto, somente quando espé-cies dominantes e subordinadas foram analisadas separadamente. Os processos determinísticos e estocásticos atuam simultaneamente nesta comunidade, mas diferem em importância para espécies dominantes e subordinadas em diferentes escalas.

Made available in DSpace on 2014-06-12T17:39:28Z (GMT). No. of bitstreams: 2 arquivo6902_1.pdf: 3068620 bytes, checksum: eb2722910d045ddc14f0488856268b0c (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2007 / Esta dissertação se propõe a investigar a representação de sinais no plano tempofreq üência e, mais especificamente, os problemas relativos à resolução de sinais. O princípio da incerteza de Gabor-Heisenberg para sinais e wavelets é analisado criteriosamente. Versões de Gnedenko-Kolmogorov do tipo Teorema Central do Limite são avaliadas, procurando elucidar sua relevância no contexto da resolução de sinais. Finalmente, o conceito de derivada Blur é usado para propor uma nova família de wavelets - as wavelets beta - construídas a partir de distribuições beta de probabilidade. Essas wavelets são atrativas por terem suporte compacto, são monocíclicas, possuem descrição analítica e podem ser consideradas como uma generalização suavizada das wavelets de Haar. Sua relevância decorre do Teorema Central do Limite aplicado a wavelets de suporte compacto. Campos promissores para aplicação destas wavelets são mencionado

O 90Y tem uma meia-vida de 2,7 dias, decaindo com 99,98% por emissão beta para o estado fundamental do 90Zr. Neste trabalho foram aplicadas duas metodologias para a padronização do 90Y. O método do traçador em um sistema de coincidência de 4πβ-γ, onde foi medido o emissor beta puro, misturado com um emissor de beta-gama, que proporciona a eficiência de detecção beta. Para este método, o radionuclídeo 24Na, que decai com meia-vida de 0,623 dia pela emissão beta, com energia beta máxima de 1393 keV, seguido por dois raios gama, foi usado como traçador. A eficiência foi obtida, selecionando-se o pico de absorção total com energia de 1369 keV no canal gama. Alíquotas conhecidas do traçador, previamente padronizadas pelo método de coincidência 4πβ-γ, foram misturadas com alíquotas conhecidas de 90Y. A atividade do emissor beta puro foi calculada por meio de um sistema de coincidência por software (SCS) usando discriminação eletrônica para alterar a eficiência de beta. O comportamento da curva de extrapolação foi predito por meio do código Esquema, que utiliza a técnica de Monte Carlo. O outro método usado foi o método CIEMAT/NIST desenvolvido para sistemas de contagem de cintilação líquida. Para este método, utilizou-se uma solução padrão de 3H. O sistema 2100TR TRICARB foi usado para as medições, o qual opera em coincidência com duas fotomultiplicadoras; uma fonte externa, colocada perto do sistema de medição foi usada para determinar o parâmetro quenching. O coquetel utilizado foi o Ultima Gold, a variação do fator de quenching foi obtida pelo uso de nitrometano. As amostras radioativas foram preparadas em frascos de vidro com baixa concentração de potássio. As atividades determinadas pelos dois métodos foram comparadas e os resultados obtidos são concordantes dentro das incertezas experimentais. Por meio deste trabalho, foi possível avaliar o desempenho do método CIEMAT/NIST, que apresenta várias vantagens em relação ao método do traçador, entre elas a facilidade para a preparação das fontes, medidas simples e rápidas sem a necessidade de determinar as curvas de extrapolação. / The 90Y has a half-life of 2.7 days, decaying with 99.98 % by beta emission to the ground state of 90Zr. In this work two methodologies for the standardization of yttrium-90 (90Y) were applied. One was the tracing method performed in a 4πβ-γ coincidence system, measuring the pure beta emitter mixed with a beta-gamma emitter, which provides the beta detection efficiency. For this method, the radionuclide 24Na, which decays with half life of 0.623 day by beta particle, with end point energy of 1393 keV followed by two gamma-rays, was used as tracer, the efficiency was obtained by selecting the 1369 keV total energy absorption peak at the gamma channel. Known aliquots of the tracer, previously standardized by 4πβ-γ coincidence, were mixed with known aliquots of 90Y. The activity was calculated by means of a Software Coincidence System (SCS) using electronic discrimination for changing the beta efficiency. The behavior of the extrapolation curve was predicted by means of the Esquema code, which uses the Monte Carlo technique. The other was the CIEMAT/NIST method developed for Liquid Scintillation Counting (LSC) systems. For this method, a 3H standard solution was used. A TRICAB 2100TR system was used for the measurements. It operates with two photomultipliers in coincidence and an external source placed near the measurement system is used for determining the quenching parameter. Ultima Gold was the liquid scintillation cocktail. In order to obtain the quenching parameter curve a nitro methane carrier solution was used. The radioactive samples were prepared in glass vials with low potassium concentration. The activities determined by the two methods were compared and they are in agreement within the experimental uncertainties. By means of this work it was possible to evaluate the performance of the CIEMAT/NIST method, which presents several advantages with respect to the tracer method, among them is the facility for the preparation of the sources, simple and fast measurements without the need of determining extrapolation curves.

Beta-blockers and non-steroidal anti-inflammatory drugs (NSAIDs) are often avoided in asthma over risk of bronchospasm which may vary according to drug selectivity and duration of administration. This thesis attempts to quantify the risk of beta-blocker and NSAID exposure in asthma by synthesising clinical trial evidence and conducting observational studies using linked electronic medical records. As part of this thesis, three systematic reviews of clinical trials were conducted evaluating: the prevalence of aspirin-exacerbated respiratory disease (AERD); risk of selective NSAIDs/COX-2 inhibitors in people with AERD; and risk of acute beta-blocker exposure in people with asthma. Electronic primary care data from the Clinical Practice Research Datalink (CPRD) was used to define a cohort of people with active asthma, measure the prevalence of beta-blocker and NSAID prescribing, and perform a series of nested case control studies evaluating asthma death, asthma hospitalisation and primary care asthma exacerbations (PCAE). A self-controlled case-series was performed for PCAE as well. Based upon work in this thesis, the prevalence of AERD in people with asthma was around 9%. Selective NSAIDs triggered respiratory symptoms in 8% of people with AERD whilst no significant changes in lung function or symptoms occurred with COX-2 inhibitors. Acute non-selective beta-blocker exposure caused a significant mean fall in FEV1 of 10%, a significant increase in respiratory symptoms in around 1 in 13 and a non-significant increase in falls in FEV1 of ≥20% in around 1 in 9. Acute selective beta-blocker exposure caused a significant mean fall in FEV1 of 7%, significant falls in FEV1 of ≥20% in around 1 in 8 and a non-significant increase in respiratory symptoms in around 1 in 33. The prevalence of selective beta-blocker prescribing in asthma rose by around 200% over the 12 year period whilst the prevalence of non-selective beta-blocker prescribing rose by around 90%. Changing trends in NSAID prescribing occurred over the 12 year period with COX-2 inhibitors now rarely prescribed. Using the nested case control design, both incident and high-dose non-selective beta-blocker exposure was associated with significantly increased risk of asthma morbidity (hospitalisation and PCAE). In contrast, no significant increased risk of asthma morbidity occurred with any type of selective beta-blocker exposure. Consistent findings were seen for PCAE using the self-controlled case series. No significantly increased risk was seen with different oral NSAIDs apart from weak evidence of an association between asthma death and non-selective NSAID exposure which is unlikely to be causal. Significant numbers of people with asthma are prescribed beta-blockers and NSAIDs. Evidence from clinical trials and observational studies demonstrate that non-selective beta-blockers significantly increase asthma morbidity with risk appearing to vary according to dose and duration of administration. Although selective beta-blockers have the potential to cause significant changes in lung function, no significant increase in asthma morbidity was observed in observational studies. Although around 9% of asthmatics may be susceptible to NSAIDs, no strong evidence was found to suggest that the current practice of NSAID prescribing increases asthma morbidity. At the same time, COX-2 inhibitors are infrequently prescribed despite apparently being well tolerated by people with AERD.

The transforming growth factor-ß (TGFß) pathway, including the bone morphogenetic protein (BMP), plays critical roles during embryogenesis and in adult tissue homeostasis. Hence, malfunctions in TGFß/BMP signalling result in several diseases. Signalling is initiated by ligand binding to cell surface receptor kinases, which phosphorylate and activate the R-SMAD transcription factors. R-SMADs translocate to the nucleus and regulate the transcription of hundreds of genes. The cellular responses to TGFß/BMP signals are tightly controlled and highly regulated. TGFß/BMP receptors and R-SMADs, as the intracellular mediators of TGFß/BMP ligands, are key targets for regulation to control duration and potency of signalling. Reversible ubiquitylation of R-SMADs and TGFß/BMP receptors is a key mechanism to control TGFß/BMP signalling. Several E3 ubiquitin ligases have been reported to regulate the turnover and activity of TGFß/BMP receptors and R-SMADs, however little is known about their cognate deubiquitylating enzymes (DUBs). A proteomic screen identified the DUBs OTUB1 and USP15 as potential novel regulators of the TGFß and BMP pathways respectively. Endogenous OTUB1 was recruited to the active phospho-SMAD2/3 complex only upon TGFß induction and OTUB1 had a crucial role in TGFß-mediated gene transcription and cellular migration. OTUB1 inhibited the ubiquitylation of phospho-SMAD2/3 by binding to and inhibiting the E2 ubiquitin-conjugating enzymes independently of its catalytic activity. Consequently, the depletion of OTUB1 in cells caused a rapid loss in levels of TGFß-induced phospho-SMAD2/3, which was rescued by the proteasomal inhibitor Bortezomib. These findings demonstrated a novel signal-induced phosphorylation-dependent recruitment of OTUB1 to its target. Hence, OTUB1 could be exploited as a target to intervene against diseases that are provoked by an imbalance in TGFß signalling. DUBs are highly regulated enzymes and recent reports have shed light into the molecular regulation OTUB1. The N-terminal region of OTUB1 harbours an ubiquitin binding domain, which is critical for its function to inhibit ubiquitylation. While investigating the role of OTUB1 in TGFß signalling, it became apparent that OTUB1 itself could be post-translationally modified by phosphorylation. Two phosphorylation sites at the OTUB1 N-terminal region have been identified by mass spectrometry. S18 of OTUB1 was phosphorylated in vitro by the type I TGFß receptor (ALK5), whereas S16 was phosphorylated by the constitutively active kinase CK2 in vitro and in vivo. Phosphorylation of the OTUB1 N-terminal region could affect its physiological function and requires further investigation. Although much is known about DUBs that target the type I TGFß receptor, no DUBs that target the type I BMP receptors had been identified. USP15 was identified in a proteomic screen as an interactor of SMAD6, which is a negative regulator of the BMP pathway. USP15 also binds to and deubiquitylates the type I BMP receptor (ALK3), thereby enhancing BMP signalling. Consequently, USP15 impacts BMP-induced SMAD1 phosphorylation, mouse osteoblastic differentiation and Xenopus embryogenesis. A proteomic approach identified O-GlcNAc transferase (OGT) as an interactor of SMAD2. SMADs have not been associated with O-GlcNAc modifications and the regulation of TGFß/BMP signalling by O-GlcNAcylation has not been investigated. Endogenous SMADs1-3 bound OGT and pulled down potential O-GlcNAc modified proteins. Furthermore, SMAD4 was possibly O-GlcNAcylated, which implies that O-GlcNAc modification could regulate TGFß/BMP signalling. Further investigation is needed to decipher the precise molecular mechanisms of this potential regulation.

O 90Y tem uma meia-vida de 2,7 dias, decaindo com 99,98% por emissão beta para o estado fundamental do 90Zr. Neste trabalho foram aplicadas duas metodologias para a padronização do 90Y. O método do traçador em um sistema de coincidência de 4πβ-γ, onde foi medido o emissor beta puro, misturado com um emissor de beta-gama, que proporciona a eficiência de detecção beta. Para este método, o radionuclídeo 24Na, que decai com meia-vida de 0,623 dia pela emissão beta, com energia beta máxima de 1393 keV, seguido por dois raios gama, foi usado como traçador. A eficiência foi obtida, selecionando-se o pico de absorção total com energia de 1369 keV no canal gama. Alíquotas conhecidas do traçador, previamente padronizadas pelo método de coincidência 4πβ-γ, foram misturadas com alíquotas conhecidas de 90Y. A atividade do emissor beta puro foi calculada por meio de um sistema de coincidência por software (SCS) usando discriminação eletrônica para alterar a eficiência de beta. O comportamento da curva de extrapolação foi predito por meio do código Esquema, que utiliza a técnica de Monte Carlo. O outro método usado foi o método CIEMAT/NIST desenvolvido para sistemas de contagem de cintilação líquida. Para este método, utilizou-se uma solução padrão de 3H. O sistema 2100TR TRICARB foi usado para as medições, o qual opera em coincidência com duas fotomultiplicadoras; uma fonte externa, colocada perto do sistema de medição foi usada para determinar o parâmetro quenching. O coquetel utilizado foi o Ultima Gold, a variação do fator de quenching foi obtida pelo uso de nitrometano. As amostras radioativas foram preparadas em frascos de vidro com baixa concentração de potássio. As atividades determinadas pelos dois métodos foram comparadas e os resultados obtidos são concordantes dentro das incertezas experimentais. Por meio deste trabalho, foi possível avaliar o desempenho do método CIEMAT/NIST, que apresenta várias vantagens em relação ao método do traçador, entre elas a facilidade para a preparação das fontes, medidas simples e rápidas sem a necessidade de determinar as curvas de extrapolação. / The 90Y has a half-life of 2.7 days, decaying with 99.98 % by beta emission to the ground state of 90Zr. In this work two methodologies for the standardization of yttrium-90 (90Y) were applied. One was the tracing method performed in a 4πβ-γ coincidence system, measuring the pure beta emitter mixed with a beta-gamma emitter, which provides the beta detection efficiency. For this method, the radionuclide 24Na, which decays with half life of 0.623 day by beta particle, with end point energy of 1393 keV followed by two gamma-rays, was used as tracer, the efficiency was obtained by selecting the 1369 keV total energy absorption peak at the gamma channel. Known aliquots of the tracer, previously standardized by 4πβ-γ coincidence, were mixed with known aliquots of 90Y. The activity was calculated by means of a Software Coincidence System (SCS) using electronic discrimination for changing the beta efficiency. The behavior of the extrapolation curve was predicted by means of the Esquema code, which uses the Monte Carlo technique. The other was the CIEMAT/NIST method developed for Liquid Scintillation Counting (LSC) systems. For this method, a 3H standard solution was used. A TRICAB 2100TR system was used for the measurements. It operates with two photomultipliers in coincidence and an external source placed near the measurement system is used for determining the quenching parameter. Ultima Gold was the liquid scintillation cocktail. In order to obtain the quenching parameter curve a nitro methane carrier solution was used. The radioactive samples were prepared in glass vials with low potassium concentration. The activities determined by the two methods were compared and they are in agreement within the experimental uncertainties. By means of this work it was possible to evaluate the performance of the CIEMAT/NIST method, which presents several advantages with respect to the tracer method, among them is the facility for the preparation of the sources, simple and fast measurements without the need of determining extrapolation curves.

Prefrontal cortex (PFC) is critical to behavioral flexibility and, hence, the top-down control over bottom-up sensory information. The mechanisms underlying this capacity have been hypothesized to involve the propagation of alpha/beta (8-30 Hz) oscillations via feedback connections to sensory regions. In contrast, gamma (30-160 Hz) oscillations are thought to arise as a function of bottom-up, feedforward stimulation. To test the hypothesis that such oscillatory phenomena embody such functional roles, we assessed the performance of nine monkeys on tasks of learning, categorization, and working memory concurrent with recording of local field potentials (LFPs) from PFC. The first set of tasks consisted of two classes of learning: one, explicit and, another, implicit. Explicit learning is a conscious process that demands top-down control, and in these tasks alpha/beta oscillations tracked learning. In contrast, implicit learning is an unconscious process that is automatic (i.e. bottom up), and in this task alpha/beta oscillations did not track learning. We next looked at dot-pattern categorization. In this task, category exemplars were generated by jittering the dot locations of a prototype. By chance, some of these exemplars were similar to the prototype (low distortion), and others were not (high distortion). Behaviorally, the monkeys performed well on both distortion levels. However, alpha/beta band oscillations carried more category information at high distortions, while gamma-band category information was greatest on low distortions. Overall, the greater the need for top-down control (i.e. high distortion), the greater the beta, and the lesser the need (i.e. low distortion), the greater the gamma. Finally, laminar electrodes were used to record from animals trained on working memory tasks. Each laminar probe was lowered so that its set of contacts sampled all cortical layers. During these tasks, gamma oscillations peaked in superficial layers, while alpha/beta peaked in deep layers. Moreover, these deep-layer alpha/beta oscillations entrained superficial alpha/beta, and modulated the amplitude of superficial-layer gamma oscillations. These laminar distinctions are consistent with anatomy: feedback neurons originate in deep layers and feedforward neurons in superficial layers. In summary, alpha/beta oscillations reflect top-down control and feedback connectivity, while gamma oscillations reflect bottom-up processes and feedforward connectivity.

The goal of this research was the development of a method to extract pure (1,3/1,6)-β-glucans from Saccharomyces cerevisiae. These β-glucans are of pharmaceutical importance because an animal’s immune system can recognize glucan molecules, and these molecules can act as immunomodulators, essentially turning on the immune system. The problem in the past has been that previously published methods produce β-glucans with low side chain lengths and few branching occurrences. This issue was tackled by a multivariable approach that reduced extraction steps, initial sample size, and concentrations of reagents used. This method has been shown to produce greater yields of β-glucans while maintaining high purity. Analyses such as 1H-NMR and GC-MS have been used to confirm the content of the extracted glucans. Ideally, this research will generate interest for further β-glucan studies and ultimately be utilized pharmacologically with immunocompromised individuals.