Thesis (Ph.D. in Neuroscience) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 145-171).

Thesis (Ph.D. in Toxicology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 137-149).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 115-136).

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2014-04-22T16:33:20Z No. of bitstreams: 1 Luciane Marieta Soares O uso da talidomida...2013.pdf: 1032465 bytes, checksum: 705c1f8374aee3a4f8a1b0f537cd33f6 (MD5) / Made available in DSpace on 2014-04-22T16:33:21Z (GMT). No. of bitstreams: 1 Luciane Marieta Soares O uso da talidomida...2013.pdf: 1032465 bytes, checksum: 705c1f8374aee3a4f8a1b0f537cd33f6 (MD5) Previous issue date: 2013 / Universidade Federal da Bahia. Faculdade de Medicina da Bahia. Salvador, BA, Brasil / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / A leptospirose é uma zoonose de importância global, causada por leptospiras patogênicas. Seu tratamento é limitado quando iniciado após quatro dias do surgimento de sintomas, portanto, novas terapias adjuvantes são necessárias. Objetivo. Testar a droga imunomoduladora talidomida como terapia adjuvante à ampicilina no modelo de tratamento tardio da leptospirose experimental em hamsters. Métodos. 60 hamsters foram infectados via intraperitoneal por Leptospirainterrogans cepa L1-130, e foram separados em grupos: nenhum tratamento (NONE), talidomida (TAL), ampicilina (AMP) e ambos (AMP-TAL). A talidomida foi administrada via sonda orogástrica na dosagem de 50 mg/Kg diluída em óleo de linhaça (2ml/Kg) por três dias. Já a ampicilina por via intramuscular: 100mg/Kg/bid por seis dias. Foram realizados dois desenhos experimentais. Experimento 1: o tratamento foi iniciado 48h após o início dos sinais clínicos da doença. Experimento 2: o tratamento foi iniciado imediatamente após a detecção do primeiro óbito entre os animais infectados. Resultados. Experimento1: todos os hamsters dos grupos AMP e AMP-TAL sobreviveram (n=8) enquanto todos os animais dos grupos NONE (n=6) e TAL (n=8) morreram. Os hamsters dos grupos AMP e AMP-TAL não mostraram lesões renais, pulmonares ou hepáticas. Baixa carga de leptospiras foi detectada nos órgãos alvo. Experimento 2: o desfecho letal foi observado em 6/6 hamsters no grupo NONE, 8/8 do grupo TAL, 6/8 do AMP e 6/8 do AMP-TAL. A talidomida usada como adjuvante não prolongou a sobrevida dos animais tratados com ampicilina. No entanto, o grupo TAL apresentou sobrevida mais longa que o grupo que não recebeu tratamento algum. Os animais tratados apresentaram maior frequência de destrabeculação de hepatócitos. Conclusão: Esse estudo abre novas perspectivas no desenvolvimento de um tratamento tardio na leptospirose, contudo observou-se que a talidomida utilizada como terapia adjuvante não teve impacto na sobrevida de hamsters como modelo de início tardio de terapia antimicrobiana. / Leptospirosis is a zoonosis of global importance, caused by pathogenic leptospira. His treatment is limited when started after four days of onset of symptoms, increasing the risk of morbidity and mortality, so new adjuvant therapies are needed.Objectives.To test the immunomodulatory drug, thalidomide, as an adjuvant therapy to antibiotics in experimental leptospirosis. Methods. Hamsters were infected by Leptospirainterrogans strain L1-130, and groups were assigned based on no treatment (NONE), thalidomide only (TAL), ampicillin only (AMP) or both (AMP-TAL). Thalidomide was administered via a gastric tube: 50 mg/kg in linseed oil and 2 ml/kg for three days. Ampicillin was administered intramuscularly at the rate of 100 mg/kg/bid for six days. Treatment was started two days after the onset of symptoms (experiment 1) and immediately after detection of the first death (experiment 2). Results. Experiment 1: all hamsters from the groups AMP and AMP-TAL survived (n=8), while all hamsters from groups NONE (n=6) and TAL (n=8) died. The AMP and the AMP-TAL groups showed no renal, lung or liver pathology and absent or very low leptospiral burden in target organs. Experiment 2: lethal outcome was observed in 6/6 hamsters in the NONE group, 8/8 in the TAL group, and 6/8 in the AMP group and the AMP-TAL group. Thalidomide showed no survival benefit when compared to hamsters treated with ampicillin alone. The TAL group, however, had a longer interval between infection and death when compared to the NONE group. Treated animals had a higher frequency of liver plate disarray. The TAL, AMP and AMP-TAL groups had very low tissue leptospiral counts. Conclusion: This study may open new perspectives to assist the development of a delayed treatment in leptospirosis, however it was observed that thalidomide used as adjuvant therapy had no impact on the survival of hamsters as late onset of antimicrobial therapy model.

In vitro disease models are powerful platforms for the development of drugs and novel therapies. Stem-cell based approaches have emerged as cutting-edge tools in disease modelling, allowing for deeper insights into previously unknown disease mechanisms. Hence the significant role of these disease-in-a-dish methods in therapeutics and translational medicine. Impedance sensing is a non-invasive, quantitative technique that can monitor changes in cellular behaviour and morphology in real-time. Bioimpedance measurements can be used to characterize and evaluate the establishment of a valid disease model, without the need for invasive end-point biochemical assays. In this work, two stem cell-based disease models-on-a-chip are proposed for acute liver failure (ALF) and age-related macular degeneration (AMD). The ALF disease model-on-a-chip integrates impedance sensing with the highly-differentiated HepaRG cell line to monitor in real-time quantitative and dynamic response to various hepatotoxins. Bioimpedance analysis and modelling has revealed an unknown mechanism of paracetamol hepatotoxicity; a temporal, dose-dependent disruption of tight junctions (TJs) and cell-substrate adhesion. This disruption has been validated using ultrastructural imaging and immunostaining of the TJ-associated protein ZO-1. Age-related macular degeneration (AMD) is the leading cause of blindness in the developed world with a need for disease models for its currently incurable forms. Human induced pluripotent stem cells (hiPSCs) technology offers a novel approach for disease modelling, with the potential to impact translational retinal research and therapy. Recent developments enable the generation of Retinal Pigment Epithelial cells from patients (hiPSC-RPE), thus allowing for human retinal disease in vitro studies with great clinical and physiological relevance. In the current study, the development of a tissue-on- a-chip AMD disease model has been established using RPE generated from a patient with an inherited macular degeneration (case cell line) and from a healthy sibling (control cell line). A reproducible Electric Cell-substrate Impedance Sensing (ECIS) electrical wounding assay was conducted to mimic RPE damage in AMD. First, a robust and reproducible real-time quantitative monitoring over a 25-day period demonstrated the establishment and maturation of RPE layers on microelectrodes. A spatially-controlled RPE layer damage that mimicked cell loss in AMD was then initiated. Post recovery, significant differences in migration rates were found between case and control cell lines. Data analysis and modelling suggested this was due to the lower cell-substrate adhesion of the control cell line. These findings were confirmed using cell adhesion biochemical assays. Moreover, different-sized, individually-addressed square microelectrode arrays with high spatial resolution were designed and fabricated in-house. ECIS wounding assays were performed on these chips to study immortalized RPE migration. Migration rates comparable to those obtained with ECIS circular microelectrodes were determined. The two proposed disease-models-on-a-chip were then used to explore the therapeutic potential of the antioxidant N-Acetyl-Cysteine (NAC) on hiPSC-RPE and HepaRG cell recovery. Addition of 10 mM NAC at the end of a 24h paracetamol challenge caused a slight increase in the measured impedance, suggesting partial cell recovery. On the other hand, no effect on case hiPSC-RPE migration has been observed. More experiments are needed to examine the effect of different NAC concentrations and incubation periods. The therapeutic potential of electrical stimulation has also been explored. A preliminary study to evaluate the effect of electrical stimulation on RPE migration has been conducted. An externally applied direct current electric field (DC EF) of 300 mV/mm was found to direct the migration of the immortalized RPE cell line (hTERT-RPE1) perpendicular to the EF. The cells were also observed to elongate and to realign their long axes perpendicular to the applied EF. The proposed tissue-on-a-chip disease models are powerful platforms for translational studies. The potential of such platforms has been demonstrated through revealing unknown effects of acetaminophen on the liver as well as providing deeper insights into the underlying mechanisms of macular degeneration. Combining stem cell technology with impedance sensing provides a high throughput platform for studying patient-specific diseases and evaluating potential therapies.

Orientador: Marcus Alexandre Finzi Corat / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T05:45:50Z (GMT). No. of bitstreams: 1 Gimenes_AnaPaula_M.pdf: 8685522 bytes, checksum: 6c82475e18242cbd1ab92daf37c6b5b5 (MD5) Previous issue date: 2014 / Resumo: A anemia falciforme é uma doença hereditária que acomete milhões de pessoas Promove destruição crônica das células vermelhas (eritrócitos) do sangue, causando anemia, vaso-oclusão, isquemia e consequentes sintomas deintensa dor, susceptibilidade à infecções e, em alguns casos, a morte precoce. Nas últimas décadas, estudos têm mostrado que o aumento dos níveis de hemoglobina fetal (HbF) inibe o processo de falcização das Hemácias melhorando os sintomas de pacientes com anemia falciforme. Neste estudo, produzimos um modelo animal humanizado com traço falciforme e expressão concomitante de HbF persistente, associando as características genéticas dos camundongos transgênicos B-HPFH195 e Berk-SCM . O primeiro é animal transgênico com persistência de hemoglobina fetal, onde aprodução de HbF continua após o nascimento. A segunda linhagem corresponde ao modelo para doença falciforme, Hbatm1Paz Hbbtm1Tow Tg (HBA - HBBS ) 41Paz / J ( Berk - SCM) e mimetiza a doença humana em sua forma mais grave. Nestes animais a reprodução está comprometida, as fêmeas não levam a gestação ao termino e a maioria dos neonatos morrem horas depois do nascimento. Isto ocorre principalmente devido ao fato de que a (HbF) presente no transgene humano destes animais ser trocada pela HbS enquanto o feto ainda esta em gestação. O novo modelo desenvolvido foi analisado clinicamente e histologicamente para a caracterização e apresentou em sua constituição sanguínea hemoglobinas totalmente humanizadas com cadeias ?, ?s e ? persistente porém, ainda mantendo alterações histopatológicas semelhantes aos modelos da doença falciforme. Este novo animal representa um modelo potencial para uso em testes de drogas no aumento de HbF onde os resultados podem ser relevantes para o estudo e tratamento de hemoglobinopatias, bem como para compreender o comportamento das mutações hereditárias para a expressão persistente da cadeia de globina Y / Abstract: Sickle cell disease is a hereditary disease that affects millions of people promotes chronic destruction of red blood cells (erythrocytes), causing anemia, vascular occlusion, ischemia, pain, and susceptibility to infections and in some instances, death premature. In recent decades, studies have shown that increased HbF levels play an important role in improving the symptoms of patients with sickle cell anemia. In this study, we produced and characterized a humanized animal model with sickle cell trait and concomitant expression of persistent HbF, using the genetic characteristics of transgenic mice B - HPFH195 with Berk¿SCM. This model is a transgenic mice model with hereditary persistence of HbF. The second strain constitutes a mouse with sickle cell disease, Hbatm1Paz Hbbtm1Tow Tg ( HBA - HBBs ) 41Paz /J ( Berk - SCM ), an animal model used to study SCD with human hemoglobin S ( HbS ) in its blood. The Berkeley mouse mimics the human disease in severe form, which compromises in its, females do not lead to pregnancy termination and most newborns die hours after birth. This occurs mainly due to the fact that fetal hemoglobin (HbF), present in the human transgene of these animals,is exchanged to HbS while the fetus still in gestation. The new model produced was analyzed clinically and histologically for proper characterization and presents in its constitution humanized blood, hemoglobin chains with ?, ? ?s and persistent, but still maintains histopathological changes similar to models of sickle cell disease. This model may be a potential model for use in drug in testing increase HbF. And results may be relevant for the study and treatment of hemoglobinopathies as well as for understanding the behavior of hereditary mutations for the persistent expression of the Y globin chain / Mestrado / Clinica Medica / Mestra em Clínica Médica

No description available.

Locomotion in vertebrates depends on proper formation and maintenance of neuronal networks in the hind-brain and spinal cord. Malformation or loss of factors required for proper maintenance of these networks can lead to severe neurodegenerative diseases limiting or preventing locomotion. A powerful tool to investigate the genetic and cellular requirements for development and/or maintenance of these networks is a collection of zebrafish mutants with defects in motility. The zebrafish mutant quetschkommode (que) harbors a previously unknown gene defect leading to abnormal locomotor behavior. Here I show that the que mutants display a seizure-like behavior starting around four days post fertilization (dpf) that is characterized by a lack of an initial high amplitude body bend (C-bend) and simultaneous contra-lateral contractions leading to a seizure-like phenotype and paralysis. Peripheral nerve recordings show a significant increase in the number of initiated swimming bouts and overlap between left and right motor neuron activity. These data suggest that the que mutation leads to defects in nervous system function, at the level of motor neurons or central control of motor neurons. I have genetically mapped the que locus to a 0.36cM interval on chromosome 22 using meiotic mapping. I identified a splice mutation in the gene `dihydrolipoamide branched-chain transacylase E2' (dbt) as defective in que mutants. An orthologous mutation in humans lead to Maple Syrup Urine Disease (MSUD), a devastating metabolic disorder leading to seizures, mental retardation, coma and neonatal death if untreated. In zebrafish, dbt is expressed throughout early development and dbt transcripts become enriched in the hind-brain as well as in the gut and liver by 96 hpf. In MSUD patients levels of branched chain amino acids (BCAA) and their keto acids are significantly increased due to the essential role of the dbt enzyme for the BCAA metabolic pathway. The que mutation causes a significant increase of branched chain amino acids in the zebrafish mutant and a strong decrease of neurotransmitters such as glutamate and GABA as well as precursors like glutamine. I hypothesize that reduced neurotransmitter levels in que lead to the observed motility phenotype. Consistent with this hypothesis, I show a tissue specific reduction of glutamate in the hind-brain and spinal cord of que mutants. To evaluate the que mutant's potential as a vertebrate model for MSUD I performed a pilot drug screen using a selection of metabolites of the pathway as well as diet additives currently evaluated in clinical trials. Conversely, application of phenylbutyrate, one of the diet additives, had a beneficial influence on swimming abilities of que mutant embryos, while the keto acid α-ketoisocaproate (KIC), one of the elevated keto acids in human patients, decreased the percentage of larvae capable of swimming. These results help establish the zebrafish que mutant as a new model for MSUD disease that can be used to further the understanding of this disorder and to help identify therapeutic agents.

<p dir="ltr">Neurodegenerative disease (ND) is a debilitating neurological disorder characterized by progressive loss of neurons in central nervous system (CNS), resulting in the decline in memory, cognition and motor functions. Alzheimer's disease (AD) and Parkinson's disease (PD) are the two of the most prevalent NDs, affecting millions of individuals in the United States. While hundreds of genetic risk factors have been identified in association with ND, familial cases with genetic origin only account for 10% and 15% of diagnosed AD and PD incidences, respectively. The majority of ND cases occur sporadically. Mounting evidence from epidemiology studies suggests that environmental stressors are one of the key ND associated risk factors where exposure to environmental stressors leads to the on-set of ND years or decades later. Little is known about the molecular mechanism facilitating the establishment of the persistent and potentially permanent neurotoxic state after exposures, particularly at a developmental stage. Hence, there is a pressing need in understanding the cellular machineries involved in establishment of a persistent neurotoxic state resulting from early-life exposure to environmental toxins. Subcellular compartments are crucial for the maintenance of neuronal homeostasis. Alterations in various subcellular compartments, including the nucleus, mitochondria, and lysosomes, have been commonly noted in cases of AD and PD; and are believed to play a crucial role in the establishment of a persistent neurotoxic state. The primary goal of my thesis is thus to uncover the dysregulation in multiple subcellular compartments and their contributes to ND pathogenesis induced by early-in-life exposure to environmental stressors, including atrazine (ATZ), per-and polyfluoroalkyl substances (PFAS), and neurofibrillary tangles.</p><p dir="ltr">I started by developing live-cell compatible tools to track cellular and sub-cellular changes. Mitochondria DNA methylation is of particular interest, due to its potential regulatory role in the expression of electron transport chain (ETC) subunits and thus mitochondrial activity. Thus, I started expanding the mitochondria probe tool set by designing a novel probe targeting methylated CpGs of mitochondrial DNA (mtDNA). We demonstrated the capability of our probe to reveal spatial distribution of methylated mtDNA and capture mtDNA methylation change at single cell level. Combined with our previously developed probe for nuclear DNA methylation, we monitored mtDNA and nuclear DNA methylation simultaneously on the single-cell level where unsynchronized dynamics of DNA methylation from nucleus and mitochondria were discovered.</p><p dir="ltr">Our tool offers a unique opportunity to understand epigenetic regulation of mtDNA and its dynamic response to microenvironment and cellular changes. Later, I further extended these efforts to develop in situ probes for tracking the formation of tau aggregates based on fluorescence resonance energy transfer (FRET); and demonstrated the superior performance of our engineered probes compared to the current state-of-the-art.</p><p dir="ltr">I explored two neuronal culture systems, namely SH-SY5Y- and human induced pluripotent stem cell (hiPSC)-derived neurons; and their feasibility in studying neurotoxic effects of developmental exposure to environmental stressors. Specifically, I used SH-SY5Y derived neuron-like cells to study the impact of pre-differentiation exposure to PFOA, abundant chemical in environment due to its historical uses in consumer products and industrial applications. hiPSC-derived neurons were used to study the effects of developmental exposure to ATZ. Both studies identified cellular changes, for example neurite morphology and expression of enzyme catalyzing the production of neurotransmitters, that last after completion of differentiation. We also identified changes of pathogenic markers aligning with increased PD risks associated with developmental PFOA and ATZ exposure. Compared to SH-SY5Y, hiPSC-derived neurons were more advantageous due to their ability to recapitulate neuronal activity and pathogenic changes related to ND, and thus were used in my follow-up studies.</p><p dir="ltr">I adopted hiPSC derived neuron model to study the molecular mechanism of ND using established ND etiology. Patients with neurodegenerative disorders (ND) exhibit varying levels and temporal patterns of aggregated β-amyloid (Aβ) and tau protein. We exposed neurons derived from hiPSC with preformed fibrils (PFFs) of Aβ, tau and Aβ+tau, respectively. These treatments result in significant alterations in neurite network morphology, nuclear morphology, chromatin compactness and synaptic density. Interestingly, Aβ and tau fibrils seem to have opposite effects on mitochondrial membrane potential on neurites. Increased quantity of lysosomes was found in neurons treated with Aβ, tau and Aβ+tau, while decrease of lysosomal acidity was only observed in neurons treated with Aβ and tau sequentially. Collectively, our data suggests the potential synergy between Aβ and tau in establishing a neurotoxic state.</p><p dir="ltr">In summary, my thesis work has developed enabling engineering tools to monitor cellular and subcellular changes in neurons; identified hiPSC-derived neurons as a promising platform for studying developmental neurotoxicity; and paved the way towards understanding multi-etiology and its molecular underpinning for ND.</p>

No description available.