Pain signaling involves transmission of nociceptive stimuli in the spinal cord where a critical balance between excitatory and inhibitory inputs determines the response to noxious stimuli. The neuropeptide, substance P (SP), mediates transmission of pain in part by binding to the tachykinin receptor (NK-1R) in the dorsal horn (DH) of the spinal cord. One of SP’s downstream effects is to modulate N-type Ca2+(N-) channels. While phospholipid breakdown is a part of the inflammatory process that accompanies tissue damage, the role of this metabolic pathway has not been completely described with respect to N-channel modulation during pain signaling. Despite the incomplete understanding of this modulation, pharmacological antagonists of both NK-1R and N-channels have been used to treat pain. In Chapter II, using whole-cell patch clamp recording techniques, the SP signaling cascade that mediates inhibition of recombinant N-channel activity was characterized. By adopting a pharmacological approach, I show that this pathway resembles the slow pathway that was earlier described for modulation of N-current by the M1 muscarinic receptor (M1R). M1R couples to Gq to stimulate phospholipid breakdown. Together with previous observations, the data presented in this chapter provide evidence for involvement of the extracellular receptor kinase (ERK1/2), phospholipase A2 and release of phospholipid metabolites in the modulation of N-current by SP. Overall, this chapter shows that phospholipid metabolism involved in modulation of N-currents is not specific to M1Rs but that other Gq-coupled receptors may also modulate N-currents via the same signal transduction pathway. In Chapter III, enhancement of N-current by SP was studied as part of a collaborative project to understand current enhancement that occurs when a palmitoylated accessory CaVβ2a subunit is co-expressed with the pore-forming subunit CaV2.2 and the accessory subunit α2δ-1. When CaVβ3 is present, SP inhibits N-current as described in Chapter II. However, when palmitoylated CaVβ2a is co-expressed with CaV2.2 (and α2δ-1), current enhancement is observed at negative test potentials, demonstrating that both M1Rs and NK-1Rs exhibit the same profile of N-current modulation. This change in modulation by muscarinic agonists is not observed in the presence of a depalmitoylated CaVβ2a. However a chimeric CaVβ2aβ1b subunit that contains the palmitoylated N-terminus from CaVβ2a confers enhancement. Normally expression of the β1b subunit resulted in current inhibition. These findings indicated that the palmitoylated CaVβ2a participates in enhancement of current. Our data support a model where inhibition dominates over enhancement; when inhibition is blocked, enhancement may be observed. Lastly, we show that N-current inhibition by SP is minimized when exogenous palmitic acid is applied to cells co-expressing CaVβ3 subunits with N-channels. These results indicate that the presence of palmitic acid can prevent N-current inhibition when SP is applied most likely by interacting with CaV2.2. We propose a model where palmitic acid occupies the inhibitory site and serves to antagonize inhibition by a lipid metabolite, which is most likely arachidonic acid. The CaVβ2a protein seems to have a role in positioning the palmitoyl groups near CaV2.2. This chapter provides a new role for protein palmitoylation where the palmitoyl groups of CaVβ2a are both necessary and sufficient to block inhibition of another protein: CaV2.2. In Chapter IV, I probe the role of the relative orientation of CaVβ2a and the pore-forming subunit of the N-channel in N-current modulation. Evidence is presented that shows that not just the presence of a palmitoylated CaVβ2a is necessary, but the relative orientation of CaVβ2a to CaV2.2 is critical for blocking inhibition. Using N-channel mutants that cause a change in the orientation of CaVβ2a relative to CaV2.2, I show that the block of inhibition is disrupted; inhibition by the slow pathway is rescued. These findings further support my model that the palmitoyl groups of CaVβ2a normally reside in a specific location that overlaps with the slow pathway inhibitory site on CaV2.2. Lastly I present data showing that the enhancement of N-current, observed when palmitoylated CaVβ2a is present, occurs via the slow pathway. In Chapter V the effect of CaVβ’s orientation on N-channel modulation by the dopamine D2 receptor is tested. In this form of modulation, inhibition is rapid and voltage-dependent. The signaling pathway is membrane-delimited since Gβγ, released after receptor stimulation, directly interacts with the N-channel at a site that overlaps with a high affinity binding site for CaVβs. While N-currents are modulated by this pathway, the deletion mutants show aberrant membrane-delimited modulation. The findings in this chapter further underscore the importance of proper positioning of CaVβ to CaV2.2 for eliciting proper N-current modulation after GPCR stimulation. Overall, the data presented in this dissertation provides a mechanistic approach into examining modulation of N-current by different GPCRs via two different signaling pathways as well as the role CaVβ subunits serve in each modulatory pathway.

Submitted by IARA BURIOLA TREVISAN null (iara_buriola@hotmail.com) on 2016-02-11T21:26:43Z No. of bitstreams: 1 Dissertação mestrado Iara B Trevisan.pdf: 1661968 bytes, checksum: 0ef9c6c9bf0a83ca5bd7671b9b047766 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-02-12T16:50:42Z (GMT) No. of bitstreams: 1 trevisan_ib_me_prud.pdf: 1661968 bytes, checksum: 0ef9c6c9bf0a83ca5bd7671b9b047766 (MD5) / Made available in DSpace on 2016-02-12T16:50:42Z (GMT). No. of bitstreams: 1 trevisan_ib_me_prud.pdf: 1661968 bytes, checksum: 0ef9c6c9bf0a83ca5bd7671b9b047766 (MD5) Previous issue date: 2016-01-26 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Introdução: A colheita da cana-de-açúcar precedida pela queima noturna expõe trabalhadores a altas concentrações de poluentes que somados aos efeitos da jornada de trabalho acarreta o aumento do trabalho cardiorrespiratório, ocasionando alterações multissistêmicas. Apesar das grandes evidências dos efeitos deletérios da exposição ao material particulado (MP), ainda tem sido dada pouca atenção aos efeitos das exposições ocupacionais a longo prazo. Objetivos: Esta dissertação teve como objetivos: avaliar os sintomas nasais, parâmetros hemodinâmicos e perfil inflamatório nasal e sistêmico de cortadores de cana-de-açúcar expostos à queima de biomassa ao longo de uma safra canavieira. Métodos: Foram avaliados no artigo I 41 indivíduos e no artigo II 45 indivíduos, cortadores de cana-de-açúcar do gênero masculino de uma usina de açúcar e álcool, divididos em grupos de acordo com o hábito tabagístico. Para avaliação da frequência e intensidade dos sintomas nasais foi aplicado questionário de sintomas respiratórios e para avaliação dos parâmetros hemodinâmicos foi registrado valores de pressão arterial (PA), frequência cardíaca (FC) e respiratória (f). Para análise do perfil inflamatório nasal (IL-6, IL-10 e IL-4) foi coletado amostras de lavado nasal e para análise de perfil inflamatório sistêmico (TNF-α, IL-6, IL-8 e IL-10) e contagem total de monócitos e neutrófilos foram realizados coletas de sangue venoso. As avaliações ocorreram durante a pré-safra (março), meio da safra (julho) e final da safra (outubro) de 2014. Resultados: Durante o meio da safra houve aumento significativo da frequência e intensidade dos sintomas espirros, congestão nasal além do aumento de relatos na dificuldade para respirar. Em relação a PA, FC e f todos os participantes apresentaram alterações significativas durante a safra. Na análise do perfil inflamatório houve alterações significativas a nível sistêmico para IL-8 e contagem de neutrófilos que obteve aumento no meio da safra seguido de um decréscimo ao final safra. Conclusões: Conclui-se que cortadores de cana-de-açúcar apresentam aumento na frequência e intensidade de sintomas nasais assim como alterações nos parâmetros hemodinâmicos principalmente no meio da safra. Além disso, todos os cortadores apresentaram diminuição de IL-8 e número de neutrófilos a nível sistêmico devido a exposição repetida ao MP. / Introduction: The harvest of sugarcane preceded by night burning exposes workers to high concentrations of pollutants added to the effects of the workday causes increased cardiorespiratory work, causing alterations multisystem. Despite the great evidences of the deleterious effects of exposure to particulate matter (PM), still has been given little attention to the effects of long-term occupational exposures. Objective: This Master’s thesis aimed to evaluated nasal symptoms, hemodynamic parameters and nasal and systemic inflammatory profile of sugarcane cutters exposed to biomass burning along a sugarcane harvest. Methods: Were evaluated in article I 41 individuals and in article II 45 individuals, sugarcane cutters, male cutters from a sugar and alcohol plant divided into groups according to smoking habit. To evaluate the frequency and intensity nasal symptoms was questionnaire applied of respiratory symptoms and to evaluate the hemodynamic parameters was registered blood pressure (BP), heart rate (HR) and respiratory (f). For analysis of the nasal inflammatory profile (IL-6, IL-10 and IL-4) was collected nasal lavage and for analysis of the systemic inflammatory profile (TNF-α, IL-6, IL-8 and IL-10) and total count monocytes and neutrophils from venous blood were performed. The evaluations occurred during the pre-harvest (March), middle of harvest (July) and end of harvest (October) in 2014. Results: During the middle of harvest there was a significant increase in the frequency and intensity of symptoms sneezing, nasal congestion beyond the increase in reports of difficulty in breathing. In relation the BP, HR and RR all participants showed significant alterations during the harvest. For analysis of inflammatory profile there were significant changes in systemic levels for IL-8 and neutrophil counts that had an increase in middle of harvest followed by a decrease at the end of harvest. Conclusions: Conclude that sugarcane cutters have increased in frequency and intensity of nasal symptoms, as well as changes in hemodynamic parameters, mainly in the middle of harvest. In addition, all cutters showed a decrease in IL-8 and neutrophil counts at systemic level at the end of harvest due to repeated exposure to PM. / FAPESP: 2014/08029-0

A pressão positiva contínua nas vias aéreas (CPAP) é o tratamento de escolha para pacientes com Apnéia Obstrutiva do Sono, mas muitos sintomas nasais conseqüentes da terapia são relatados. Vazamentos aéreos pela boca e alterações do epitélio respiratório são importantes no desenvolvimento de sintomas nasais e a umidificação aquecida é utilizada no alívio destes sintomas. O objetivo deste trabalho foi o de investigar os efeito agudos do nCPAP e o impacto da umidificação aquecida e vazamento aéreo no transporte mucociliar e inflamação nasal de indivíduos sadios. Para este fim avaliamos o transporte mucociliar nasal in vivo (através do Teste da Sacarina), a transportabilidade in vitro do muco nasal (através do Método Palato de Rã), lavado nasal e sintomas respiratórios (através de uma Escala Visual Analógica) de dezesseis indivíduos sadios antes e após aplicação aguda do CPAP sobre diferentes condições: CPAP com e sem umidificação aquecida e CPAP com e sem vazamento aéreo. O transporte mucociiar nasal in vivo aumentou significativamente após todas as intervenções com CPAP. Não houve diferença significativa da transportabilidade do muco, contagem total e diferencial de células inflamatórias provenientes do lavado nasal após nenhuma das intervenções com o CPAP. Houve um aumento significante da percepção subjetiva dos sintomas respiratórios estudados após o uso do CPAP sem umidificação e com vazamento aéreo. Concluimos que o uso agudo do CPAP independente da umidificação ou vazamento aéreo, aumenta significativamente o transporte mucociliar nasal in vivo, não altera significativamente a transportabilidade do muco nasal nem a composição celular de amostras de lavado nasal. Já o uso do CPAP sem umidificação e com vazamento aéreo causa aumento significativo dos sintomas de ressecamento nasal e de garganta, coriza e obstrução nasal. / Continuous positive airway pressure (CPAP) is the treatment of choice for patients with Obstructive Sleep Apnea but yet nasal symptoms are often reported. Air leaks and changes of the respiratory epithelium are important in the development of nasal symptoms and heated humidification is used to alleviate these symptoms. The aim of this study was to investigate the acute effects of CPAP and the impact of heated humidification and air leak on the nasal mucociliary clearance and nasal inflammation of healthy volunteers. To this end we evaluated nasal mucociliary clearance in vivo (through the Saccharin Test), in vitro nasal mucus transportability (through the Frog Palate Model), nasal lavage and respiratory symptoms (through a Visual Analogue Scale) of sixteen healthy volunteers before and after acute CPAP application under different conditions: CPAP with and without heated humidification and with and without air leak. In vivo nasal mucociliary clearance increased significantly after all CPAP interventions. There was no significant difference in mucus transportability, total or differential inflammatory cell count from the nasal lavage after any CPAP intervention. There was a significant increase in the subjective perception of the respiratory symptoms studied after the use of CPAP without humidification and with air leak. We conclude that the acute use of CPAP independently of humidification or air leak significantly increases in vivo nasal mucociliary clearance, doesn\'t change mucus transportability and total or differential cell count. However, the use o CPAP without humidification and with air leak significantly increased nasal and throat dryness, coryza and nasal obstruction subjective perception.

Introdução: O aumento das taxas de incidência e mortalidade por câncer colorretal (CCR) no Brasil pode ter sido consequência do processo de transição socioeconômica do país, com maior exposição aos fatores de risco. No entanto, fatores como o desconhecimento a respeito das estratégias de prevenção primária e secundária podem ter contribuído para o aumento do impacto da doença. Objetivos: Avaliar o grau de conhecimento a respeito do CCR em pacientes do Sistema Único de Saúde (SUS) e alunos da Faculdade de Medicina de Ribeirão Preto (FMRP) e caracterizar a realidade do programa de rastreamento no município de Ribeirão Preto. Métodos: Trata-se de um estudo transversal, onde se aplicou um questionário próprio, elaborado a partir de um caso clínico contendo sinais de alarme do CCR a fim de investigar o conhecimento sobre a doença. Foram entrevistados 1000 pacientes do SUS de Ribeirão Preto e 134 alunos da FMRP, no período de janeiro de 2015 a março de 2016. Resultados: Comparados aos alunos, os pacientes entrevistados apresentaram menor capacidade de diagnosticar o CCR (8% x 94,8%; p< 0,001). Notou-se, ainda, diferença no número médio de fatores de risco do CCR (0,76±1,3 x 4,18±1,72; p < 0,001) e no número médio de exames complementares para o diagnóstico do CCR (0,1±0,3x 2,5±1,12; p < 0,001) citados pelos pacientes e alunos. Apenas 3,7% dos entrevistados conseguiram identificar o coloproctologista como responsável pelo tratamento do caso. A análise multivariada mostrou que, no grupo de pacientes, idade >= 50 anos, sexo feminino, história familiar prévia de CCR e nível de escolaridade mais elevado foram variáveis independentes associadas a um maior grau de conhecimento a respeito da doença. Na amostra de pacientes com idade >= 50 anos, apenas 11,1% havia realizado algum teste de rastreamento e apenas 0,2% havia recebido informações prévias sobre a doença. Conclusões: O nível de conhecimento a respeito do diagnóstico, fatores de risco e métodos complementares de prevenção do CCR foi baixo entre os pacientes e adequado no grupo de alunos. Idade maior ou igual a 50 anos, sexo feminino, história familiar prévia de CCR e nível de escolaridade elevado foram fatores associados a um maior grau de conhecimento sobre a doença. Observou-se baixa taxa de realização de exames de rastreamento para o CCR. / Introduction: The increasing burden of colorectal cancer (CRC) in Brazil may be a consequence of the socio-economic transition with higher exposure to risk factors. In addition, low levels of CRC awareness and lack of a screening program may have been responsible for the high CRC incidence and mortality rates in Ribeirão Preto. Objectives: (1) to evaluate CRC awareness among patients from the Unified Health System and medical students from Ribeirao Preto Medical School, and (2) to investigate screening practices in the city. Methods: We conducted face-to-face interviews with a questionnaire prepared by the authors, from January 2015 to March 2016. A total of 1000 users and 134 medical students were interviewed. Results: Compared to medical students, the ability to diagnose CRC was lower among patients (8% x 94.8%; p< 0.001). Patients identified lower mean number of risk factors (0.76±1.3 x 4.18±1.72; p< 0.001) and screening methods (0.1±0.3 x 2.5±1.12; p< 0.001). Thirty-seven subjects (3.7%) identified the proctologists as the most appropriate specialist to treat CCR. On a multivariate analysis, age >= 50 years old, sex (female), family history of CCR and higher education levels were significantly associated with increased CRC awareness. Four hundred and seven patients were considered eligible to CRC screening; however, only 11.1% had already performed any method. Only 0.2% of the patients had been previously exposed to any kind of information about CRC. Conclusions: CRC awareness was very low among Health System users and adequate among medical students. Age >= 50 years old, sex (female), family history of CCR and higher education levels were independent predictors of increased knowledge among patients. Low rates of screening were observed in the city.

Made available in DSpace on 2014-07-29T15:04:32Z (GMT). No. of bitstreams: 1 Dissertacao Agueda Maria Ruiz Zimmer Cavalcante.pdf: 1035136 bytes, checksum: b4a1536a8353c2d355d3e8be5cf0bef5 (MD5) Previous issue date: 2009-06-19 / Upon the multiple ilnesses that aflict the elderly, respiratory problems stand out as cause of hospital admitance and re-admitance, and consequent worsening of funtional capacity. The nursing interventions directed to the human responses in evidence allow clinical improvement, visibility, and quality of nursing assistance to elderly patients with respiratory problems. The goal was to analyse the nursing interventions performed by the nursing staff to the nursing diagnosis ineffective breathing pattern in elderly patients. It is a descriptive study, developed in the internal medicine section of a scholl hospital in Goiânia, done in three stages, from July to December of 2008. The first stage consisted in the search of activities performed by the nursing staff to the ineffective breathing pattern in the elderly. The second consisted in the crossed mapping between the quoted activities and the interventions and activities priorized by the NIC to the ineffective breathing pattern diagnosis. And the third stage consisted in the new collection of data analyse the priorized NIC activities to ineffective breathing pattern in the elderly. If was adapted the descriptive statistic with distribution of the activities in simple frequence. 43 nursing workers took part in the study, after having read andsigned the form of free consent and understanding. Of the participants, 83,7% were women and 16,3% were men; 23,8% were between 36 and 40 years-old; 46,6% refered having completed high school, 11,6% had college education and 18,6% were in college, but not yet graduated; 18,6% had some complementary study after college and 2,3% had master degree; 60,5% were 1 to 5 years in the institution and 74,4% were 1 to 5 years in the same hospital section. From the 62 activities referred and mapped as NIC s interventions and activities, the ones witch presented a frequency higher than 50% were: Positioning (99,9%), Oxygen Therapy (99,8%) e Medication Administrantion (65,8%). Lower than 50%: Airway Manegement (45,0%), Anxiety Reduction (42,9%), Ventilation Assistance (40,8%), Respiratory Monitoring (36,8%), Vital Signs Monitoring (36,2%), Energy Management (31,6%), Emotional Support (27,2%), Pain Management (24,9%) e Surveillance (20,4%). Lower than 20%: Chest Physiotherapy (13,6%), Neurologic Monitoring (9,09%), Intravenous Therapy (6,8%), Airway Suctioning (6,8%), Intravenous Insertion (4,5%), Smoking Cessation Assistance (2,2%), Medication Management (2,2%), Fluid Monitoring (2,2%), Emergency Care (2,2%), Aspiration Precautions (2,2%) e Tube Care: Chest (2,2%).The drug administration activity present accordance as of the necessity of medical prescription; there was no accordance of the activities that are exclusive of the nurse. The activities concerning positioning, drug, administration, oxygen administration, aerosol administration and vital signs monitoring were referred as being frequently perfomed. The concerning monitoring of values of pulmonary function, fisioterapy and the ones that demanded, a physical exam evaluation were referred as not performed, and the main reason was being performed by another professional . The study made it possible to identify: the need to consider the interdisciplinarity in the interventions, the gaps in the assistance provided by the nursing staff, the abilities and skills required from the nurse to the assistance of the elderly patients that present ineffective breathing pattern , with focus a the prevention of disorders and on the integrality of the assistance, / Diante dos múltiplos agravos que acometem os idosos, destacam-se os problemas respiratórios como causa de internação e reinternação e consequente diminuição da capacidade funcional. As intervenções de enfermagem direcionadas às respostas humanas evidenciadas, possibilitam identificar a melhora clínica, tornando visível a qualidade da assistência de enfermagem à clientela idosa com problemas respiratórios. Objetivou-se analisar as intervenções de enfermagem realizadas pela equipe de enfermagem para o diagnóstico de enfermagem padrão respiratório ineficaz em idosos. Trata-se de um estudo descritivo realizado, nos meses de julho a dezembro de 2008, em três etapas, na Clínica Médica de um hospital escola de Goiânia. A primeira etapa consistiu na busca das atividades realizadas pela equipe de enfermagem para o padrão respiratório ineficaz em idosos. A segunda no mapeamento cruzado entre as atividades citadas e as intervenções e atividades prioritárias da NIC preconizadas para o padrão respiratório ineficaz . A terceira consistiu em uma nova coleta de dados para análise das atividades prioritárias da NIC preconizadas para o padrão respiratório ineficaz em idosos. Foi adotada a estatística descritiva com distribuição de frequência simples das atividades. Fizeram parte do estudo, 43 trabalhadores de enfermagem que atenderam os critérios de inclusão e exclusão. Dos participantes, 83,7% eram do sexo feminino e 16,3% masculino; 25,8% tinham entre 36 a 40 anos; 46,6% referiam ter o segundo grau completo, 11,6% tinham concluído e 18,6% estavam em processo de conclusão do terceiro grau; 18,6% eram pós-graduados e 2,3% mestres; 60,5% tinham de 1 a 5 anos de instituição e 74,4%, de 1 a 5 anos de atuação no setor. Das 62 atividades referidas pelos profissionais, foram mapeadas as intervenções e atividades da NIC. Apresentaram frequência maior que 50%: Posicionamento (99,9%), Oxigenoterapia (99,8%) e Administração de medicamentos (65,8%). As intervenções e atividades com frequência menor que 50% foram: Controle de Vias Aéreas (45,0%), Redução da Ansiedade (42,9%), Assistência Ventilatória (40,8%), Monitoração Respiratória (36,8%), Monitoração de Sinais Vitais (36,2%), Controle de Energia (31,6%), Suporte Emocional (27,2%), Controle da Dor (24,9%) e Supervisão (20,4%). Inferior a 20%: Fisioterapia Respiratória (13,6%), Monitoração Neurológica (9,09%), Terapia Endovenosa (6,8%), Aspiração de Vias Aéreas (6,8%), Punção venosa (4,5%), Assistência para parar de fumar (2,2%), Controle de Medicamentos (2,2%), Controle Hídrico (2,2%), Cuidados de Emergência (2,2%), Precauções contra Aspiração (2,2%) e Ventilação mecânica (2,2%). A atividade de administração de medicamentos apresentou concordância entre os profissionais quanto à necessidade da prescrição médica; não foi evidenciada concordância entre as atividades exclusivas do enfermeiro. As de posicionamento, de administração de medicamentos, de administração de oxigênio, de administração de aerossóis e de monitoração de sinais clínicos foram referidas por serem frequentemente realizadas. As atividades de monitoração de valores de função pulmonar, de realização de fisioterapia e aquelas que exigiam a propedêutica do exame físico foram referidas como não realizadas, cujo motivo principal foi ser realizada por outro profissional . O estudo possibilitou identificar: a necessidade de considerar a interdisciplinaridade nas intervenções, as lacunas na assistência prestada pela equipe de enfermagem, as habilidades e competências requeridas pelo enfermeiro para atendimento da clientela idosa que apresenta padrão respiratório ineficaz com foco na prevenção de agravos e na integralidade da assistência.

The human Philadelphia chromosome (Ph) arises from a translocation between chromosomes 9 and 22 [t(9;22)(q34;q11)]. The resulting chimeric BCR-ABLoncogene encodes a constitutively activated, oncogenic tyrosine kinase that induces chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL). The BCR-ABL tyrosine kinase inhibitor (TKI), imatinib mesylate, induces a complete hematologic and cytogenetic response in the majority of CML patients, but is unable to completely eradicate BCR-ABL–expressing leukemic cells, suggesting that leukemia stem cells are not eliminated. Over time, patients frequently become drug resistant and develop progressive disease despite continued treatment. Two major reasons cause the imatinib resistance. The first one is the BCR-ABL kinase domain mutations which inhibit the interaction of BCR-ABL kinase domain with imatinib; the second one is the residual leukemia stem cells (LSCs) in the patients who are administrated with imatinib. To overcome these two major obstacles in CML treatment, new strategies need further investigation. As detailed in Chapter II, we evaluated the therapeutic effect of Hsp90 inhibition by using a novel water-soluble Hsp90 inhibitor, IPI-504, in our BCR-ABL retroviral transplantation mouse model. We found that BCR-ABL mutants relied more on the HSP90 function than WT BCR-ABL in CML. More interestingly, inhibition of HSP90 in CML leukemia stem cells with IPI-504 significantly decreases the survival and proliferation of CML leukemia stem cells in vitro and in vivo. Consistent with these findings, IPI-504 treatment achieved significant prolonged survival of CML and B-ALL mice. IPI-504 represents a novel therapeutic approach whereby inhibition of Hsp90 in CML patients and Ph+ ALL may significantly advance efforts to develop a cure for these diseases. The rationale underlying the use of IPI-504 for kinase inhibitor–resistant CML has implications for other cancers that display oncogene addiction to kinases that are Hsp90 client proteins. Although we proved that inhibition of Hsp90 could restrain LSCs in vitro and in vivo, it is still unclear how to define specific targets in LSCs and eradicate LSCs. In Chapter III, we took advantage of our CML mouse model and compared the global gene expression signature between normal HSCs and LSCs to identify the downregulation of Pten in CML LSCs. CML develops faster when Pten is deleted in Ptenfl/fl mice. On the other hand, Pten overexpression significantly delays the CML development and impairs leukemia stem cell function. mTOR is a major downstream of Pten-Akt pathway and it is always activated or overepxressed when Pten is mutated or deleted in human cancers. In our study, we found that inhibition of mTOR by rapamycin inhibited proliferation and induced apoptosis of LSCs. Notably, our study also confirmed a recent clinical report that Pten has been downregulated in human CML patient LSCs. In summary, our results proved the tumor suppressor role of Pten in CML mouse model. Although the mechanisms of Pten in leukemia stem cells still need further study, Pten and its downstream, such as Akt and mTOR, should be more attractive in LSCs study.

Cytoplasmic polyadenylation element-binding protein (CPEB) is a sequence-specific RNA-binding protein that promotes polyadenylation-induced translation. While a CPEB knockout (KO) mouse is sterile but overtly normal, embryo fibroblasts derived from this mouse (MEFs) do not enter senescence in culture as do wild-type MEFs, but instead are immortal. Exogenous CPEB restores senescence in the KO MEFs and also induces precocious senescence in wild-type MEFs. CPEB cannot stimulate senescence in MEFs lacking the tumor suppressors p53, p19ARF, or p16INK4A; however, the mRNAs encoding these proteins are unlikely targets of CPEB since their expression is the same in wild-type and KO MEFs. Conversely, Ras cannot induce senescence in MEFs lacking CPEB, suggesting that it may lie upstream of CPEB. One target of CPEB regulation is myc mRNA, whose unregulated translation in the KO MEFs may cause them to bypass senescence. Thus, CPEB appears to act as a translational repressor protein to control myc translation and resulting cellular senescence. CPEB is a sequence-specific RNA binding protein that regulates cytoplasmic polyadenylation-induced translation. We report here that CPEB KO mice are hypersensitive to LPS-induced endotoxic shock, which correlates with elevated serum levels of the proinflammatory cytokines IL-6, IL-8 and IL-12. Peritoneal macrophages from the KO mice, as well as a CPEB-depleted macrophage cell line, not only secrete more IL-6 than control cells in response to LPS, but also have prolonged retention of NFϰB in the nucleus, which is responsible for elevated IL-6 transcription. The amount of nuclear NFϰB correlates with reduced levels of IϰBα, which is hyperphosphorylated and rapidly degraded. Collectively, these data suggest that CPEB deficiency enhances the inflammatory response via delayed resolution of NFϰB signaling.

The hypothalamo-neurohypophysial model has been studied for many years. Of note, when the axons of the magnocellular, peptidergic neurons of the supraoptic nucleus (SON) and paraventricular nucleus (PVN) are transected or crushed, varying degrees of polydipsia and polyuria ensue as the result of measurable losses of vasopressin (AVP) within the organism's circulation. Following insult, these hypothalamic cells show a remarkable capacity to reorganize themselves within the proximal areas of the infundibular stalk and median eminence and form what has come to be known as a new 'mini neural lobe' . While the surviving neurons sprout new projections toward the level of the external zone, vascular hypertrophy is marked throughout the new neurohypophysis and new neurohemal contacts have been identified (at the ultrastructural level) associated with these vessels. In parallel with this vascular hypertrophy is a measurable re-release of vasopressin into the circulation. This new 'mini neural lobe' now has the morphological and physiological appearance of an intact neural lobe and is capable of releasing AVP in response to changes in water balance. While the ability of these axons to reorganize is more characteristic of the peripheral nervous system (PNS), this model system provides an unique opportunity to study axonal regeneration of the central nervous system (CNS). Not only the mechanisms underlying the restoration of AVP function following axotomy but the extent to which various magnocellular neuron populations are involved in the regenerative process may also be analyzed. Before attempting to identify putative markers associated with this regenerative process, it was necessary to carefully characterize the system following axonal injury. Using Sprague Dawley rats, we repeated previous physiological studies which had examined the intake of water and output of urine following hypophysectomy. In addition, we also correlated the restoration of water balance with the return of AVP release, as measured by radioimmunoassay. These data defined a temporal framework in which magnocellular AVP regeneration occurs. As a result of repeating these physiological studies, we noted several inconsistencies between other previously published work. First, the time course of AVP recovery did not agree with other published results, nor did the first appearance of AVP immunoreactivity . We did not observe a complete recovery of water balance as previously reported and the degree of magnocellular death was inconsistent with other reports. In light of these many conflicting observations between several historical reports and our own results, we did a basic physiological re-characterization of the hypothalamo-neurohypohysial system following hypophysectomy. By means of immunohistochemistry, we also demonstrated the re-appearance of AVP within the new the 'mini neural lobe ' concomitant with the increased appearance of synapsin I, a marker associated with the presence of mature and presumably functioning synapses to be no sooner than 28 days following surgical removal of the hypophysis. Immunocytochemistry was also used in conjunction with retrograde fluorescent labeling to extend the previous studies and include a 2-D analysis of cell survival throughout the PVN and SON following hypophysectomy or neurohypophysectomy. As reported previously, magnocellular neuronal loss is greater within the SON, particularly the hypophysectomized subject, and less so within the PVN; again with the greater loss in the PVN of the hypophysectomized animal. Based upon our observations and other recent reports, we suggest the possibility that some cells of the hypothalamo-neurohypophysial system or some other extrahypothalamic cell population may be capable of expressing vasopressin in response to neurohypophysectomy. We provide initial evidence that glial cells of the third ventricle may indeed be involved. Finally, one of the ultimate goals of using this as a model system of CNS regeneration is to understand the underlying mechanisms and components essential to central nervous tissue regeneration. Toward that end I have been involved with the initial studies to optimize an adenovirus delivery system which will be capable of incorporating various putative neurotransmitter and/or peptide anti-sense messages, being injected into the neurohypophysis and transported back into the cells of the hypothalamo-neurohypophysial system. Once these antisense sequences are expressed by the cells following axotomy, the sequence of expression of various proteins in response to injury may be elucidated.

Diabetes mellitus is a group of metabolic disorders characterized by hyperglycemia. The pathogenesis of these diseases involves β-cell dysfunction and death. The primary function of β-cells is to tightly regulate the secretion, production, and storage of insulin in response to blood glucose levels. In order to manage insulin biosynthesis, β-cells have an elaborate endoplasmic reticulum (ER). The ER is an essential organelle for the proper processing and folding of proteins such as proinsulin. Proteins fold properly when the ER protein load balances with the ER folding capacity that handles this load. Disruption of this ER homeostasis by genetic and environmental stimuli leads to an accumulation of misfolded and unfolded proteins, a condition known as ER stress. Upon ER stress, the unfolded protein response (UPR) is activated. The UPR is a signaling network that aims to alleviate ER stress and restore ER homeostasis promoting cell survival. Hence, the UPR allows β-cells to handle the physiological fluctuations of insulin demand. However upon severe unresolvable ER stress conditions such as during diabetes progression, the UPR switches to pathological outputs leading to β-cell dysfunction and apoptosis. Severe ER stress may also trigger inflammation and accumulating evidence suggests that inflammation also contributes to β-cell failure, but the mechanisms remain elusive. In this dissertation, we demonstrate that thioredoxin interacting protein (TXNIP) mediates ER stress induced β-cell inflammation and apoptosis. During a DNA microarray analysis to identify novel survival and death components of the UPR, we identified TXNIP as an interesting proapoptotic candidate as it has been linked to glucotoxicity in β-cells. During our detailed investigation, we discovered that TXNIP is selectively expressed in β-cells of the pancreas and is strongly induced by ER stress through the IRE1α and PERK-eIF2α arms of the UPR and specifically its transcription is regulated by activating transcription factor 5 (ATF5) and carbohydrate response element binding protein (ChREBP) transcription factors. As TXNIP has been shown to activate the Nod-like receptor protein 3 (NLRP3) inflammasome leading to the production of the inflammatory cytokine interleukin-1β (IL- 1β), we hypothesized that perhaps TXNIP has a role in IL-1β production under ER stress. We show that ER stress can induce IL-1β production and that IL-1β is capable of binding to IL-1 type 1 receptor (IL-1R1) on the surface of β-cells stimulating its own expression. More importantly, we demonstrate that TXNIP does indeed play a role in ER stress mediated IL-1β production through the NLRP3 inflammasome. Furthermore, we also confirmed that TXNIP is a mediator of β-cell apoptosis under ER stress partially through IL-1β signaling. Collectively, we provide significant novel findings that TXNIP is a component of the UPR, mediates IL-1β production and autostimulation, and induces cell death under ER stress in β-cells. It is becoming clear that TXNIP has a role in the pathogenesis of diabetes and is a link between ER stress, oxidative stress and inflammation. Understanding the molecular mechanisms involved in TXNIP expression, activity, and function as we do here will shed light on potential therapeutic strategies to tackle diabetes.

Dengue fever (DF) and dengue hemorrhagic fever (DHF) are emerging infectious diseases which are endemic in many regions of the globe, many of which are resource-poor areas. DHF and DF impose a severe economic health burden in tropical and subtropical areas. Dengue virus causes an acute febrile illness that can be a self-limited febrile illness, as seen in most cases of DF, or a life-threatening illness with plasma leakage and shock, as seen in cases of DHF. A systematic review of the literature revealed gaps in the knowledge base of clinical laboratory findings of dengue illness with regards to longitudinal dynamics and classification and predictive modeling of disease severity. The objective of this thesis was to investigate the utility of clinical laboratory variables for classification and prediction of disease outcomes. The data used in this investigation was derived from a prospective study of Thai children presenting to either of two study hospitals within 72 hours of onset of an acute febrile illness. Systematic data collection, including clinical laboratory parameters, and routine clinical management continued each day until 24 hours after the fever had subsided. A final diagnosis of DHF, DF, or other febrile illness (OFI) was assigned by an expert physician after chart review. The first research objective of this study was to describe the temporal dynamics of clinical laboratory parameters among subjects with DHF, DF, or OFI. Data were analyzed using lowess curves and population-average models. Quadratic functions of clinical variables over time were established and demonstrated significantly divergent patterns between the various diagnostic groups. The second research objective was to establish and validate tools for classification of illness severity using easily obtained clinical laboratory measures. Bivariate logistic regression models were established using data from one hospital in an urban area of Thailand as a training data set and validated with a second data set from a hospital in a rural area of Thailand. The validated models maintained a high sensitivity and specificity in distinguishing severe dengue illnesses without using the hallmark indicators of plasma leakage. The third research objective used classification and regression tree (CART) analysis to established diagnostic decisions trees using data obtained on the day of study enrollment, within the first 3 days of acute illness. Decision trees with high sensitivity were established for severe dengue defined either as: 1) DHF with evidence of shock (dengue shock syndrome, DSS); or 2) DSS or dengue with significant pleural effusion. This study expands existing knowledge of the potential utility of clinical laboratory variables during different phases of dengue illness. The application of the results of these studies should lead to promising opportunities in the fields of epidemiological research and disease surveillance to reduce the health burden, and improve the clinical management, of dengue illness. Future directions involve application of these algorithms to different study populations and age groups. Additionally, other analytical techniques, such as those involving CART analysis, can be explored with these data.