Thesis (Ph.D. (Biochemistry)) - Rhodes University, 2007.

Proefschrift Rijksuniversiteit Limburg, Maastricht. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

This thesis attempts to analyze the impact of disaster capitalism on the areas affected by the Great Flood of 1993. Using Naomi Klein’s book, the Shock Doctrine, I selected three variables that can be indicators of disaster capitalism. Unemployment rates, new private housing units authorized by permit, and employment in the mining, logging, and construction industry are used. I use a comparison of means test and a difference-in- differences estimate to find if the variables were changed as a result of the flood. Unemployment rates seemed to be affected by the crisis and strongly support Klein’s theories of disaster capitalism.

Orientador: Eliana Aguiar Petri Nahas / Resumo: Objetivo: Avaliar os fatores de risco cardiovascular em mulheres na pós-menopausa tratadas de câncer de mama comparadas a mulheres na pós-menopausa sem câncer de mama e a associação das heat shock proteins (HSP) 60 e 70 com o câncer de mama em mulheres na pós-menopausa. Métodos: Realizou-se estudo clínico de corte transversal com 96 mulheres tratadas de câncer de mama comparadas a 192 mulheres na pós-menopausa (controle), com idades entre 45 e 75 anos. Foram incluídas no grupo principal mulheres com amenorréia > 12 meses e idade ≥ 45 anos, com diagnóstico histológico de câncer de mama, sem doença metastática e sem doença cardiovascular (DCV) estabelecida. O grupo controle foi constituído por mulheres com amenorréia > 12 meses, idade ≥ 45 anos, sem câncer de mama e DCV. Os grupos foram pareados por idade, tempo de menopausa e índice de massa corpórea (IMC) na proporção 1 caso para 2 controles, conforme cálculo amostral, com o mínimo de 92 pacientes tratadas de câncer de mama. Dados clínicos e antropométricos (IMC e circunferência da cintura) foram coletados por meio de entrevista e exame físico. Para análise bioquímica foram solicitados colesterol total, HLD, LDL, triglicerídeos, glicose e insulina. Foram consideradas com síndrome metabólica (SM), as mulheres que apresentaram três ou mais critérios diagnósticos: circunferência da cintura (CC) > 88 cm; TG ³ 150 mg/dL; HDL colesterol < 50 mg/dL; pressão arterial ³ 130/85 mmHg; glicose ³ 100 mg/dL. Para determinação das concentra... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: To evaluate cardiovascular risk factors in postmenopausal breast cancer survivors as compared to postmenopausal women without breast cancer and the association of heat shock proteins (HSP) 60 and 70 with breast cancer in postmenopausal women. Methods: In this cross-sectional study, 96 postmenopausal breast cancer survivors were compared with 192 postmenopausal women (controls), aged 45 to 75 years. The principal group included women with amenorrhea > 12 months and age ≥45 years, with histological diagnosis of breast cancer, without metastatic disease and without established cardiovascular disease (CVD). The control group consisted of women with amenorrhea > 12 months, age ≥45 years, without breast cancer and CVD. The groups were matched by age, time since menopause, and body mass index (BMI) in the proportion 1 case for 2 controls, according to the sample calculation, with a minimum of 92 breast cancer survivors. Clinical and anthropometric data (BMI and waist circumference) were collected by interview and physical examination. For biochemical analysis, total cholesterol, HLD, LDL, triglycerides, glucose and insulin levels were measured. Women who presented three or more of the following criteria were diagnosed as having metabolic syndrome (MetS): waist circumference (WC)> 88 cm; TG≥ 150 mg/dL; HDL cholesterol <50 mg/dL; blood pressure ≥ 130/85 mmHg; glucose ≥ 100 mg/dL. For measuring plasma concentrations of HSP 60 and 70, immunoassays by ELISA technique were used. Atheroscl... (Complete abstract click electronic access below) / Doutor

Heat shock protein 90 (HSP90), a conserved molecular chaperone, has become a potential molecular target for cancer therapeutics. HSP90 inhibition (HSP90i) causes inhibition of several oncogenic pathways simultaneously and leads to anti-cancer activities in multiple cancers including in triple-negative breast cancer (TNBC). TNBC is a subtype of breast cancer with poor prognosis and lack of approved targeted therapies. Although HSP90i has shown promising initial clinical data, resistance to HSP90i can still arise in TNBC patients and its resistance mechanisms are not yet understood. In this study, using an in vitro system, we report for the first time the isolation of TNBC cells with acquired resistance to HSP90i. Proteome and whole transcriptome profiling, and a bioactive small molecule screen were performed to identify the molecular basis of resistance to HSP90i and potential therapeutic approaches to overcome acquired resistance to HSP90i in TNBC cells. Two independent HSP90i-resistant clones were acquired through prolonged exposure of a TNBC cell line (Hs578T) to HSP90i. The clones showed significant resistance to HSP90i, notably to resorcinol-based HSP90i. The HSP90i-resistant clones also shared genomic sequence variants, suggesting a pre-existing population of resistant cells within the parental cells. We demonstrate that upregulated expression of UGT1A9, possibly due to an increased intrinsic oxidative stress, is associated with acquired resistance to resorcinol-based HSP90i in TNBC cells, and sensitivity to HSP90i can be restored with a competitive inhibitor of UGT1A9. The HSP90i-resistant clones also exhibited slower growth and upregulated IL6- mediated JAK2-STAT3 survival signalling pathway, which might contribute to the crossresistance to chemotherapeutics and other targeted therapies seen in the clones. Finally, we demonstrate that inhibition of JAK2-STAT3 signalling pathway is able to increase the cytotoxic effects of HSP90i to TNBC cells. We conclude that by using in vitro assays, we are able to identify potential mechanisms of acquired resistance to HSP90i in TNBC cells. We propose that expression of UGT1A9 or STAT3 might be a potential biomarker of sensitivity to HSP90i in TNBC cells. A combined inhibition of HSP90 and JAK2 might be a potential therapeutic approach for the development of effective targeted therapies in TNBC patients.

Orientador: Paulo José Paupitz Gonçalves / Co-orientador: Edson Antônio Capello Sousa / Banca: Marcos Silveira / Banca: Fabricio Cesar Lobato de Almeida / Banca: Douglas Domingues Bueno / Banca: Laercio Javarez Junior / Resumo: O objetivo principal desta tese é a análise de amortecedores não lineares, incluindo os efeitos da folga, assimetria e histerese. A tese também apresenta um estudo sobre a influência da temperatura sobre esses efeitos. O estudo baseia-se em testes experimentais realizados em três diferentes amortecedores, incluindo um de atrito seco, um hidráulico e um do tipo magnetoreológico. Esses amortecedores foram testados em uma máquina de teste universal utilizando excitação cíclica para diferentes condições de amplitude, frequência e corrente elétrica no caso do amortecedor magnetoreológico. Três modelos paramétricos foram utilizados para representar as forças de amortecimento que foram medidas durante os ensaios experimentais, onde os parâmetros que definem esses modelos foram ajustados utilizando um algoritmo de otimização. A tese propõe uma contribuição para o campo de engenharia ao representar fenômenos não lineares típicos de amortecedores através de testes experimentais e descrevendo uma metodologia para representar as forças de amortecimento não linear e os efeitos da temperatura nestes modelos. / Abstract: The main objective of this thesis is the analysis of nonlinear dampers including the effects of clearance, asymmetry and hysteresis. The thesis also presents a study on the temperature influence over these effects. The study is based on experimental tests performed on three different damping devices (dashpots), including a dry friction, a hydraulic and a magnetorheological dashpots. Theses dampers were tested on an universal testing machine using cyclic excitation for different conditions of amplitude, frequency and current in the case of the magnetorheological damper. Three parametric models have been used to represent the damping forces that were measured during the experimental testes, where the parameters defining these models were adjusted using an optimization algorithm. The thesis proposes a contribution to the engineering field by representing typical nonlinear phenomena of dampers through experimental tests and describing a methodology to represent the nonlinear damping forces and the effects of temperature on these models / Doutor

Heat shock proteins (HSPs) play important roles in the process of maintaining proteostasis in a cell. HSP72, the inducible form of the HSP70 family, is expressed in response to stress on the cell or tissue, including those stresses caused by tumor growth. Increasing evidence suggests that HSP72 is necessary for a cancerous cell to survive under the stresses of a tumor microenvironment. This has naturally raised interest in identifying an inhibitor selective for HSP72. The Haystead Laboratory at Duke University identified such a small-molecule inhibitor, referred to as HS-72, and proposed the scaffold as an ideal starting point to develop a family of therapeutic agents targeting HSP72. This work examines the potency and effectiveness of HS-72 and a number of its analogs developed by the Haystead Laboratory. These results suggest that HS-159 is a more effective inhibitor of HSP72 on a range of human tumor cell lines than HS-72. Further studies are needed to quantify how much more potent HS-159 is than HS-72 and potentially identify even more potent compounds.

In high impact human activities, much of the impact shock wave is dissipated through internal body structures, preventing excessive accelerations from reaching vital organs. Mechanisms responsible for this attenuation, including lower limb joint compression and spinal compression have been neglected in existing whole-body simulation models. Accelerometer data on one male subject during drop landings and drop jumps from four heights revealed that peak resultant acceleration tended to decrease with increasing height in the body. Power spectra contained two major components, corresponding to the active voluntary movement (2 Hz 14 Hz) and the impact shock wave (16 Hz 26 Hz). Transfer functions demonstrated progressive attenuation from the MTP joint towards the C6 vertebra within the 16 Hz 26 Hz component. This observed attenuation within the spine and lower-limb joint structures was considered within a rigid body, nine-segment planar torque-driven computer simulation model of drop jumping. Joints at the ankle, knee, hip, shoulder, and mid-trunk were modelled as non-linear spring-dampers. Wobbling masses were included at the shank, thigh, and trunk, with subject-specific biarticular torque generators for ankle plantar flexion, and knee and hip flexion and extension. The overall root mean square difference in kinetic and kinematic time-histories between the model and experimental drop jump performance was 3.7%, including ground reaction force root mean square differences of 5.1%. All viscoelastic displacements were within realistic bounds determined experimentally or from the literature. For an equivalent rigid model representative of traditional frictionless pin joint simulation models but with realistic wobbling mass and foot-ground compliance, the overall kinetic and kinematic difference was 11.0%, including ground reaction force root mean square differences of 12.1%. Thus, the incorporation of viscoelastic elements at key joints enables accurate replication of experimentally recorded ground reaction forces within realistic whole-body kinematics and removes the previous need for excessively compliant wobbling masses and/or foot-ground interfaces. This is also necessary in cases where shock wave transmission within the simulation model must be non-instantaneous.

Submitted by ISADORA PIMENTEL DE SOUZA (euisaps@yahoo.com.br) on 2018-08-10T17:48:48Z No. of bitstreams: 1 DISSERTACAO 10.08.pdf: 1322071 bytes, checksum: bb198d40ebd35093636b6713a6bab637 (MD5) / Approved for entry into archive by Sulamita Selma C Colnago null (sulamita@btu.unesp.br) on 2018-08-13T18:02:13Z (GMT) No. of bitstreams: 1 souza_ip_me_bot.pdf: 1322071 bytes, checksum: bb198d40ebd35093636b6713a6bab637 (MD5) / Made available in DSpace on 2018-08-13T18:02:13Z (GMT). No. of bitstreams: 1 souza_ip_me_bot.pdf: 1322071 bytes, checksum: bb198d40ebd35093636b6713a6bab637 (MD5) Previous issue date: 2018-07-23 / Introdução: O choque séptico é uma importante causa de morbimortalidade em recém-nascidos prematuros. O lactato vem sendo estudado como marcador diagnóstico e prognóstico do choque, mas os estudos no período neonatal são escassos. Objetivo: Investigar se os níveis séricos de lactato podem predizer o risco de morte em prematuros com choque séptico. Métodos: Estudo retrospectivo, longitudinal, realizado na UTI Neonatal do Hospital das Clinicas da Faculdade de Medicina de Botucatu no período de janeiro de 2014 a dezembro de 2016, após aprovação do CEP. Foram incluídos todos os prematuros menores que 37 semanas, internados na UTI, com mais de 72 horas de vida, que apresentaram diagnóstico de choque séptico e dosagem do lactato sérico nas primeiras 48 horas do choque. Foram excluídos aqueles com malformações múltiplas, infecções congênitas e erros inatos do metabolismo. Variáveis estudadas: maternas, gestacionais, neonatais, agente etiológico e dosagem do lactato. Os prematuros foram comparados em dois grupos: sobrevida e óbito. Desfecho: óbito. Análise estatística: testes paramétricos e não paramétricos com significância estatística se p<0.05. Acurácia do lactato: sensibilidade, especificidade, valor preditivo positivo e negativo. Resultados: Dentre os 456 prematuros sobreviventes por mais de 72 horas, 130 apresentaram sepse tardia (28,5%) e destes, 36 choque séptico (28%). A mortalidade no choque foi de 42%. A positividade em hemocultura foi de 36% com predomínio de Gram-positivos no grupo sobrevida e de Gram-negativos no grupo óbito. A comparação entre os grupos sobrevida e óbito, respectivamente mostrou: idade gestacional 29,5sem vs 27,5sem (p=0.05); peso de nascimento 950g vs 900g (p=0.386), idade no diagnóstico do choque 11 dias vs 7 dias (p=0.071), uso de drogas vasoativas 52% vs 93% (p=0.011). Os níveis de lactato foram maiores no grupo óbito (1,2mmol/L vs 1,7 mmol/L; p=0.043). O lactato  4mmol/L apresentou boa acurácia na predição de morte diretamente relacionada ao choque (89%) com baixa sensibilidade, 33%, mas com especificidade e valor preditivo positivo de 100% e valor preditivo negativo de 88%. Conclusão: A incidência e mortalidade do choque séptico em prematuros foram altas, sendo os muito prematuros e os de extremo baixo peso os mais acometidos. Valores de lactato  4 mmol/L apresentaram boa acurácia na predição de morte, alta especificidade e alto valor preditivo positivo. / Introduction: Septic shock is an important cause of morbidity and mortality in premature infants. Lactate has been studied as a diagnostic and prognostic marker of shock, but studies in the neonatal period are scarce. Objective: To investigate if serum lactate levels can predict the risk of death in preterm infants with septic shock. Methods: Retrospective, longitudinal study performed at the Neonatal Intensive Care Unit (NICU) of the Clinics Hospital – Botucatu School of Medicine, from January 2014 to December 2016, after approval of the Ethics Committee. All preterm infants less than 37 weeks gestational age, with more than 72 hours of life, admitted at the NICU with diagnosis of septic shock and serum lactate dosage in the first 48 hours of shock were included. Those with multiple malformations, congenital infections and inborn errors of metabolism were excluded. Variables studied: maternal, gestational, neonatal, etiologic agent and lactate dosage. The preterm infants were compared in two groups: survival and death. Outcome: death. Statistical analysis: parametric and non-parametric tests with statistical significance if p<0.05. Lactate accuracy: sensitivity, specificity, positive and negative predictive value. Results: Among the 456 preterm infants who survived for more than 72 hours, 130 had late onset sepsis (28.5%) and of these 36 septic shock (28%). The shock mortality was 42%. The positivity in blood cultures was 36%, with a predominance of Gram-positive in the survival group and Gram-negative in the death group. The comparison between survival and death groups, respectively, showed: gestational age 29.5weeks vs 27.5 weeks (p=0.05); birth weight 950g vs 900g (p=0.386), age at shock diagnosis 11 days vs 7 days (p=0.071), vasoactive drugs 52% vs 93% (p=0.011). Lactate levels were higher in the death group (1.2mmol/L vs 1.7 mmol/L, p=0.043). Lactate ≥4 mmol/L showed good accuracy in predicting death directly related to shock (89%) with low sensitivity 33%, but with specificity and a positive predictive value of 100% and a negative predictive value of 88%. Conclusion: The incidence and mortality of septic shock in premature infants were high, and very premature and extremely low birth weight were the most affected. Values of lactate  4 9 mmol/L showed good accuracy in predicting death, high specificity and high positive predictive value.

Pacientes críticos são indivíduos internados em Unidades de Terapia Intensiva (UTI) e se caracterizam por apresentar um quadro patológico crítico e complexo, decorrente de fragilidades fisiológicas graves, podendo evoluir para sepse, sepse severa, choque séptico ou óbito. Muitos recursos financeiros são investidos no controle da sepse em hospitais públicos e privados, e a sepse é a maior causa de morte entre as UTIs brasileiras. O desfecho de um paciente crítico é influenciado por vários fatores, entre eles, os genéticos. A molécula HLA-G apresenta variabilidade proteica limitada e expressão tecidual restrita. A interação entre moléculas de HLA-G e os receptores KIR e LILR desencadeia diversas atividades imunomodulatórias. Na região 3’ UTR localizam-se os polimorfismos +2960INDEL, +3142C>G e +3187A>G que podem regular a expressão de HLA-G. Até o momento, apenas um estudo investigou a expressão de sHLA-G5 em pacientes críticos com choque séptico, e se observou que o aumento dos níveis de sHLA-G5 foram preditivos de sobrevivência entre os pacientes que evoluíram para choque séptico. O objetivo do presente trabalho foi determinar as frequências alélicas do polimorfismo +2960INDEL e dos seguintes SNPs: +3003C>T, +3010C>G, +3027A>C,+3142 C>G e +3187A>G, bem como avaliar a influência dessas variantes na evolução para sepse, choque séptico e óbito entre pacientes críticos. Foram analisadas 698 amostras provenientes de pacientes críticos do Hospital São Lucas – PUCRS. O éxon 8 da região 3’ UTR foi amplificado por PCR e encaminhado para sequenciamento direto (ABI 3730 XL DNA Sequencer). A inferência haplotípica foi determinada pelo software PHASE versão 2.1. A frequência haplotípica, desequilíbrio de ligação (DL), Equilíbrio De Hardy-Weinberg (EHW) e teste de heterozigosidade foram estimados pelo software ARLEQUIN versão 3.5. Os dados foram submetidos ao pacote estatístico SPSS versão 18.0. Observaram-se as seguintes frequências alélicas e genotípicas, respectivamente: +2960INDEL, DEL= 0,578 e IN= 0,421, DELDEL= 0,310, DELIN= 0,536 e ININ= 0,152; +3003C>T, C= 0,109 e T= 0,890, CC= 0,012, CT= 0,194 e TT= 0,793; +3010C>G, CC= 0,521 e G= 0,478, CC= 0,250, CG= 0,540 e GG= 0,208; +3027A>C, A= 0,034 e C= 0,965, AA= 0,001, AC= 0,006 e CC= 0,932; +3035C>T, C= 0,861 e T= 0,141, CC= 0,733, CT= 0,255 e TT= 0,010; +3142C>G, C= 0,463 e G= 0,536, CC= 0,180, CG= 0,565 e GG= 0,253 e +3187A>G, A= 0,699 e G= 0,304, AA= 0,448, AG= 0,503 e GG= 0,048. Os polimorfismos +2960INDEL, +3010C>G, +3142C>G e +3187A>G não corresponderam ao EHW e apresentaram heterozigosidade observada maior do que a esperada. Os dois haplótipos mais frequentes foram DTGCCCG (27,90%) e ITCCCGA (26,65%), confirmando a heterogeneidade da população brasileira. A região 3’ UTR está sob seleção balanceadora, em que a seleção dos heterozigotos pode ser vantajosa, permitindo o balanço entre altas e baixas expressões de HLA-G, conforme o contexto biológico. Todos os sítios polimórficos apresentaram forte DL entre si. Entre os pacientes críticos que evoluíram para sepse, observou-se uma associação entre os portadores do alelo +2960IN e choque séptico (Chi-Square, p= 0,036). Entre os pacientes críticos, observou-se uma associação entre os portadores do haplótipo +2960IN_+3142G_+3187A e choque séptico (Chi-Square, p=0,038). E os pacientes críticos com sepse e portadores do haplótipo +2960IN_+3142G_+3187A (Chi-Square, p=0,023) também foram mais suscetíveis a evoluírem para choque séptico. Observou-se, pela primeira vez, uma associação entre o haplótipo +2960IN_+3142G_+3187A e os desfechos de pacientes críticos. Mais estudos são necessários para confirmação desses resultados e outros parâmetros poderiam ser considerados, como níveis de IL-10 e glicocorticóides endógenos, além do perfil de citocinas liberado por pacientes críticos. Devido às propriedades imunomodulatórias descritas para a molécula de HLA-G, futuramente os seus polimorfismos e sua expressão poderiam ser empregadas em abordagens clínicas nos mais variados contextos imunológicos. A amostra estudada está de acordo com as freqüências alélicas, genotípicas e haplotípicas observadas na população brasileira e novos polimorfismos poderiam ser investigados no éxon 8 da região 3’ UTR do HLA-G. / Critically ill patients are individuals hospitalized in Intensive Care Units (ICU) and are characterized by presenting pathologic complex conditions due to serious physiological weaknesses that may evolve to sepsis, septic shock or even death. Massive resources have been invested in sepsis control both in public and private sectors, although sepsis is still the major cause of death in Brazilians ICUs. Several factors influence the outcome of a critically ill patient, including genetic factors. HLA-G molecule is characterized by limited protein variability and restricted tissue expression. HLA-G binding to KIR and LILR receptors triggers various immunoregulatory activities. The HLA-G gene 3’ UTR presents a 14 bp insertion/deletion at +2960, a SNP at +3142C>G and a SNP at +3187A>G that may regulate HLA-G expression. So far, only one study has investigated HLA-G expression in critically ill patients with septic shock, observing that increased sHLA-G5 levels were predictive of survival among septic shock patients. The aim of this study was to assess the frequencies of 14 pb insertion/deletion polymorphism at +2960INDEL, and the followings SNPs: +3003C>T, +3010C>G, +3027A>C, +3035C>T, +3142C>G and +3187A>G in exon 8 at the 3’ UTR of the HLA-G gene and correlate these variants with different clinical outcomes in critically ill patients. 698 samples were obtained from ICU patients from São Lucas-PUCRS Hospital and the exon 8 at the 3’ UTR of the HLA-G gene was sequenced. The haplotype inference was determinated by PHASE version 2.1 software. Haplotype frequency, linkage disequilibrium (LD), Hardy-Weinberg Equilibrium (HWE) and heterozigosity test were estimated by ARLEQUIN version 3.5 software. The data were submitted to the package SPSS version 18.0 software. The followings allelic and genotypic frequencies were observed, respectively: +2960INDEL, DEL= 0.578 and IN= 0.421, DELDEL= 0.310, DELIN= 0.536 and ININ= 0.152; +3003C>T, C= 0.109 and T= 0.890, CC= 0.012, CT= 0.194 and TT= 0.793; +3010C>G, CC= 0.521 and G= 0.478, CC= 0.250, CG= 0.540 and GG= 0.208; +3027A>C, A= 0.034 and C= 0.965, AA= 0.001, AC= 0.006 and CC= 0.932; +3035C>T, C= 0.861 and T= 0.141, CC= 0.733, CT= 0.255 and TT= 0.010; +3142C>G, C= 0.463 and G= 0.536, CC= 0.180, CG= 0.565 and GG= 0.253 and +3187A>G, A= 0.699 and G= 0.304, AA= 0.448, AG= 0.503 and GG= 0.048. The +2960INDEL, +3010C>G, +3142C>G and +3187A>G polymorphisms were not on HWE and had higher observed heterozygosity than expected. The two most frequent haplotypes were DTGCCCG (27.90%) and ITCCCGA (26.65%). The Brazilian population is a heterogeneous population, and these data agree with this statement. The 3’ UTR is under balancing selection and heterozygote selection is advantageous, allowing the balance between high and low expression of HLA-G, according to the biological context. All polymorphic sites showed strong LD between them. Among critically ill patients who developed sepsis, there was an association between the allele +2960IN and septic shock (Chi-Square, p = 0.036). Among all critically ill patients, there was an association between carriers of the haplotype +2960IN_+3142 G_+3187A and septic shock (Chi-Square, p = 0.038). Critically ill patients with sepsis who carrier the haplotype +2960IN_+3142 G_+3187A were also more susceptible to progression to septic shock (Chi-Square, p = 0.023). For the first time, an association between +2960IN_+3142G_+3187A haplotype and outcomes of critically ill patients was observed. More studies are needed to confirm these results and other parameters could be considered, as IL-10 and endogenous glucocorticoids levels and cytokine profile released by critically ill patients. Because of the immunomodulatory properties described for the HLA-G molecule, in future its polymorphisms and its expression could be used in clinical approaches in various immune contexts. These results are according to allelic, genotypic and haplotypic frequencies observed in Brazilian population, and new polymorphisms could be investigated in exon 8 at the 3 'UTR of HLA-G gene.