Individer med homozygot familjär hyperkolesterolemi(FH), har höga halter av Low Density Lipoprotein (LDL) vilket leder till ökad risk för kardiovaskulära sjukdomar. Behandling av dessa individer kan göras med extrakorporal elimination av LDL med hjälp av specifika reningskolonner. Syftet med studien var att utvärdera några agarosmodifierade adsorbenter för denna applikation. Adsorbenterna, modifierad polyakrylat (DALI), agaros (Zetaros), direkt sulfateradZetarose och taurin immobiliserad Zetarose, inkuberades med humant plasma spädd med PBS, och en volyms förhållande mellan matris och plasman på 1:5. Inkubering utfördes i rumstemperatur under 60 min med kontinuerlig blandning i rotator. Efter inkubation centrifugerades proverna och LDL bestämdes i såväl supernatant som pellet. Totalmängd adsorberade proteiner analyserades också i eluat från erhållen pellet. LDL bestämdes indirekt med hjälp av Friedewaldsformel (LDL = totalkolesterol (TC) –highdensitylipoprotein (HDL) - (0,45 x Triglycerider(TG)). TC och TG bestämdes enzymatiskt medan HDL kvantifierades som TC efter utfällning av LDL med dextransulfat. Resultaten visar tydligt att DALI har god adsorptionsförmåga.Dock uppvisar de modifierade Zetaroserna begränsad adsorptionskapacitet för LDL. Vid desorption av adsorbenterna visar SDS en bättre elueringsförmåga än NaCl relaterad till protein, vilket tyder hydrofoba proteiner. Metodiken som används i studien är lämplig för vidare studier av andra adsorbenter som förväntas användas i kliniska applikationer för elimination av LDL hos FH patienter. / Individuals that suffer from homozygote Familiar Hyperkolesterolemia (FH), has increased amounts of Low Density Lipoproteins (LDL) which leads to a higher risk of cardiovascular diseases. Treatment of these individuals can be achieved by extracorporeal elimination of LDL using specific columns. The aim of this study was to evaluate different agarose-modified adsorbents ability to adsorb LDL from human plasma. The adsorbents (DALI, Zetarose, sulphonated Zetarose and taurine immobilized onto Zetarose) were incubated for 60 minutes with human plasma diluted with PBS, in a ratio of 1:5 between the matrix and the plasma during rotation with a rotator. After incubation the samples were centrifuged and the LDL content was determined in both the supernatant and the pellet. The amount proteins adsorbed were assayed by eluting the pellets. LDL was determined indirectly using Friedwalds equation; LDL= Total cholesterol (TC) - High density lipoprotein (HDL)-(0,45x Triglycerides (TG). The values of TC and TG in the sample were determined enzymatically, whilst HDL was quantified as TC after LDL-precipitation by dextran sulfate. The results clearly show that DALI has good adsorption capacity, but none of the modified Zetaroses shows any capacity to absorb LDL from human plasma. Desorption of the adsorbents using SDS gave higher amounts of eluated protein compared to NaCl elution, indicating hydrofobic proteins. However, the methods used in this study could be used to evaluate new adsorbents for LDL-elimination applications in patients with chronic hyperlipemia.

Os tumores malignos apresentam um aumento da expressão dos receptores de lipoproteínas, devido ao aceleramento da proliferação celular com consequente aumento da necessidade de lípides para a síntese das membranas celulares. Esse aumento da expressão dos receptores de LDL no câncer pode ser utilizado para concentrar fármacos de ação antineoplásica em tecido tumoral, utilizando lipoproteínas ou nanoemulsões semelhantes a lipoproteínas como veículo. No presente estudo, foram investigados os efeitos da quimioterapia convencional na expressão dos receptores de LDL e LRP-1 em 16 pacientes com carcinoma de mama estádios II ou III, não candidatas à cirurgia conservadora e com indicação de tratamento quimioterápico neoadjuvante. A expressão dos receptores LDLR e LRP-1 foi avaliada por imunoistoquimica em tecido mamário normal e em tecido neoplásico antes e depois da quimioterapia neoadjuvante. Quatro pacientes que apresentaram resposta completa à quimioterapia foram retiradas da análise da expressão de receptores por não existir tumor no fragmento cirúrgico. Em relação ao LDLR, a expressão desse receptor no tecido neoplásico foi maior em comparação ao tecido normal em 8 das 11 pacientes. Após a quimioterapia, a expressão do receptor de LDL diminuiu em 6, aumentou em 4 e não se alterou em 2 pacientes. Do mesmo modo, a expressão do receptor LRP-1 no tecido tumoral estava aumentada em relação ao tecido normal em 4 pacientes das 12 avaliadas. Em comparação com o tecido tumoral antes da quimioterapia, a expressão do receptor LRP-1 diminuiu em 6, aumentou em 4 e permaneceu inalterada em 2 pacientes após a quimioterapia. Esses dados mostram que o efeito da quimioterapia na expressão dos receptores de lipoproteínas foi heterogêneo. A redução da expressão dos receptores não foi o padrão observado, o que indica que o uso de sistemas de carreamento de fármacos via receptores de LDL para o tratamento do câncer pode ser de grande importância. Esses resultados podem contribuir para o desenho de futuros estudos clínicos / Proliferative tumor cells present a high expression of LDL receptors due to accelerated mitosis rates which takes to increased need of lipids internalization for building new membranes. Upregulation of LDL receptors may be used as a gate to deliver anticancer drugs to tumor tissues using lipoproteins or artificial nanoemulsions as vehicle. This study investigated the effects of conventional chemotherapy on the expression of LDL and LRP-1 receptors in 16 patients with breast cancer in stage II or III who were not candidates to conservative surgery and with indication of neo-adjuvant chemotherapy. Expression of LDL and LRP-1 receptor was evaluated by immunohistochemistry in normal and neoplastic breast tissue before and after chemotherapy. For absence of tumor in the surgical fragments, 4 patients who presented complete response to chemotherapy were excluded from this analysis. In relation of LDLR, the expression in neoplastic tissue was higher than in normal tissue in 8 of 11 patients. After chemotherapy, LDL receptor expression diminished in 6, increased in 4 and unchanged in 2 patients. Expression of LRP-1 in tumor tissue was higher in 4 of 12 patients when compared to normal tissue. After chemotherapy, the expression of LRP-1 diminished in 6, increased in 4 and showed no difference in 2 patients. These data show that the chemotherapy effects on the tumor expression of LDL receptors were very heterogeneous. The diminution of the receptor expression is not the post-chemotherapy pattern, allowing the use of drug carrier systems that target cancer cells via the LDL receptor pathway. These results may contribute for the design of future clinical assays

Os tumores malignos apresentam um aumento da expressão dos receptores de lipoproteínas, devido ao aceleramento da proliferação celular com consequente aumento da necessidade de lípides para a síntese das membranas celulares. Esse aumento da expressão dos receptores de LDL no câncer pode ser utilizado para concentrar fármacos de ação antineoplásica em tecido tumoral, utilizando lipoproteínas ou nanoemulsões semelhantes a lipoproteínas como veículo. No presente estudo, foram investigados os efeitos da quimioterapia convencional na expressão dos receptores de LDL e LRP-1 em 16 pacientes com carcinoma de mama estádios II ou III, não candidatas à cirurgia conservadora e com indicação de tratamento quimioterápico neoadjuvante. A expressão dos receptores LDLR e LRP-1 foi avaliada por imunoistoquimica em tecido mamário normal e em tecido neoplásico antes e depois da quimioterapia neoadjuvante. Quatro pacientes que apresentaram resposta completa à quimioterapia foram retiradas da análise da expressão de receptores por não existir tumor no fragmento cirúrgico. Em relação ao LDLR, a expressão desse receptor no tecido neoplásico foi maior em comparação ao tecido normal em 8 das 11 pacientes. Após a quimioterapia, a expressão do receptor de LDL diminuiu em 6, aumentou em 4 e não se alterou em 2 pacientes. Do mesmo modo, a expressão do receptor LRP-1 no tecido tumoral estava aumentada em relação ao tecido normal em 4 pacientes das 12 avaliadas. Em comparação com o tecido tumoral antes da quimioterapia, a expressão do receptor LRP-1 diminuiu em 6, aumentou em 4 e permaneceu inalterada em 2 pacientes após a quimioterapia. Esses dados mostram que o efeito da quimioterapia na expressão dos receptores de lipoproteínas foi heterogêneo. A redução da expressão dos receptores não foi o padrão observado, o que indica que o uso de sistemas de carreamento de fármacos via receptores de LDL para o tratamento do câncer pode ser de grande importância. Esses resultados podem contribuir para o desenho de futuros estudos clínicos / Proliferative tumor cells present a high expression of LDL receptors due to accelerated mitosis rates which takes to increased need of lipids internalization for building new membranes. Upregulation of LDL receptors may be used as a gate to deliver anticancer drugs to tumor tissues using lipoproteins or artificial nanoemulsions as vehicle. This study investigated the effects of conventional chemotherapy on the expression of LDL and LRP-1 receptors in 16 patients with breast cancer in stage II or III who were not candidates to conservative surgery and with indication of neo-adjuvant chemotherapy. Expression of LDL and LRP-1 receptor was evaluated by immunohistochemistry in normal and neoplastic breast tissue before and after chemotherapy. For absence of tumor in the surgical fragments, 4 patients who presented complete response to chemotherapy were excluded from this analysis. In relation of LDLR, the expression in neoplastic tissue was higher than in normal tissue in 8 of 11 patients. After chemotherapy, LDL receptor expression diminished in 6, increased in 4 and unchanged in 2 patients. Expression of LRP-1 in tumor tissue was higher in 4 of 12 patients when compared to normal tissue. After chemotherapy, the expression of LRP-1 diminished in 6, increased in 4 and showed no difference in 2 patients. These data show that the chemotherapy effects on the tumor expression of LDL receptors were very heterogeneous. The diminution of the receptor expression is not the post-chemotherapy pattern, allowing the use of drug carrier systems that target cancer cells via the LDL receptor pathway. These results may contribute for the design of future clinical assays

The field of proteomics studies the structure and function of proteins in a large scale and high throughput manner. My work in the field of proteomics focuses on identifying interactions between proteins and discovering novel interactions. The identification of these interactions provides new information on metabolic and disease pathways and the working proteome of a cell. Cells are lysed and purified using antibody based affinity purification followed by digestion and identification using an HPLC coupled to a mass spectrometer. In my studies, I looked at the interaction networks of several AD related genes (Apolipoprotein E, Clusterin variant 1 and 2, Low-density lipoprotein receptor, Phosphatidylinositol binding clathrin assembly protein, Alpha-synuclein and Platelet-activating factor receptor) and an endosomal recycling pathway involved in cholesterol metabolism (Eps15 homology domain 1,2 and 4, Proprotein convertase subtilisin/kexin type 9 and Low-density lipoprotein receptor). Several novel and existing interactors were identified and these interactions were validated using co-immunopurification, which could be the basis for future research.

Heat shock protein (HSP)27 is traditionally described as an intracellular chaperone and signaling molecule, but growing evidence suggests it is released from immune cells where it plays an anti-inflammatory role during atherogenesis. Previously, the O’Brien lab found that overexpression of HSP27 led to augmented HSP27 serum levels in female apolipoprotein E knockout (ApoE-/-) mice, attenuated atherogenesis, and inhibited macrophage foam cell formation via physical binding with scavenger receptor (SR)-A. However, the precise mechanism of atheroprotection remained elusive. This thesis sought to ascertain the mechanism(s) by which HSP27 prevents foam cell formation, and determine if SR-A, a key receptor involved in the uptake of lipid into macrophages, plays an important role in HSP27-mediated atheroprotection. Pre-treatment of human macrophages with recombinant HSP27 (rHSP27) inhibited acytelated low density lipoprotein (acLDL) binding and uptake independent from receptor competition effect. Reduction in uptake was associated with attenuation of expression of SR-A mRNA, total protein, and cell surface expression. To explore the signaling mechanism by which HSP27 modulated SR-A expression it was hypothesized that nuclear factor-kappa B (NF-kB), a major regulator of many atherosclerosis gene programs, is altered by extracellular HSP27. Indeed, rHSP27 markedly activated NF-kB signaling in macrophages. Using an inhibitor of NF-kBsignaling there was an attenuation of rHSP27-induced inhibition of SR-A gene and protein expression, as well as lipid uptake, suggesting that SR-A expression is regulated by NF-kB activation. Lastly, to investigate if SR-A is required for HSP27-mediated atheroprotection in vivo, ApoE-/- and ApoE-/-SR-A-/- mice fed a high fat diet were treated with rHSP25, the mouse orthologue of HSP27, or PBS for 3 weeks. While rHSP25 therapy equally reduced serum cholesterol levels in the mouse cohorts, aortic atherogenesis, assessed using en face and sinus cross-sectional analyses, was attenuated in ApoE-/- mice but not ApoE-/-SR-A-/- mice. In conclusion, rHSP27 inhibits foam cell formation by downregulating SR-A expression. This effect may be associated with NF-kB activation. Reductions in atherosclerotic burden by rHSP27 require SR-A, and are independent of changes in serum cholesterol levels, highlighting the importance of macrophage lipid uptake in atherogenesis. Results presented in this thesis demonstrate that SR-A is a major target for HSP27 atheroprotection in the vessel wall, and provide an impetus for further studies that investigate the potential therapeutic value of HSP27.

The field of proteomics studies the structure and function of proteins in a large scale and high throughput manner. My work in the field of proteomics focuses on identifying interactions between proteins and discovering novel interactions. The identification of these interactions provides new information on metabolic and disease pathways and the working proteome of a cell. Cells are lysed and purified using antibody based affinity purification followed by digestion and identification using an HPLC coupled to a mass spectrometer. In my studies, I looked at the interaction networks of several AD related genes (Apolipoprotein E, Clusterin variant 1 and 2, Low-density lipoprotein receptor, Phosphatidylinositol binding clathrin assembly protein, Alpha-synuclein and Platelet-activating factor receptor) and an endosomal recycling pathway involved in cholesterol metabolism (Eps15 homology domain 1,2 and 4, Proprotein convertase subtilisin/kexin type 9 and Low-density lipoprotein receptor). Several novel and existing interactors were identified and these interactions were validated using co-immunopurification, which could be the basis for future research.

Heat shock protein (HSP)27 is traditionally described as an intracellular chaperone and signaling molecule, but growing evidence suggests it is released from immune cells where it plays an anti-inflammatory role during atherogenesis. Previously, the O’Brien lab found that overexpression of HSP27 led to augmented HSP27 serum levels in female apolipoprotein E knockout (ApoE-/-) mice, attenuated atherogenesis, and inhibited macrophage foam cell formation via physical binding with scavenger receptor (SR)-A. However, the precise mechanism of atheroprotection remained elusive. This thesis sought to ascertain the mechanism(s) by which HSP27 prevents foam cell formation, and determine if SR-A, a key receptor involved in the uptake of lipid into macrophages, plays an important role in HSP27-mediated atheroprotection. Pre-treatment of human macrophages with recombinant HSP27 (rHSP27) inhibited acytelated low density lipoprotein (acLDL) binding and uptake independent from receptor competition effect. Reduction in uptake was associated with attenuation of expression of SR-A mRNA, total protein, and cell surface expression. To explore the signaling mechanism by which HSP27 modulated SR-A expression it was hypothesized that nuclear factor-kappa B (NF-kB), a major regulator of many atherosclerosis gene programs, is altered by extracellular HSP27. Indeed, rHSP27 markedly activated NF-kB signaling in macrophages. Using an inhibitor of NF-kBsignaling there was an attenuation of rHSP27-induced inhibition of SR-A gene and protein expression, as well as lipid uptake, suggesting that SR-A expression is regulated by NF-kB activation. Lastly, to investigate if SR-A is required for HSP27-mediated atheroprotection in vivo, ApoE-/- and ApoE-/-SR-A-/- mice fed a high fat diet were treated with rHSP25, the mouse orthologue of HSP27, or PBS for 3 weeks. While rHSP25 therapy equally reduced serum cholesterol levels in the mouse cohorts, aortic atherogenesis, assessed using en face and sinus cross-sectional analyses, was attenuated in ApoE-/- mice but not ApoE-/-SR-A-/- mice. In conclusion, rHSP27 inhibits foam cell formation by downregulating SR-A expression. This effect may be associated with NF-kB activation. Reductions in atherosclerotic burden by rHSP27 require SR-A, and are independent of changes in serum cholesterol levels, highlighting the importance of macrophage lipid uptake in atherogenesis. Results presented in this thesis demonstrate that SR-A is a major target for HSP27 atheroprotection in the vessel wall, and provide an impetus for further studies that investigate the potential therapeutic value of HSP27.

Orientador: Wilson Nadruz Junior / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T13:58:57Z (GMT). No. of bitstreams: 1 Paim_LaydeRosane_M.pdf: 776318 bytes, checksum: f8e1046411d4b45041c7d37ca207a89e (MD5) Previous issue date: 2014 / Resumo: Estudos prévios mostram que indivíduos com lesão crônica na medula espinhal (LM) apresentam maior risco cardiovascular em comparação com indivíduos fisicamente normais. O presente estudo investigou a relação entre os níveis plasmáticos da lipoproteína de baixa densidade oxidada (LDLox), as metaloproteinases de matriz (MMP) e seus inibidores teciduais (TIMPs) e o remodelamento vascular em pacientes com LM, e o papel da atividade física nesta relação. Foram estudados 42 homens com LM (? 2 anos), [18 sedentários (S-LM) e 24 fisicamente ativos (A-LM)] e 16 homens fisicamente saudáveis por meio de análise clínica, antropométrica, laboratorial e de espessura íntima-média da carótida (EIM). Todos os participantes estudados eram normotensos, não diabéticos, não fumantes e normolipêmicos. As concentrações plasmáticas de LDLox, MMP-2, MMP-8, MMP-9, TIMP-1 e TIMP-2 foram determinados por ensaio imunoenzimático (ELISA). Os resultados mostraram que a EIM da carótida, razão EIM/diâmetro e as concentrações de LDLox dos A-LM e dos indivíduos fisicamente normais não foram diferentes estatisticamente. Por outro lado, indivíduos com S-LM apresentaram maior EIM, razão EIM/diâmetro e concentrações aumentados de LDLox em comparação com A-LM (p<0,01, p<0,001 e p=0,01, respectivamente) e indivíduos controles (p<0,001 para todos). Os resultados da análise de correlação bivariada, incluindo todos os indivíduos com LM, demonstrou que o EIM de carótida e a razão EIM/diâmetro se correlacionaram apenas com LDLox, MMP-8 e com a relação MMP-8/TIMP-1. Além disso, a análise de regressão ajustada para a presença ou não de atividade física e idade mostrou que a LDLox foi associada à EIM carotídea e com a relação EIM/diâmetro, enquanto que MMP-8 foi associado com o índice EIM/diâmetro em indivíduos com LM. Em conclusão, as concentrações plasmáticas de LDLox e MMP-8 estão associados com aterosclerose carotídea e há interação entre a inatividade física, aterosclerose e LDLox em indivíduos com LM / Abstract: Previous reports have indicated that subjects with chronic spinal cord injury (SCI) exhibit increased cardiovascular risk compared to able-bodied individuals. This study investigated the relationship between plasmatic oxidized low-density lipoprotein (OxLDL), matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) levels and vascular remodeling in SCI subjects and the role of physical activity in this regard. We studied 42 men with chronic (?2 years) SCI [18 sedentary (S-SCI) and 24 physically active (PA-SCI)] and 16 able-bodied men by clinical, anthropometric, laboratory, and carotid intima-media thickness (IMT) analysis. All enrolled subjects were normotensive, non-diabetics, non-smokers and normolipemic. Plasmatic OxLDL, MMP-2, MMP-8, MMP-9, TIMP-1 and TIMP-2 levels were determined by enzyme-linked immunosorbent assay. The results showed that carotid IMT, IMT/diameter ratio and OxLDL levels of PA-SCI and able-bodied subjects were statistically similar. Conversely, S-SCI subjects exhibited higher IMT, IMT/diameter ratio and OxLDL levels compared to PA-SCI (p<0.01, p<0.001 and p=0.01, respectively) and able-bodied (p<0.001 for all) individuals. Results of bivariate correlation analysis including all injured subjects showed that carotid IMT and IMT/diameter ratio only correlated with OxLDL, MMP-8 and MMP-8/TIMP-1 ratio. Further stepwise regression analysis adjusted for the presence or not of physical activity and age showed that OxLDL was associated with carotid IMT and IMT/diameter ratio, while MMP-8 was associated with IMT/diameter ratio in SCI individuals. In conclusion, plasmatic OxLDL and MMP-8 levels are associated with carotid atherosclerosis and there is an interaction among physical inactivity, atherosclerosis and OxLDL in SCI individuals / Mestrado / Clinica Medica / Mestra em Clínica Médica

Heat shock protein (HSP)27 is traditionally described as an intracellular chaperone and signaling molecule, but growing evidence suggests it is released from immune cells where it plays an anti-inflammatory role during atherogenesis. Previously, the O’Brien lab found that overexpression of HSP27 led to augmented HSP27 serum levels in female apolipoprotein E knockout (ApoE-/-) mice, attenuated atherogenesis, and inhibited macrophage foam cell formation via physical binding with scavenger receptor (SR)-A. However, the precise mechanism of atheroprotection remained elusive. This thesis sought to ascertain the mechanism(s) by which HSP27 prevents foam cell formation, and determine if SR-A, a key receptor involved in the uptake of lipid into macrophages, plays an important role in HSP27-mediated atheroprotection. Pre-treatment of human macrophages with recombinant HSP27 (rHSP27) inhibited acytelated low density lipoprotein (acLDL) binding and uptake independent from receptor competition effect. Reduction in uptake was associated with attenuation of expression of SR-A mRNA, total protein, and cell surface expression. To explore the signaling mechanism by which HSP27 modulated SR-A expression it was hypothesized that nuclear factor-kappa B (NF-kB), a major regulator of many atherosclerosis gene programs, is altered by extracellular HSP27. Indeed, rHSP27 markedly activated NF-kB signaling in macrophages. Using an inhibitor of NF-kBsignaling there was an attenuation of rHSP27-induced inhibition of SR-A gene and protein expression, as well as lipid uptake, suggesting that SR-A expression is regulated by NF-kB activation. Lastly, to investigate if SR-A is required for HSP27-mediated atheroprotection in vivo, ApoE-/- and ApoE-/-SR-A-/- mice fed a high fat diet were treated with rHSP25, the mouse orthologue of HSP27, or PBS for 3 weeks. While rHSP25 therapy equally reduced serum cholesterol levels in the mouse cohorts, aortic atherogenesis, assessed using en face and sinus cross-sectional analyses, was attenuated in ApoE-/- mice but not ApoE-/-SR-A-/- mice. In conclusion, rHSP27 inhibits foam cell formation by downregulating SR-A expression. This effect may be associated with NF-kB activation. Reductions in atherosclerotic burden by rHSP27 require SR-A, and are independent of changes in serum cholesterol levels, highlighting the importance of macrophage lipid uptake in atherogenesis. Results presented in this thesis demonstrate that SR-A is a major target for HSP27 atheroprotection in the vessel wall, and provide an impetus for further studies that investigate the potential therapeutic value of HSP27.

The field of proteomics studies the structure and function of proteins in a large scale and high throughput manner. My work in the field of proteomics focuses on identifying interactions between proteins and discovering novel interactions. The identification of these interactions provides new information on metabolic and disease pathways and the working proteome of a cell. Cells are lysed and purified using antibody based affinity purification followed by digestion and identification using an HPLC coupled to a mass spectrometer. In my studies, I looked at the interaction networks of several AD related genes (Apolipoprotein E, Clusterin variant 1 and 2, Low-density lipoprotein receptor, Phosphatidylinositol binding clathrin assembly protein, Alpha-synuclein and Platelet-activating factor receptor) and an endosomal recycling pathway involved in cholesterol metabolism (Eps15 homology domain 1,2 and 4, Proprotein convertase subtilisin/kexin type 9 and Low-density lipoprotein receptor). Several novel and existing interactors were identified and these interactions were validated using co-immunopurification, which could be the basis for future research.