Gene therapy techniques delivering exogenous angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2), are currently being investigated as potential treatments for ischemia resulting from a variety of conditions, such as peripheral artery disease (PAD) and chronic wounds. Despite these intense efforts, a viable clinical option to promote therapeutic neovascularization remains elusive. Electroporation is a simple in vivo method to deliver normally impermeable molecules, such as plasmid DNA, to a variety of tissues including skin and muscle. This study investigated intradermal injection of plasmids encoding angiogenic growth factors with electroporation as a novel therapeutic approach to increase perfusion in areas of ischemia. Two common animal models of ischemia were employed: a skin flap model, used to study wound healing, and a hindlimb ischemia model, used to investigate potential therapies for PAD. In the skin flap model, delivery of plasmid VEGF with electroporation significantly increased VEGF expression for 5 days after delivery compared to injection of the plasmid alone. While the increase in VEGF expression was short-term, it significantly increased expression of the downstream angiogenic growth factor endothelial nitric oxide synthase, as well as perfusion and healing in the distal area of the skin flap. To facilitate the translation of electroporation to the clinic, a novel electrode configuration was previously designed for cutaneous delivery of plasmids to a large surface area. The design of the Multielectrode Array allows for delivery to a large surface area without the need to increase the applied voltage. Conditions for plasmid delivery with this electrode were optimized and it was then utilized to deliver plasmid FGF-2 (pFGF) to the hindlimb ischemia model. FGF-2 expression, perfusion, and angiogenesis were assessed. FGF-2 expression was significantly higher for 10 days after treatment with pFGF with electroporation compared to injection of pFGF alone. This increase in FGF-2 expression induced a significant increase in perfusion and angiogenesis in the ischemic limb. The research presented here suggests intradermal injection of plasmids encoding angiogenic factors by electroporation is a novel potential therapeutic approach to increase perfusion to areas of ischemia and promote wound healing.

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<p>  </p> <p>Breast cancer is a disease that impacts nearly one out of three women at some point in their life. Although the scientific community’s understanding of breast cancer development is actively researched, there is still a low 5-year survival rate of 30% following distant metastasis compared to the near 100% survival rate for localized disease. Epithelial to mesenchymal transition (EMT) is a known contributor to metastasis. Cells that undergo EMT shed cell-to-cell junctions and become fibroblastic like cells with differential extracellular matrix organization and increased mesenchymal gene expression. This change allows for greater cell motility and invasive potential, critical for metastasis. Our recent studies with single cell RNA sequencing demonstrate distinct populations of epithelial and mesenchymal cells. Several components of fibroblast growth factor receptor (FGFR) signaling are regulated during EMT. Fibroblast growth factor binding protein 1 (FGFBP1) is a known developmental factor that was observed at low expression in mesenchymal cells, with an unknown role in breast cancer. This study utilizes immunoblotting, mRNA analyses, immunofluorescence staining and novel 3D culture platform to investigate the regulation of FGFBP1 during EMT. FGFBP1 was consistently downregulated in HER2 transformed human mammary epithelial cells (HME2) during transforming growth factor β (TGF-Beta) induced EMT. Since FGFBP1 is acts as a secretory chaperone protein, secretion rate analysis was conducted at time periods throughout EMT showing rapid downregulation of secretion. Characterization of FGFBP1 regulation during EMT could lead to greater understanding of EMT and possibly a more sensitive marker for EMT relative to the current known markers.</p>

A mixed self assembling monolayer (mSAM) chip with attached heparin was developed to analyze heparin-protein interactions using a Reichert Inc, SR7000, surface plasmon resonance (SPR) instrument. The heparin was attached via streptavidin-biotin linkage where the streptavidin was covalently coupled to the mSAM and biotinylated heparin bound to it. These chips were then used to quantify the interactions of fibroblast growth factor-2 (FGF-2) with the surface bound heparin. Kinetic rate constants of association and disassociation were calculated. The association data of FGF-2 with heparin was fit to a single compartment, well-mixed model as the data did not exhibit mass transfer limitations. The results suggested that rebinding was prevalent and observed disassociation rates differed significantly in the presence of competing soluble heparin during disassociation. Our results indicate that the Reichert instrument and mSAM chips can be used to analyze heparin-protein interactions but that a careful protocol, outlined in this thesis, should be followed to obtain optimal data. / Master of Science

NOVEL METABOLIC REGULATORS AND PROINFLAMMATORY FACTORS IN THE ETIOPATHOGENESIS OF TYPE 2 DIABETES MELLITUS AND OBESITY: THE INFLUENCE OF PHARMACOLOGICAL AND DIETARY INTERVENTIONS MUDr. Miloš Mráz Doctoral Thesis ABSTRACT (EN) Identifying novel factors involved in the etiopathogenesis of obesity, diabetes mellitus and their complications has become one of the primary scopes of metabolic research in the last years. The aim of the present study was to evaluate the role of recently discovered metabolic and inflammatory regulators including fibroblast growth factors 19 and 21 and chemotactic cytokines in the development of obesity and type 2 diabetes mellitus (DM2). A total number of 182 patients were included into the study. They were divided into 3 groups - patients with obesity but without type 2 diabetes mellitus, individuals with obesity and type 2 diabetes mellitus and healthy control normal-weight subjects. Selected interventions included 2 to 3 weeks of very-low-calorie diet (VLCD - energy content 2500 kJ/day), 3 months of administration of PPARα receptor agonist fenofibrate and acute hyperinsulinemia during hyperinsulinemic isoglycemic clamp. Our results indicate that the increase of circulating FGF-21 levels after VLCD and fenofibrate treatment could contribute to positive metabolic effects of these...

NOVEL METABOLIC REGULATORS AND PROINFLAMMATORY FACTORS IN THE ETIOPATHOGENESIS OF TYPE 2 DIABETES MELLITUS AND OBESITY: THE INFLUENCE OF PHARMACOLOGICAL AND DIETARY INTERVENTIONS MUDr. Miloš Mráz Doctoral Thesis ABSTRACT (EN) Identifying novel factors involved in the etiopathogenesis of obesity, diabetes mellitus and their complications has become one of the primary scopes of metabolic research in the last years. The aim of the present study was to evaluate the role of recently discovered metabolic and inflammatory regulators including fibroblast growth factors 19 and 21 and chemotactic cytokines in the development of obesity and type 2 diabetes mellitus (DM2). A total number of 182 patients were included into the study. They were divided into 3 groups - patients with obesity but without type 2 diabetes mellitus, individuals with obesity and type 2 diabetes mellitus and healthy control normal-weight subjects. Selected interventions included 2 to 3 weeks of very-low-calorie diet (VLCD - energy content 2500 kJ/day), 3 months of administration of PPARα receptor agonist fenofibrate and acute hyperinsulinemia during hyperinsulinemic isoglycemic clamp. Our results indicate that the increase of circulating FGF-21 levels after VLCD and fenofibrate treatment could contribute to positive metabolic effects of these...

O carcinoma hepatocelular (HCC) é a sexta neoplasia mais comum e a terceira causa de mortalidade por câncer no mundo. Está relacionado, principalmente, à exposição a diversos fatores de risco, como a doença hepática gordurosa não alcoólica (NAFLD). O HCC apresenta mau prognóstico; neste sentido, é importante a adoção de medidas de controle, como a quimioprevenção. A quimioprevenção é o método mais apropriado para se evitar o câncer e consiste na prevenção, inibição ou reversão das etapas iniciais da carcinogênese, pela administração de um ou mais compostos químicos sintéticos ou naturais. Diversos compostos presentes nos alimentos podem apresentar atividade quimiopreventiva, dentre esses, a tributirina (TB), pró-fármaco do ácido butírico. Dessa forma, propôs-se avaliar o efeito quimiopreventivo da TB nas etapas de iniciação/promoção inicial, em ratos submetidos ao modelo de hepatocarcinogênese do hepatócito resistente (RH) associado à NAFLD. Ratos Wistar machos foram distribuídos em Grupo dos animais não tratados (NT), e dois outros grupos experimentais, o grupo RH + NAFLD + maltodextrina (grupo controle isocalórico, CI), e o grupo RH + NAFLD + tributirina (grupo TB). Os animais do grupo CI foram tratados diariamente, por gavagem, com emulsão hiperlipídica [1 mL/100 g de peso corpóreo (p. c)], e maltodextrina (300 mg/ 100 g p.c.), enquanto que os animais do Grupo TB foram tratados diariamente, por gavagem, com emulsão hiperlipídica [1 mL/100 g de p. c.], e TB (200 mg/ 100 g de p.c.), durante 13 semanas consecutivas quando foram eutanasiados. Após esse período, a análise por cromatografia a gás revelou que o grupo dos animais tratados com a tributirina apresentou uma concentração de AB 2.118 vezes superior (p < 0.05) às concentrações de AB constatadas no grupo controle. Em relação aos dados de morfometria de LPNs [lesões pré-neoplásicas persistentes (pLPN) ou em remodelação (rLPN)], a atividade quimiopreventiva da tributirina foi observada a partir da redução (p < 0,05) significativa do número de lesões pré-neoplásicas persistentes (pLPN) em comparação ao grupo controle. A área das pLPNs, contudo, foi maior (p < 0,05) no Grupo TB quando comparada à do grupo controle isocalórico. Não foram observadas diferenças estatisticamente significativas (p >= 0,05) no número e na área de rLPN, bem como na % da área do corte do fígado ocupada por LPNs entre os dois grupos experimentais. A quantificação da apoptose por H&E revelou maior (p < 0,05) índice apoptótico em pLPN e em rLPN nos animais tratados com TB, quando comparados aos animais do grupo 6 controle. Nesse sentido, observou-se que os animais tratados com a tributirina apresentaram maior (p < 0,05) ativação de Caspase 3 do que os ratos do grupo controle isocalórico. Em relação à avaliação da proliferação celular, o Grupo TB apresentou redução (p < 0,05) do índice de proliferação tanto em pLPNs como em rLPNs, quando comparado ao do grupo controle isocalórico. Não foi observada diferença estatisticamente significativa (p > 0,05) entre os surroundings dos dois grupos experimentais. A determinação do perfil lipídico sérico revelou que os animais tratados com a tributirina apresentaram menores (p < 0,05) concentrações séricas de LDL-colesterol e maiores (p < 0,05) concentrações séricas de HDL-colesterol, quando comparados às do grupo controle. Além disso, foram observadas menores (p < 0,05) concentrações hepáticas de colesterol no Grupo TB quando comparadas às do Grupo CI. Em relação a análise por PAS, embora, o presente estudo não tenha revelado diferença estatisticamente significativa (p >= 0,05) entre os dois grupos experimentais, o score entre os graus de marcação por PAS apresentou tendência (p = 0,07) a ser maior nos animais tratados com a tributirina quando comparados aos ratos do grupo controle. Em relação à expressão gênica de FGF-21, o Grupo TB apresentou maior (p < 0,05) expressão em comparação a seu grupo controle (Grupo CI). Além disso, esses dados são corroborados por meio da marcação imunoistoquímica de FGF-21, que revelou que os animais tratados com a tributirina apresentaram maior (p < 0,05) score desta marcação quando comparados aos ratos do grupo controle isocalórico. A análise de expressão em nível proteico de PPAR-&#945; revelou que o Grupo TB apresentou maior (p < 0,05) expressão em comparação ao Grupo CI. O presente estudo demonstrou que o tratamento com a tributirina apresentou um efeito quimiopreventivo quando administrada nas etapas de iniciação/promoção inicial em modelo de hepatocarcinogênese associado à NAFLD. Adicionalmente, sugere-se que a atividade como HDACi da TB poderia modular a expressão de genes supressores de tumor, bem como a daqueles relacionados ao metabolismo lipídico, atenuando os fatores de risco relacionados à NAFLD. / Hepatocellular carcinoma (HCC) is the sixth most common neoplasm and the third leading cause of cancer mortality in the world. It is mainly related to exposure to several risk factors, such as non-alcoholic fatty liver disease (NAFLD). HCC presents poor prognosis; In this sense, it is important to adopt control measures, such as chemoprevention. Chemoprevention is the most appropriate method to avoid cancer and consists in the prevention, inhibition or reversal of the early stages of carcinogenesis by the administration of one or more synthetic or natural chemical compounds. Several compounds present in foods may present chemopreventive activity, among them, tributyrin (TB), a prodrug of butyric acid. Thus, it was proposed to evaluate the chemopreventive effect of TB in the initiation / initial promotion stages in rats submitted to the hepatocyte-resistant hepatocyte model (HR) associated with NAFLD. Male Wistar rats were divided into two groups: RH + NAFLD + maltodextrin (isocaloric control group, CI), and RH + NAFLD + tributyrin group (TB group). The animals of the CI group were treated daily with gavage with hyperlipid emulsion [1 mL / 100 g body weight (wt.)], and maltodextrin (300 mg / 100 g body wt.), while the animals of the TB Group were treated daily by gavage with hyperlipid emulsion [1 mL / 100 g of body wt.], and TB (200 mg / 100 g body wt.) for 13 consecutive weeks when they were euthanized. After this period, the gas chromatographic analysis revealed that the group of animals treated with tributyrin had a concentration of AB 2184 times higher (p < 0.05) than the concentrations of AB found in the control group. Regarding the morphometry data of PNLs [persistent pre-neoplastic lesions (pPNL) or remodeling (rPNL)], the chemopreventive activity of tributyrin was observed from a significant (p < 0.05) reduction in the number of pPNL compared to the control group. The area of the pPNLs, however, was higher (p < 0.05) in the TB Group when compared to the isocaloric control group. There were no statistically significant differences (p >= 0.05) in the number and area of rPNL, as well as in the area of the cut of the liver occupied by LPNs between the two experimental groups. The quantification of apoptosis by H&E revealed a higher (p < 0.05) apoptotic index in pPNL and rPNL in the TB treated animals, when compared to the animals in the control group. In this sense, animals treated with tributyrin showed greater (p < 0.05) activation of Caspase 3 than the rats of the isocaloric control group. In relation to the evaluation of cell proliferation, the TB group presented a reduction (p < 0.05) in the proliferation index in both pPNLs and rPNLs, when compared to the isocaloric control group. No statistically significant difference (p> 0.05) was observed between the two experimental groups. The determination of serum lipid profile revealed that the animals treated with tributyrin showed lower (p < 0.05) serum LDL-cholesterol concentrations and higher (p < 0.05) serum HDL-cholesterol concentrations when compared to those in the group control. In addition, lower hepatic concentrations (p < 0.05) of cholesterol were observed in the TB Group when compared to those in the CI Group. Regarding the SBP analysis, although the present study did not reveal a statistically significant difference (p >= 0.05) between the two experimental groups, the score between the PAS presented a tendency (p = 0.07) to be greater in animals 8 treated with tributyrin compared to the rats in the control group. In relation to the gene expression of FGF-21, the TB Group presented higher (p <0.05) expression in comparison to its control group (CI Group). In addition, these data are corroborated by FGF-21 immunohistochemical labeling, which revealed that animals treated with tributyrin showed a higher (p <0.05) score for this marker when compared to the isocaloric control rats. Analysis of protein-level expression of PPAR-&#945; revealed that the TB Group had higher (p <0.05) expression compared to the CI Group. The present study demonstrated that treatment with tributyrin had a chemopreventive effect when administered in the initiation / initial promotion steps in a hepatocarcinogenesis model associated with NAFLD. In addition, it is suggested that the activity as HDACi of TB could modulate the expression of tumor suppressor genes, as well as those related to lipid metabolism, attenuating the risk factors related to NAFLD.