Skeletal development is a complex event that requires a delicate balance between bone formation and bone resorption. Multiple transcription factors expressed in the bone-forming cells, osteoblasts, play crucial roles during the process of bone formation. Among them, ATF4 (Activating Transcription Factor 4) is a basic domain-leucine zipper transcriptional activator that is responsible for osteoblast differentiation, osteoblast-specific genes expression, synthesis of type I collagen, and osteoclast differentiation. Mice deficient for ATF4 are runted and exhibit severe skeletal dysplasia. Our laboratory has discovered Factor Inhibiting ATF4-mediated Transcription (FIAT), whose name was coined for its interaction with ATF4 and subsequent repression of ATF4-mediated osteocalcin gene transcription. FIAT is a leucine zipper nuclear molecule lacking a basic domain for DNA binding. We hypothesize that FIAT suppresses the bone-forming activities of osteoblasts by interacting with ATF4 and thereby blocking ATF4 attachment to the DNA to mediate downstream signalling pathways. To prove this hypothesis, we monitored the expression profiles of FIAT in parallel with ATF4 during osteoblastogenesis. Mechanism of FIAT repression of ATF4 was investigated through structure-function and mutation analysis. The physiological significance of FIAT expression in osteoblasts was studied through silencing FIAT in osteoblasts by RNA interference, as well as through characterization of two genetic mouse models: FIAT transgenic mice which overexpress FIAT in osteoblasts, and osteoblast-specific FIAT knockout mice. These studies showed that FIAT and ATF4 are co-expressed in osteoblasts, and that FIAT inhibition of matrix mineralization requires dimerization with ATF4 through the second leucine zipper. Furthermore, transgenic mice overexpressing FIAT exhibited osteopenia whereas FIAT knockout mice showed enhanced bone formation. These results support our hypothesis and demonstrate that FIAT is a key transcriptional repressor that modulates osteoblast function.

Solute carriers (SLCs) comprise the largest group of transporters in humans and there are currently 52 SLC families. They are embedded in cellular membranes and transport numerous molecules; defects in many of the genes encoding SLCs have been connected to pathological conditions, and several SLCs are potential drug targets. The SLC38 family has in total eleven members in humans and they encode transporters called SNATs. In paper I and paper II, we reported molecular and functional characterization of Slc38a7 and Slc38a8, two of the previous orphan members in the family which we suggested to be named SNAT7 and SNAT8, respectively. Using in situ hybridization and immunohistochemistry, these transporters showed similar expression pattern and localized to neurons in the brain For functional characterization proteins were overexpressed in X. laevis oocytes and an Uptake Assay and electrophysiological recordings showed preferred transport of L-glutamine, L-histidine, L-alanine, L-asparagine, L-aspartate and L-arginine for SNAT7. A similar pattern was seen for SNAT8 in a slightly different order of affinities. We classified SNAT7 as a system N transporter and SNAT8 as belonging to system A, and suggests that SNAT7 and SNAT8 could play a role in the glutamine/glutamate(GABA) cycle (GGC) in the brain. Furthermore, we studied the vesicular B0AT3 (Slc6a17) transporter in paper III, and the sodium-coupled amino acid transporter B0AT2 (Slc6a15) in paper IV. Tissue expression studies showed similar localization of Slc6a17 and Slc6a15 mRNA using in situ hybridization and real-time PCR. In paper III, vesicular localization of B0AT2 was shown in both excitatory and inhibitory neurons. When challenging the monoaminergic system with drugs both Slc6a17 and Slc6a15 were upregulated. Suggested roles for the transporters are thereby in synaptic remodeling by regulating the availability of free amino acids used as precursors needed in neurotransmitter synthesis. Moreover, in paper IV, immunohistochemistry showed B0AT3 localization to neurons, astrocytes and epithelial cells of the choroid plexus. Leucine injections caused a smaller reduction of food intake as well as higher neuronal activation in the paraventricular hypothalamic nucleus in Slc6a15 KO mice, compared with wild type mice. This suggests B0AT2 involvement in the anorexigenic effects of leucine.

Studies had shown that leucine supplementation increases insulin sensitivity and it has been studied that n-3 PUFA may have an anti-inflammatory effect in adipocytes. However, the extent to which dietary sources such as leucine and/or n-3 PUFA act through PFKFB3/iPFK2 to suppress adipocyte inflammatory response has not been studied; PFKFB3/iPFK2 is a regulator that links adipocyte metabolism and inflammatory responses. In this study, the involvement of PFKFB3/iPFK2 in the effects of insulin sensitizing and anti-inflammatory effect of leucine and/or n-3 PUFA are explored using cultured 3T3-L1 adipocytes including wild-type cells, PFKFB3-control cells (iPFK2-Ctrl) and PFKFB3-knockdown cells (iPFK2-KD). In iPFK2-Ctrl cells, leucine supplementation appears to have insulin-sensitizing effects through improving p-Akt/Akt insulin signaling, but have no effect on adiponectin expression, and appear to have limited anti-inflammatory effects. n-3 PUFA supplementation appears to have limited effects on both insulin sensitizing and anti-inflammatory effects in iPFK2-Ctrl. In contrast, n-3 PUFA exhibit pro-inflammatory expression in iPFK2-KD. The results of this study support the hypothesis that PFKFB3/iPFK2 is critically involved in insulin-sensitizing effects of leucine. This role of PFKFB3/iPFK2, however, appears to be independent of anti-inflammatory responses. Given this, it is likely that PFKFB3/iPFK2 only account, in part, for the beneficial effects of leucine. n-3 PUFA stimulate PFKFB3/iPFK2 activity in wild-type adipocytes. However, PUFA do not exhibit anti-inflammatory and insulin-sensitizing effects in controls. In contrast, n3-PUFA exhibit proinflammatory effects in iPFK2-KD cells. Taken together, PFKFB3/iPFK2 is involved, at least in part, in the effects of insulin sensitization of leucine and appears to protect adipocytes from inflammatory responses, which could be exacerbated by n-3 PUFA when PFKFB3/iPFK2 is disrupted.

No description available.

Thesis (Ph.D.)--Baylor University, 2007. / Includes bibliographical references (p. 134-146).

Thesis (Ph.D.)--Baylor University, 2007. / Includes bibliographical references (p. 145-152).

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Illinois State University, 2004. / Title from title page screen, viewed May 23, 2006. Dissertation Committee: Brian J. Wilkinson (chair), Radheshyam K. Jayaswal, Anthony J. Otsuka, David L. Williams, Wade A. Nichols. Includes bibliographical references (leaves 105-113) and abstract. Also available in print.

Antimalarial drug discovery remains a challenging endeavour as malaria parasites continue to develop resistance to drugs, including those which are currently the last line of defence against the disease. Plasmodium falciparum is the most virulent of the malaria parasites and it delivers its deadliest impact during the erythrocytic stages of the parasite’s life cycle; a stage characterised by elevated catabolism of haemoglobin and anabolism of parasite proteins. The present study investigates the use of nanotechnology in the form of metallic silver nanoparticles (AgNPs) against P. falciparum leucine aminopeptidase (PfLAP), a validated biomedical target involved in haemoglobin metabolism. AgNPs were also tested against the human homolog cytosolic Homo sapiens leucine aminopeptidase (HsLAP) to ascertain their selective abilities. PfLAP and HsLAP were successfully expressed in Escherichia coli BL21(DE3) cells. PfLAP showed optimal thermal stability at 25 °C and optimal pH stability at pH 8.0 with a Km of 42.7 mM towards leucine-p-nitroanilide (LpNA) and a Vmax of 59.9 μmol.ml⁻¹.min⁻¹. HsLAP was optimally stable at 37 °C and at pH 7.0 with a Km of 16.7 mM and a Vmax of 17.2 μmol.ml⁻¹.min⁻¹. Both enzymes exhibited optimal activity in the presence of 2 mM Mn²⁺. On interaction with polyvinylpyrrolidone (PVP) stabilised AgNPs, both enzymes were inhibited to differing extents with PfLAP losing three fold of its catalytic efficiency relative to HsLAP. These results show the ability of AgNPs to selectively inhibit PfLAP whilst having much lesser effects on its human homolog. With the use of available targeting techniques, the present study shows the potential use of nanotechnology based approaches as “silver bullets” that can target PfLAP without adversely affecting the host. However further research needs to be conducted to better understand the mechanisms of AgNP action, drug targeting and the health and safety issues associated with nanotechnology use.

Orientador: Everardo Magalhães Carneiro / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T11:51:38Z (GMT). No. of bitstreams: 1 Russo_MorganaRejaneRabeloRosa_D.pdf: 4218123 bytes, checksum: b0799db6946f29ef482adbc56a8add73 (MD5) Previous issue date: 2011 / Resumo: O aminoácido leucina é conhecido por ativar a via de síntese protéica muscular. Pesquisas recentes relatam alterações moleculares de proteínas envolvidas na via de síntese protéica após sessões agudas de exercício. No entanto, o efeito da leucina associada ao treinamento aeróbio foi pouco investigado. Sendo assim, o presente estudo teve como objetivo avaliar o papel da suplementação crônica com leucina, durante um programa de exercício aeróbio, sobre proteínas sinalizadoras da via de sensibilidade à insulina e síntese protéica em músculo esquelético e fígado de camundongos adultos. Os animais foram divididos em 4 grupos: controle (C), controle suplementado com leucina (CS), grupo de natação treinado (T) e grupo de natação treinado suplementado com leucina (TS). Após 12 semanas de exercício (1h30, 5 vezes/semana), o peso corporal do grupo T estava menor em relação ao outros grupos. O conteúdo de glicogênio muscular e hepático aumentou nos grupos CS, T e TS comparados ao grupo C. Os animais treinados (T e TS) apresentaram atividade da Cis e sensibilidade à insulina aumentada e menor porcentagem de gordura comparado aos animais controle (C e CS). A suplementação aumentou acentuadamente o teor de insulina e leucina plasmática no CS e TS comparado ao C e T. Em músculo esquelético, a suplementação com leucina, apesar de aumentar a fosforilação do IR, não alterou o contéudo total de IR e IRS-1 e a fosforilação do IRS-1, no entanto, a expressão gênica, o conteúdo total da mTOR e a fosforilação da p70s6k estavam aumentados. O treinamento aeróbio elevou o conteúdo total do IR e a fosforilação do IR e IRS-1, entretanto, reduziu o contéudo da p70s6k e não alterou o contéudo total da mTOR e fosforilação da p70s6k. A associação dos tratamentos elevou o conteúdo total e a fosforilação do IR acima dos valores encontrados quando os tratamentos foram administrados de forma individual e a fosforilação do IRS1 em relação aos grupos controle. Houve elevação da expressão gênica e conteúdo total da mTOR e conteúdo total e fosforilação da p70s6k acima dos valores encontrados para o grupo T, porém abaixo dos valores do grupo CS. No músculo esquelético, os resultados da associação dos tratamentos sugerem que a leucina diminuiu o efeito do exercício aeróbio sobre a via da mTOR/p70s6k. No fígado, a suplementação com leucina aumentou o conteúdo de IR e IRS-1 e a fosforilação do IR, assim como o conteúdo total da mTOR e fosforilação da p70s6k. O treinamento aeróbio, apesar de ter aumentado a fosforilação do IR, diminuiu o conteúdo total de IR, IRS-1, mTOR e p70s6k, assim como, a fosforilação do IRS-1. Quando os tratamentos foram associados, a leucina reverteu o efeito atenuador do exercício sobre a via da mTOR/p70s6k e sobre o IR e IRS-1. Estes resultados indicam que a administração de suplementação crônica com leucina para camundongos adultos durante um programa de exercício aeróbio pode ser importante por proporcionar maior ativação de proteínas da via de síntese protéica em músculo esquelético e fígado quando comparadas à atividade expressa somente em função do exercício aeróbio / Abstract: The amino acid leucine is known to activate the pathway of muscle protein synthesis. Recent surveys have reported molecular alterations after acute exercise sections. However, the effect of leucine associated with the aerobic training was seldom investigated. Thus, this study aimed to evaluate the role of chronic leucine supplementation during a program of aerobic exercise on protein signaling pathway in insulin sensitivity and protein synthesis in skeletal muscle and liver of adult mice. The animals were divided into four groups: control (C), control supplemented with leucine (CS), swim trained group (T) and swim-trained group supplemented with leucine (TS). After 12 weeks of exercise (1h30, 5 times / week), body weight of group T was lower than in the other groups. The glycogen content in muscle and liver increased in T and TS compared with group C. Trained animals (T and TS) had increased activity of Cis, insulin sensitivity and lower fat percentage compared to control animals (C and CS). The supplementation increased significantly the level of plasma insulin and leucine in CS and TS compared to C and T. In skeletal muscle, supplementation with leucine, despite increasing the phosphorylation of IR, did not alter the total content of IR and IRS-1 and phosphorylation of IRS-1; however, gene expression, the entire contents of mTOR and phosphorylation of p70s6k were increased. Aerobic training increased the total content of IR and phosphorylation of IR and IRS-1, however, reduced the content of p70s6k and did not alter the content of total mTOR and phosphorylation of p70s6k. The combination treatment increased the total content and phosphorylation of IR above the values found when the treatments were administered individually and phosphorylation of IRS-1 above the values control groups. There was an elevation of mRNA expression and mTOR total content, and phosphorylation of p70s6k above the values found for the T group, but below the CS group. In skeletal muscle, the results of the association, suggest that leucine reversed the effect of aerobic exercise on the pathway of mTOR/p70s6k. In the liver, leucine supplementation increased the content of IR and IRS-1 and phosphorylation of IR, the entire contents of phosphorylation of mTOR and p70s6k. The aerobic training, although it increased the phosphorylation of IR, decreased the total content of IR, IRS-1, mTOR and p70s6k, as well as the phosphorylation of IRS-1. When the treatments were associated leucine, it reversed the negative effect of exercise on the path of mTOR/p70s6k and the IR and IRS-1. These results indicate that chronic administration of leucine supplementation to adult mice during an aerobic exercise program may be important for making proteins pathways more active to protein synthesis in skeletal muscle and liver when compared to the activity expressed only in terms of aerobic exercise / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular