Dietary vitamin E, highly expressed in palm oil, exists as either tocopherols or tocotrienols. Evidence indicates that vitamin Es maybe potent cancer preventive agents. In this study, the y- and O- isoforms of vitamin E were found to he the most effective at cancer cell growth inhibition, with the tocotrienols being more effective than the tocopherols in androgen-independent PC-3 prostate cancer cells. To assure that these compounds were selective toward cancer cells, the growth arrest of PrEC normal prostate cells was compared to PC-3 cells. At concentrations of -30 iM dietary, y-vitamin Es showed no signficant growth arrest on PrEC cell growth, hut selectively inhibited growth in the PC-3 cancer cells. Moreover y-Tocotrienol demonstrated a greater potential to inhibit growth in cancer cells at these lower concentrations than did y-Tocopherol. Two independent pathways important in carcinogenesis were tested: PPAR y and NFicB. The PPAR y was up regulated by both dietary y-vitamin Es by the modulation of the endogenous ligand 15-S-HETE, while NFicB was only regulated by y-Tocotrienol. The modulation of NFicB was confirmed by the down regulation of the pro-Apoptotic proteins clAP, xIAP, and BcL-2 which potentiate apoptosis and are down stream effectors of NFicB.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Jakub Draský Supervisor: prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Pharmacological influence of nuclear receptors in diabetes mellitus therapy Nuclear receptors belong to the superfamily of transcription factors, their main functions include regulating the expression of target genes. In my work I focused mainly on the group of orphan receptors, namely the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR). A common feature of these receptors is their activation by a specific ligand. Both CAR and PXR have an essential function as biological sensors of hydrophobic xenobiotics when they induce enzymes I and II. phase of metabolism. They are also essential in the regulation of gluconeogenesis, insulin response, adipogenesis, cholesterol homeostasis, fatty acids, triglycerides and glycogen. The aim of this experimental work was to introduce a luciferase reporter assay method for two DNA constructs containing the promoter region of the PEPCK and CYP7A1 genes. We used the known agonist rifampicin and the antagonist SPA70 to activate/deactivate PXR. We used CITCO as a CAR receptor agonist. We first verified the functionality of the luciferase reporter gene assay...

This research proposes a new construct, post-purchase anticipated regret (PPAR), to expand the existing retailing and regret theory literature streams. Specifically, the purpose of this research is to understand the antecedents and consequences of post-purchase anticipated regret in an online retailing context. Study 1 uses qualitative data drawn from depth-interviews with online shoppers to investigate the pre-purchase factors that contribute to PPAR formation. Building from these results, studies 2 and 3 empirically test an original conceptual model to holistically understand PPAR. Additionally, the role of online shopping self-affirmation and regret coping strategies are explored as means to combat PPAR formation. Managerial and theoretical implications are offered, as well as research limitations and future research directions.

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La résistance à l'insuline associée à l'obésité est une condition qui favorise les troubles métaboliques tels que le diabète de type 2 (T2D) et la stéatose hépatique non alcoolique (NAFLD). Des altérations de l'homéostasie des lipides et du glucose, ainsi qu'une inflammation chronique de bas grade sont les caractéristiques du T2D et de la NAFLD. SHP-1 (codé par le gène *PTPN6*) est une tyrosine phosphatase avec deux domaines SH2 et est connu pour agir comme modulateur du métabolisme du glucose et des lipides. SHP-1 régule également la signalisation des cytokines et l'expression des gènes inflammatoires. En plus d'être localisé dans le cytoplasme, SHP-1 se trouve également dans le noyau des cellules épithéliales. La fonction de ce SHP-1 nucléaire reste inconnue. Ici, nous avons étudié les fonctions dépendantes et indépendantes de la tyrosine phosphatase de SHP-1 dans le contrôle des voies métaboliques. Des découvertes antérieures de notre laboratoire ont établi un lien entre SHP-1 et l'activité du récepteur activé par les proliférateurs de peroxysomes γ2 (PPARγ2). PPARγ2 est un facteur de transcription activé par des ligands et contrôle le métabolisme des lipides. Dans le chapitre II, nous avons constaté que SHP-1 interagit avec PPARγ2 principalement via son domaine SH2 en N-terminal et peut déphosphoryler PPARγ2 *in vitro*. Nos données suggèrent que PPARγ2 est phosphorylé sur le résidu tyrosine 78 (Y78) et que la déphosphorylation catalysée par SHP-1 est associée à la stabilité de PPARγ2. L'invalidation génétique de SHP-1 dans des cellules exprimant PPARγ2 augmente l'expression des cibles transcriptionnelles de PPARγ2 telles que *FABP4* et *CD36* en plus d'augmenter l'adipogenèse. Ces effets étaient atténués dans des cellules exprimant une forme mutée de PPARγ2 où la tyrosine 78 avait été remplacée par une phénylalanine ne pouvant être phosphorylée (Y78F). Collectivement, ces résultats indiquent que l'activité phosphatase de SHP-1 contrôle la stabilité de PPARγ2 et donc affecte l'adipogenèse. SHP-1 est un modulateur de la signalisation de l'insuline. L'invalidation génétique de SHP-1 spécifiquement dans les hépatocytes chez la souris (SHP-1 KO) est associée à une glycémie à jeun plus basse que celle de leurs congénères non transgéniques. Les souris SHP-1 KO présentaient également une diminution importante de la production hépatique de glucose, suggérant donc l'existence d'une autre fonction de SHP-1 sur l'homéostasie du glucose, possiblement indépendante de l'insuline. Dans le chapitre III, nous avons découvert que SHP-1 agit comme coactivateur pour contrôler la transcription du gène phosphoénolpyruvate carboxykinase 1 (PCK1) et donc régule la gluconéogenèse. SHP-1 est recruté à la région régulatrice du gène *PCK1*, le cite potentiel où il interagit avec l'ARN polymérase II (RNAPII). Le recrutement de SHP-1 à la chromatine est dépendant du facteur de transcription transducteur de signal et activateur de transcription 5 (STAT5). L'épuisement de STAT5 ainsi que SHP-1 résulte en une diminution du niveau de transcrit de *PCK1* et une réduction de la gluconéogenèse. Ensemble, ces résultats indiquent que nous avons découvert une nouvelle fonction de SHP-1 où la phosphatase agit comme un co-régulateur transcriptionnel clef du gène *PCK1* et exerce un contrôle sur la gluconéogenèse. / Insulin resistance coupled with obesity is a condition that promotes metabolic disorders such as type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD). Alterations in lipid and glucose homeostasis, as well as low-grade chronic inflammation are the hallmarks of T2D and NAFLD. SHP-1 (encoded by the gene Protein Tyrosine Phosphatase Non-Receptor Type 6, *PTPN6*) is a tyrosine phosphatase with two SH2 domains and is known to act as a modulator of glucose and lipid metabolism. In addition, SHP-1 also regulates cytokine signaling and inflammatory gene expression. Besides being localized in the cytoplasm, SHP-1 is also found in the nucleus of epithelial cells. The function of this nuclear SHP-1 remains elusive. Here, we investigated tyrosine phosphatase dependent and independent functions of SHP-1 in controlling metabolic pathways. Previous findings from our laboratory established a link between SHP-1 and peroxisome proliferator-activated receptor γ2 (PPARγ2) activity. PPARγ2 is a ligand-activated transcription factor that controls lipid metabolism. In chapter II, we found that SHP-1 interacts with PPARγ2 mainly via its N-terminal SH2 domain and can dephosphorylate PPARγ2 *in vitro*. Our data suggest that PPARγ2 is tyrosine phosphorylated mainly on tyrosine residue 78 (Y78) and SHP-1-mediated dephosphorylation of PPARγ2 is associated with its stability. The knockdown of SHP-1 in PPARγ2 expressing cells resulted in enhanced expression of the classical PPARγ2 targets *FABP4* and *CD36* coupled with increased adipogenesis. These effects were blunted in cells expressing mutant PPARγ2 where tyrosine 78 has been replaced with the non-phosphorylatable phenylalanine (Y78F). Collectively, phosphatase activity of SHP-1 controls the stability of PPARγ2 thereby affecting lipid metabolism. SHP-1 is a modulator of insulin signaling. Hepatocyte-specific SHP-1 KO mice compared to their wild type control littermates exhibited lower fasting glucose and markedly decreased hepatic glucose production suggesting the existence of an additional insulin-independent effect of SHP-1 on glucose homeostasis. In Chapter III, we found that SHP-1 acts as a co-activator for controlling the transcription of the phosphoenolpyruvate carboxykinase 1 (*PCK1*) gene thereby regulating gluconeogenesis. SHP-1 is recruited to the regulatory region of the *PCK1* gene, the potential site where it interacts with RNA polymerase II (RNAPII). The recruitment of SHP-1 to the chromatin was mediated by the transcription factor signal transducer and activator of transcription 5 (STAT5). Depletion of STAT5 as well as SHP-1 resulted in a decrease in *PCK1* transcript levels and blunted gluconeogenesis. Taken together, we discovered a novel function of SHP-1 whereby it acts as a key transcriptional co-regulator of the *PCK1* gene and exerts control over gluconeogenesis. Collectively, findings from these studies provide novel functions of SHP-1 in controlling lipid and glucose metabolism.

Receptores ativados da proliferação de peroxissomos (PPAR) são receptores nucleares que regulam o metabolismo de gordura e glicose, adipogênese e polarização de macrófagos, e são os mediadores da ação de uma grande classe de fármacos usada no tratamento de diabetes tipo 2, as tiazolidinadionas (TZD). Enquanto as TZDs reduzem a glicose do sangue e aumentam efetivamente a sensibilidade à insulina, elas podem também apresentar efeitos colaterais como aumento do risco de complicações cardiovasculares, ganho de peso, retenção de fluido e toxicidade hepática. Por causa disso, novos fármacos que possuem respostas mais favoráveis devem ser desenvolvidos, e o mecanismo de ativação do PPAR por ligantes vem sendo intensamente examinado. Para entender a relação entre a ligação de agonistas ao PPAR e a ativação transcricional, pretendíamos primeiramente obter cristais de PPAR-LBD (domínio de ligação ao ligante) humano na forma apo. Porém, surpreendentemente, a análise do sítio de ligação ao ligante revelou a presença de três pequenas moléculas, identificadas como ácidos nonanoicos e octanoicos. Este trabalho reporta a análise da estrutura cristalográfica do PPAR LBD complexado simultaneamente com três ácidos graxos de cadeia média (AGCM), provindos de bactérias (organismo de expressão), localizados no sítio de ligação ao ligante. A análise estrutural e funcional sugere que os AGCM são agonistas parciais que estabilizam a conformação do LBD do PPAR por mecanismo independente da hélice 12. / PPARs (peroxisome proliferator activated receptors) are nuclear receptors that regulate glucose and fat metabolism, adipogenesis and macrophage polarization and mediate actions of a major class of drugs that are used to treat type 2 diabetes, the thiazolidinediones. While TZDs reduce blood glucose and improve insulin sensitivity effectively, they can also exhibit deleterious side effects such as increased cardiovascular risk, weight gain, fluid retention and liver toxicity. Because it is desirable to develop new PPAR drugs with more favorable spectrums of response, mechanisms of PPAR ligand activation have come under intense scrutiny. To understand relationships between PPAR ligand binding and transcriptional activation, we sought to obtain apo human PPAR-LBD (ligand binding domain) crystals that diffract to high resolution. More surprisingly, close analysis of the ligand binding pocket revealed the presence of three small molecules, identified as nonanoic acid and octanoic acid. Here, we report the X-ray structural analysis of the PPAR LBD complexed with three bacterial (expression organism) medium chain fatty acids (MCFAs) that simultaneously occupy the buried ligand binding pocket (LBP). Structural and functional analysis suggests that MCFAs are partial agonists that stabilize PPAR LBD conformation, through a helix 12 independent mechanism.