<p><em>Panax ginseng</em> is a traditional herb that has been used for over 2000 years to promote health and longevity. Active components of ginseng include ginsenosides, polysaccharides, flavonoids, polyacetylenes, peptides, vitamins, phenols and enzymes, of which the ginsenosides are considered to be the major bioactive constituents. Although widely used, the exact mechanisms of ginseng and its compounds remain unclear. In this thesis we use melanophores from <em>Xenopus laevis</em> to investigate the effects of <em>Panax ginseng</em> extract G115 and its constituents on organelle transport and signalling. Due to coordinated bidirectional movement of their pigmented granules (melanosomes), in response to defined chemical signals, melanophores are capable of fast colour changes and provide a great model for the study of intracellular transport. The movement is regulated by alterations in cyclic adenosine 3’:5’-monophosphate (cAMP) concentration, where a high or low level induce anterograde (dispersion) or retrograde (aggregation) transport respectively, resulting in a dark or light cell. Here we demonstrate that <em>Panax ginseng</em> and its constituents ginsenoside Rc and Rd and flavonoid quercetin induce a concentration-dependent anterograde transport of melanosomes. The effect of ginseng is shown to be independent of cAMP changes and protein kinase A activation. Upon incubation of melanophores with a combination of Rc or Rd and quercetin, a synergistic increase in anterograde movement was seen, indicating cooperation between the ginsenoside and flavonoid parts of ginseng. Protein kinase C (PKC) inhibitor Myristoylated EGF-R Fragment 651-658 decreased the anterograde movement stimulated by ginseng and ginsenoside Rc and Rd. Moreover, ginseng, but not ginsenosides or quercetin, stimulated an activation of 44/42-mitogen activated protein kinase (MAPK), previously shown to be involved in both aggregation and dispersion of melanosomes. PKC-inhibition did not affect the MAPK-activation, suggesting a role for PKC in the ginseng- and ginsenoside-induced dispersion but not as an upstream activator of MAPK.</p> / <p><em>Panax ginseng </em>är ett av de vanligaste naturläkemedlen i världen och används traditionellt för att öka kroppens uthållighet, motståndskraft och styrka. Ginseng är ett komplext ämne bestående av ett antal olika substanser, inklusive ginsenosider, flavonoider, vitaminer och enzymer, av vilka de steroidlika ginsenosiderna anses vara de mest aktiva beståndsdelarna. Flavonoider (som finns i till exempel frukt och grönsaker) och ginseng har genom forskning visat sig motverka bland annat hjärt-och kärlsjukdomar, diabetes, cancer och demens. Trots den omfattande användningen är dock mekanismen för hur ginseng verkar fortfarande oklar. I den här studien har vi använt pigmentinnehållande celler, melanoforer, från afrikansk klogroda för att undersöka effekterna av <em>Panax ginseng</em> på pigment-transport och dess maskineri. Melanoforer har förmågan att snabbt ändra färg genom samordnad förflyttning av pigmentkorn fram och tillbaka i cellen, och utgör en utmärkt modell för studier av intracellulär transport. Förflyttningen regleras av förändringar i halten av cykliskt adenosin-monofosfat (cAMP) i cellen, där en hög eller låg koncentration medför spridning av pigment över hela cellen (dispergering) eller en ansamling i mitten (aggregering), vilket resulterar i mörka respektive ljusa celler. Här visar vi att <em>Panax ginseng</em>, ginsenosiderna Rc och Rd samt flavonoiden quercetin stimulerar en dispergering av pigmentkornen. När melanoforerna inkuberades med en kombination av ginsenosid Rc eller Rd och quercetin, kunde en synergistisk ökning av dispergeringen ses, vilket tyder på en samverkan mellan ginsenosid- och flavonoid-delarna av ginseng. Ett protein som tidigare visats vara viktigt för pigmenttransporten är mitogen-aktiverat protein kinas (MAPK), och här visar vi att också melanoforer stimulerade med ginseng, men dock inte med ginsenosider eller quercetin, innehåller aktiverat MAPK. Genom att blockera enzymet protein kinas C (PKC) (känd aktivator av dispergering), minskade den ginseng- och ginsenosid-inducerade dispergeringen, medan aktiveringen av MAPK inte påverkades alls. Detta pekar på en roll för PKC i pigment-transporten men inte som en aktivator av MAPK.</p>

Panax ginseng is a traditional herb that has been used for over 2000 years to promote health and longevity. Active components of ginseng include ginsenosides, polysaccharides, flavonoids, polyacetylenes, peptides, vitamins, phenols and enzymes, of which the ginsenosides are considered to be the major bioactive constituents. Although widely used, the exact mechanisms of ginseng and its compounds remain unclear. In this thesis we use melanophores from Xenopus laevis to investigate the effects of Panax ginseng extract G115 and its constituents on organelle transport and signalling. Due to coordinated bidirectional movement of their pigmented granules (melanosomes), in response to defined chemical signals, melanophores are capable of fast colour changes and provide a great model for the study of intracellular transport. The movement is regulated by alterations in cyclic adenosine 3’:5’-monophosphate (cAMP) concentration, where a high or low level induce anterograde (dispersion) or retrograde (aggregation) transport respectively, resulting in a dark or light cell. Here we demonstrate that Panax ginseng and its constituents ginsenoside Rc and Rd and flavonoid quercetin induce a concentration-dependent anterograde transport of melanosomes. The effect of ginseng is shown to be independent of cAMP changes and protein kinase A activation. Upon incubation of melanophores with a combination of Rc or Rd and quercetin, a synergistic increase in anterograde movement was seen, indicating cooperation between the ginsenoside and flavonoid parts of ginseng. Protein kinase C (PKC) inhibitor Myristoylated EGF-R Fragment 651-658 decreased the anterograde movement stimulated by ginseng and ginsenoside Rc and Rd. Moreover, ginseng, but not ginsenosides or quercetin, stimulated an activation of 44/42-mitogen activated protein kinase (MAPK), previously shown to be involved in both aggregation and dispersion of melanosomes. PKC-inhibition did not affect the MAPK-activation, suggesting a role for PKC in the ginseng- and ginsenoside-induced dispersion but not as an upstream activator of MAPK. / Panax ginseng är ett av de vanligaste naturläkemedlen i världen och används traditionellt för att öka kroppens uthållighet, motståndskraft och styrka. Ginseng är ett komplext ämne bestående av ett antal olika substanser, inklusive ginsenosider, flavonoider, vitaminer och enzymer, av vilka de steroidlika ginsenosiderna anses vara de mest aktiva beståndsdelarna. Flavonoider (som finns i till exempel frukt och grönsaker) och ginseng har genom forskning visat sig motverka bland annat hjärt-och kärlsjukdomar, diabetes, cancer och demens. Trots den omfattande användningen är dock mekanismen för hur ginseng verkar fortfarande oklar. I den här studien har vi använt pigmentinnehållande celler, melanoforer, från afrikansk klogroda för att undersöka effekterna av Panax ginseng på pigment-transport och dess maskineri. Melanoforer har förmågan att snabbt ändra färg genom samordnad förflyttning av pigmentkorn fram och tillbaka i cellen, och utgör en utmärkt modell för studier av intracellulär transport. Förflyttningen regleras av förändringar i halten av cykliskt adenosin-monofosfat (cAMP) i cellen, där en hög eller låg koncentration medför spridning av pigment över hela cellen (dispergering) eller en ansamling i mitten (aggregering), vilket resulterar i mörka respektive ljusa celler. Här visar vi att Panax ginseng, ginsenosiderna Rc och Rd samt flavonoiden quercetin stimulerar en dispergering av pigmentkornen. När melanoforerna inkuberades med en kombination av ginsenosid Rc eller Rd och quercetin, kunde en synergistisk ökning av dispergeringen ses, vilket tyder på en samverkan mellan ginsenosid- och flavonoid-delarna av ginseng. Ett protein som tidigare visats vara viktigt för pigmenttransporten är mitogen-aktiverat protein kinas (MAPK), och här visar vi att också melanoforer stimulerade med ginseng, men dock inte med ginsenosider eller quercetin, innehåller aktiverat MAPK. Genom att blockera enzymet protein kinas C (PKC) (känd aktivator av dispergering), minskade den ginseng- och ginsenosid-inducerade dispergeringen, medan aktiveringen av MAPK inte påverkades alls. Detta pekar på en roll för PKC i pigment-transporten men inte som en aktivator av MAPK.

L'objectif de cette thèse était de caractériser les éléments appartenant à la voie de signalisation énergétique AMP-activated protein kinase chez l'huître creuse Crassostrea gigas afin de comprendre son implication dans la gestion de l'énergie, en particulier en réponse à des conditions physiologiques qui sollicitent de l'énergie telles que la reproduction, ou à des stress environnementaux comme l'hypoxie ou le jeûne. Au niveau génomique, les trois sous-unités constitutives du trimère AMPK ainsi que plusieurs éléments impliqués dans cette voie de signalisation et dans les métabolismes glucidiques et lipidiques, potentiellement cibles de l'AMPK, ont été décrits. Au niveau protéique, plusieurs anticorps hétérologues ciblant les isoformes de la sous-unité α et la phosphorylation du résidu thréonine 172 de la sous-unité α, témoin indirect de l'activité AMPK, ont été utilisés. Deux sous-unités α tronquées dans le domaine kinase ont été caractérisées principalement dans les tissus musculaires suggérant leurs implications dans la fonction musculaire. Au cours d'un stress hypoxique, une augmentation significative des quantités de sous-unités α tronquées a été observée dans le muscle lisse. Ce résultat suggère que pendant une durée d'au moins 6 h, ces protéines tronquées sont nécessaires au maintien du métabolisme aérobie dans le muscle lisse, lui permettant ainsi de remplir son rôle de fermeture statique des valves. Nous avons suggéré une hypothèse indiquant que l'accumulation in vivo de ces sous-unitésα tronquées pourrait exercer un rôle de modulation ou de transdomination négative de l'activité de la sous-unité α entière. Dans la gonade, nous avons observé une activation de l'AMPK tout au long du processus de gamétogénèse afin de supporter les processus cataboliques de création de gamètes. Une diminution de cette activation a été observée lors du stade anabolique de mise en réserve des ovocytes. Enfin, lors d'un conditionnement en milieu contrôlé, une approche physiologique par privation de nourriture et une approche pharmacologique par injection d'AICAR ont été réalisées pour provoquer une modulation de l'AMPK. Les analyses ont montré que ni le jeûne ni l'AICAR n'ont induit une augmentation de la phosphorylation de la sous-unité α. Cependant, plusieurs changements liés à l'injection de l'AICAR ont été observés sur la physiologie de l'huître : la modification du rapport AMP:ATP chez les huîtres nourries en comparaison aux huîtres à jeun, et une mortalité dépendante de la dose injectée d'AICAR chez les huîtres mises à jeun. La caractérisation de l'AMPK chez C. gigas ouvre de nombreuses perspectives exigeant des études fonctionnelles poussées afin de démontrer le rôle pivot de cette kinase dans la gestion de l'énergie, comme démontré chez de nombreuses espèces de vertébrés, et ainsi décrypter le métabolisme énergétique de l'huître.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 95-113).

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 106-122).

Le contrôle de la masse musculaire est sous la dépendance d'un équilibre entre les processus de synthèse et de dégradation. Sur le plan cellulaire, deux voies signalétiques majeures sont impliquées : la voie des facteurs de transcription de la famille FoxO qui contrôle l'expression des gènes impliqués dans les systèmes de dégradation (système ubiquitine-protéasome et autophagie), et la voie IGF-1/Akt/mTORC1 qui représente la voie majeure de la synthèse protéique. Nos travaux mettent en évidence, sur des cellules musculaires le rôle de la protéine kinase AMP-dépendante (AMPK) qui inhibe l'activité de la voie mTOR et régule les systèmes ubiquitine-protéasome et autophagiques de manière FoxO3 dépendante. Une nouvelle cible de l'AMPK a également été identifiée : la protéine Ulk1 qui possède une fonction clé dans l'activation de l'autophagie. Par ailleurs, nous avons montré le rôle centraldu facteur d'initiation à la traduction eIF3f dans l'induction de l'hypertrophie, et dans l'augmentation de l'activité de la voie mTORC1 associée. De plus, nous montrons que la surexpression d'un mutant d'eIF3f résistant à la dégradation est associée à une protection effective contre l'atrophie. / Skeletal muscle mass is depending upon a dynamic balance between anabolic and catabolic processes. At a cellular level, two major signaling pathways are involved: the transcription factors FoxO related pathway, implicated in the control of protein breakdown systems(ubiquitin-proteasome system and autophagy), and the IGF-1/Akt/mTORC1 pathway associated with the canonic pathway of protein synthesis. We show in muscle cells that theAMP-activated protein kinase (AMPK) decreases the mTORC1 pathway activity and simulate subiquitin-proteasome and autophagy systems in a FoxO3-dependant manner. Furthermore,we identify Ulk1 as a new interacting partner of AMPK, which plays a major role in the autophagy induction. Moreover, we demonstrate the key role of the eukaryotic translation initiation factor eIF3f in hypertrophy induction and in the associated increase of the mTORC1activity. In addition, we show that the overexpression of an eIF3f mutant resistant to the degradation is associated with a protection against muscle atrophy.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Anna Zolkiewska / Cancer cells exploit key signaling pathways in order to survive, proliferate, and metastasize. Understanding the intricacies of the aberrant signaling in cancer may provide new insight into how to therapeutically target tumor cells. The goal of my research was to explore the role of two modulators of transmembrane signaling, the secretory pathway and cell surface proteolysis, in the aggressiveness of breast cancer cells. To study the role of the secretory pathway, I focused on the family of endoplasmic reticulum (ER) chaperones. I found that several ER chaperones were upregulated in breast cancer cells grown under anchorage-independent conditions as mammospheres versus those grown under adherent conditions. Furthermore, certain members of the protein disulfide isomerase (PDI) family were consistently upregulated in two different cell lines at both the mRNA and protein levels. Knocking down these PDIs decreased the ability of the cells to form mammospheres. I demonstrated that the requirement for PDI chaperones in mammosphere growth is likely due to an increased flux of extracellular matrix (ECM) components through the ER. Next, I examined the role of cell surface proteolysis in modulating the aggressiveness of breast cancer cells. Cell-surface metalloproteases release soluble growth factors from cells and activate the corresponding growth factor receptors. I determined that specific metalloproteases (ADAM9 or ADAM12), modulate the activation of Epidermal Growth Factor Receptor (EGFR). I demonstrated that EGFR activation enhances the CD44⁺/CD24⁻ cell surface marker profile, which is a measure of cancer cell aggressiveness. I found that the MEK/ERK pathway, which is a downstream effector of EGFR activation, modulates the CD44⁺/CD24⁻ phenotype. When DUSP4, a negative regulator of the MEK/ERK pathway, is lost, activation of EGFR by metalloproteases no longer plays a significant role in cancer cell aggressiveness. This indicates that the ligand dependent activation of the EGFR/MEK/ERK pathway is a critical step in DUSP4-positive aggressive breast cancer. Finally, I examined the importance of metalloproteases in the regulation of Programmed-death ligand 1 (PD-L1), a transmembrane protein expressed by some cancer cells that plays a major role in suppressing the immune system. I demonstrated that cell-surface metalloproteases have the ability to cleave PD-L1 and release its receptor-binding domain to the extracellular environment. Collectively, these data indicate that (a) ER chaperones support anchorage-independent cell growth, (b) metalloproteases are important in regulation of an aggressive phenotype through the EGFR/MEK/ERK pathway, and (c) metalloproteases cleave PD-L1, a key component of immunosuppression in cancer.

Orientadores: Licio Augusto Velloso, Marcio Alberto Torsoni / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T11:00:58Z (GMT). No. of bitstreams: 1 Oliveira_MaristelaCesquinide_D.pdf: 2056313 bytes, checksum: fe162225f6402886dd228faa021438b9 (MD5) Previous issue date: 2006 / Resumo: O aumento da prevalência da obesidade e diabete tipo 2 nas sociedades modernas está fortemente associado às doenças cardiovasculares, hipertensão, infarto e aterosclerose. Estas patologias têm sido relacionadas com ingestão calórica excessiva e diminuição do gasto energético. Muitos fatores regulam a ingestão alimentar, incluindo hormônios como insulina e leptina, alimentos e comportamento. Estes fatores são integrados no hipotálamo que media um sistema de sinalização celular para regular o comportamento alimentar e o balanço energético da célula. A proteína quinase ativada por AMP (AMPK) tem sido proposta como capaz de mediar as adaptações celulares às variações nutricionais ambientais. O resultado da ativação da AMPK é a inibição de vias biossintetizantes que consomem energia, tais como as vias de síntese de ácidos graxos e proteínas, e a ativação de vias catabólicas, como a via de oxidação de ácidos graxos e a glicólise. No hipotálamo, a AMPK desempenha o papel de mediar os efeitos hormonais na ingestão alimentar e no balanço energético. Outras moléculas, como a acetil-CoA carboxilase (ACC) e o seu produto, o malonil-CoA têm sido propostas como possíveis intermediários na sinalização hipotalâmica que monitora o estado energético do corpo. No presente trabalho, nós administramos citrato, um ativador alostérico da ACC, no hipotálamo de ratos a fim de investigarmos o efeito deste composto na atividade hipotalâmica e hepática da AMPK. O resultado desta modulação no metabolismo energético, metabolismo de glicose e comportamento alimentar foi comparado aos ratos tratados com salina. Nós observamos que o tratamento com citrato inibiu a fosforilação da AMPK hipotalâmica seguido de inibição da fosforilação da ACC hipotalâmica, indicando menor atividade da AMPK neste tecido. A inibição da AMPK promoveu diminuição da ingestão alimentar, perda de peso corpóreo e aumento da expressão dos neuropeptídeos anorexigênicos POMC e CRH. No grupo de ratos tratados com citrato, a captação de glicose foi maior do que nos ratos que receberam solução salina, como mostrada pelo teste tolerância à glicose (GTT) e pelo clamp hiperinsulinêmico-euglicêmico. Consistente com estes resultados, no fígado, os ratos tratados com citrato mostraram maior fosforilação das proteínas da cascata de sinalização da insulina, reduzidos níveis de fosforilação da AMPK e ACC e expressão aumentada da PEPCK e G6Pase, se comparado aos animais controle. O efeito central do citrato na melhora da sinalização da insulina nos tecidos periféricos, na ativação da produção de glicose pelo fígado e na inibição da AMPK hepática foi bloqueado por antagonista ?-adrenérgico. De acordo com estes resultados, nós podemos sugerir que a modulação da AMPK por intermediários metabólicos possa ser um mecanismo importante de controle da homeostase energética e consequentemente do diabetes e obesidade / Abstract: The increased prevalence of obesity and type II diabetes in modern societies is largely linked to cardiovascular disease, hypertension, stroke and atherosclerosis. These pathologies have been associated to excessive caloric intake and decreased energy expenditure. Many factors regulate food intake, including hormones such as insulin and leptin, fuels and behavior. These factors are integrated in the hypothalamus that mediates a signaling system to regulate food behavior and cellular energy balance. The AMPactivated protein quinase (AMPK) has been proposed to be capable of mediating the cellular adaptations to nutritional environmental variation. The result of AMPK activation is the inhibition of nergy-consuming biosynthetic pathways, such as fatty acid and protein synthesis, and activation of ATP-catabolic pathways, such as fatty acid oxidation and glycolysis. In the hypothalamus, AMPK mediates hormonal effects on food intake and energy balance. Other molecules, such as acetyl-CoA carboxylase (ACC) and its product malonyl-CoA have been proposed as possible intermediaries in the hypothalamic signaling pathway that monitors energy status. In the present work we administrated citrate, an allosteric activator of ACC, in the hypothalamus to investigate its effect on hypothalamic and hepatic AMPK activity and the result of this modulation in the energetic cellular metabolism, such as glucose metabolism and food behavior. Results were compared to saline-treated rats. Here we show that citrate treatment inhibited hypothalamic AMPK phosphorylation followed by an inhibition of hypothalamic ACC phosphorylation, indicating lower AMPK activity in this tissue. The AMPK inhibition promoted decrease in food intake, loss of body weight and increased expression of anorexigenic neuropeptides (POMC and CRH). In the citrate group, the glucose uptake was higher than in animals receiving saline according to GTT and hyperinsulinemic-euglycemic clamp. Consistent with these results, in the liver, citrate-treated rats showed also higher phosphorylation of insulin signaling proteins than control rats, reduced AMPK and ACC phosphorylation and increased PEPCK and G6Pase expression. Supported by these results, we suggest that AMPK modulation by citrate can be an important mechanism to understand deregulated glucose metabolism involved in diabetes and obesity. The central effect of citrate in the improvement of peripheral insulin signaling, in the activation of liver glucose production, and in the inhibition of hepatic AMPK was blocked by the ?-adrenergic antagonist. According to these results, we suggest that AMPK modulation by metabolic intermediaries can be an important mechanism controlling the energetic homeostase, diabetes and obesity / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Prevalence of obesity and associated diseases like type 2 diabetes has increased rapidly during last years. These diseases closely relate to each other. Obesity leads to insulin resistence, which directly precedes type 2 diabetes. Metformin is the most prescribed medicament for type 2 diabetic patients and insulin resistant people. It improves glucose tolerance and insulin resistance. Enzyme AMP-activated protein kinase (AMPK) is strogly involved in metformin action. The latest studies using transgenic models lacking AMPK suggest, that notable part of mechanisms involved in metformin action is independent on AMPK. n-3 polyunsaturated fatty acids (n-3 PUFA), namely eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are abundant in sea fish, have beneficial effects on metabolism. These fatty acids lower plasma lipids and exert cardioprotective effects. n-3 PUFA also prevent development of insulin resistence and type 2 diabetes in rodents. The aim of this thesis was to characterise acute effects of metformin on glucose homeostasis, impact of short term diet intervention with diet rich in n-3 PUFA on metformin action and the role of insulin stimulated signalling pathways and AMPK. Results suggest that early effect of metformin is dose dependent and that single dose of metformin...