[EN] The Saccharomyces cerevisiae protein kinases Sat4 (Hal4) and Hal5 are required for the plasma membrane stability of the high affinity K+ transporter Trk1 and some amino acid and glucose permeases. A transcriptomic analysis of the hal4 hal5 strain revealed that the absence of these genes causes general alterations in the metabolism of amino acids and glucose. This data is confirmed by the following approaches: activity of the Gcn2-Gcn4 pathway, uptake of methionine and leucine, activity of succinate dehydrogenase (SDH), glucose consumption and ethanol production of this mutant. In this thesis, we demonstrated that the high affinity permease Mup1 is internalized and degraded in the vacuole in the absence of potassium supplementation, like other plasma membrane permeases such as Hxt1 (glucose), Can1 (arginine), Fur4 (uracil) and Gap1 (amino acids). This destabilization of the Mup1 permease is likely to explain the reduction in the uptake of methionine in the double mutant hal4 hal5 and suggests that Hal4 and Hal5 are involved in a general mechanism of regulation of the stability of permeases in the plasma membrane. This hypothesis was corroborated by studies with inhibitors of endocytosis and mutant isoforms of the E3 ubiquitin ligase Rsp5, which is responsible for the ubiquitination and subsequent vacuolar degradation of the permeases studied. The process of Rsp5-mediated ubiquitination requires, in many cases, specific adapters for recognition of the target protein. So far, 19 Rsp5 adapter proteins have been described, among which there are 9 ARTs proteins (Arrestin-Related Trafficking adaptor). In this study, we investigated whether there is a functional connection between Hal4 and Hal5 kinases and ARTs, since this mechanism could explain the observed phenotypes. We studied whether Art1, a regulator of Mup1 and Can1 endocytosis, is involved in the internalization of these permeases in hal4 hal5 strains. Our data indicates that Art1 is not necessary for internalizing Mup1 in the hal4 hal5 strain in the absence of potassium supplementation, therefore suggesting a new role for the Hal4 and Hal5 kinases. We extended the study to include the transporter of aspartic and glutamic acid, Dip5, whose endocytosis is mainly mediated by Aly2 (Art3). The results were positive, providing support for a more general mechanism of regulation of the permeases of the plasma membrane by these kinases. It has been proposed that Npr1, a kinase that is an effector of the Target of Rapamycin Complex 1 (TORC1), controls the activity of Art1, Aly1 (Art6) and Aly2 (Art3), leading to the accumulation of some permeases in the plasma membrane. We observe lower expression levels of Npr1 in hal4 hal5 strains. We also observe a state of constitutive hyperphosphorylation, similar to WT cells under limiting potassium. Furthermore, overexpression of the Npr1 kinase partially rescues the growth defects and instability of the permeases in the plasma membrane described in the hal4 hal5 mutant. Therefore, we identify part of the pathway regulated by the Hal4 and Hal5 kinases. In eukaryotes, TOR (Target of Rapamycin) exists in two distinct multiprotein complexes, TOR complex 1 (TORC1) and TOR complex 2 (TORC2). We have analyzed the direct substrates of TORC1 (Sch9) and TORC2 (Ypk1) in hal4 hal5 and trk1 trk2 mutants in potassium limiting conditions, observing alterations in the phosphorylation levels of both effectors. Finally, we observed that hal4 hal5 and trk1 trk2 mutants are highly sensitive to the TORC1 inhibitor, rapamycin, and that this sensitivity is rescued by increased external potassium. We confirmed that cells treated with rapamycin had lower internal potassium levels, an effect which is dependent on TORC1 and independent of Trk1 and Trk2. Therefore, our data indicates that the Hal4 and Hal5 kinases have a more specific effect on Npr1 and that there is a reciprocal regulation between potassium and the TOR signaling pathway. / [ES] Las proteínas quinasa de Saccharomyces cerevisiae Sat4 (Hal4) y Hal5 son necesarias para la estabilidad del transportador de K+ de alta afinidad Trk1 y de algunas permeasas de aminoácidos y de glucosa. El análisis transcriptómico del mutante hal4 hal5 reveló que la ausencia de estos genes origina alteraciones generales en el metabolismo de aminoácidos y de glucosa, datos que confirmamos mediante la medida de la ruta Gcn2-Gcn4, de la toma de metionina y de leucina, de la actividad de la succinato deshidrogenasa (SDH), del consumo de glucosa y de la producción de etanol. En esta tesis, hemos demostrado que la permeasa de alta afinidad de metionina, Mup1 se degrada en la vacuola en ausencia de un suplemento de potasio en el mutante hal4 hal5, igual que otras permeasas de la membrana plasmática como Hxt1, Can1, Fur4 y Gap1. Esta desestabilización de Mup1 podría explicar el defecto en la toma de metionina observado y sugiere que Hal4 y Hal5 están implicadas en un mecanismo general de regulación de la estabilidad de las permeasas en la membrana plasmática. Esta hipótesis fue corroborada mediante estudios con inhibidores de la endocitosis y mutantes en la E3 ubiquitina ligasa Rsp5, responsable de la ubiquitinación y posterior degradación vacuolar de las permeasas estudiadas. El proceso de ubiquitinación, en muchos casos, precisa de adaptadores específicos para reconocer la proteína diana. Se han descrito 19 proteínas adaptadoras de Rsp5, entre las que se encuentran 9 proteínas ARTs (Adaptadores de tráfico relacionados con arrestina). En este trabajo, hemos investigado si existe una conexión funcional entre las quinasas Hal4 y Hal5 y los ARTs; este mecanismo podría explicar los fenotipos observados. Estudiamos si Art1, regulador de la endocitosis de Mup1 y Can1, está implicado en la internalización de estas permeases en la cepa hal4 hal5. Nuestros datos indican que Art1 no es necesario para la internalización de Mup1 y Can1 en una cepa hal4 hal5 en ausencia de un suplemento de potasio, sugiriendo un papel novedoso de las quinasas Hal4 y Hal5. Ampliamos el estudio al transportador de ácido aspártico y glutámico, Dip5, cuya endocitosis viene mediada principalmente por Aly2 (Art3). Los resultados fueron positivos apoyando un mecanismo más general de regulación de las permeasas de la membrana plasmática por parte de estas quinasas. Se ha propuesto que Npr1, una quinasa efectora del Target of Rapamycin Complex 1 (TORC1), controla la actividad de Art1, Aly1 (Art6) y Aly2 (Art3) generando la acumulación de algunas permeasas en la membrana plasmática. Observamos menores niveles de expresión de Npr1 en mutantes hal4 hal5, además de un estado de hiperfosforilación constitutivo similar al de células WT en condiciones de potasio limitante. Además, la sobreexpresión de NPR1 rescata los defectos de crecimiento observados en medios con baja disponibilidad de potasio e inestabilidad de permeasas de la membrana plasmática descritos en el mutante hal4 hal5. Por tanto, identificamos parte de la ruta regulada por las quinasas Hal4 y Hal5. En los organismos eucariotas TOR (Target of Rapamycin) existe en dos complejos multiproteicos distintos, complejo TOR1 (TORC1) y complejo TOR2 (TORC2). Hemos analizado los sustratos directos de TORC1 (Sch9) y TORC2 (Ypk1) en mutantes hal4 hal5 y trk1 trk2 y en condiciones de potasio limitante observando alteraciones en los niveles de fosforilación de ambos efectores. Finalmente, hemos observado que los mutantes hal4 hal5 y trk1 trk2 son altamente sensibles al inhibidor de TORC1, rapamicina y que esta sensibilidad se rescata con un exceso de potasio en el medio. Comprobamos que células tratadas con rapamicina presentan una disminución del potasio interno dependiente de TORC1 e independiente de Trk1 y Trk2. Por tanto, nuestros datos indican que las quinasas Hal4 y Hal5 tienen un efecto más específico sobre Npr1 y que hay una regulación reciproca entre el potasio y la r / [CA] Les proteïnes quinasa de Saccharomyces cerevisiae Sat4 (Hal4) i Hal5 són necessàries per a l'estabilitat del transportador de K+ d'alta afinitat Trk1 i d'algunes permeases d'aminoàcids i de glucosa. L'anàlisi transcriptòmic d'una soca mutant hal4 hal5 revela que l'absència d'aquests gens origina alteracions generals en el metabolisme d'aminoàcids i de glucosa, dades que confirmem mitjançant la mesura de la ruta Gcn2-Gcn4, de la presa de metionina i leucina, de l'activitat de succinat deshidrogenasa (SDH), del consum de glucosa i de la producció d'etanol d'aquest mutant. En aquesta tesi, hem demostrat que la permeasa d'alta afinitat de metionina, Mup1 es degrada en el vacúol en absència d'un suplement de potassi en el mutant hal4 hal5, igual que altres permeases de la membrana plasmàtica com Hxt1, Can1, Fur4 i Gap1. Aquesta desestabilització de Mup1 podria explicar el defecte en la presa de metionina observat i suggereix que Hal4 i Hal5 estan implicades en un mecanisme general de regulació de l'estabilitat de les permeases a la membrana plasmàtica. Aquesta hipòtesi va ser corroborada mitjançant estudis amb inhibidors de l'endocitosi i mutants en l'E3 ubiquitina ligasa Rsp5, responsable de la ubiquitinació i posterior degradació vacuolar de les permeases estudiades. El procés d'ubiquitinació, en molts casos, precisa d'adaptadors específics per reconèixer la proteïna diana. Fins al moment s'han descrit 19 proteïnes adaptadores de Rsp5, entre les quals es troben 9 proteïnes ART (Adaptadors de trànsit relacionats amb arrestina). En aquest treball hem investigat si existeix una connexió funcional entre les quinases Hal4 i Hal5 i els ARTs; aquest mecanisme podria explicar els fenotips observats. Estudiem si Art1, regulador de l'endocitosi de Mup1 i Can1, està implicat en la internalització d'aquestes permeases a la soca hal4 hal5. Les nostres dades indiquen que Art1 no és necessari per a la internalització de Mup1 i Can1 en una soca hal4 hal5 en absència d'un suplement de potassi, suggerint un paper nou de les quinases Hal4 i Hal5. Ampliem l'estudi per incloure el transportador d'àcid aspàrtic i glutàmic, Dip5, en l'endocitosi del qual intervé principalment Aly2 (Art3). Els resultats van ser positius recolzant un mecanisme més general de regulació de les permeases de la membrana plasmàtica per part d'aquestes quinases. S'ha proposat que Npr1, una quinasa que és un efector del Target of rapamycin Complex 1 (TORC1), controla l'activitat de Art1, Aly1 (Art6) i Aly2 (Art3) i genera l'acumulació d'algunes permeases a la membrana plasmàtica. Observem nivells menors d'expressió de Npr1 en mutants hal4 hal5, a més d'un estat d'hiperfosforilació constitutiu semblant al de cèl·lules WT en condicions de potassi limitant. A més, la sobreexpressió de la quinasa Npr1 rescata parcialment els defectes de creixement observats en medis amb baixa disponibilitat de potassi i la inestabilitat de permeases de la membrana plasmàtica descrits en el mutant hal4 hal5. Per tant, identifiquem part de la ruta regulada per les quinases Hal4 i Hal5. En els organismes eucariotes, TOR (Target of rapamycin) existeix en dos complexos multiproteics diferents, complex TOR1 (TORC1) i complex TOR2 (TORC2). Hem analitzat els substrats directes de TORC1 (Sch9) i TORC2 (Ypk1) en mutants hal4 hal5 i trk1 trk2 i en condicions de potassi limitant observant alteracions en els nivells de fosforilació dels dos efectors. Finalment, hem observat que els mutants hal4 hal5 i trk1 trk2 són altament sensibles a l'inhibidor de TORC1, rapamicina, i que aquesta sensibilitat es rescatada amb un excés de potassi en el medi. Vam comprovar que cèl·lules tractades amb rapamicina presenten una disminució del potassi intern dependent de TORC1 e independent de Trk1 i Trk2. Per tant, les nostres dades indiquen que les quinases Hal4 i Hal5 tenen un efecte més específic sobre Npr1 i que hi ha una regulació recíproca entre el potassi i / Primo Planta, C. (2015). Mecanismos de regulación post-traduccional de transportadores de la membrana plasmática: Papel de las quinasas Hal4 y Hal5 en el tráfico de transportadores de nutrientes e iones en el organismo modelo Saccharomyces cerevisiae [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/52697

Yes / Metabolic syndrome is a global public health problem and predisposes individuals to obesity, diabetes and cardiovascular disease. Although the underlying mechanisms remain to be elucidated, accumulating evidence has uncovered a critical role of adipokines. Chemerin, encoded by the gene Rarres2, is a newly discovered adipokine involved in inflammation, adipogenesis, angiogenesis and energy metabolism. In humans, local and circulating levels of chemerin are positively correlated with body mass index and obesity-related biomarkers. In this review, we discuss both peripheral and central roles of chemerin in regulating body metabolism. In general, chemerin is upregulated in obese and diabetic animals. Previous studies by gain or loss of function show an association of chemerin with adipogenesis, glucose homeostasis, food intake and body weight. In the brain, the hypothalamus integrates peripheral afferent signals including adipokines to regulate appetite and energy homeostasis. Chemerin increases food intake in seasonal animals by acting on hypothalamic stem cells, the tanycytes. In peripheral tissues, chemerin increases cell expansion, inflammation and angiogenesis in adipose tissue, collectively resulting in adiposity. While chemerin signalling enhances insulin secretion from pancreatic islets, contradictory results have been reported on how chemerin links to obesity and insulin resistance. Given the association of chemerin with obesity comorbidities in humans, advances in translational research targeting chemerin are expected to mitigate metabolic disorders. Together, the exciting findings gathered in the last decade clearly indicate a crucial multifaceted role for chemerin in the regulation of energy balance, making it a promising candidate for urgently needed pharmacological treatment strategies for obesity.

Iveta Šteflová Drug induced imbalance of sodium Diploma thesis Charles University in Prague, Faculty of Pharmacy in Hradec Králové Pharmacy Department of Biological and Medical Science Supervisor: Doc.MUDr. Josef Herink, DrSc. Sodium (Na+ ) is the major extracellular cation. It plays an important role in maintaining membrane potential and depolarization that is the basic mechanism of transmission of the nerve impulse. It is involved in maintaining acid-base balance, osmotic pressure, water retention in the body. The largest part of the sodium is in the extracellular fluid where it is stored about 50 % of sodium. Plasma concentration of sodium is 140 ± 5 mmol/l. Drug-induced electrolyte disorders are increasingly reported and may be associated with considerable morbidity and mortality. The risk of drug-induced hyponatremia is generally higher than the risk of drug-induced hypernatremia. Hyponatremia is a common electrolyte disorder defined as a decrease plasma sodium concentration below 135 mmol/l. It is classified by the state of volume - hypovolemic, euvolemic and hypervolemic hyponatremia. It reflects the relative rate between sodium and water in the body. The most common cause is the syndrome of inappropriate secretion of antidiuretic hormone that induces euvolemic hyponatremia. Hypernatremia is...

Hepcidin is a well-known iron-regulatory hormone that plays a key role in maintaining peripheral iron homeostasis. The presence and wide-spread distribution of hepcidin in the brain suggests that this peptide may also be an important player in brain iron homeostasis. In this study, we tested the hypothesis that hepcidin exerts an important role in the regulation of brain iron content, which might benefit iron-associated NDs such as PD. We also examined the hypothesis that hepcidin could control iron transport processes via regulating iron transport proteins in the brain cells, thus maintaining brain iron homeostasis. / In conclusion, the results of the present study implied that hepcidin plays an important role in maintaining brain iron homeostasis. Hepcidin is beneficial for PD and this effect is related to its iron-regulatory effect via inhibiting iron accumulation in the substantia nigra. Hepcidin effectively controls iron uptake and release through regulating iron transport proteins expressions in the brain, which would contribute to brain iron homeostasis. Therefore, manipulation of hepcidin level in the brain has a potential to be developed into a novel preventive approach for the iron-associated NDs such as PD. / In the second part, we investigated the effect of hepcidin on the processes of iron uptake and release in the cultured brain cells including neurons, astrocytes and brain vascular endothelial cells (BVECs). The expressions of iron uptake proteins such as transferrin receptor 1 (TfR1) and divalent metal transporter 1 (DMT1) as well as the iron exporter ferroportin 1 (Fpn1) were also observed. We found that hepcidin reduced both iron uptake and release via decreasing iron transport proteins expressions in these brain cells, which would contribute to its iron regulatory effect. Finally, we further explored the mechanisms underlying the regulatory effect of hepcidin on the iron transporters in the last part, and found that the action of hepcidin in reducing TfR1 expression is a direct and cAMP-PKA (Cyclic Adenosine 3', 5'-monophosphate/ Protein Kinase-A) pathway-dependent event. / Iron is a transition trace metal essential for mammalian cellular and tissue viability. It also plays important roles in the central nervous system (CNS), including embryonic brain development, myelination, and neurotransmitters synthesis. However, abnormal iron accumulation has been demonstrated in a number of neurodegenerative diseases (NDs) such as Parkinson's (PD), Alzheimer's (AD) and Huntington's diseases (HD). Currently very little is known about the mechanisms involved in brain iron homeostasis and therefore it is not known why and how iron is abnormally increased in the brain. However, given the essential role that excess iron plays in the pathological processes in the NDs, to suppress the accumulated iron is expected to be an effective strategy to prevent and treat these NDs. / To investigate whether hepcidin could benefit iron-associated NDs including PD and whether this beneficial role is related to its iron-regulatory function in the brain, in the first part of study, we investigated the effects of hepcidin on the 6-hydroxydopamine (6-OHDA) in vitro and in vivo PD models. We found that in primary cultured mesencephalic (MES) neurons, hepcidin overexpression via adenovirus-hepcidin (Ad-hepcidin) infection prevented 6-OHDA-induced increase in cellular iron content and protected the MES neurons. In the 6-OHDA model of PD in vivo, overexpression of hepcidin in the substantia nigra via Ad-hepcidin intranigral injection significantly prevented iron accumulation and dopaminergic neurons loss in the pars compacta of substantia nigra (SNc). These effects were accompanied by a marked improvement in motor performance of the PD animals. These findings indicate that hepcidin could benefit iron-associated NDs such as PD and via its iron-regulatory role in the brain. / Du, Fang. / Adviser: Ya Ke. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 152-173). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Li, Sui Mui. / On t.p. "beta" appears as the Greek letter. / Thesis submitted in: December 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 182-189). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese) --- p.iii / Acknowledgements --- p.v / Table of contents --- p.vi / List of figures --- p.xii / List of appendices --- p.xix / Abbreviations --- p.xx / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Role of PPARP in adipocyte differentiation - an in vitro study --- p.20 / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Materials and Methods --- p.23 / Chapter 2.2.1 --- Preparation ofPPARβ (+/+) and PPARβ (-/-) MEFs --- p.23 / Chapter 2.2.1.1 --- Materials --- p.23 / Chapter 2.2.1.2 --- Methods --- p.23 / Chapter 2.2.1.2.1 --- Isolation of MEFs --- p.23 / Chapter 2.2.1.2.2 --- Passage ofMEF culture --- p.25 / Chapter 2.2.2 --- Genotyping of PPARβ (+/+) and PPARβ (-/-) MEFs --- p.25 / Chapter 2.2.2.1 --- Materials --- p.26 / Chapter 2.2.2.2 --- Methods --- p.26 / Chapter 2.2.2.2.1 --- Primer design --- p.26 / Chapter 2.2.2.2.2 --- Genomic DNA extraction --- p.27 / Chapter 2.2.2.2.3 --- PCR reaction --- p.29 / Chapter 2.2.3 --- Western blotting of PPARβ(+/+) and PPARβ (-/-) MEFs --- p.30 / Chapter 2.2.3.1 --- Materials --- p.30 / Chapter 2.2.3.2 --- Methods --- p.31 / Chapter 2.2.3.2.1 --- Preparation of nuclear extracts --- p.31 / Chapter 2.2.3.2.2 --- Western blot --- p.32 / Chapter 2.2.4 --- Induction of adipocyte differentiation of PPARβ (+/+) and PPARβ(-/-) MEFs --- p.33 / Chapter 2.2.4.1 --- Materials --- p.34 / Chapter 2.2.4.2 --- Methods --- p.34 / Chapter 2.2.4.2.1 --- Seeding ofMEFs --- p.34 / Chapter 2.2.4.2.2 --- Adipocyte differentiation --- p.35 / Chapter 2.2.5 --- Oil Red O staining of differentiated PPARβ(+/+) and PPARβ(-/-) MEFs --- p.36 / Chapter 2.2.5.1 --- Materials --- p.36 / Chapter 2.2.5.2 --- Method --- p.37 / Chapter 2.2.5.2.1 --- Oil Red O staining --- p.37 / Chapter 2.2.6 --- Determination of triglyceride-protein assay of differentiated PPARβ (+/+) and PPARβ (-/-) MEFs --- p.37 / Chapter 2.2.6.1 --- Materials --- p.39 / Chapter 2.2.6.2 --- Methods --- p.39 / Chapter 2.2.6.2.1 --- Lysis of differentiated MEFs --- p.39 / Chapter 2.2.6.2.2 --- Measurement of triglyceride concentration in cell lysate --- p.40 / Chapter 2.2.6.2.3 --- Measurement of protein concentration in cell lysate --- p.41 / Chapter 2.2.7 --- Preparation of PPARβ(+/+) and PPARβ (-/-) MEF RNA for RT-PCR and Northern blot analysis --- p.42 / Chapter 2.2.7.1 --- Materials --- p.42 / Chapter 2.2.7.2 --- Method --- p.42 / Chapter 2.2.7.2.1 --- RNA isolation --- p.42 / Chapter 2.2.8 --- RT-PCR analysis of differentiated PPARβ(+/+) and PPARβ (-/-) MEFs --- p.44 / Chapter 2.2.8.1 --- Materials --- p.45 / Chapter 2.2.8.2 --- Methods --- p.45 / Chapter 2.2.8.2.1 --- Primer design --- p.45 / Chapter 2.2.8.2.2 --- RT-PCR --- p.46 / Chapter 2.2.9 --- Northern blot analysis of differentiated PPARβ(+/+) and PPARβ (-/-) MEFs --- p.47 / Chapter 2.2.9.1 --- Materials --- p.48 / Chapter 2.2.9.2 --- Methods --- p.49 / Chapter 2.2.9.2.1 --- Preparation of cDNA probes for Northern blotting --- p.49 / Chapter 2.2.9.2.1.1 --- RNA extraction --- p.49 / Chapter 2.2.9.2.1.2 --- Primer design --- p.49 / Chapter 2.2.9.2.1.3 --- RT-PCR of extracted mRNA --- p.50 / Chapter 2.2.9.2.1.4 --- Subcloning of amplified cDNA products --- p.50 / Chapter 2.2.9.2.1.5 --- Screening of recombinant clones by phenol-chloroform extraction --- p.51 / Chapter 2.2.9.2.1.6 --- Confirmation of the recombinant clones by restriction enzyme site mapping --- p.52 / Chapter 2.2.9.2.1.7 --- Confirmation of the recombinant clones by PCR method --- p.52 / Chapter 2.2.9.2.1.8 --- Mini-preparation of plasmid DNA from the selected recombinant clones --- p.54 / Chapter 2.2.9.2.1.9 --- Preparation of cDNA probes --- p.54 / Chapter 2.2.9.2.1.10 --- Formaldehyde agarose gel electrophoresis of RNA --- p.55 / Chapter 2.2.9.2.1.11 --- Hybridization and color development --- p.56 / Chapter 2.3 --- Results --- p.58 / Chapter 2.3.1 --- Confirmation of PPARβ(+/+) and PPARβ (-/-) MEFs genotypes --- p.58 / Chapter 2.3.2 --- PPARβ (-/-) MEFs differentiated similarly to PPARβ(+/+) MEFs as measured by Oil Red O staining --- p.61 / Chapter 2.3.3 --- PPARβ (-/-) MEFs differentiated similarly to PPARβ(+/+) MEFs as reflected by their intracellular triglyceride contents --- p.64 / Chapter 2.3.4 --- PPARβ(-/-) MEFs expressed the adipocyte differentiation marker genes similarly to PPARβ (+/+) MEFs --- p.66 / Chapter 2.4 --- Discussion --- p.77 / Chapter Chapter 3 --- Role of PPARβ in adipocyte differentiation and lipid homeostasis - an in vivo study --- p.82 / Chapter 3.1 --- Introduction --- p.83 / Chapter 3.2 --- Materials and Methods --- p.85 / Chapter 3.2.1 --- Animal and high fat diet treatment --- p.85 / Chapter 3.2.1.1 --- Materials --- p.85 / Chapter 3.2.1.2 --- Method --- p.86 / Chapter 3.2.1.2.1 --- Animal treatment --- p.86 / Chapter 3.2.2 --- Tail-genotyping of PPARβ (+/+) and PPARβ (-/-) mice --- p.87 / Chapter 3.2.2.1 --- Materials --- p.87 / Chapter 3.2.2.2 --- Methods --- p.88 / Chapter 3.2.2.2.1 --- DNA extraction from tail --- p.88 / Chapter 3.2.2.2.2 --- PCR tail-genotyping --- p.89 / Chapter 3.2.3 --- "Measurement of serum triglyceride, cholesterol and glucose levels by enzymatic and spectrophometric methods" --- p.89 / Chapter 3.2.3.1 --- Materials --- p.90 / Chapter 3.2.3.2 --- Methods --- p.91 / Chapter 3.2.3.2.1 --- Serum preparation --- p.91 / Chapter 3.2.3.2.2 --- Measurement of serum triglycerides --- p.91 / Chapter 3.2.3.2.3 --- Measurement of serum cholesterol --- p.92 / Chapter 3.2.3.2.3 --- Measurement of serum glucose --- p.93 / Chapter 3.2.4 --- Measurement of serum insulin and leptin levels by ELISA --- p.94 / Chapter 3.2.4.1 --- Materials --- p.95 / Chapter 3.2.4.2 --- Methods --- p.95 / Chapter 3.2.4.2.1 --- Measurement of serum insulin --- p.95 / Chapter 3.2.4.2.2 --- Measurement of serum leptin --- p.97 / Chapter 3.2.5 --- "Histological studies of liver, interscapular BF and gonadal WF pads" --- p.99 / Chapter 3.2.5.1 --- Materials --- p.100 / Chapter 3.2.5.2 --- Methods --- p.100 / Chapter 3.2.5.2.1 --- "Fixation, dehydration, embedding in paraffin and sectioning" --- p.100 / Chapter 3.2.5.2.2 --- H&E staining --- p.101 / Chapter 3.2.6 --- Analyses of fecal lipid contents --- p.102 / Chapter 3.2.6.1 --- Materials --- p.102 / Chapter 3.2.6.2 --- Method --- p.103 / Chapter 3.2.6.2.1 --- Extraction of lipid contents from stools --- p.103 / Chapter 3.2.7 --- Statistical analysis --- p.104 / Chapter 3.3 --- Results --- p.105 / Chapter 3.3.1 --- Confirmation of genotypes by PCR --- p.105 / Chapter 3.3.2 --- PPARβ (-/-) mice were more resistant to high fat diet-induced obesity --- p.105 / Chapter 3.3.3 --- PPARβ (-/-) mice consumed similarly as to PPARβ (+/+) counterparts… --- p.122 / Chapter 3.3.4 --- Effect of high fat diet on organ weights --- p.128 / Chapter 3.3.4.1 --- PPARβ (-/-) mice were more resistant to high fat diet-induced liver hepatomegaly --- p.134 / Chapter 3.3.4.2 --- PPARβ (-/-) mice were resistant to high fat diet-induced increased white fat depots --- p.134 / Chapter 3.3.4.3 --- PPARβ (-/-) mice were resistant to high fat diet-induced increased brown fat mass --- p.137 / Chapter 3.3.5 --- Effect of high fat diet on organ histology --- p.142 / Chapter 3.3.5.1 --- PPARβ(-/-) mice were more resistant to high fat diet-induced liver steatosis --- p.143 / Chapter 3.3.5.2 --- No defect in white adipocyte expansion in PPARβ(-/-) mice upon high fat diet feeding --- p.153 / Chapter 3.3.5.3 --- No defect in brown adipocyte expansion in PPARβ (-/-) mice upon high fat diet feeding --- p.159 / Chapter 3.3.6 --- "Effect on high fat diet on serum cholesterol, triglyceride, glucose, insulin and leptin levels" --- p.164 / Chapter 3.3.6.1 --- "PPARβ (-/-) mice had a lower serum cholesterol level, but a similar triglyceride level as compared to PPARβ (+/+) mice upon high fat diet feeding" --- p.165 / Chapter 3.3.6.2 --- PPARβ (-/-) mice were resistant to high fat diet-induced insulin resistance --- p.167 / Chapter 3.3.6.3 --- PPARβ (-/-) mice had a similar serum leptin level as PPARβ (+/+) mice --- p.170 / Chapter 3.3.7 --- No decision made in fecal lipid content of PPARβ (+/+) and PPARβ (-/-) mice --- p.173 / Chapter 3.4 --- Discussion --- p.176 / References --- p.182 / Appendices --- p.190

This dissertation is an initial study of how modern engineering control may be applied to reverse engineer homeostasis in metabolic pathways using high-throughput biological data. This attempt to reconcile differences between engineering control and biological homeostasis from an interdisciplinary perspective is motivated not only by the observation that robust behavior in metabolic pathways resembles stabilized dynamics in controlled systems, but also by the challenges forewarned in achieving a true meeting of minds between engineers and biologists. To do this, a comparator model is developed and applied to model the effect of single-gene (SPT) overexpression on C16:0 sphingolipid de novo biosynthesis in vitro, specifically to simulate and predict potential homeostatic pathway interactions between the sphingolipid metabolites. Sphingolipid de novo biosynthesis is highly regulated because its pathway intermediates are highly bioactive. Alterations in sphingolipid synthesis, storage, and metabolism are implicated in human diseases. In addition, when variation in structure is considered, sphingolipids are one of the most diverse and complex families of biomolecules. To complete the modeling paradigm, wild type cells are defi ned as the reference that exhibits the "desired" pathway dynamics that the treated cells approach. Key model results show that the proposed modern engineering control approach using a comparator to reverse engineer homeostasis in metabolic systems is: (a) eff ective in capturing observed pathway dynamics from experimental data, with no signifi cant di fference in precision from existing models, (b) robust to potential errors in estimating state-space parameters as a result of sparse data, (c) generalizable to model other metabolic systems, as demonstrated by testing on a separate independent dataset, and (d) biologically relevant in terms of predicting steady-state feedback as a result of homeostasis that is verifi ed in literature and with additional independent data from drug dosage experiments.

The mammalian brain is the most cholesterol-rich organ of the body, requiring in situ de novo cholesterol synthesis to maintain its cholesterol requirement. Defects in brain cholesterol homeostasis are implicated in cognitive deficits related to aging and in neurodegenerative diseases such as Alzheimer's Disease (AD). Oxysterol-binding protein (OSBP) - related proteins are highly conserved cytosolic proteins that coordinate lipid homeostasis by regulating cell signaling, inter-organelle membrane contact sites and non-vesicular transport of cholesterol. Previously, ORP6, a poorly characterized member of this family, was found to be part of complex transcriptional cascade coordinated by SBREP2 and emerged as a novel regulator of intracellular cholesterol trafficking in hepatocytes and macrophages. Yet how ORP6 regulates these pathways and its function in the brain where it is most highly expressed is unknown. Here, we show that ORP6 is highly expressed in the brain, where it exhibits spatial and cell-type specific expression. ORP6 expression is enriched in the hippocampus and caudal-putamen brain regions, specifically within neurons and astrocytes. ORP6 knockdown in astrocytes altered the expression of cholesterol biosynthesis, cholesterol efflux and cholesterol esterification genes, resulting in the accumulation of esterified cholesterol within cytoplasmic lipid droplets and reduced cholesterol efflux highlighting a role for ORP6 in astrocytic cholesterol metabolism. We also present in this thesis, the newly generated second viable ORP family member knockout mouse. ORP6 ablation in mice results in the dysregulation of brain and whole-body lipid homeostasis, increased Aβ deposition in the brain and neuroanatomical alterations. Together, our findings highlight a critical role for cholesterol trafficking proteins in brain cholesterol homeostasis and identify ORP6 as a potential novel target for AD.

D.Diac. (Play Therapy) / Teachers in South Africa experience strain and tension, unique to their specific work description, which is evident in the career-related stress symptoms experienced by the individual teachers, the high rate of absenteeism amongst teachers, as well as the high attrition rate. Although factors causing teacher-stress, and the consequences thereof, have been extensively researched, a limited number of empirical evaluations of the effectiveness and accessibility of stress-management programmes have been conducted. Due to the holistic nature of Gestalt therapy, its emphasis on the here-and-now and the Gestalt principles of awareness, dialogue and process, a Gestalt therapeutic process model was developed to empower teachers to regain homeostasis. The aim and objectives of the research were the design, development, presentation and evaluation of a Gestalt therapeutic process model for teachers suffering from career-related stress, in their quest to regain homeostasis. The model was developed to be implemented within the school environment by a trained member of the school management team. The process of intervention research was used for the research study. A functional Gestalt therapeutic programme, based on the theoretical Gestalt therapeutic model, was developed and presented to ten teachers, selected through purposive sampling, and again to five different teachers, selected through theoretical sampling. The teachers identified were representative regarding age, gender, race and years in education. Triangulation was used and qualitative and quantitative data were collected simultaneously. The hypothesis stated for the research was that if teachers, suffering from career-related stress, were exposed to a Gestalt therapeutic model, they would regain homeostasis. Both the qualitative and quantitative data supported the hypothesis. The effect of the variables on each other was compared to confirm the reliability, applicability and neutrality of the research data. At the end of the three month research period the teachers who were exposed to the said model experienced less stress-related symptoms, as well as growth towards maturity and self-support, which would ultimately result in the regaining of homeostasis. A further objective of the research was to determine the feasibility of a trained school management team member implementing the Gestalt therapeutic process model at school. The qualitative data collected, indicated the feasibility thereof on condition that the school management team member did receive the necessary Gestalt therapeutic training. / Social Work

Wong Yin-Mei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 119-135). / Abstracts in English and Chinese. / Abstract (English version) --- p.i / Abstract (Chinese version) --- p.iii / Publications --- p.v / Acknowledgements --- p.vi / Table of contents --- p.viii / List of tables --- p.xi / List of figures --- p.xii / Abbreviations --- p.xiv / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Osteoporosis --- p.1 / Chapter 1.1.1 --- Consensus statement --- p.1 / Chapter 1.1.2 --- Epidemiology and outcomes --- p.4 / Chapter 1.1.2.1 --- Hip fractures --- p.4 / Chapter 1.1.2.2 --- Vertebral fractures --- p.5 / Chapter 1.1.2.3 --- Wrist fractures --- p.7 / Chapter 1.1.3 --- Postmenopausal osteoporosis --- p.8 / Chapter 1.1.3.1 --- Pathogenesis --- p.8 / Chapter 1.1.3.1.1 --- Genetics --- p.11 / Chapter 1.1.3.1.2 --- Bone remodeling --- p.14 / Chapter 1.1.3.1.3 --- Calcium homeostasis --- p.21 / Chapter 1.1.3.1.4 --- Life style 一 nutrition and exercise --- p.26 / Chapter 1.1.3.2 --- Current pharmacological treatment --- p.27 / Chapter 1.1.3.2.1 --- Introduction --- p.27 / Chapter 1.1.3.2.2 --- Limitations --- p.31 / Chapter 1.2 --- Traditional Chinese medicine --- p.33 / Chapter 1.2.1 --- The Kidney --- p.33 / Chapter 1.2.2 --- Kidney-tonifying herbs --- p.33 / Chapter 1.3 --- Aim of the studies --- p.36 / Chapter Chapter 2. --- Materials and methods --- p.38 / Chapter 2.1 --- Kidney-tonifying herbs and herbal formula --- p.38 / Chapter 2.1.1 --- Sources --- p.38 / Chapter 2.1.2 --- Herbal extract preparation --- p.38 / Chapter 2.2 --- Animal study --- p.40 / Chapter 2.2.1 --- Reagents --- p.40 / Chapter 2.2.2 --- Animal care --- p.40 / Chapter 2.2.3 --- Herbs and herbal formula preparations for animal studies --- p.41 / Chapter 2.2.4 --- Experimental design --- p.41 / Chapter 2.2.5 --- Gene expression study --- p.44 / Chapter 2.2.5.1 --- Tissue preparation --- p.44 / Chapter 2.2.5.2 --- Isolation of total RNA --- p.45 / Chapter 2.2.5.3 --- Complementary DNA synthesis --- p.47 / Chapter 2.2.5.4 --- Real-time polymerase chain reaction analysis --- p.47 / Chapter 2.3 --- Cell culture study --- p.49 / Chapter 2.3.1 --- Reagents --- p.49 / Chapter 2.3.2 --- Cell lines --- p.49 / Chapter 2.3.2.1 --- "Rat osteosarcoma cell line, UMR-106" --- p.49 / Chapter 2.3.2.2 --- "Human breast cancer cell line, MCF-7" --- p.50 / Chapter 2.3.2.3 --- Cell culture techniques --- p.50 / Chapter 2.3.3 --- Herbs preparations for cell culture --- p.51 / Chapter 2.3.4 --- Cell viability assay --- p.51 / Chapter 2.3.5 --- Cellular alkaline phosphatase activity assay --- p.52 / Chapter 2.3.6 --- Matrix mineralization assay --- p.54 / Chapter 2.3.7 --- Competitive estrogen receptor binding assay --- p.56 / Chapter 2.4 --- Statistical analyses --- p.58 / Chapter Chapter 3. --- Results --- p.59 / Chapter 3.1 --- Extraction yields of Kidney-tonifying herbs and herbal formula --- p.59 / Chapter 3.2 --- Effects of Kidney-tonifying herbs and herbal formula on the gene expressions of calcium absorption and reabsorption related genes --- p.61 / Chapter 3.2.1 --- Gene expression of 25-hydroxyvitamin D3-1 alpha-hydroxylasein the kidney --- p.62 / Chapter 3.2.2 --- Gene expression of vitamin D receptor in the duodenum --- p.65 / Chapter 3.2.3 --- Gene expression of calbindin D9K in the duodenum --- p.67 / Chapter 3.2.4 --- Gene expression of vitamin D receptor in the kidney --- p.69 / Chapter 3.2.5 --- Gene expression of calbindin D28K in the kidney --- p.71 / Chapter 3.3 --- Effects of Kidney-tonifying herbs on osteoblastic UMR-106 cell line --- p.73 / Chapter 3.3.1 --- Effects of Kidney-tonifying herbs on the cell viability of UMR-106 cells --- p.73 / Chapter 3.3.2 --- Effects of Kidney-tonifying herbs on the osteoblastic differentiation of UMR-106 cells --- p.76 / Chapter 3.3.2.1 --- Cellular alkaline phosphatase activity --- p.76 / Chapter 3.3.2.2 --- Degree of matrix mineralization --- p.80 / Chapter 3.4 --- Estrogen receptor binding activities of Kidney-tonifying herbs --- p.85 / Chapter Chapter 4. --- Discussion --- p.89 / Chapter 4.1 --- Safety of Kidney-tonifying herbs and herbal formula --- p.89 / Chapter 4.2 --- Kidney-tonifying herbs and herbal formula preserve bone mineral density --- p.93 / Chapter 4.3 --- Kidney-tonifying herbs and herbal formula modulate calcium homeostasis --- p.97 / Chapter 4.3.1 --- "Roles in renal synthesis of the hormonally active form of vitamin D: 1,25-dihydroxyvitamin D3" --- p.97 / Chapter 4.3.2 --- Roles in calcium absorption in the duodenum --- p.99 / Chapter 4.3.3 --- Roles in calcium reabsorption in the kidney --- p.102 / Chapter 4.3.4 --- Summary --- p.104 / Chapter 4.4 --- Kidney-tonifying herbs modulate bone formation --- p.106 / Chapter 4.4.1 --- Effects on osteoblast proliferation --- p.106 / Chapter 4.4.2 --- Effects on osteoblastic differentiation --- p.107 / Chapter 4.4.3 --- Summary --- p.108 / Chapter 4.5 --- Kidney-tonifying herbs interact with estrogen receptor --- p.110 / Chapter 4.6 --- Active ingredients of Kidney-tonifying herbs --- p.111 / Chapter 4.7 --- Limitations of the present studies --- p.115 / Chapter 4.8 --- Conclusion and future prospect --- p.117 / References --- p.119

D.Diac. (Play Therapy) / Teachers in South Africa experience strain and tension, unique to their specific work description, which is evident in the career-related stress symptoms experienced by the individual teachers, the high rate of absenteeism amongst teachers, as well as the high attrition rate. Although factors causing teacher-stress, and the consequences thereof, have been extensively researched, a limited number of empirical evaluations of the effectiveness and accessibility of stress-management programmes have been conducted. Due to the holistic nature of Gestalt therapy, its emphasis on the here-and-now and the Gestalt principles of awareness, dialogue and process, a Gestalt therapeutic process model was developed to empower teachers to regain homeostasis. The aim and objectives of the research were the design, development, presentation and evaluation of a Gestalt therapeutic process model for teachers suffering from career-related stress, in their quest to regain homeostasis. The model was developed to be implemented within the school environment by a trained member of the school management team. The process of intervention research was used for the research study. A functional Gestalt therapeutic programme, based on the theoretical Gestalt therapeutic model, was developed and presented to ten teachers, selected through purposive sampling, and again to five different teachers, selected through theoretical sampling. The teachers identified were representative regarding age, gender, race and years in education. Triangulation was used and qualitative and quantitative data were collected simultaneously. The hypothesis stated for the research was that if teachers, suffering from career-related stress, were exposed to a Gestalt therapeutic model, they would regain homeostasis. Both the qualitative and quantitative data supported the hypothesis. The effect of the variables on each other was compared to confirm the reliability, applicability and neutrality of the research data. At the end of the three month research period the teachers who were exposed to the said model experienced less stress-related symptoms, as well as growth towards maturity and self-support, which would ultimately result in the regaining of homeostasis. A further objective of the research was to determine the feasibility of a trained school management team member implementing the Gestalt therapeutic process model at school. The qualitative data collected, indicated the feasibility thereof on condition that the school management team member did receive the necessary Gestalt therapeutic training. / Social Work