UCP1 is a member of the mitochondrial transmembrane anion carrier protein superfamily and is required to mediate adaptive thermogenesis in brown adipose tissue (BAT). Once activated, UCP1 uncouples mitochondrial respiration from ATP synthesis, thereby wasting the protonmotive force formed across the mitochondrial inner membrane as heat. It is hypothesized that proton leaks through UCP1 could be a molecular target to combat certain forms of obesity. Although it is well established that UCP1 is regulated by allosteric mechanisms, alternative methods such as post-translational modification still remain to be explored. The aims of the present study were to confirm the phosphorylation of UCP1 and the physiological relevance of this modification. Using isoelectric focusing, we confirmed that UCP1 displayed acidic shifts consistent with phosphorylation in BAT mitochondria isolated from cold exposed versus warm acclimated mice. A mouse model that ectopically expressed UCP1 in skeletal muscle was used to explore the link between the mitochondrial redox status and UCP1 function. Our results show that the expression of UCP1 in skeletal muscle led to decreases in body and tissues weights. In contrast, glucose uptake into skeletal muscle, food intake and energy expenditure was increased with the expression of UCP1. Finally, proton leaks through UCP1 were determined to be increased in isolated mitochondria from transgenic versus wild-type mice. Taken together these results indicate a complex interplay between mitochondrial redox status, post-translational modification and UCP1 function. Elucidation of novel mechanisms regulating UCP1 offers alternatives strategies that can be explored in order to modulate BAT thermogenesis.

Aerobic exercise (AE) may interfere with muscle adaptations induced by resistance exercise (RE). Three experimental campaigns were conducted to explore the influence of AE on molecular, functional and muscular adaptations to acute and chronic RE. Twenty-nine men performed unilateral knee extensor RE preceded by AE (AE+RE). The contralateral leg did RE only. First, the influence of acute AE on muscle molecular responses to RE performed 6 h later was studied. Subsequently, this exercise regimen was implemented over 5 weeks training. The relationships between acute and chronic outcomes were examined and molecular responses to acute exercise were assessed in untrained and trained muscle. Finally, acute and chronic responses to AE+RE, interspersed by only 15 min recovery, were investigated.Phosphorylation of mTOR and p70S6K was greater after AE+RE than after RE. In parallel, myostatin was suppressed for a longer time after AE+RE. These results suggest that AE+RE enhance skeletal muscle anabolic environment more than RE alone (Paper I). After 5 weeks training, improvements in muscle strength and power were similar across legs. However, AE+RE prompted a greater increase in muscle size than RE, suggesting that AE potentiates the hypertrophic stimulus to RE training without altering muscle function progress (Paper II). Consistent with changes in whole-muscle size, AE+RE showed greater anabolic molecular responses than RE. As chronic training blunted this effect, it appears that AE offers a synergistic hypertrophic stimulus to RE only during short-term training (Paper III). Although putative regulators of hypertrophy such as p70S6K, myostatin and PGC-1a4 were examined, no molecular marker correlated with changes in muscle size, strength or power induced by training. Hence, this study challenges the concept that single molecular markers are viable predictors of training-induced muscle adaptations (Paper III–IV). When recovery time between exercise bouts was reduced to 15 min, AE+RE still produced a more substantial increase in muscle size than RE. However, progression of concentric strength was blunted. Thus, while restored muscle function between exercise bouts is a prerequisite for achieving maximal gains in strength and power, incomplete recovery appears not to compromise muscle hypertrophy (Paper V).Collectively, the results suggest that outcomes of AE+RE are impacted by chronic training and time allowed for recovery between exercise modes. Yet, the current study offers no support to the view that AE interferes with muscle hypertrophy induced by RE.

Le séquençage intégral du génome de Mycobacterium tuberculosis a permis de mettre en évidence l'existence de onze Sérine/Thréonine Protéine-Kinases (STPKs) chez cette bactérie. Bien que la quasi-totalité des STPKs aient été biochimiquement caractérisées, très peu de substrats endogènes ont pu être identifiés. Par conséquent, le rôle physiologique de ces couples kinase/substrat reste à élucider. Tout d'abord, les études réalisées au cours de ce travail ont concerné la caractérisation biochimique de la protéine-kinase PknL, ainsi que l'identification de ses substrats potentiels, et notamment la protéine Rv2175c. En effet, l'analyse de l'environnement génétique du gène pknL de la kinase a révélé la présence du gène adjacent rv2175c, pouvant ainsi représenter un substrat éventuel de PknL. Les différentes approches mises en oeuvre ont permis d'identifier cinq sites de phosphorylation sur PknL, et de mettre en évidence le caractère essentiel des résidus K48, T173 et T175 dans les mécanismes d'autophosphorylation de PknL et de phosphorylation de Rv2175c, confirmant ainsi Rv2175c comme substrat spécifique de PknL. Par ailleurs, la caractérisation par RMN de la structure de Rv2175c a permis de déterminer la fonction de cette protéine. Rv2175c possède toutes les caractéristiques structurales d'une protéine capable de fixer l'ADN. Des études fonctionnelles ont permis de confirmer la capacité de Rv2175c de fixer l'ADN et ont mis en évidence le mécanisme de régulation via phosphorylation régissant son activité de fixation. Ensuite, nous avons mis en évidence la phosphorylation des protéines chaperonnes mycobactériennes et, plus particulièrement, caractérisé GroEL1. Nous avons démontré que GroEL1 était phosphorylée par PknF, et identifié les résidus T25 et T54 comme étant les sites de phosphorylation de GroEL1. L'ensemble de cette étude nous a donc permis de caractériser de nouveaux substrats de phosphorylation chez M. tuberculosis, de mieux appréhender les interactions kinase/substrat et d'impliquer la phosphorylation dans la régulation de l'activité de ces substrats

Proteins play important roles in most cellular functions, such as, replication, transcription regulation, signal transduction, for catalyzing chemical reaction, etc. Technologies developed to identify proteins rely either on observing their own properties such as charge, size, mass to charge ratio or sequence composition; or on using affinity reagents that recognize specific protein targets. Immunoassays utilizing functionalized affinity reagents are powerful for targeted proteomics. Among them, DNA-assisted immunoassays in which affinity reagents are labeled with DNA molecules, offer some unique advantages. In this thesis, I will present works to improve current DNA-assisted immunoassays such as proximity ligation assays (PLA), as well as to take advantage of DNA reactions to adress other problems. In paper I, a new solid support (MBC-Ts) was functionalized with antibodies and used in the solid-phase PLA for detection of VEGF. The assay using MBC-Ts was compared among the commercially available solid supports in different matrices and it was shown to exhibit enhanced limit of detection in complex matrices. In paper II, a two-step protocol was described to prepare high-quality probes used in homogeneous and in situ PLA by purifying DNA-labeled affinity reagents from unconjugated affinity reagents and excess oligonucleotides. In paper III, PLA was applied on a capillary western blotting instrument so that both the sensitivity and specificity of the original assay were improved. In paper IV, a new method was introduced to profile protein components in individual protein complexes by DNA-barcoded antibodies. This method has been used to profile protein complexes such as surface proteins on individual secreted vesicles.

Perilla L. are significant for multi-pharmacological effect. The aim of this work is to study Perilla L. growth and develop¬ment tendencies, productivity; composition of biologically active compounds and their variations during the vegetation period, and the biological effect of the extracts; to select perspective plants for cultivation in Lithuania and for production of medicinal preparations. The objectives of the Study: To investigate and determine growth dynamics of Perilla L. species and varieties during the vegetation period and to assess the influence of climate conditions on the vegetation rhythmic. To assess the quantity dynamics of Perilla L. species and varieties herbal medicinal raw material and to compare the productivity of Perilla L. species and varieties. To determine the quantity of the essential oil in Perilla L. species and varieties and to identify the chemotypes of the plants. To determine the qualitative composition and variation dynamics of the phenolic acids in Perilla L. species and varieties. To determine the composition and variation dynamics of the flavone complex in Perilla L. species and varieties. To determine the composition and variation dynamics of antho¬cyanidines in Perilla L. species and varieties. To assess the radical scavenging activity of the extracts of Perilla L. species and varieties raw materials. To research the effect of Perilla L. extracts on the oxidative phosphorylation in the rat heart mitochondria. / Perilla L. genties vienmečiai vaistiniai augalai yra augaliniai imunomoduliatoriai, pasižymintys daugeliu farmakologinių poveikių. Darbo tikslas: Ištirti Vidurio Lietuvoje auginamų Perilla L. rūšių ir varietetų augimo ir vystymosi dėsningumus, biologiškai aktyvių junginių sudėtį ir jų įvai¬ravimą vegetacijos metu bei ekstraktų biologinį poveikį; atrinkti perspek¬tyvius augalus auginimui Lietuvoje. Uždaviniai: Ištirti ir nustatyti Perilla L. augimo dinamiką vegetacijos metu ir įvertinti klimatinių veiksnių įtaką augalų vegetacijai. Įvertinti Perilla L. vaistinės augalinės žaliavos kiekio dinamiką vege¬tacijos metu ir palyginti Perilla L. rūšių ir varietetų produk¬tyvumą. Nustatyti Perilla L. rūšių ir varietetų eterinio aliejaus kiekį auga¬luose vegetacijos metu ir identifikuoti augalų chemotipus. Nustatyti Perilla L. rūšių ir varietetų fenolinių rūgščių kokybinę sudėtį ir jų kitimo dėsningumus vegetacijos metu. Nustatyti Perilla L. rūšių ir varietetų flavonų komplekso sudėtį ir kitimo dinamiką vegetacijos metu. Nustatyti Perilla L. rūšių ir varietetų antocianidinų sudėtį bei kitimo dinamiką vegetacijos metu. Įvertinti Perilla L. rūšių ir varietetų žaliavų ekstraktų antiradikalinį aktyvumą. Ištirti Perilla L. rūšių ir varietetų ekstraktų poveikį žiurkės širdies mitochondrijų oksidaciniam fosforilinimui. Tyrimų rezultatai ir poveikiu pasižyminčių junginių identifikavimas atveria perspektyvas ateities tyrimams, kurie reikalingi kuriant preparatus iš perilių augalinių žaliavų.

Vieni populiariausių vaistinių preparatų Europoje yra dviskiaučių ginkmedžių (Ginkgo biloba L.) lapų preparatai. Nors Ginkgo biloba lapų ekstraktai pasižymi plačiu farmakologiniu pritaikymu, tačiau nėra daug duomenų apie jų poveikį mitochondrijoms. Todėl darbo tikslas buvo ištirti dviskiaučių ginkmedžių lapų ekstraktų poveikį žiurkės širdies ir kepenų mitochondrijų oksidacinio fosforilinimo sistemai. Darbo uždaviniai: 1. Ištirti dviskiaučių ginkmedžių lapų ekstraktų poveikį žiurkės širdies permeabilizuotų skaidulų bei izoliuotų širdies ir kepenų mitochondrijų kvėpavimui. 2. Ištirti dviskiaučių ginkmedžių lapų tinktūros poveikio oksidaciniam fosforilinimui širdies mitochondrijose mechanizmą. 3. Įvertinti dviskiaučių ginkmedžių lapų tinktūros poveikį izoliuotos žiurkės širdies elektromechaniniam aktyvumui ir nustatyti kaip greit tinktūros komponentai patenka į ląstelę ir mitochondrijas. 4. Ištirti ar dviskiaučių ginkmedžių lapų tinktūra apsaugo širdies mitochondrijas nuo žalingo išemijos/reperfuzijos poveikio. 5. Ištirti dviskiaučių ginkmedžių lapų tinktūros poveikį mitochondrijų oksidacinio fosforilinimo sistemai in vivo, įvedant ją per os. / Ginkgo biloba-derived preparations have become widely used in medical practice. Thougt extracts of Ginkgo biloba leaves have a wide pharmacological application, little is known about extract effects on mitochondria. Therefore, the aim of this study was to investigate the influence of extracts of Ginkgo biloba leaves on mitochondrial oxidative phosphorylation system. The tasks of the study were following: 1. To investigate the effects of extract of Ginkgo biloba leaves on the respiration of isolated heart and liver mitochondria and permeabilized heart fibers. 2. To analyze the mechanism(s) of extract of Ginkgo biloba leaves on mitochondrial oxidative phosphorylation system. 3. To determine the effect of extract of Ginkgo biloba leaves on perfused rat heart electromechanical activity and to analyze how GBE given to isolated perfused rat hearts readily can penetrate into the heart cells and mitochondria. 4. To test whether perfusion with Ginkgo biloba extract protects heart mitochondria against ischemia/reperfusion damage. 5. To investigate the effect of extract of Ginkgo biloba leaves on mitochondrial oxidative phosphorylation system in vivo.

Seed formation consists of two major stages: embryo pattern formation and maturation. During seed maturation, the embryo accumulates storage material, acquires desiccation tolerance, and enters a stage of dormancy. Genetic analyses have identified several master regulators that orchestrate late embryogenesis, including the B3-domain transcription factor FUSCA3 (FUS3). In Arabidopsis, FUS3 has been shown to be a central regulator of hormonal pathways; it positively regulates late embryogenesis by increasing abscisic acid (ABA) level while repressing gibberellin (GA) synthesis. In turn, FUS3 protein level is positively and negatively regulated by ABA and GA, respectively. However, the mechanism of how this regulation occurs has not been well characterized. In this study, FUS3 has been shown to be an unstable protein rapidly degraded by the proteasome through a PEST instablility motif. To further characterize the mechanisms involved in FUS3 homeostasis, FUS3-interacting proteins were identified. The SnRK1 kinase AKIN10 was shown to interact with and phosphorylate FUS3 at its N-terminus. Furthermore, overexpression of AKIN10 delays FUS3 degradation, suggesting AKIN10 positively regulates FUS3 protein accumulation. Overexpression of AKIN10 delays developmental phase transitions, and causes defects in lateral organ development. These defects were partially rescued by the loss-of-function fus3-3 mutation, suggesting FUS3 and AKIN10 genetically interact to regulate these developmental processes. SnRK1/AMPK/Snf1 kinases are regulators of energetic stress responses. Overexpression studies suggest both FUS3 and AKIN10 positively regulate ABA signaling, but differ in sugar responses during germination; AKIN10 mediates glucose sensitivity, while FUS3 regulates osmotic stress responses. Overexpression of AKIN10 and FUS3 results in glucose and osmotic stress hypersensitivities, respectively, both of which are partially dependent on de novo ABA synthesis. Thus, FUS3 and AKIN10 act in overlapping pathways and combine different environmental signals to generate a common ABA-dependent response. In summary, novel mechanisms that regulate FUS3 homeostasis and function were identified. A model explaining the interaction between FUS3 and AKIN10 during embryonic and vegetative development, and the function of these two central developmental regulators in hormonal and stress signaling pathways is discussed.

Seed formation consists of two major stages: embryo pattern formation and maturation. During seed maturation, the embryo accumulates storage material, acquires desiccation tolerance, and enters a stage of dormancy. Genetic analyses have identified several master regulators that orchestrate late embryogenesis, including the B3-domain transcription factor FUSCA3 (FUS3). In Arabidopsis, FUS3 has been shown to be a central regulator of hormonal pathways; it positively regulates late embryogenesis by increasing abscisic acid (ABA) level while repressing gibberellin (GA) synthesis. In turn, FUS3 protein level is positively and negatively regulated by ABA and GA, respectively. However, the mechanism of how this regulation occurs has not been well characterized. In this study, FUS3 has been shown to be an unstable protein rapidly degraded by the proteasome through a PEST instablility motif. To further characterize the mechanisms involved in FUS3 homeostasis, FUS3-interacting proteins were identified. The SnRK1 kinase AKIN10 was shown to interact with and phosphorylate FUS3 at its N-terminus. Furthermore, overexpression of AKIN10 delays FUS3 degradation, suggesting AKIN10 positively regulates FUS3 protein accumulation. Overexpression of AKIN10 delays developmental phase transitions, and causes defects in lateral organ development. These defects were partially rescued by the loss-of-function fus3-3 mutation, suggesting FUS3 and AKIN10 genetically interact to regulate these developmental processes. SnRK1/AMPK/Snf1 kinases are regulators of energetic stress responses. Overexpression studies suggest both FUS3 and AKIN10 positively regulate ABA signaling, but differ in sugar responses during germination; AKIN10 mediates glucose sensitivity, while FUS3 regulates osmotic stress responses. Overexpression of AKIN10 and FUS3 results in glucose and osmotic stress hypersensitivities, respectively, both of which are partially dependent on de novo ABA synthesis. Thus, FUS3 and AKIN10 act in overlapping pathways and combine different environmental signals to generate a common ABA-dependent response. In summary, novel mechanisms that regulate FUS3 homeostasis and function were identified. A model explaining the interaction between FUS3 and AKIN10 during embryonic and vegetative development, and the function of these two central developmental regulators in hormonal and stress signaling pathways is discussed.

Muscle may experience hypoglycemia during ischemia or insulin infusion. During severe hypoglycemia energy production is blocked and an increase in AMP:ATP activates the energy sensor and putative insulin-sensitizer AMP-dependent protein kinase (AMPK). AMPK promotes energy conservation and survival by shutting down anabolism and activating catabolic pathways. We investigated the molecular mechanism of a unique glucose stress defense pathway involving AMPK-dependent, insulin-independent activation of the insulin signaling pathway. Results from my work showed that the central insulin signaling pathway is rapidly activated when cardiac and skeletal myocytes are subjected to conditions of glucose starvation. The effect occurred independently of insulin receptor ligands (insulin and IGF-1). There was a >10-fold increase in the activity of Akt as determined by phosphorylation on both Thr308 and Ser473. Phosphorylation of glycogen synthase 3 beta (GSK3b) increased in parallel, but phosphorylation of ribosomal 70S subunit-S6 protein kinase (S6K) and the mammalian target of rapamycin complex 1 (mTORC1) decreased. We identified AMPK as an intermediate in this signaling network; AMPK was activated by glucose starvation and many of the effects were mimicked by the AMPK-selective activator aminoimidazole carboxamide ribonucleotide (AICAR) and blocked by AMPK inhibitors. Glucose starvation increased the phosphorylation on IRS-1 on Ser789, but phosphomimetics revealed that this conferred negative regulation. Glucose starvation enhanced tyrosine phosphorylation of IRS-1 and the insulin receptor, effects that were blocked by AMPK inhibition and mimicked by AICAR. In vitro kinase assays using purified proteins confirmed that the insulin receptor is a direct target of AMPK. Insulin receptor kinase activity was essential for cardiac myocytes to survive gluose starvation as inhibition of the IR led to increased cell death in glucose-starved myocytes. Selective activation of mTORC2 by glucose starvation to increase Akt-Ser473 phosphorylation was dependent on the presence of rictor. SIN1 also seemed to be instrumental in the activation of mTORC2 as its levels and binding to rictor increased under glucose starvation. AMPK-mediated activation of the insulin signaling pathway conferred significant protection against the stresses of glucose starvation. Glucose starvation promoted energy conservation, augmented glucose uptake and enhanced insulin sensitivity in an AMPK- and Akt-dependent manner. My results describe a novel ligand-independent and AMPK-dependent activation of the insulin signaling pathway via direct phosphorylation and activation of the IR followed by activation of PI3K and Akt. These results may be relevant in conditions of myocardial ischemia superimposed with type 2 diabetes where AMPK could directly modify the IR to promote cell survival and confer protection.

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