Heat stress (HS) causes significant losses to the U.S. swine industry in several production and health areas including efficient lean tissue accretion. Perturbations in skeletal muscle metabolism may participate in this defect. The study objectives were to examine the cellular bioenergetic profile in skeletal muscle of piglets subjected to thermal stress in utero and/or during postnatal life. To accomplish this, 96 offspring from 14 sows were prenatally exposed to 1 of 4 environmental treatments involving thermal neutral (TN, 25°C) or HS conditions (cyclical 28-34°C). Sows exposed to TN or HS throughout gestation are denoted TNTN and HSHS, respectively whereas sows heat-stressed for the first or second half of gestation are denoted HSTN and TNHS, respectively. At 14 weeks of age, offspring were exposed to one of two postnatal thermal environments, constant TN (21°C) or HS (35°C) for 24 hrs (acute study) or 5 weeks (chronic study). Pigs were sacrificed after treatment and longissimus dorsi skeletal muscle samples collected for molecular analyses. Differences (p<0.05) were observed in protein abundance of p-4eBP1 and total Rs6 and gene expression of Cox5B, CytB, EEF2, HK2, MURF, ND1, PGC-1α, SDHA, and TFAM during the acute heat stress study. Differences (p<0.05) were observed in protein abundance of 4eBP1, total Akt, and p-Rs6 and gene expression of CytB, MURF, and PGC-1α during the chronic heat stress study. These data indicate that acute postnatal HS alters skeletal muscle metabolism, which may favor a reduction in mitochondrial respiration and protein synthesis potentially via the mTOR pathway. / Master of Science

Master of Science / Department of Animal Sciences and Industry / Evan C. Titgemeyer / Two experiments were conducted to determine effects of postruminal amino acid (AA) supplementation on protein deposition and signaling of the mammalian target of rapamycin (mTOR) pathway. For both experiments, 7 ruminally cannulated Holstein steers (172.7 ± 3.7 and 201.7 ± 3.8 kg initial BW in Exp. 1 and 2, respectively) were utilized in 6  6 Latin square designs with 7 d periods. A basal AA solution containing all essential AA, with the exception of lysine, were provided to all steers in each study in order to meet growth requirements, while making lysine the only limiting AA. Steers were fed 2.8 kg/d of a pelleted soyhull diet designed to be low in ruminally undegradable protein. Glucose was infused abomasally and volatile fatty acids were infused ruminally to prevent energy from being limiting. Steers were housed in metabolism crates to obtain total collection of both urine and feces. Blood and muscle biopsies of the longissimus lumborum were collected on the last day of each period. In experiment 1, treatments consisted of 2 levels of lysine (0 or 6 g/d) and 3 levels of leucine (0, 15, or 30 g/d) infused abomasally. Nitrogen retention increased with supplemental lysine. Leucine linearly decreased plasma concentrations of total AA. Plasma urea N (PUN) decreased with supplemental lysine. Total, phosphorylated, and the percent phosphorylated Akt were unaffected by treatments. The percentage of 4E-BP1 phosphorylated decreased linearly when leucine was supplemented. A tendency for a lysine x quadratic leucine effect was observed for the ratio of phosphorylated RPS6²⁴⁰/²⁴⁴ in which the intermediate level of leucine led to a decrease in the percent of RPS6²⁴⁰/²⁴⁴ phosphorylated when no lysine was supplied but increased when 6 g lysine/d was supplied. No differences were observed in the abundance of total, phosphorylated, or percent phosphorylated mTOR or in total abundance of E3 ubiquitin ligase proteins, MuRF1 or MAFbx. Experiment 2 was conducted similarly as experiment 1. Treatments consisted of 2 levels of lysine (0 or 6 g/d) and 3 mixtures of supplemental essential AA [none (control), 103 g/d essential AA (EAA), or EAA plus 30 g/d leucine (EL)] abomasally infused. Supplementation with essential AA, with or without leucine, increased the percentage of RPS6 phosphorylated, with a greater increase when leucine was included as part of the supplement. A lysine x (control vs. EAA+EL) interaction was observed for N retention in which the EAA and EL treatments did not improve N retention when no lysine was supplemented, but they increased it when 6 g lysine/d was provided. PUN increased above control when EAA or EL was provided, but PUN decreased when lysine was supplied. Supplementation of EAA or EL increased plasma total AA concentrations, but EL led to lower total plasma AA than EAA; however, concentrations were greater for EL than for control. In summary, leucine supplementation alone did not yield effects on whole-body protein deposition or on regulatory factors known to affect muscle protein synthesis, whereas a mixture of excess essential AA improved both lysine utilization and phosphorylation of RPS6²⁴⁰/²⁴⁴. These studies demonstrate the effects of essential AA, both limiting and nonlimiting, on protein deposition in growing cattle.

The PI3K/Akt/mTOR and MAPK pathways are frequently altered in ovarian cancer cells, making them potential candidates for targeted therapy. A more complete understanding of the complex interactions between the different proteins within the two pathways would assist in developing more effective treatment strategies to help overcome therapy resistance. The purpose of this project was to understand the phosphoproteomic changes in response to PI3K/mTOR inhibition in ovarian cancer cells and to identify potential mechanisms that may lead to targeted therapy resistance. To investigate the effect of inhibiting PI3K/mTOR at the cellular level in ovarian cancer, PI3K (LY294002), mTOR (rapamycin) and dual PI3K/mTOR (BEZ235) inhibitors were used to treat a panel of ovarian cancer cell lines. All tested cells, irrespective of PI3K/Akt/mTOR and MAPK pathways mutational status, responded to the three inhibitors. BEZ235 treatment produced greater cell inhibition than the monotargeted agents, while PTENmutated cell lines were more responsive to mTOR blockade than inhibition of PI3K alone. The phosphoproteomic changes in the cell lines were evaluated over a time course after treatment with the inhibitors, stimulated by heregulin, and studied using reverse phase protein array analysis. The results revealed that the decreased expression of pAkt (Thr308) appears to be a biomarker of sensitivity for LY294002 and BEZ235 in both PEO4 and A2780 cells, while upregulation of pAkt (Ser473) is an indicator for effective rapamycin treatment within the same cell lines. Increased pAkt (Ser473) expression after rapamycin treatment in PEO4 cells is believed to be due to the S6K1-mTORC2-Akt feedback loop. It was observed that pERK was upregulated upon BEZ235 treatment, which suggested the presence of cross talk between the PI3K/Akt/mTOR and MAPK pathways. A combination of BEZ235 and PD-0325901 (MEK inhibitor) treatments inhibited both pAkt (Ser473) and pERK, which also produced significant inhibition in cell proliferation compared to monotherapy treatment. The data also revealed a novel finding in ovarian cancer that prolonged (24h) treatment with rapamycin sensitises mTORC2 in PEO4 cells under heregulin stimulation. Moreover, network correlation and clustering analysis using the phosphoproteomic data identified significant correlations between the expression of pmTOR (Ser2481), and both p-cRaf (Ser259 and Ser338). Sin1 knockdown was performed in PEO4 cells and showed significant downregulation in the expression of pAkt (Ser473) and upregulation in pERK expression, indicating the role of Sin1 to regulate both the PI3K/Akt/mTOR and MAPK pathways potentially via mTORC2 and Ras. Phosphoproteomic profiling was performed on 469 ovarian cancer tissue samples using TMA and IHC analysis. Several significant associations were discovered between the phosphoproteomic data and the different clinicopathological parameters. High expression of pmTOR (Ser2448) was correlated with poorer overall survival in patients with ovarian endometrioid carcinoma compared to patients with low expression (p < 0.024). This implies that pmTOR (Ser2448) expression may potentially be a prognostic marker for patients with ovarian endometrioid carcinoma. In conclusion, I present dynamic phosphoproteomic profiling of the PI3K/Akt/mTOR and MAPK pathways in ovarian cancer, suggesting novel feedback loops and cross talk that could play a role in resistance mechanisms to these therapies. Combination treatment showed an additive effect on cell growth offering an approach to overcome drug resistance.

Recent research suggests that [beta]-hydroxy-[beta]-methylbutyrate (HMB), a metabolite of leucine (Leu), increases muscle mass and attenuates muscle damage during resistance training. Although Leu acts as a potent stimulator of protein synthesis, HMB, but not Leu, has been reported to be effective in suppressing proteolysis in skeletal muscle. However, mechanisms for the effects of HMB on cell signaling pathways controlling muscle protein turnover during rest and after endurance exercise are still poorly understood. Furthermore, the effects of HMB on cell signaling pathways controlling protein synthesis and degradation under normal in vivo conditions warrant further investigation. For optimal gains in muscle mass, the appropriate type and amount of protein (PRO) is required for positive protein balance to occur in skeletal muscle. Therefore, this dissertation was designed to determine the effect of HMB, PRO and Leu, individually and in combination, on the regulation of cellular signaling pathways controlling muscle protein turnover during sedentary and post-exercise conditions. Study 1 demonstrated that, compared with HMB and PRO alone, the combination of HMB and PRO was more effective in activating the mTOR signaling pathway, which controls protein synthesis, and inhibiting FOXO3A, a major regulator of the ubiquitin-proteasome proteolytic signaling pathway. Study 2 demonstrated that, compared with its individual components, a novel HMB/PRO/Leu supplement better activated protein-synthetic signals and inhibited proteolytic signals in skeletal muscle, and these effects were better sustained. Finally, Study 3 demonstrated that adding Leu to PRO-enriched mixtures after exercise additively activated protein-synthetic signals in a fiber type-specific manner, and adding HMB clearly inhibited proteolytic signaling proteins. Furthermore, provision of an HMB/PRO/Leu supplement after exercise was found to favorably modulate signaling pathways controlling both protein synthesis and degradation. Taken together, the results of these studies suggest that a novel nutrient supplement, composed of HMB, Leu and PRO, additively enhances the intracellular signaling proteins controlling protein synthesis and attenuates signaling proteins controlling proteolysis in skeletal muscle during sedentary and post-exercise recovery. Therefore, such a supplement may be beneficial for both athletic and therapeutic purposes. / text

Traditionally, drug discovery programs have focused on prioritising compounds by their affinity to a specific target in isolation, which was hypothesised to be the cause of a particular disease. Through chemical inhibition, the disease could, thus, be prevented or at the very least, controlled. These hypotheses require significant validation before drug screening can begin which relates to lengthy and expensive programs. Furthermore, drug screening against a single target in isolation is not a realistic model of cellular behaviour and is not appropriately tailored to more complex diseases such as cancer. Phenotypic drug discovery, on the other hand, bypasses any involvement of known targets, instead focusing on the desired outcome – the phenotype. In this way, drugs are biased by their potency on the phenotype and not against any particular targets. The molecular mechanism of action need not be known at all, however, it can be useful to later reveal the target(s) involved by various deconvolution methods. This thesis describes a cooperative ligand based phenotypic drug discovery approach, undertaken in order to develop more suitable small molecule drugs for cancer treatment. For this purpose, the promiscuous pyrazolopyrimidine inhibitor PP1 was chosen as a starting model compound. Modification of PP1 on the N1 position allowed a series of water solubilising groups to be incorporated into the pyrazolopyrimidine scaffold which created an initial 12-membered library. Testing against MCF7 breast cancer cells and looking at phenotypic end points such as cell proliferation, cell mobility and cell cycle, generated early target-agnostic structure/anti-proliferative activity relationships. These early results, along with compounds published in recent literature, were used to generate further libraries. Profiling lead compounds against a selection of 18 kinases known to be targeted by PP1, showed the compounds were inhibiting either SRC family or mTOR kinases which enabled the creation of two, structure specific, groups of inhibitors. Further lead optimisation led to the rapid discovery of preclinical candidates with excellent drug-like properties and potencies in both cellular assays and against their respective targets. Compounds also showed improved selectivity profiles compared to PP1 and commonly known inhibitors of SRC and mTOR kinases. Reported, herein, is the discovery of the first sub-nanomolar SRC inhibitor which does not inhibit the kinase ABL and shows excellent properties suitable for further preclinical development.

Positionnée en aval des voies PI3K/AKT et RAS/MAPK, la protéine kinase mTOR joue un rôle déterminant dans le développement et la progression tumorale des cancers colorectaux où elle est fortement surexprimée. Par ailleurs, les cancers colorectaux comme toutes les tumeurs solides, ont un microenvironnement hypoxique. L’adaptation des cellules tumorales à l’hypoxie est notamment régulée par la voie PI3K/AKT/mTOR ainsi que par les facteurs de transcription HIFs dont l’expression protéique et l’activité transcriptionnelle est en partie régulée par mTOR. Dans cette étude, nous avons montré que l’inhibition verticale et complète de l’axe PI3K/AKT/mTOR/HIF-1α par l’utilisation combinée d’irinotecan à faible dose et d’inhibiteurs catalytiques de mTOR inhibe significativement la prolifération cellulaire de lignées coliques humaines, la croissance tumorale et le développement de métastases de xénogreffes de tumeurs coliques dérivées de patients.En parallèle, une étude de cohorte de tumeurs coliques humaines de stade III par Tissue Micro Array montre que les facteurs HIFs sont fortement exprimés dans l’épithélium et le stroma de cancers du côlon de stade III, qu’une faible expression nucléaire de HIF-1α dans les cellules épithéliales confère une mauvaise survie aux patients et qu’elle a une valeur prédictive de moins bonne réponse au traitement 5-FU. / Downstream of the PI3K/AKT and RAS/MAPK pathways, mTOR protein kinase plays a decisive role in the development and tumor progression of colorectal cancers. Furthermore, the microenvironment of colorectal cancers is hypoxic. The adaptation of the tumor cells to hypoxia is regulated by the PI3K/AKT /mTOR pathway as well as by the HIFs transcription factors whose protein expression and transcriptional activity is partially regulated by mTOR. In this study, we showed that the vertical and complete inhibition of the PI3K / AKT / mTOR /HIF-1α axis by the combined use of low-dose irinotecan and mTOR catalytic inhibitors significantly inhibits human colon cancer cell proliferation, as well as the growth and metastatic development of xenografted human colon tumors. In parallel, a Tissue Micro Array study on a cohort of stage III human colic tumors shows that the HIFs are strongly expressed in the epithelium and stroma of the tumors and a low nuclear expression of HIF-1α in epithelial cells provides with poor survival to patients and has a predictive value of worse response to 5-FU treatment.

Natural killer (NK) cells were so named for their uniqueness in killing certain tumor and virus-infected cells without prior sensitization unlike T lymphocytes. NK cells possess a myriad of activation receptors and cytokine receptors that allow them to recognize stress ligands on infected/tumor cells and respond to the cytokines produced during the inflammatory process. Upon activation, NK cells produce pro-inflammatory cytokines, cytotoxic granules and chemokines to recruit other cells which ultimately result in killing of target cells. These functions of NK cells are modulated in vivo by several immune mediators; IL-15 being the most potent in enabling NK cell homeostasis, maturation and activation. Indeed, IL-15 knockout mice have no detectable NK cells. During microbial infections, NK cells stimulated with IL-15 display enhanced cytokine responses. This priming effect has previously been shown with respect to increased IFN-γ production in NK cells upon IL-12 and IL-15/IL-2 co-stimulation. In this study, I explored if this effect of IL-15 priming can be extended to other cytokines and observed enhanced NK cell responses to stimulation with IFN-, IL-21, IL-2 and IL-4 in addition to IL-12. Notably, we also observed elevated IFN-γ production in primed NK cells upon stimulation through the Ly49H activation receptor. IL-15 treatments induced NK cell proliferation, enhanced NK cell responses to activating stimuli and equipped them with cytotoxic granules thereby “readying” them for battle against infections and tumors. Here, we try to understand the signaling mechanisms underlying IL-15 treatments that activate NK cells. Currently, the fundamental processes required for priming and whether these signaling pathways work collaboratively or independently for NK cell functions are poorly understood. To identify the key signaling events, we examined IL-15 priming on NK cells in which the pathways emanating from IL-15 receptor activation were blocked with specific inhibitors. Our results demonstrate that the PI3K-AKT-mTOR pathway is indispensable for cytokine responses in IL-15 primed NK cells. Furthermore, this pathway is also implicated in a broad range of IL-15 induced NK cell effector functions such as proliferation and cytotoxicity. Given that NK cells are critical for control of viral infections like murine cytomegalovirus (MCMV), we decided to analyze the consequences of blocking the PI3K-AKT-mTOR pathway in NK cells on its anti-viral responses. Likewise, NK cells from mice treated with rapamycin to block the mTOR pathway displayed defects in proliferation, IFN-γ and granzyme B production resulting in elevated viral burdens upon MCMV infection. Taken together, our data demonstrates the requirement of PI3K-mTOR pathway for enhanced NK cell functions by IL-15. It also shows that IL-15 primes NK cell responses to several cytokines and to Ly49H activation receptor stimulation. To our knowledge this is first report to demonstrate the requirement of mTOR activity in NK cells for efficient control of acute MCMV infections; thereby coupling the metabolic sensor mTOR to NK cell anti-viral responses.

Acute myeloid leukemia (AML) is a hematological disorder characterized by blockage of differentiation of myeloblasts. To date, the main therapy for AML is chemotherapy. Yet, studies are seeking a better treatment to enhance the survival rate of patients and minimize the relapsing of the disease. Since the major problem in these cells is that they are arrested in cellular differentiation, drugs that could induce their differentiation have proven to be efficient and of major interest for AML therapy. CD44 triggering appeared as a promising target for AML therapy as it has been shown that specific monoclonal antibodies, such as A3D8 and H90, reversed the blockage of differentiation, inhibited the proliferation of all AML subtypes, and in some cases, induced cell apoptosis. Studies conducted in our laboratory have added strength to these antibodies as potential treatment for AML. Indeed, our laboratory found that treating HL60 cells with A3D8 shows a decrease in the phosphorylation of the mammalian target of Rapamycin (mTOR) kinase correlated with the inhibition of proliferation/induction of differentiation of AML cells.The relationship between the induction of differentiation and the inhibition of proliferation and the decrease of mTOR phosphorylation remains to be clarified. To study the importance of the de-phosphorylation of mTOR and the observed effect of CD44 triggering on differentiation and/or proliferation, we sought to prepare phospho-mimic mutants of the mTOR kinase that will code for a constitutively phosphorylated form of mTOR and used two main methods to express this mutant in HL60 cells: lentiviral and simple transfection (cationic-liposomal transfection).

Indiana University-Purdue University Indianapolis (IUPUI) / Key regulatory decisions of protein synthesis and autophagy are controlled by the lysosomal nutrient sensing complex (LYNUS). To engage protein synthesis signaling, LYNUS requires cellular availability of amino acids, adenosine triphosphate (ATP), growth factors, and docking at the lysosomal membrane. The molecular determinants of LYNUS signaling and docking are not completely elucidated and may involve regulators of the lipid membrane structure and function of the lysosome. Since ceramides are both bioactive second messengers and determinants of lipid membrane stiffness, we investigated the role of the ceramide-producing lysosomal acid sphingomyelinase (ASM) in the homeostatic function of mammalian target of rapamycin (mTOR) signaling and autophagy. Using ASM inhibition with either imipramine or siRNA against SMPD1, in primary human lung cells or Smpd1+/- mice, we demonstrated that ASM is an endogenous inhibitor of autophagy. ASM was necessary for physiological mTOR signaling and maintenance of sphingosine levels. Whereas overstimulation of ASM has been shown to trigger autophagy with impaired flux, inhibition of ASM activity during homeostatic, non-stressed conditions triggered autophagy with degradative potential, associated with enhanced transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis genes, translocation to the nucleus and decreased sphingosine levels. These findings suggest LYNUS signaling and autophagy are partially regulated by ASM.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19227号 / 医博第4026号 / 新制||医||1011(附属図書館) / 32226 / 京都大学大学院医学研究科医学専攻 / (主査)教授 小川 誠司, 教授 坂井 義治, 教授 松原 和夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM