Abstract The polyamines putrescine, spermidine and spermine are highly regulated polycations present in virtually all cells of higher eukaryotes. They are essential for proper cell growth and differentiation by participating in various physiological processes including DNA, RNA and protein synthesis, apoptosis and interactions with ion-channels. The complexity of polyamine metabolism and the multitude of compensatory mechanisms that are invoked to maintain polyamine homeostasis argue that these molecules are critical for cell survival. The primary aim of this study was to gain a better understanding of the mode of action of polyamines and the regulatory mechanisms in which they are involved. Transgenic mice overexpressing the polyamine biosynthetic enzymes S-AdoMetDC and ODC were found to maintain their polyamine pools by acetylation of spermidine and spermine and an increased export of these acetylated compounds. The expression of various genes was studied as a response to polyamine deprivation in cell- and kidney organ culture. Among these genes acetyl-CoA synthetase and ornithine decarboxylase were demonstrated to be developmentally regulated. Changes in gene expression patterns, with most of the transcripts upregulated in the polyamine-depleted samples, indicated selective stabilization of mRNAs. Polyamines were shown to play an important role in kidney organogenesis as their depletion results in a reduction of ureteric branching and retardation of tubule formation. The selective changes of various genes in the ureteric bud and mesenchyme indicate that polyamines might have a role in the regulation of epithelial-mesenchymal interactions during mouse kidney development.

Potassium ions are the most abundant cation and potassium transport is essential in maintaining cellular homeostasis through the regulation of cell turgor and cytoplasmic pH. It allows bacteria to grow and survive, therefore, the potassium pool needs to be strictly controlled, which is mainly performed by transport systems that contain a KTN domain. The potassium efflux system, Kef, is such a KTN-bearing system and it is widespread among Gram negative bacteria. The system provides protection against harmful electrophiles through cytoplasmic acidification. Kef is a glutathione-regulated protein: it is inhibited by glutathione (GSH), but it becomes activated by binding glutathione-S-conjugates (GSX), that are formed in the presence of electrophiles. GSH or GSX are bound in the same pocket that is located in a cytosolic regulatory domain which controls the K+ flux. Previous studies already showed that bacterial growth is inhibited when the gating of Kef is manipulated, which makes Kef a potential target for developing novel antibacterial drugs. Structure-Function studies have already lead to a better understanding of the regulation of potassium efflux activity, but no quantitative analyses had been performed until now. A simplified model Kef system (SdKef) is presented and a novel assay was developed that provided new insights into the structural components necessary for the gating of Kef. This assay makes the search for modulators of Kef, and therefore potential novel antibacterial drugs, more easily accessible. Another objective was to identify the nucleotide(s) bound and to determine its role in controlling the Kef system. This nucleotide was identified as AMP which is essential for stability of the Kef system.

Memoria para optar al Titulo Profesional de Médico Veterinario / El cultivo de células C2C12 de músculo esquelético responde a la depolarización de membrana producida por estimulación eléctrica, generando una señal lenta de calcio. Esto se traduce en un aumento de la concentración de calcio en el núcleo mediada por el receptor de Inositol 1, 4, 5 - trifosfato (IP3R), activando factores transcripcionales y regulando la expresión de ciertos genes tempranos. Esta Memoria de Título relaciona la síntesis proteica en cultivo celular C2C12 con la generación de la señal lenta de calcio intracelular inducida por estimulación eléctrica. En el desarrollo de esta investigación se utilizaron cultivo celular C2C12 diferenciados a miotubos, estimulación eléctrica directa y electroforesis bidimensional en geles de poliacrilamida (2D-PAGE). El análisis de los geles se realizó mediante el programa Flicker; y el análisis estadístico (t de Student con un 95% de confianza) mediante el programa StatGraphics Plus 5.1TM. La identificación de las manchas de proteína de realizó mediante espectrometría de masas (MS) y búsqueda en bases de datos específicas (MASCOT, SwissProt, MS – Fit y ProFound). Los resultados obtenidos permitieron detectar cambios significativos en la expresión proteica de células musculares C2C12 sometidas a estimulación eléctrica. La proteína expresada diferencialmente fue la Hsp70 o proteína de estrés calórico de 70KDa, la que aumentó su expresión en respuesta al estrés producido por la estimulación eléctrica. Esta proteína cumple un importante rol en el metabolismo proteico, facilita la regeneración y reparación del músculo esquelético dañado, previene la agregación y la apoptosis celular y favorece la mantención de la homeostasis en células musculares. / Fondecyt no. 1061154, Centro FONDAP de Estudios Moleculares de la Célula

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21992号 / 医博第4506号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 坂井 義治, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / Many signaling pathways which are classically understood for their roles in early development are also known to be involved in tissue maintenance and adult energy homeostasis. Furthermore, dysfunction of these signaling pathways results in human diseases such as cancer. An in depth understanding of how developmentally important signaling pathways function in the adult will provide mechanistic insights into disease and potential new therapeutic targets. Here in Chapter 1, the Wnt, fibroblast growth factor (FGF), and Hedgehog (Hh) signaling pathways are discussed and examples of their relevance in development, adult homeostasis, and disease are provided. Wnt signaling provides an example of this concept as it has well described roles during both development and adult metabolism. Work included in Chapter 2, investigates the regulation of adult energy homeostasis by a member of the endocrine FGF family, FGF19. The three endocrine FGFs, FGF19 (FGF15 in mice), FGF21, and FGF23 have well described roles in the regulation of metabolic processes in adults. While FGF23 is primarily involved in the regulation of phosphate and vitamin D homeostasis, FGF19 and FGF21 have shown similar pharmacological effects on whole body metabolism. Here, the importance of adaptive thermogenesis for the pharmacological action of FGF19 is explored. Using UCP1KO animals we show that whole-body thermogenesis is dispensable for body weight loss following FGF19 treatment. Finally, the potential involvement of Hh signaling in mediating the hyperphagia driven obesity observed in certain ciliopathies is explored in Chapter 3. Emerging evidence suggests cilia play an important role in the regulation of feeding behavior. In mammals, the hedgehog pathway is dependent on the primary cilium as an organizing center and defects in hedgehog signaling share some clinical symptoms of ciliopathies. Here, we characterized the expression of core pathway components in the adult hypothalamus. We show that neurons within specific nuclei important for regulation of feeding behavior express Hh ligand and members of its signaling pathway. We also demonstrate that the Hh pathway is transcriptionally upregulated in response to an overnight fast. This work provides an important foundation for understanding the functional role of Hh signaling in regulation of energy homeostasis. In its entirety, this work highlights the emerging clinical relevance of developmentally critical pathways in diseases associated with dysfunction of adult tissue homeostasis, such as obesity.

Au cours de ma thèse, j'ai eu plusieurs projets différents portant sur l'importance de l’homéostasie énergétique chez Drosophila melanogaster. L'homéostasie énergétique est le maintien constant des réserves énergétiques de la cellule. Mon projet principal de thèse porte sur le lien entre le métabolisme et la croissance dépendante des voies mTOR (mechanistic Target-Of-Rapamycin) chez Drosophila melanogaster. Il existe deux voies mTOR très conservées au cours de l’évolution : la voie mTORC1 et la voie mTORC2. Les voies mTOR sont des voies de signalisation qui activent à la fois la croissance et le métabolisme. Cependant les effets de l’activation des voies mTOR sur le métabolisme invivo sont encore à l’étude. De plus, on ne sait pas si l’activation du métabolisme est nécessaire à la croissance dépendante des voies mTOR. J’ai montré que l’activation des voies mTOR in vivo diminue le stockage des réserves énergétiques (tréhalose,glycogène) lors d'une alimentation sucrée.L'activation de la voie mTORC1 diminue aussi le stockage des triglycérides en milieu sucré. En utilisant des outils génétiques, j’ai également montré que la croissance dépendante de la voie mTORC2 dépend au niveau autonome cellulaire de la synthèse d’acides gras, de l’activité pyruvate déshydrogénase et de l’activité lactate déshydrogénase. De plus, la croissance induite par mTORC2 est inhibée par le sucre alimentaire. La croissance induite par l’activation de la voie mTORC1 ne dépend ni du métabolisme du glucose, ni de celui des acides gras.Mais elle diminue si on combine une inhibition de la synthèse des acides gras avec un ajout de sucres alimentaires. J’ai également contribué à deux autres projets au cours de ma thèse : un qui étudie l’importance de l’homéostasie énergétique dans le déclenchement de l’autophagie, et un autre qui étudie l’importance du métabolisme des acides gras dans la physiologie de l’intestin. / The main topic of my PhD is theimportance of energy homeostasis in Drosophilamelanogaster. Energy homeostasis is a biologicalprocess that holds energy stores at a steady state. Mymain PhD topic investigates the importance ofmetabolic homeostasis during growth in Drosophilamelanogaster. I focused on growth induced by themTOR (mechanistic Target-Of-Rapamycin)pathways. There are two mTOR pathways : themTORC1 and mTORC2 pathway. They are veryconserved throughout evolution. The mTORpathways are signaling pathways that activate bothgrowth and metabolism. Howevever the effects ofmTOR activation on metabolism in vivo is still underinvestigation. Moreover, it is not known ifmetabolism activation is necessary to sustain mTORdependent growth. I showed that the activationof the mTOR pathways in vivo decreases energystorage (trehalose, glycogen) during a dietsupplemented with sugar. Moreover, the activation ofthe mTORC1 pathway decreases triglyceride storageduring this diet. Using genetic tools, I also showedthat mTORC2-dependent growth on the autonomouscellular level depends on fatty acid synthesis,pyruvate dehydrogenase and lactate dehydrogenase.Moreover, mTORC2-dependent growth is inhibitedby dietary sugar. mTORC1-dependent growth is notsensitive to glucose or fatty acid metabolism alone.However, mTORC1-dependent growth decreaseswhen fatty acid synthesis inhibition is combined withan increase in dietary sugar. I also contributed to twoother projects : one investigating the effect of energyhomeostasis on autophagy onset and an otherinvestigating the effect of fatty acid metabolism ongut physiology.

Tesis presentada a la Universidad de Chile para optar al grado de Doctor en Farmacología / El músculo esquelético es un tejido esencial en la mantención de la homeostasis de la glucosa en el organismo. Alteraciones en la respuesta a la hormona insulina en este tejido se relacionan directamente con el desarrollo de resistencia a insulina y diabetes tipo 2. Los mecanismos moleculares involucrados en el desarrollo de resistencia a insulina en el músculo esquelético no se conocen con claridad. En este sentido, trabajos previos de nuestro laboratorio muestran que las alteraciones en la homeostasis del calcio (Ca2+) intracelular disminuyen la respuesta a insulina en el músculo esquelético y cardíaco. HERPUD1 es una proteína ubicada en la membrana del retículo endoplasmático (RE), involucrada en la mantención de la homeostasis del Ca2+ intracelular en condiciones de estrés celular. Se ha descrito que el ratón knockout general para la proteína Herpud1 es intolerante a la glucosa, sin mostrar alteraciones en la secreción de insulina. Dado que el músculo esquelético es principal encargado de la incorporación de glucosa dependiente de insulina en condiciones postprandiales, en esta tesis proponemos que la proteína Herpud1 es necesaria para la adecuada respuesta a insulina en el músculo esquelético. Proponemos además que Herpud1 regula la respuesta a insulina a través de la mantención de la homeostasis del Ca2+ intracelular y su efecto sobre la actividad de la serina-treonina fosfatasa activada por Ca2+ calcineurina. Previamente se ha descrito que esta fosfatasa interactúa con Akt, disminuyendo la señalización de insulina en cardiomiocitos. Los resultados obtenidos en este trabajo muestran que el silenciamiento de la proteína Herpud1 disminuye la captación de glucosa, la translocación del transportador de glucosa GLUT4 hacia la membrana plasmática y la señalización inducida por insulina en la línea celular de músculo esquelético de rata L6 (miotubos). También observamos una disminución en la fosforilación de Akt (Ser- 473) inducida por insulina en el músculo sóleo del ratón knockout general para la proteína Herpud1. El silenciamiento de Herpud1 también se asocia a un aumento en la respuesta de Ca2+ citosólico y a una disminución en la respuesta de Ca2+ mitocondrial dependientes del IP3R en los miotubos L6. Este desbalance en la respuesta de Ca2+ intracelular fue acompañado de un aumento en la actividad de calcineurina. Demostramos además que calcineurina regula la respuesta a insulina en los miotubos L6, y que la inhibición de calcineurina restablece la respuesta a insulina en las células L6 knockdown para Herpud1. Basado en estos resultados, concluimos que la proteína Herpud1 es necesaria para la respuesta a insulina en los miotubos L6 a través de la regulación del eje Ca2+-calcineurina / Skeletal muscle is an essential tissue in the maintenance of glucose homeostasis in the body. Alterations in the response to insulin in skeletal muscle are directly related to the development of insulin resistance and type 2 diabetes. The molecular mechanisms involved in the development of insulin resistance in this tissue are incompletely understood. In this sense, previous works from our laboratory show that alterations in intracellular calcium (Ca⁠2+) homeostasis decreases the insulin response in skeletal and cardiac muscle. Herpud1 is a protein located in the membrane of endoplasmic reticulum (ER), involved in the maintenance of intracellular Ca⁠2+ homeostasis under stress conditions. It has been reported that the general Herpud1 knockout mice display intolerance to a glucose load without showing altered insulin secretion. Given that skeletal muscle is primarily responsible for insulin-dependent glucose incorporation in postprandial conditions, in this thesis we propose that Herpud1 is necessary for the adequate insulin response in this tissue. We also propose that Herpud1 regulates the insulin response through maintenance of intracellular Ca⁠2+ homeostasis and its effect on the activity of the serine-threonine phosphatase activated by Ca⁠2+ calcineurin. Previously it has been described that calcineurin interacts with Akt, decreasing insulin signaling in cardiomyocytes The results obtained in this work show that the silencing of Herpud1 decreases glucose uptake, GLUT4 translocation to plasma membrane and insulin-dependent intracellular signaling in rat derived skeletal muscle cell line L6 (myotubes). We also observed a decrease in insulin-induced Akt (Ser⁠-473) phosphorylation in soleus muscle from general Herpud1-knockout mice. The silencing of Herpud1 is also associated with an increase in the cytosolic Ca⁠2+ response and a decrease in the mitochondrial Ca⁠2+ response induced by IP3R agonist histamine in L6 myotubes. This imbalance in the intracellular Ca⁠2+ response was accompanied by an increase in calcineurin activity. We also show that calcineurin regulates insulin response in L6 myotubes, and that the inhibition of calcineurin restores the insulin response in Herpud1-depleted L6 myotubes. Based on these findings, we conclude that Herpud1 is necessary for the insulin response in L6 myotubes through its role in the regulation of Ca2+-calcineurin axis

Murine Zfp106 is an 1,888 amino acid protein with two N-terminal and two C-terminal zinc finger domains with a beta-propeller upstream of the C-terminal zinc fingers. The transcription of the protein was found to be controlled through two promoter regions, leading to two isoform families, P1 and P2 Zfp106. The splice variants from each promoter are thought to have distinct starting exons and n-terminal regions. However, the isoforms are not well studied. Since its identification, Zfp106 has been implicated in RNA metabolism, transcription control, immune response, and muscle and testis development. It has been found to be capable of binding C9ORF72 repeats as well as being associated with TDP43 and FUS. However, its function is unknown. The aim of this study is to understand the role of Zfp106 in vivo through the use and development of various mouse models targeting exons specific to either the P1 or P2 family of isoforms. To begin with, we studied the Zfp106LacZ mouse model whose homozygous mice showed severe muscle atrophy beginning at 4 weeks leading to a premature death by 16 weeks. Research has supported the theory that the muscle atrophy is due to a motor neuron dysfunction potentially stemming from perturbed mitochondrial and spliceosome function. We, along with other researchers, found that this mouse model is not a complete disruption of Zfp106 through qPCR and RNAseq. We then found that this mouse model is an effective depletion of Zfp106 exon 2 and 3 which are exclusive to the P1 Zfp106 isoform family. Additionally, the Zfp106LacZ mouse model has an increased amount of the 1b exon associated with P2-Zfp106 in the skeletal muscle. Next, we established a CRISPR mouse line (ΔZfp106) targeting an exon common to the full-length splice variants of both the P1 and P2 family of isoforms, exon 5. This was in an attempt to dissect whether or not the muscle atrophy in the Zfp106LacZ mice was due to the interruption of exons 2 and 3 or from the increase in the P2 Zfp106 isoforms. Motor neurons derived from homozygous ΔZfp106 mouse embryonic stem cells, were found to be susceptible to CPA-induced endoplasmic reticulum stress and rotenone induced mitochondrial stress. Interestingly, the in vivo penetration rate of the muscle atrophy phenotype of homozygous ΔZfp106 mice is 60% for male and 12.5% for female mice. This is in stark contrast to the 100% penetration rate of the Zfp106LacZ mice. The reason behind this is currently unclear but may be due to either the incomplete backcrossing of the mouse model, a difference in the splice variants affected by the Zfp106 targeting, or because the muscle atrophy in the Zfp106LacZ mouse model is caused in part by the increase in the expression the P2 Zfp106 family of isoforms. These two mouse models show that affecting the expression of the full-length isoforms of P1-Zfp106 can lead to muscle atrophy. In an attempt to see if the Zfp106LacZ muscle atrophy was due to a lack of Zfp106 in the skeletal muscle, spinal cord, or necessitated its depletion in both, we derived a mouse line from the Zfp106LacZ that conditionally depletes exon 3 which impairs the expression of full length P1 Zfp106. This was used to target exon 3 removal to the skeletal muscle (Myf5), cholinergic neurons (ChAT), simultaneously (Myf5/ChAT), or a whole body depletion (Ella2). Surprisingly, the whole body depletion of Zfp106 exon 3 did not lead to muscle atrophy even though its removal leads to a frame shift and premature stop codon. The lack of a muscle atrophy phenotype may be because of the expression of a splice variant without exon 3, thereby rescuing the neuromuscular pathology. Lastly, to better understand the role of the P2 Zfp106 in vivo, we created a mouse line with a CRISPR mediated knockout of exon 1b (ΔP2). Exon 1b is the start exon of the P2 Zfp106 isoform family and the introduction of a destructive INDEL should independently affect the P2 isoform family. Interestingly, this mouse model showed no observed neuromuscular dysfunction or metabolic disorder, responding to a glucose bolus similarly with controls. The lack of a phenotype may be due to compensation by other Zfp106 isoforms or that the P2 isoform family is important in other biological roles.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第18901号 / 医科博第57号 / 新制||医科||4(附属図書館) / 31852 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 長澤 丘司, 教授 長田 重一, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Communication between epithelial and immune cells is critical for the maintenance and repair of mucosal tissues, with dysregulated epithelia contributing to pulmonary diseases including chronic obstructive pulmonary disease (COPD), asthma, cancer and pulmonary fibrosis. While the role of immune cells in regulating epithelial maintenance and repair has been extensively studied in the distal lung, relatively little is known about the immune microenvironment in the proximal conducting airways, including the function of these immune cells in epithelial regeneration and repair. To study the biology of the human conducting airways, we utilized the mouse trachea as a model tissue and sought to characterize the global immune landscape at homeostasis by multi-parameter flow cytometry and single cell RNA sequencing (scRNA-seq). We then utilized a well-characterized model of tracheal epithelial injury to study changes in the immune landscape in response to injury. These studies revealed that neutrophils are early responders to airway epithelial injury and may function to phagocytose epithelial cell debris. Monocytes and macrophages are then recruited to the injured airway and adopt an activated anti-inflammatory phenotype to participate in tissue repair. Finally, we examined the impact of severe combined immunodeficiency on epithelial cells at homeostasis and following injury. Airway basal stem cells in immunodeficient animals have altered expression of genes related to cytoskeletal support, epithelial adhesion and critical signaling pathways that may impact airway epithelial morphology, barrier integrity, and proliferation and differentiation following injury. Together, these data suggest a functional tracheal immune microenvironment is critical for both the normal development and functional regeneration of the airway epithelium. Macrophages are heterogenous and adaptable immune cells that have has important functions in pulmonary homeostatic maintenance and tissue repair. Distinct subtypes of macrophages have important implications for injury response and repair in the lungs, though relatively little is known about the phenotypes and roles of macrophages in the proximal conducting airways. To address this gap, we characterized murine tracheal macrophages relative to more distal pulmonary macrophages using scRNA-seq and flow cytometry. Tracheal macrophages have a cell surface signature distinct from any previously characterized pulmonary macrophage subtype and were shown to be largely monocyte-derived macrophages generated via fetal liver kinase-2 (Flk2)-dependent adult hematopoiesis. Following polidocanol airway injury, these specialized monocyte-derived tracheal macrophages are recruited to the trachea to become pro-regenerative activated macrophages to aid in regeneration and repair. This macrophage injury response is largely dependent on the chemokine receptor CCR2, with CCR2-deficient mice showing decreased tracheal macrophage recruitment and activation, abnormal epithelial morphology, altered proliferation of airway stem cells, and delayed epithelial repair. Overall, this work highlights the importance of tissue-specific injury-responsive macrophages in airway epithelial regeneration and repair. / 2025-02-12T00:00:00Z