Axonal regeneration within the mammalian central nervous system following traumatic damage is limited and interventions to enable regrowth is a crucial goal in regenerative medicine. The nematode Caenorhabditis elegans is an excellent model to identify the intrinsic genetic programs that govern axonal regrowth. Here we demonstrate that alterations in O-linked N- beta-acetylglucosamine (O-GlcNAc) post-translational modifications of proteins can increase the regenerative potential of individual neurons. O-GlcNAc are single monosaccharide protein modifications that occur on serines/threonines in nucleocytoplasmic compartments. Changes in O-GlcNAc levels serve as a sensor of cellular nutrients and acts in part through the insulin-signaling pathway. Loss of O-GlcNAc via mutation of the O-GlcNAc Transferase (OGT), the enzyme that adds O-GlcNAc onto target proteins, enhances regeneration by 70%. Remarkably, hyper-O-GlcNAcyation via mutation of the O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc from target proteins, also enhances regeneration by 40%. Our results shed light on this apparent contradiction by demonstrating that O-GlcNAc enzyme mutants differentially modulate the insulin-signaling pathway. OGT mutants act through AKT1 to modulate glycolysis. In contrast, OGA mutants act through the FOXO/DAF-16 transcription factor to improve the mitochondrial stress response. These findings reveal for the first time the importance of O-GlcNAc post-translational modifications in axon regeneration and provide evidence that regulation of metabolic programs can dictate the regenerative capacity of a neuron. / 2021-02-20T00:00:00Z

The O-linked N-acetylglucosamine post-translational modification (O-GlcNAcylation) is the dynamic and reversible attachment of N-acetylglucosamine to serine and threonine residues of target proteins. It is abundant in metazoa, involving hundreds of proteins linked to a plethora of biological functions with implications in human diseases. The process is catalysed by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), that add and remove the sugar moiety, respectively. Ogt gene knock-out is embryonic lethal in a range of animal models, hampering the study of the biological role of O-GlcNAc. O-GlcNAcylation of nuclear and cytoplasmic proteins has been extensively studied, however little is known about the role of O-GlcNAc in mitochondria. A previous report suggested the presence of a mitochondrial OGT isoform (mOGT) in human cell lines in addition to the well-characterised nucleocytoplasmic one (ncOGT). Since this report more than one decade ago, this putative mOGT has not been studied further. Similarly, hundreds of O-GlcNAcylated nucleocytoplasmic proteins have been identified by high-throughput proteomic screens, whereas only a few mitochondrial proteins have been detected. Nevertheless, several studies suggest that altered O-GlcNAc signalling affects mitochondrial function and morphology, with potential clinical implications. The aim of this thesis work was to study and characterise the biological role of mOGT and determine the mitochondrial O-GlcNAc proteome. Firstly, the presence of mOGT in human cell lines and mouse tissues was investigated. Surprisingly, analysis of genomic sequences indicates that this isoform cannot be expressed at protein level in most of the species analysed, except human and some primates. In fact, the putative mOGT cDNA in most of the genomes analysed contains a stop codon that excludes the presence of such isoform. In addition, mOGT was not detected at protein level in a wide range of human cell lines. Knock-down experiments and Western blot analysis of all the predicted OGT isoforms suggested the expression of only a single OGT isoform. In agreement with this, overexpression of ncOGT in HEK 293 suspension cells led to increased O-GlcNAcylation of mitochondrial proteins, suggesting that ncOGT is necessary and sufficient for the generation of the mitochondrial O-GlcNAc proteome. These data point to a model where O-GlcNAc cycling of mitochondrial proteins occurs in the cytosol, followed by their import into mitochondria. Alternatively, ncOGT itself might be transported into mitochondria where it can take part to O-GlcNAc cycling inside the organelle. In parallel, some advance in determining the O-GlcNAc mitochondrial proteome has been undertaken. Different mitochondrial fractionation protocols, combined with O-GlcNAc enrichment methods have been explored in order to map novel glycosylation sites on mitochondrial proteins. A novel technique established in our research group, employing a bacterial OGA orthologue as a bait to trap O-GlcNAcylated proteins, has been applied to crude mitochondrial fractions allowing the identification of several hits, although site mapping has not been yet achieved. The second chapter describes the work that has been done to improve and optimise novel O-GlcNAc inhibitors previously designed in the laboratory, called goblins. The original objective was to make these molecules cell-permeable and possibly target them to mitochondria in order to inhibit mOGT. Several strategies were explored to deliver the compounds into living cells, including the use of transfection reagents and covalent linkage to linear cell-penetrant peptides. The above methods did not achieve cellular uptake, although recently designed cyclic cell-penetrant peptides, linked to fluorescein, were internalised by HeLa cells with immediate diffuse nucleocytoplasmic staining. These molecules will be linked to goblins aiming to use the inhibitors for cell biology studies. A different approach, based on the permeabilisation of Drosophila embryos, enabled the penetration of goblins into the organisms with consequent reduction of global O-GlcNAc levels. This method allowed the use of these novel bisubstrate inhibitors in vivo for the first time, with potential applications in studying the role of O-GlcNAc in Drosophila development and possibly for future therapeutic purposes after further development of the scaffold.

<p> 0-GlcNAc modification is a single N-acetylglucosamine (GlcNAc) modification on Ser or Thr residue on protein. The addition and removal of the 0GlcNAc molecule are controlled by two enzymes (OGT and NCOAT). In this study, I expressed and purified the two enzymes involved in the 0-GlcNAc modification. A method was developed for the synthesis and purification of the peptide substrate YSDSPSTST for in vitro glycosylation and characterized the OGT enzyme activity by the in vitro glycosylation and H3 labeling. A method was developed based on detection of glycosylation peptide by mass spectrometry after separation by capillary liquid chromatography (CapLC). The optimization of mass spectrometry parameters was done using synthesized standard glycopeptide YSDSPSgTST ("Sg" represents 0-GlcNAc modified Serine). The in vitro modification site was determined by CID after alkaline J3-elimination. Furthers experiment could include detection of 0-GlcNAc modification of protein substrate both in vitro and in vivo. This will give a better understanding of the dynamics of 0-GlcNAc modification. </p> / Thesis / Master of Science (MSc)

Yes / Cardiovascular diseases (CVDs) are the leading cause of death globally. While the major focus of pharmacological and non-pharmacological interventions has been on targeting disease pathophysiology and limiting predisposing factors, our understanding of the cellular and molecular mechanisms underlying the pathogenesis of CVDs remains incomplete. One mechanism that has recently emerged is protein O-GlcNAcylation. This is a dynamic, site-specific reversible post-translational modification of serine and threonine residues on target proteins and is controlled by two enzymes: O-linked β-N-acetylglucosamine transferase (OGT) and O-linked β-N-acetylglucosaminidase (OGA). Protein O-GlcNAcylation alters the cellular functions of these target proteins which play vital roles in pathways that modulate vascular homeostasis and cardiac function. Through this review, we aim to give insights on the role of protein O-GlcNAcylation in cardiovascular diseases and identify potential therapeutic targets in this pathway for development of more effective medicines to improve patient outcomes.

A O-glicosilação com N-acetil-glucosamina (O-GlcNAc) é uma modificação pós-translacional altamente dinâmica que modula diversas vias de sinalização. O processo de O-GlcNAc é controlado por duas enzimas: a enzima OGT é responsável por catalisar a adição de N-acetil-glucosamina no grupo hidroxila dos resíduos de serina e treonina, enquanto a OGA catalisa a remoção de O-GlcNAc das proteínas modificadas. Proteínas com importante papel na função vascular são alvo de O-GlcNAc e o aumento da expressão de proteínas modificadas por O-GlcNAc promove aumento da reatividade vascular para estímulos contráteis. Um dos mecanismos de extrema importância no controle do tônus vascular está ligado à regulação da concentração de cálcio (Ca2+) intracelular, onde destacamos a participação do sistema STIM1/Orai1. As moléculas de interação estromal (STIM) atuam como sensores dos estoques intracelulares de Ca2+ e as proteínas Orai representam as subunidades que formam os canais de Ca2+ ativados pela liberação de Ca2+ (CRAC). Neste estudo investigamos a hipótese de que o aumento dos níveis vasculares de proteínas glicosiladas aumenta a resposta contrátil em aorta de ratos, por mecanismos relacionados ao controle da concentração intracelular de Ca2+.Em nossos experimentos, utilizamos aortas torácicas de ratos incubadas com PugNAc (inibidor seletivo da OGA, ), por 24h. Utilizando protocolo experimental que permite avaliar contrações induzidas pelo influxo de Ca2+ e liberação de Ca2+ intracelular, demonstramos que a incubação com PugNAc aumentou a resposta contrátil à PE bem como a contração durante o período de influxo de Ca2+, induzida pela reintrodução de solução fisiológica contendo Ca2+ (1,56 mM). O bloqueio dos canais CRAC com 2-APB (100 ) e gadolíneo (Gd3+, 100 ) diminuiu significativamente as contrações induzidas pelo influxo de Ca2+ em aortas incubadas com PugNAc. Além disso, estas aortas apresentaram aumento da expressão protéica de STIM1, o que resultaria em maior influxo de Ca2+. A contração induzida por cafeína (20 mM) e serotonina (10 ), a qual reflete a capacidade funcional do retículo sarcoplasmático (RS) em captar Ca2+, foi maior em aortas incubadas com PugNAc. O papel da Ca2+-ATPase (SERCA) foi avaliado com a utilização de tapsigargina, bloqueador da SERCA. O efeito da tapsigargina foi semelhante em artérias incubadas com PugNAc e veículo, apesar do aumento de expressão proteica da SERCA em aortas incubadas com PugNAc. Como a proteína cinase C (PKC) é ativada por aumentos de Ca2+ intracelular, determinamos se a atividade de proteínas alvo da PKC estavam aumentadas. A incubação com PugNAc aumentou a expressão das formas fosforiladas da CPI-17, MYPT-1 e MLC. Em conjunto, estes resultados sugerem que a ativação de STIM1/Orai1, aumento da liberação de Ca2+ intracelular e ativação da via de sinalização da PKC podem representar mecanismos que modulam as alterações vasculares em resposta ao aumento de proteínas glicosiladas por O-GlcNAc. / Glycosylation with O-linked -N-acetyl-glucosamine (O-GlcNAc) is a highly dynamic post-translational modification. The process of O-GlcNAc is controlled by two enzymes: the OGT enzyme catalyses the addition of N-acetyl-glucosamine to the hydroxyl group of serine and threonine residues of a target protein, while OGA catalyzes the cleavage of O-GlcNAc from post-translationally-modified proteins. Proteins with an important role in vascular function are targets of O-GlcNAc and increased levels of proteins modified by O-GlcNAc increase vascular reactivity to contractile stimuli. The regulation of intracellular calcium (Ca2+) concentration, including the activation of STIM1/Orai1, is key in the control of vascular tone. The stromal interaction molecules (STIM) act as sensors of intracellular Ca2+ stores whereas the Orai proteins represent subunits of the Ca2+ release-activated Ca2+ channels (CRAC). We hypothesized that increased levels of vascular O-GlcNAc proteins augment vascular contractile responses by altering mechanisms that regulate the intracellular Ca2+. Rat thoracic aortas were incubated with PugNAc (OGA selective inhibitor, ) for 24h. Using an experimental protocol that evaluates contractions induced by Ca2+ influx and release, we demonstrated that incubation with PugNAc increases contractile responses to phenylephrine (PE) as well as the contraction induced by Ca2+ influx, after depletion of intracellular Ca2+ stores. The CRAC channel blockers, 2-APB (100 ) and gadolinium (Gd3+, 100 ), significantly reduced the contractions induced by Ca2+ influx in aortas incubated with PugNAc. Furthermore, these aortas showed increased STIM1 protein expression, which could result in increased influx of Ca2+ and, in turn, increase vascular contraction. The contraction induced by the release of intracellular Ca2+ stores, stimulated by caffeine (20 mM) and serotonin (10 ), was increased in aortas incubated with PugNAc. The Ca2+-ATPase (SERCA) inhibitor thapsigargin produced similar effects in arteries incubated with PugNAc or vehicle, despite the increased SERCA protein expression in aortas incubated with PugNAc. Since PKC is activated by increases in intracellular Ca2+ and arteries incubated with PugNAc show activation of PKC, we determined whether the activity of proteins that are targets of PKC was increased in PugNAc-treated aortas. Incubation with PugNAc increased the expression of phosphorylated forms of CPI-17, MYPT-1 and MLC. Together, these results suggest that activation of STIM1/Orai1, increased release of intracellular Ca2+ and PKC activation may represent mechanisms that modulate vascular responses upon increased O-GlcNAc proteins.

A O-glicosilação com N-acetil-glucosamina (O-GlcNAc) é uma modificação pós-translacional altamente dinâmica que modula diversas vias de sinalização. O processo de O-GlcNAc é controlado por duas enzimas: a enzima OGT é responsável por catalisar a adição de N-acetil-glucosamina no grupo hidroxila dos resíduos de serina e treonina, enquanto a OGA catalisa a remoção de O-GlcNAc das proteínas modificadas. Proteínas com importante papel na função vascular são alvo de O-GlcNAc e o aumento da expressão de proteínas modificadas por O-GlcNAc promove aumento da reatividade vascular para estímulos contráteis. Um dos mecanismos de extrema importância no controle do tônus vascular está ligado à regulação da concentração de cálcio (Ca2+) intracelular, onde destacamos a participação do sistema STIM1/Orai1. As moléculas de interação estromal (STIM) atuam como sensores dos estoques intracelulares de Ca2+ e as proteínas Orai representam as subunidades que formam os canais de Ca2+ ativados pela liberação de Ca2+ (CRAC). Neste estudo investigamos a hipótese de que o aumento dos níveis vasculares de proteínas glicosiladas aumenta a resposta contrátil em aorta de ratos, por mecanismos relacionados ao controle da concentração intracelular de Ca2+.Em nossos experimentos, utilizamos aortas torácicas de ratos incubadas com PugNAc (inibidor seletivo da OGA, ), por 24h. Utilizando protocolo experimental que permite avaliar contrações induzidas pelo influxo de Ca2+ e liberação de Ca2+ intracelular, demonstramos que a incubação com PugNAc aumentou a resposta contrátil à PE bem como a contração durante o período de influxo de Ca2+, induzida pela reintrodução de solução fisiológica contendo Ca2+ (1,56 mM). O bloqueio dos canais CRAC com 2-APB (100 ) e gadolíneo (Gd3+, 100 ) diminuiu significativamente as contrações induzidas pelo influxo de Ca2+ em aortas incubadas com PugNAc. Além disso, estas aortas apresentaram aumento da expressão protéica de STIM1, o que resultaria em maior influxo de Ca2+. A contração induzida por cafeína (20 mM) e serotonina (10 ), a qual reflete a capacidade funcional do retículo sarcoplasmático (RS) em captar Ca2+, foi maior em aortas incubadas com PugNAc. O papel da Ca2+-ATPase (SERCA) foi avaliado com a utilização de tapsigargina, bloqueador da SERCA. O efeito da tapsigargina foi semelhante em artérias incubadas com PugNAc e veículo, apesar do aumento de expressão proteica da SERCA em aortas incubadas com PugNAc. Como a proteína cinase C (PKC) é ativada por aumentos de Ca2+ intracelular, determinamos se a atividade de proteínas alvo da PKC estavam aumentadas. A incubação com PugNAc aumentou a expressão das formas fosforiladas da CPI-17, MYPT-1 e MLC. Em conjunto, estes resultados sugerem que a ativação de STIM1/Orai1, aumento da liberação de Ca2+ intracelular e ativação da via de sinalização da PKC podem representar mecanismos que modulam as alterações vasculares em resposta ao aumento de proteínas glicosiladas por O-GlcNAc. / Glycosylation with O-linked -N-acetyl-glucosamine (O-GlcNAc) is a highly dynamic post-translational modification. The process of O-GlcNAc is controlled by two enzymes: the OGT enzyme catalyses the addition of N-acetyl-glucosamine to the hydroxyl group of serine and threonine residues of a target protein, while OGA catalyzes the cleavage of O-GlcNAc from post-translationally-modified proteins. Proteins with an important role in vascular function are targets of O-GlcNAc and increased levels of proteins modified by O-GlcNAc increase vascular reactivity to contractile stimuli. The regulation of intracellular calcium (Ca2+) concentration, including the activation of STIM1/Orai1, is key in the control of vascular tone. The stromal interaction molecules (STIM) act as sensors of intracellular Ca2+ stores whereas the Orai proteins represent subunits of the Ca2+ release-activated Ca2+ channels (CRAC). We hypothesized that increased levels of vascular O-GlcNAc proteins augment vascular contractile responses by altering mechanisms that regulate the intracellular Ca2+. Rat thoracic aortas were incubated with PugNAc (OGA selective inhibitor, ) for 24h. Using an experimental protocol that evaluates contractions induced by Ca2+ influx and release, we demonstrated that incubation with PugNAc increases contractile responses to phenylephrine (PE) as well as the contraction induced by Ca2+ influx, after depletion of intracellular Ca2+ stores. The CRAC channel blockers, 2-APB (100 ) and gadolinium (Gd3+, 100 ), significantly reduced the contractions induced by Ca2+ influx in aortas incubated with PugNAc. Furthermore, these aortas showed increased STIM1 protein expression, which could result in increased influx of Ca2+ and, in turn, increase vascular contraction. The contraction induced by the release of intracellular Ca2+ stores, stimulated by caffeine (20 mM) and serotonin (10 ), was increased in aortas incubated with PugNAc. The Ca2+-ATPase (SERCA) inhibitor thapsigargin produced similar effects in arteries incubated with PugNAc or vehicle, despite the increased SERCA protein expression in aortas incubated with PugNAc. Since PKC is activated by increases in intracellular Ca2+ and arteries incubated with PugNAc show activation of PKC, we determined whether the activity of proteins that are targets of PKC was increased in PugNAc-treated aortas. Incubation with PugNAc increased the expression of phosphorylated forms of CPI-17, MYPT-1 and MLC. Together, these results suggest that activation of STIM1/Orai1, increased release of intracellular Ca2+ and PKC activation may represent mechanisms that modulate vascular responses upon increased O-GlcNAc proteins.

The ability of cells to respond to their microenvironment is controlled by a complex communication system. Cell signaling utilizes a series of post-translational events to regulate and coordinate cellular activities. Although phosphorylation is thought to be the key regulator of these events, recent findings implicate the O-GlcNAc modification as an additional control mechanism. Modulation of signal transduction requires compartmentalization of the kinases and phosphatases. Based on the evidence of subcellular localization of OGT isoforms, the diversity of O-GlcNAcylated proteins upon stimulation, and its role during insulin signaling, it can be hypothesized that O-GlcNAc is involved and regulates signal transduction in a compartmentalized manner. To investigate the spatio-temporal dynamics of O-GlcNAc in cell signaling, we have generated a series of genetically encoded O-GlcNAc reporters based on fluorescence resonance energy transfer (FRET). These reporters and localized variants have allowed compartment specific visualization of O-GlcNAc activity in the nucleus, cytoplasm and plasma membrane. Herein we describe these reporters and their use to examine O-GlcNAc dynamics in signaling using serum stimulation and environmentally relevant concentrations of arsenite. Acute exposure to arsenite through drinking water has become an environmental health concern worldwide. Our results imply a complex regulation of O-GlcNAc on a fast timescale that is tied to more canonical kinase pathways. / text

There are many tools available for the study of post-translational modifications. The majority of these tools is specific towards the individual modification and involves separation of modified proteins from non-modified ones. The drawback of using a modification specific method is that there is a lack of flexibility in its usage for other modifications. The goal of these studies was to investigate the possibility of obtaining a similar separation effect by fractionating post-translationally modified proteins based on the physical properties of proteins. The post-translational modification chosen to be the basis of this study was the O-GlcNAc modification. Using the C2C12 mouse myoblast cell line, it was determined that the optimal conditions for producing lysates containing increased yields of O-GlcNAc modified proteins was to treat differentiated C2C12 cells with 10nM insulin, 12g/L glucose and 2mM of the O-GlcNAcase inhibitor Streptozotocin for 24 hours. Using the optimized lysis buffer, it was shown that protein separation by surface charge using standard anion exchange separation did not provide enough resolution or material to obtain any identifications of modified proteins. However, when a chromatofocusing method which separates proteins on the basis of their isoelectric points was used, a separation scheme with larger capacity and higher resolution was possible. Using this separation method followed by gel electrophoresis of individual fractions, proteins which are potentially O-GlcNAc modified were identified by mass spectrometry. It was evident from the number of protein bands observed per fraction on the Coomassie stained gels and the number of proteins identified per protein band by mass spectrometry that further reduction in sample complexity was required to assist in the positive identification of O-GlcNAc modified proteins. Among the identified proteins, 32 percent were metabolic proteins, 21 percent were protein processing proteins, 16 percent were structural proteins and the remainder a mix of other proteins. Unfortunately, it was not possible to validate the presence or absence of the O-GlcNAc modification on these proteins using available methodologies such as immunoprecipitation. As such, further work is required to optimize the separation strategy and to verify the usefulness of this separation strategy in identifying O-GlcNAc/post-translationally modified proteins.

There are many tools available for the study of post-translational modifications. The majority of these tools is specific towards the individual modification and involves separation of modified proteins from non-modified ones. The drawback of using a modification specific method is that there is a lack of flexibility in its usage for other modifications. The goal of these studies was to investigate the possibility of obtaining a similar separation effect by fractionating post-translationally modified proteins based on the physical properties of proteins. The post-translational modification chosen to be the basis of this study was the O-GlcNAc modification. Using the C2C12 mouse myoblast cell line, it was determined that the optimal conditions for producing lysates containing increased yields of O-GlcNAc modified proteins was to treat differentiated C2C12 cells with 10nM insulin, 12g/L glucose and 2mM of the O-GlcNAcase inhibitor Streptozotocin for 24 hours. Using the optimized lysis buffer, it was shown that protein separation by surface charge using standard anion exchange separation did not provide enough resolution or material to obtain any identifications of modified proteins. However, when a chromatofocusing method which separates proteins on the basis of their isoelectric points was used, a separation scheme with larger capacity and higher resolution was possible. Using this separation method followed by gel electrophoresis of individual fractions, proteins which are potentially O-GlcNAc modified were identified by mass spectrometry. It was evident from the number of protein bands observed per fraction on the Coomassie stained gels and the number of proteins identified per protein band by mass spectrometry that further reduction in sample complexity was required to assist in the positive identification of O-GlcNAc modified proteins. Among the identified proteins, 32 percent were metabolic proteins, 21 percent were protein processing proteins, 16 percent were structural proteins and the remainder a mix of other proteins. Unfortunately, it was not possible to validate the presence or absence of the O-GlcNAc modification on these proteins using available methodologies such as immunoprecipitation. As such, further work is required to optimize the separation strategy and to verify the usefulness of this separation strategy in identifying O-GlcNAc/post-translationally modified proteins.

There are many tools available for the study of post-translational modifications. The majority of these tools is specific towards the individual modification and involves separation of modified proteins from non-modified ones. The drawback of using a modification specific method is that there is a lack of flexibility in its usage for other modifications. The goal of these studies was to investigate the possibility of obtaining a similar separation effect by fractionating post-translationally modified proteins based on the physical properties of proteins. The post-translational modification chosen to be the basis of this study was the O-GlcNAc modification. Using the C2C12 mouse myoblast cell line, it was determined that the optimal conditions for producing lysates containing increased yields of O-GlcNAc modified proteins was to treat differentiated C2C12 cells with 10nM insulin, 12g/L glucose and 2mM of the O-GlcNAcase inhibitor Streptozotocin for 24 hours. Using the optimized lysis buffer, it was shown that protein separation by surface charge using standard anion exchange separation did not provide enough resolution or material to obtain any identifications of modified proteins. However, when a chromatofocusing method which separates proteins on the basis of their isoelectric points was used, a separation scheme with larger capacity and higher resolution was possible. Using this separation method followed by gel electrophoresis of individual fractions, proteins which are potentially O-GlcNAc modified were identified by mass spectrometry. It was evident from the number of protein bands observed per fraction on the Coomassie stained gels and the number of proteins identified per protein band by mass spectrometry that further reduction in sample complexity was required to assist in the positive identification of O-GlcNAc modified proteins. Among the identified proteins, 32 percent were metabolic proteins, 21 percent were protein processing proteins, 16 percent were structural proteins and the remainder a mix of other proteins. Unfortunately, it was not possible to validate the presence or absence of the O-GlcNAc modification on these proteins using available methodologies such as immunoprecipitation. As such, further work is required to optimize the separation strategy and to verify the usefulness of this separation strategy in identifying O-GlcNAc/post-translationally modified proteins. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate