Skeletal muscle injury results in functional deficits that can take several weeks to fully recover. Ultimate recovery of function is dependent on the muscle’s ability to regenerate, a highly coordinated process that involves transient muscle inflammation and the replacement of damaged myofibers. Instrumental in the inflammatory response, is the pro-inflammatory cytokine TNF-α. Expression of TNF-α is thought to be regulated, in part, by the stress sensing 70 kDa heat shock protein (Hsp70). However, it remains unclear how Hsp70 alters TNF-α following injury, and if so, how these changes affect skeletal muscle repair. Therefore, we up-regulated Hsp70 expression using 17-allylamino-17-demethoxygeldanamycin (17-AAG) prior to and following BaCl2-induced injury, and assessed TNF-α and myogenin content. Regenerating fiber cross-sectional area (CSA) and in vivo isometric torque were also analyzed in the weeks following the injury. Treatment of 17-AAG resulted in a ~5 fold increase in Hsp70 of the uninjured muscle, but did not affect any other biochemical, morphological or functional variables compared to controls. In the days following the injury, TNF-α and myogenin were elevated and directly correlated. At these earlier time points (≤7 days), treatment of 17-AAG increased TNF-α above that of the injured controls and resulted in a sustained increase in myogenin. However, no differences were observed in regenerating fiber CSA or in vivo torque production between the groups. Together, these data suggest that Hsp70 induction increases TNF-α and myogenin content following BaCl2-induced injury, but does not appear to alter skeletal muscle regeneration or attenuate functional deficits in otherwise healthy young mice.

Sleep fragmentation induces acute inflammation and increases glucocorticosteroids in vertebrates. Obesity and sleep fragmentation are often concurrent pro-inflammatory conditions in patients with obstructive sleep apnea. Despite the association between the two, their simultaneous effects on immune and endocrine profiles have not been explored. In the first experiment, we investigated changes in proinflammatory (IL-1β, TNF-α) and anti-inflammatory (TGF-β1) cytokine gene expression in the periphery (liver, spleen, fat, and heart) and brain (hypothalamus, prefrontal cortex, and hippocampus) in mice exposed to various intervals of sleep fragmentation. Serum corticosterone concentration was also assessed. Sleep was disrupted in male C57BL/6J mice using an automated sleep fragmentation chamber (Lafayette Industries), which involved movement of a sweeping bar at specified intervals. Mice were exposed to bar sweeps every 20 sec (high sleep fragmentation; HSF), 120 sec (low sleep fragmentation; LSF), or the bar remained stationary (control). Trunk blood and tissue samples were collected after 24 h of SF. It was found that HSF is a potent inducer of inflammation in the periphery (IL-1β: adipose, heart, and hypothalamus), but leads to upregulation of antiinflammatory cytokines in the brain (TGF-β1: hypothalamus and hippocampus), despite elevated serum corticosterone. Due to the association between obesity and SF, this experiment was replicated in male C57BL/6J mice (lean) and ob/ob KO mice (obese) using the previously described methods. We predicted the acute inflammatory response resulting from HSF would be different for the lean and the obese mice, with the greatest cytokine gene expression levels in the OB HSF group, due to a summative effect of the pro-inflammatory conditions. Obesity was the factor that most affected cytokine gene expression profiles. Additionally, the pro- vs anti-inflammatory gene expression profile varied with tissue type. While obesity resulted in neuroinflammation (hypothalamus, prefrontal cortex, hippocampus), it led to decreases in pro-inflammatory cytokine gene expression in the periphery (spleen, fat, heart). Serum corticosterone concentration was significantly elevated due to SF, but was not affected by obesity. As a result, the obese mice likely had neuroendocrine adaptations to combat the pre-existing pro-inflammatory condition of obesity, which impacted the acute inflammatory response to sleep loss.

Each year, over 50 million Americans suffer from persistent pain, including debilitating headaches, joint pain, and severe back pain. Although morphine is amongst the most effective analgesics available for the management of severe pain, prolonged morphine treatment results in decreased analgesic efficacy (i.e., tolerance). Despite significant headway in the field, the mechanisms underlying the development of morphine tolerance are not well understood. The midbrain ventrolateral periaqueductal gray (vlPAG) is a primary neural substrate for the analgesic effects of morphine, as well as for the development of morphine tolerance. A growing body of literature indicates that activated glia (i.e., microglia and astrocytes) facilitate pain transmission and oppose morphine analgesia, making these cells important potential targets in the treatment of chronic pain. Morphine affects glia by binding to the innate immune receptor toll-like receptor 4 (TLR4), leading to the release of proinflammatory cytokines and opposition of morphine analgesia. Despite the established role of the vlPAG as an integral locus for the development of morphine tolerance, to date, no studies have examined the role of glia activation within this region. Additionally, the role of TLR4 in the development of tolerance has not been elucidated. This dissertation seeks to address the lack of knowledge regarding the role of vlPAG glia and TLR4 in the development of morphine tolerance by (1) Characterizing the effects of chronic morphine and peripheral inflammatory pain on vlPAG glial cell activity; (2) Investigating the role of glia activation within the vlPAG in the development of morphine tolerance; (3) Characterizing the role of the glial receptor TLR4 within the vlPAG in the development of morphine tolerance; and (4) Characterizing the glia to neuron signaling mechanisms involved in the development of morphine tolerance. These experiments, together, provide novel information about the mechanism by which central nervous system glia regulate morphine tolerance, and identify a potential therapeutic target for the enhancement of analgesic efficacy in the clinical treatment of chronic pain.

This work involves the characterization of two genes using mouse models of gene deficiency and thus has two focuses: Focus I: Innate immune responses are vital for pathogen defence but can result in septic shock when excessive. A key mediator of septic shock is TNFα, which is shed into intercellular spaces after cleavage from the plasma membrane by the protease TACE. Here we report that the rhomboid family member iRhom2 interacts with TACE and regulates TNFα shedding in vitro and in vivo. Compared to controls, gene-targeted iRhom2-deficient mice showed reduced serum TNFα after LPS challenge survived a lethal LPS dose. Furthermore, iRhom2-deficient mice failed to adequately control the replication of Listeria monocytogenes and thus succumbed to even mild infections. Our study has identified iRhom2 as a novel regulator of innate immunity that may be an important target for modulating sepsis and pathogen defence. Focus II: Smg1 is a phosphatidylinositol 3-kinase-related kinase (PIKK) associated with multiple cellular functions, including DNA damage responses, telomere maintenance, and nonsense-mediated mRNA decay (NMD). NMD degrades transcripts that harbour premature termination codons (PTCs) due to events such as mutation or alternative splicing (AS). Recognition of PTCs during NMD requires the action of the Upstream frameshift protein Upf1, which must first be phosphorylated by Smg1. However, the physiological function of mammalian Smg1 is not known. Using a gene-trap model of Smg1 deficiency, we show that this kinase is essential for mouse embryogenesis such that Smg1 loss is lethal at embryonic day 8.5 (E8.5). High-throughput RNA sequencing (RNA-Seq) of RNA from cells of Smg1-deficient embryos revealed that Smg1 depletion led to pronounced accumulation of PTC-containing splice variant transcripts from ~9% of genes predicted to contain AS events capable of eliciting NMD. Among these genes are those involved in splicing itself, as well as genes not previously known to be subject to AS-coupled NMD, including several involved in transcription, intracellular signalling, membrane dynamics, cell death and metabolism. Our results demonstrate a critical role for Smg1 in early mouse development and link the loss of this NMD factor to major and widespread changes in the mammalian transcriptome.

This work involves the characterization of two genes using mouse models of gene deficiency and thus has two focuses: Focus I: Innate immune responses are vital for pathogen defence but can result in septic shock when excessive. A key mediator of septic shock is TNFα, which is shed into intercellular spaces after cleavage from the plasma membrane by the protease TACE. Here we report that the rhomboid family member iRhom2 interacts with TACE and regulates TNFα shedding in vitro and in vivo. Compared to controls, gene-targeted iRhom2-deficient mice showed reduced serum TNFα after LPS challenge survived a lethal LPS dose. Furthermore, iRhom2-deficient mice failed to adequately control the replication of Listeria monocytogenes and thus succumbed to even mild infections. Our study has identified iRhom2 as a novel regulator of innate immunity that may be an important target for modulating sepsis and pathogen defence. Focus II: Smg1 is a phosphatidylinositol 3-kinase-related kinase (PIKK) associated with multiple cellular functions, including DNA damage responses, telomere maintenance, and nonsense-mediated mRNA decay (NMD). NMD degrades transcripts that harbour premature termination codons (PTCs) due to events such as mutation or alternative splicing (AS). Recognition of PTCs during NMD requires the action of the Upstream frameshift protein Upf1, which must first be phosphorylated by Smg1. However, the physiological function of mammalian Smg1 is not known. Using a gene-trap model of Smg1 deficiency, we show that this kinase is essential for mouse embryogenesis such that Smg1 loss is lethal at embryonic day 8.5 (E8.5). High-throughput RNA sequencing (RNA-Seq) of RNA from cells of Smg1-deficient embryos revealed that Smg1 depletion led to pronounced accumulation of PTC-containing splice variant transcripts from ~9% of genes predicted to contain AS events capable of eliciting NMD. Among these genes are those involved in splicing itself, as well as genes not previously known to be subject to AS-coupled NMD, including several involved in transcription, intracellular signalling, membrane dynamics, cell death and metabolism. Our results demonstrate a critical role for Smg1 in early mouse development and link the loss of this NMD factor to major and widespread changes in the mammalian transcriptome.

Reconstructive flaps are a mainstay form of treatment for anatomical defects in plastic surgery, and despite extensive progress in the areas of flap anatomy and design, the mechanism of flap healing and the factors that regulate this process are poorly understood. This thesis investigates the regulation of flap healing, and tests the hypothesis that the introduction of genes for angiogenic growth factors can be used to augment the vascularisation and wound healing of ischaemic flaps. Using a modified McFarlane ischaemic skin flap model in Sprague Dawley rats, endogenous angiogenic regulatory factors that included the VEGF and angiopoietin families and their receptors were investigated. Twelve specific quantitative real-time PCR assays documented a general up-regulation of angiogenic genes and their receptors with time following flap elevation. There was not a readily identifiable “master regulator”. Angiogenic protein levels were more variable with a decrease VEGF-A and TNF-α levels along the flap. Debridement studies of the necrotic distal flaps demonstrated for the first time that VEGF-A164 and TNF-α protein levels stabilised, while angiogenic genes of VEGF-A164, VEGF-A120, angiopoietins and their receptors were down-regulated and VEGF-B186 and HIF-1α mRNA increased, compared to non-debrided flaps. Leucocyte proteolysis in devitalised tissue is discussed as a possible mechanism for reduced angiogenic proteins levels in ischaemic flaps. The impact of angiogenic gene therapy using adenoviral vectors in the flap model revealed for the first time that recombinant adenoviruses containing the VEGF-B186 transgene could significantly augment neovascularisation and improve flap survival. This neovascularisation correlated with up-regulation of the expression of multiple endogenous angiogenic genes that included VEGF-A164, the angiopoietins and their receptors. Erythematous plaques were documented as a side effect of Ad VEGF-A165 and Ad VEGF-B186 treatment of rat skin, although in the latter treatment they were very mild. Weals induced by the presence of VEGF-A165 transgene were associated with a marked acute inflammatory cell infiltrate and oedema consistent with the increased vascular permeability effects of VEGF-A165. Ad VEGF-A165 plus Ad ANG-1* induced weals were less prominent with reduced oedema highlighting the stabilising effect of Ad ANG-1* on vascular permeability.

NAD+ has previously been shown to regulate TNF-α synthesis and TNF-α has been shown to regulate NAD+ homeostasis, thus providing a link between a pro-inflammatory response and redox status. Despite the well-established link between TNF-α and NAD+, the mechanism as to how NAD+ modulates TNF-α release is not fully understood. To achieve this, this link was investigated using THP-1 cell line-derived M1-like (pro-inflammatory) and M2-like (anti-inflammatory) macrophages using PMA and vitamin D3, respectively. NAD+ levels differed markedly between M1-like and M2-like macrophages, with M1-like having much higher basal levels. LPS increases NAD+ levels and TNF-α secretion in M1-like but not M2-like cells. In an effort to investigate the source of the NAD+ levels and the association with TNF-α release, three inhibitors (FK866, DPI and 1D-MT) were used. Following stimulation, NAD+ is produced partially via NADH oxidation and partially through NAD+ synthesis. Both DPI and FK866 reduced TNF-α secretion with DPI showing the largest effect. The two phenotypes showed differential profiles of NAD+ homeostasis gene expression compared with each other and with the progenitor THP-1 in both resting and activated states. While IDO expression was induced in both phenotypes, CD38 and NAMPT were upregulated in M1-like cells whereas CD157 was upregulated in M2-like cells. LPS induced M1-like cells to up-regulate CD38 and CD157 and down-regulate NAMPT unlike M2-like cells which up-regulated NAMPT and CD38 and down-regulated CD157. M1s increased glycolysis activity whereas conversely, decreased oxidative metabolism during LPS stimulation confirming previous findings showing that classical M1s are predominantly glycolytic. Collectively, these data suggest that the relationship between NAD+ levels and pro-/anti-inflammatory responses is complex and may be regulated via a combination of pathways. These findings open the possibility of pharmacological manipulation of NAD+ synthesis as a way of selectively modulating macrophage responses which may be beneficial for the development of therapeutics targeting inflammatory diseases.

La biotecnología ha adquirido un rol fundamental en la industria de producción de biofármacos, la cual ofrece nuevas alternativas para el tratamiento de diversas enfermedades. Entre los más utilizados están los anticuerpos monoclonales, los cuales se utilizan para el tratamiento de enfermedades autoinmunes, cáncer, rechazo de transplante renal, tratamiento de cardiopatía coronaria, prevención de enfermedades infecciosas, diagnóstico clínico, aplicaciones industriales e investigación. Son producidos principalmente por células modificadas genéticamente, como por ejemplo la línea celular CHO-K1. En este caso, se produce el anticuerpo monoclonal recombinante anti factor de necrosis tumoral alfa, el cual se utiliza en el tratamiento de artritis reumatoide y otras enfermedades. Es por ello que se planteó desarrollar un cultivo celular en suspensión libre de componentes animales para realizar ensayos preclínicos, para la posterior administración de este anticuerpo a pacientes que lo necesitan. Se purificaron desde un cultivo bacteriano de Escherichia coli DH5α los plasmidios que contienen los genes que codifican para las cadenas del anticuerpo, se co-transfectaron células CHO-K1 con aquellos plasmidios, se seleccionó el mejor clon productor, y se caracterizó su crecimiento en adherencia suplementado con 10%, 0% v/v suero fetal bovino (FBS), y en suspensión en matraces Erlenmeyer sin FBS, proceso que fue realizado luego de muchos pasajes sucesivos. El estado metabólico de cada condición se puede apreciar en la razón lactato producido sobre glucosa consumida. Se observó que al cambiar el FBS por diversos suplementos alimenticios, la concentración de células viables máxima (de 1,87∙106 cels/ml a 1,17∙106 cels/ml), viabilidad y tasa específica de crecimiento (de 0,038 hr-1 a 0,027 hr-1) disminuyeron ligeramente, y la productividad de anticuerpo medida en absorbancia a 405 nm (de 0,155 a 0,414) y la tasa específica de absorbancia (de 0,0083 [106 /(hr x cels)] a 0,012 [106 /(hr x cels)]) aumentaron ligeramente, lo cual muestra que el cambio de medio de cultivo hizo que el clon creciera más lentamente, pero fuera un mejor productor. Los cambios más drásticos en las variables medidas fueron observados en el cultivo en suspensión sin FBS, las cuales siguieron la misma tendencia anterior (concentración de células viables máxima de 0,34∙106 cels/ml y tasa específica de crecimiento de 0,009 hr-1), con la excepción de la productividad de anticuerpo, ya que la concentración de células viables fue demasiado baja para mostrar una producción de anticuerpo significativa. Además, la concentración de lactato estuvo en niveles inhibitorios al segundo día de cultivo. Analizando los perfiles metabólicos, se observó que las células en suspensión utilizaron la energía sólo para mantención, crecieron muy poco y luego murieron. Este fenómeno pudo haber ocurrido porque no se utilizó el medio de cultivo, concentración de nutrientes, suplementos, estrategia de alimentación, modo de operación del biorreactor, protocolo de adaptación y/o vectores de expresión adecuados, con lo cual queda propuesta una serie de estrategias para mejorar el comportamiento de los clones en cultivos futuros.

?rea de concentra??o: Neuroimunoendocrinologia. / Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2016-01-04T18:24:18Z No. of bitstreams: 2 wellington_fabiano_gomes.pdf: 3823362 bytes, checksum: 39e0250b96c5bd95724cca2eef404fd7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2016-01-04T18:24:49Z (GMT) No. of bitstreams: 2 wellington_fabiano_gomes.pdf: 3823362 bytes, checksum: 39e0250b96c5bd95724cca2eef404fd7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-01-04T18:24:49Z (GMT). No. of bitstreams: 2 wellington_fabiano_gomes.pdf: 3823362 bytes, checksum: 39e0250b96c5bd95724cca2eef404fd7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014 / Funda??o de Amparo ? Pesquisa do estado de Minas Gerais (FAPEMIG) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (Capes) / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / A osteartrite de joelho (OAj) ? uma doen?a que afeta principalmente os idosos e pode levar a grandes limita??es f?sicas e funcionais. No entanto, os efeitos espec?ficos da terapia por exerc?cios, especialmente a caminhada sobre o sistema imunol?gico, s?o desconhecidas. Portanto, este estudo teve como objetivo analisar o efeito de 12 semanas de caminhada (3x / semana) no perfil de leuc?citos, nos n?veis plasm?ticos de interleucina (IL-6), do fator de necrose tumoral alfa (TNF-?), dos receptores sol?veis de TNF-? (sTNFR1 e sTNFR2), e do fator neurotr?fico derivado do c?rebro (BDNF) a partir de plasma retirado do sangue perif?rico de mulheres idosas com OAj. Al?m disso, as avalia??es cl?nicas e funcionais (teste de WOMAC, teste de caminhada de 6 minutos, SF-36, a percep??o da dor) foram realizadas. Dezesseis mulheres (idade: 67 ? 4 anos, ?ndice de massa corporal: 28,07 ? 4,16 kg/m2) participaram de um programa de caminhada (36 sess?es de fisioterapia) e de um esfor?o f?sico (01 sess?o de fisioterapia). As vari?veis foram avaliadas antes e ap?s 12 semanas de treinamento com dura??o (30-55 min) e intensidade (70-80% da FCm?x) progressivamente maiores. As amostras de sangue coletadas foram analisadas com um contador de c?lulas, cit?metro de fluxo e pelo m?todo ELISA. As sess?es de fisioterapia resultaram em um aumento de 47% da qualidade de vida (p <0,05) e um aumento de 21% no VO2max (p <0,0001) em mulheres idosas com OAj. Al?m disso, houve uma redu??o nas c?lulas T CD4 + (antes 46,59 ? 7%, depois 44,58 ? 9%, p = 0,0189) e uma intensidade de fluoresc?ncia mais elevada para CD18 + CD4 + (antes 45,30 ? 10, depois de 64,27 ? 33, p = 0,0256) e CD18 + CD8 + (antes: 64,2 ? 27, depois de 85,02 ? 35, p = 0,0130). A ET aumentou a concentra??o plasm?tica de sTNR1; no entanto, diminuiu a concentra??o de plasma de sTNFR2, quando comparado com os n?veis em repouso de pacientes. O exerc?cio agudo afetou diferencialmente os n?veis de sTNFR1 dependente de quando as amostras foram tomadas, antes e ap?s o treinamento aer?bico. No entanto, os n?veis de sTNFR2 n?o foram afetados pelo treinamento. O exerc?cio agudo aumentou os n?veis de BDNF apenas antes do per?odo de treinamento de 12 semanas (p <0,001). Al?m disso, houve aumento das concentra??es plasm?ticas de BDNF (p <0,0001) e melhora em par?metros cl?nicos (funcional p <0,001; percep??o da dor p <0,01). A varia??o dos n?veis de receptores sol?veis correlacionou-se com a melhora funcional; no entanto, os marcadores inflamat?rios de osteoartrite (IL-6 e TNF-?) n?o foram afetados pelas 36 sess?es de fisioterapia. / Tese (Doutorado) ? Programa Multic?ntrico de P?s-Gradua??o em Ci?ncias Fisiol?gicas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2014. / ABSTRACT Osteoarthritis of the knee (kOA) is a disease that mainly affects the elderly and can lead to major physical and functional limitations. However, the specific effects of exercise therapy (ET), specially walking and particularly on the immune system, are unknown. Therefore, this study aimed to analyze the effect of 12 weeks of walking (3x/week) on the leukocyte profile, levels of interleukin 6 (IL-6), tumor necrosis factor alpha, (TNF-?), soluble forms of the TNF-? receptor (sTNFR1 and sTNFR2), and brain-derived neurotrophic factor (BDNF) from plasma taken from the peripheral blood of elderly women with kOA. Additionally, clinical and functional assessments (WOMAC test, 6-min walk, SF-36, pain perception) were performed. Sixteen women (age: 67 ? 4 years, body mass index: 28.07 ? 4.16 kg/m2) participated in a walking program and physical exertion. The variables were assessed before and after 12 weeks of training with a progressively longer duration (30-55 min) and higher intensity (70-80% of HRmax). The blood samples were collected for analysis with a cell counter, the Scan Fac flow cytometer and were measured by ELISA. The ET resulted in a 47% enhancement of the self-reported quality of life (p <0.05) and a 21% increase in the VO2max (p <0.0001) in elderly women with kOA. Furthermore, there was a reduction in CD4+ cells (before 46.59?7%, after 44.58?9%, p=0.0189) and a higher fluorescence intensity for CD18+CD4+ (before 45.30 ? 10, after 64.27 ? 33, p=0.0256) and CD18+CD8+ (before: 64.2 ?27, after 85.02 ?35, p=0.0130). Aerobic training increased the plasma concentration of sTNR1; however, it decreased the plasma concentration of sTNFR2, when compared with levels of resting patients. Acute exercise differentially affects the levels of sTNFR1 dependent on when the samples were taken, before and after aerobic training. However, the levels of sTNFR2 were not affected by training. The acute exercise increased the levels of BDNF only before the 12-week training period (p<0.001). Moreover, the training augmented the plasma concentrations of BDNF (p<0.0001) and improved clinical parameters (functional p<0.001; pain perception p<0.01). The variation in the levels of soluble receptors correlated with functional improvement; however, the inflammatory osteoarthritis markers (IL-6 and TNF-?) were unaffected by the walking exercises, in physical therapy.

No description available.