Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Doutor em Genética e Bioquímica / CHAPTER II: Purpose: We performed a meta-analysis of randomized trials to assess the effect of metformin on inflammatory markers and metabolic parameters in subjects with diabetes. Methods: We performed comprehensive searches on NCBI, Cochrane, Science Direct databases from 1966 to Jun of 2015. We included randomized trials of at least 4 weeks duration that compared groups with diabetes before and after the treatment with metformin or metformin plus other drugs, and evaluated body mass index, blood glucose, HbA1c and inflammatory parameters such as C-reactive protein, tumor necrosis factor and adiponectin. Results: Pooled results of the 26 trials, with 1760 participants at the end of treatment reduce BMI in 0.9% p=0,0043, as well as, decrease of blood glucose level [SMD -0,411 mg/dL, 95%CI -0,463 to -0,369, I2= 56.62%], HbA1c [SMD -0.479%, 95%CI -0,568 to -0,390, I2= 55.02%], CRP levels [SMD -0,274mg/dL, 95%CI -0,419 to -0,129, I2= 72.78%], TNFα concentration [SMD -0,103pg/ml, 95%CI -0,514 to 0,309, I2= 87.67%] and increase of adiponectin [SMD 0,171μg/ml, 95%CI 0,098 to 0,440, I2= 81.09%] compared with pretreatment. Conclusion: The long-treatment with metformin monotherapy or metformin plus other drugs improves metabolic parameters and induced changes in inflammatory markers in diabetic subject. CHAPTER III: Background: Metformin increases insulin sensitivity by decreasing hepatic glucose production and increasing glucose disposal in skeletal muscle. However, modulation of inflammatory response and CaMKKβ/AMPK/Myosin V activation in gastrocnemius muscle by metformin treatment has not been demonstrated in hypoinsulinemic diabetic rats. Objective: The present study investigated how the metformin improve insulin sensitivity in skeletal muscle of hypoinsulinemic diabetic rats. Methods: Diabetes was induced by streptozotocin (45 mg/kg, intraperitoneally) 10 days prior treatments. On 11th day, diabetic rats were treated with metformin (500 mg/kg, oral gavage), insulin (2U at 08:00 h and 4U at 17:00 h, subcutaneously) or untreated. After 20 days, glycemia was measured and insulin sensitivity was determined by KITT. Serum Insulin, GLUT4, IRSthr, inflammatory markers (NF-κB, IκB, TNF-α and p-JNK) and CAMKK, AMPK and Myosin V in gastrocnemius muscle were determined by ELISA. Results: As expected, insulin and metformin improved the insulin sensitivity. Besides, metformin treatment promoted reduction in inflammatory response mediated by NF-κB, IκB, TNF-α and p-JNK, and that was accompanied by increased CaMKKβ/AMPK/Myosin V/GLUT4 pathway activity in gastrocnemius muscle of diabetic rats. Conclusion: Our findings suggest that metformin induces significant reductions in several inflammatory markers in skeletal muscle of diabetic rats. Metformin-induced increase in CaMKKβ/AMPK/Myosin V/GLUT4 pathway activity was associated with higher insulin sensitivity. CHAPTER IV: Diabetes is characterized by a proinflammatory state which can activate TLR2 and TLR4, and these receptors could induce NF-κB and JNK activation in skeletal muscle. In this study, we investigated the inflammatory and apoptotic signaling pathways triggered by TLRs/NF-κB and JNK activation in skeletal muscle of diabetic rats treated with metformin before and after an insulin tolerance test. Metformin treatment decreased p-JNK and NF-κB, and increased IκB concentrations. This attenuation leads to a decrease of TNFα and CXCL1/KC, and an increase of p-AMPK, BAX and Bcl2 concentration. Furthermore, KITT revealed an improvement of the insulin sensitivity in the diabetic rats treated with metformin. In addition, metformin was not capable of attenuating the changes in the inflammatory pathway triggered by insulin injection as the increase of TNFα and TLR4 in metformin treated rats, and IκB, CXCL1/KC, TNFα and p-AMPK increase in the untreated group. Taken together, these results point out that metformin may attenuate the activation of the inflammatory pathway TLRs/NF-κB/TNFα/CXCL1/KC and the apoptotic signaling BAX/Bcl2/p-JNK, which could be accompanied by a reduction of the inflammatory damage caused by hyperglycemia and an improvement of insulin sensitivity in diabetic rats.

The Tumor Necrosis Factor derived-TIP peptide is a small 17 amino acids cyclic peptide with lectin-like activity, that possesses several therapeutically relevant biological activities, among which is activation of alveolar liquid clearance in both healthy and injured lungs in vivo. Accumulation of fluid in the lungs? alveoli and interstitial spaces is a life-threatening condition called pulmonary edema. The mortality rate due permeability pulmonary edema, accompanied by a dysfunction of the alveolar/capillary barrier, is high because no effective treatment lacking side effects exists nowadays. It is known that the TIP peptide is able to activate vectorial Na+ transport ? which mediates lung liquid clearance. However, the mechanism of action of remains elusive. The aim of this thesis was to investigate the initial steps of interaction between the TIP peptide and airway epithelial cells. Numerous novel methods and single-molecule techniques were used to unravel: (i) how the TIP peptide interacts with the molecules on the apical side of the lung epithelial cells; (ii) whether the TIP peptide need to be internalized inside of the cells to trigger its effects; (iii) the nature of the interaction between the TIP peptide and its putative receptor(s); (iv) the putative receptor(s) for the TIP peptide on the apical surface of the lung epithelial cells.

A alveolite é uma complicação pós-operatória de carácter inflamatório que acomete alvéolos de dentes recém-extraídos. A incidência dessa complicação varia de 1 a 4% e pode chegar a 30%. O objetivo deste estudo foi analisar os mecanismos biológicos envolvidos no processo de reparo de alvéolos intencionalmente infectados, em ratos; comparar diferentes modalidades de tratamento e correlacionar os resultados encontrados através de duas análises (microscópica e molecular). Foram utilizados 84 ratos, divididos nos grupos: I: alvéolo não infectado; II: alvéolo infectado sem nenhum tratamento; III: alvéolo infectado tratado com irrigação de solução de iodeto de sódio a 2% e peróxido de hidrogênio a 3% na proporção de 1:1; e IV: alvéolo infectado submetido à curetagem, irrigação com soro fisiológico e preenchimento com uma pasta à base de metronidazol. Os animais foram eutanasiados aos 6, 15 e 28 dias pós-operatório. Foi realizada a análise quantitativa da expressão de genes envolvidos no processo de reparo [colágeno tipo I (COL-I), fator de crescimento do endotélio vascular (VEGF), osteocalcina (OCN), fosfatase alcalina (ALP), runt-related transcription factor 2 (RUNX2) e fator de necrose tumoral alfa (TNF-\'alfa\')], através da RealTimePCR, correlacionando sua expressão com as características microscópicas observadas qualitativa e quantitativamente. Com base nos resultados da análise microscópica e molecular, podemos concluir que os marcadores RUNX2, OCN e TNF-\'alfa\' podem ser usados como indicadores para avaliar a neoformação óssea e a quantidade de infiltrado inflamatório em alveolite. Os marcadores ALP e VEGF não representaram adequadamente o que se observou microscopicamente. Embora o tratamento da alveolite com a pasta à base de metronidazol promova maior densidade de neoformação óssea aos 28 dias, não há diferenças entre os tratamentos. / Dry socket is an inflammatory postoperative complication that undertakes sockets of recently extracted teeth. The incidence of such complication varies from 1 to 4% and might reach up to 30%. The objective of this study was to analyze the biological mechanisms involved in the repair process of intentionally infected sockets in mice; compare different treatment conditions and correlate the results of two different analysis (microscopic and molecular). 84 mice were used in this study, divided according the following groups: I: uninfected socket; II: infected socket without any treatment; III: infected socket treated with irrigation of 2% sodium iodide and 3% hydrogen peroxide solution at 1:1 proportion; and IV: infected socket submitted to curettage, physiological saline solution irrigation and fulfillment with metronidazole base paste. The animals were killed at a postoperative period of 6, 15 and 28 days. A quantitative analysis was performed using a RealTimePCR to evaluate the genes expression involved [Collagen Type I (COL-I), vascular endothelial growth factor (VEGF), osteocalcin (OCN), alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2) and tumor necrosis factor-alpha (TNF-\'alpha\')], in the repair process, correlating its expression with the microscopic characteristics observed in both qualitative and quantitative manner. Based in the results of the microscopic and molecular analysis, it can be concluded that the RUNX2, OCN and TNF-\'alpha\' markers can be used as indicators to evaluate the dry socket bone neoformation and inflammatory infiltrate quantity. The ALP and VEGF markers did not represented appropriately what was observed microscopically. Although the dry socket treatment with metronidazole base paste promotes an increase in the bone neoformation density at 28 days, no difference was found among the treatments.

Alterações metabólicas, inflamatórias e do sistema nervoso autônomo estão presentes em pacientes com insuficiência cardíaca. No entanto, não há até o momento, consenso de que tais alterações são decorrentes da disfunção ventricular ou da síndrome de insuficiência cardíaca. Objetivo: Avaliacão do metabolismo e atividade inflamatória em pacientes com miocardiopatia dilatada chagásica e idiopática e sua correlação com medidas de funcão do sistema nervoso autônomo. Casuística: Foram avaliados 46 pacientes divididos em três grupos: pacientes com miocardiopatia dilatada idiopática, chagásica e controle, pareados entre si de acordo com sexo e idade e índice de massa corpórea. Critérios de inclusão: miocardiopatia chagásica com sorologia positiva em dois métodos diferentes (imunofluorescência indireta e ELISA) e miocardiopatia dilatada idiopática com idade superior a 18 anos; ambos os sexos; Índice de massa corporal (IMC) entre 18,5 e 25 kg/m2 e fração de ejeção < 40% pelo método de Simpson ao ecocardiograma. Metodologia: Todos pacientes foram submetidos a medidas antropométricas: índice de massa corporal e medida da porcentagem de gordura corporal através de bioimpedância. Coletado sangue para dosagens sanguíneas de leptina, adiponectina, interleucina-6, fator de necrose tumoral, glicose, insulina, colesterol total, HDL-Colesterol, LDL-Colesterol e triglicerídeos após jejum de 12 horas e realizados: Holter de 24 horas para avaliação da função autonômica, ecocardiograma transtorácico bidimensional complementado com modo-M, Doppler pulsátil, tecidual e colorido. Resultados: Não houve diferenças entre os grupos com relação a dosagem de glicemia, colesterol total, LDL-colesterol, HDL-colesterol e triglicerídeos. Com relação à leptina e adiponectina e o índice HOMA-IR, não houve diferença entre os grupos. As dosagens de insulina foram menores no grupo chagásico em comparação ao grupo controle e de idiopático (5,4; 8,0; 9,9) respectivamente (p = 0,007). As dosagens de interleucina-6 e fator de necrose tumoral-alfa foram maiores no grupo chagásico em relação aos outros grupos. A insulina correlacionou positivamente no grupo chagásico com leptina (r = 0,579; p = 0,024) e sistema nervoso autônomo ( atividade simpática) BF/AF ( r = 0,562; p = 0,029) e BF (r = 0,562; p = 0,029) e negativamente com adiponectina (r = -0,603; p = 0,017). Na análise multivariada, apenas a adiponectina foi significante. A adição de uma unidade de adiponectina reduziu a média de insulina em 0,332. Conclusões: Os níveis de insulina foram menores nos pacientes com miocardiopatia chagásica em comparação aos pacientes com miocardiopatia dilatada idiopática e controle. Os níveis das citocinas inflamatórias (TNF-alfa e interleucina-6) foram maiores nos pacientes com miocardiopatia chagásica em comparação aos pacientes com miocardiopatia dilatada idiopática e controle. A insulina correlacionou negativamente no grupo chagásico com adiponectina / Metabolic, inflammatory and autonomic nervous system changes are present in patients with heart failure. However, there is so far, consensus that these changes are due to ventricular dysfunction or heart failure syndrome. Objective: Evaluation of metabolism and inflammatory activity in patients with Chagas\' disease and idiopathic dilated cardiomyopathy and its correlation with user function measures the autonomic nervous system. Patients: A total of 46 patients divided into three groups: patients with idiopathic dilated cardiomyopathy, Chagas and control, matched each other according to sex and age and body mass index. Inclusion criteria: serology for Chagas\' disease in two different methods (indirect immunofluorescence and ELISA); idiopathic dilated cardiomyopathy; older than 18 years; both sexes; Body mass index (BMI), ie between 18.5 and 25 kg / m2 and ejection fraction < 40% by Simpson method by echocardiography. Methods: All patients underwent anthropometric measurements: body mass index and measure the percentage of body fat by bioimpedance. Blood was collected for blood leptin dosages, adiponectin, interleukin-6, tumor necrosis factor, glucose, insulin, total cholesterol, HDLcholesterol, LDL-cholesterol and triglyceride levels after fasting for 12 hours and performed: 24 hours for evaluation Holter autonomic function, two-dimensional transthoracic echocardiography complemented by M-mode, pulsed Doppler, tissue and colorful. Results: There were no differences between groups with respect to blood glucose levels, total cholesterol, LDL-cholesterol, HDLcholesterol and triglycerides. With respect to leptin and adiponectin and HOMAIR, there was no difference between groups. Insulin doses were lower in the chagasic group compared to the control group and Idiopathic: 5.4; 8.0; 9.9, respectively (p = 0.007). Dosages of interleukin-6 and tumor necrosis factoralpha were higher in the chagasic group compared to other groups. Insulin positively correlated in the chagasic group with leptin (r = 0.579; p = 0.024) and autonomic nervous system (sympathetic activity) LF / HF (r = 0.562; p = 0.029) and BF (r = 0.562; p = 0.029) and negatively with adiponectin (r = -0.603; p = 0.017). In multivariate analysis, only adiponectin was significant. The addition of an adiponectin unit reduced the average insulin 0.332. Conclusions: Insulin levels were lower in patients with Chagas\' heart disease compared to patients with idiopathic dilated cardiomyopathy and control. The levels of inflammatory cytokines (TNF-alpha and interleukin-6) were higher in patients with Chagas\' heart disease compared to patients with idiopathic dilated cardiomyopathy and control. Insulin negatively correlated with adiponectin in the chagasic group

Le cancer colorectal (CCR) est la troisième maladie maligne la plus fréquente et la deuxième cause de décès par cancer. La famille des récepteurs tyrosine kinases transmembranaires ErbB a été identifiée comme l'un des principaux moteurs du développement et de la progression du CCR et l'un de ses membres les plus connus, le récepteur du facteur de croissance épidermique (EGFR / ERBB1 / Her1), considéré comme l'une des cibles les plus importantes en traitement CRC. Deux autres membres de la famille ErbB, les récepteurs Her2 et Her3, apparaissent également comme de nouvelles cibles importantes pour le CRC en raison de la mutation somatique, de l’amplification génique ou de la résistance aux traitements anti-EGFR. La protéine transmembranaire, Fas (TNFRSF6 / CD95), est un membre de la superfamille des récepteurs du facteur de nécrose tumorale (TNFRSF). Il peut transmettre des signaux multiples qui mènent à des destins de cellules complètement différents. Selon les contextes cellulaires, Fas initie la mort cellulaire par apoptose, essentielle pour arrêter les réponses immunitaires chroniques et prévenir l'auto-immunité et le cancer, ou pour stimuler la survie, la prolifération et la motilité des cellules, ce qui favorise l'auto-immunité, la croissance cancéreuse et les métastases. Avec des preuves de plus en plus nombreuses de la signalisation pro-survie médiée par Fas, les activités de promotion du cancer chez les patients atteints de cancer sont maintenant reconnues comme étant significatives et cliniquement pertinentes. Bien que cette polyvalence de signalisation ait été particulièrement bien démontrée dans le cancer du côlon, les mécanismes moléculaires qui sous-tendent les voies de survie sont encore largement inconnus. Dans ce contexte, l'objectif principal de mon doctorat Le projet visait à étudier l’importance du crosstalks entre les membres de la famille Fas et ErbB et, plus particulièrement, à déterminer si la signalisation Fas pouvait influencer la signalisation de l’EGFR favorisant le cancer.Plus précisément, je décris comment l’état de phosphorylation de la tyrosine Fas influence fortement la signalisation de la voie EGFR dans les cellules colorectales. Mes données démontrent que Fas dans son état prosurvival, phosphorylé à Y291 (pY291-Fas), interagit en effet avec EGFR et que cette interaction intensifie significativement la signalisation de l'EGFR dans les cellules cancéreuses colorectales anti-EGFR via la voie Yes-1 / STAT3. Le pY291-Fas s'accumule dans le noyau lors du traitement par EGF et favorise la localisation nucléaire du phospho-EGFR et du phospho-STAT3, l'expression de la cycline D1, l'activation des voies Akt et MAPK médiées par STAT3 et enfin la prolifération et la migration cellulaires. De plus, je découvre également le rôle potentiel que Her3 pourrait jouer avec Fas dans la libération des cellules cancéreuses colorectales de l'inhibition anti-EGFR.Tous ensemble mon doctorat des études permet de mieux comprendre le rôle des voies de survie de Fas dans la signalisation ErBb dans le CRC. Fait important, en démontrant un lien entre l'émergence d'une résistance aux traitements anti-ErbB et le signal de Fas pro-survie, mon travail justifie le développement d'une thérapie ciblée Fas / phospho-Fas comme nouvelle option thérapeutique pour surmonter les anti-EGFR, chez les patients présentant une résistance anti-EGFR secondaire. / Colorectal cancer (CRC) is the third most common malignant disease and the second most frequent cause of cancer-related death. The ErbB family of transmembrane receptor tyrosine kinases has been identified as a major driver of the development and progression of CRC and one its best-known member, the epidermal growth factor receptor (EGFR /ERBB1/Her1), considered one of the most important targets in CRC treatment. Two others members of the ErbB family, the receptors Her2 and Her3, also emerge as important new targets for CRC due to the somatic mutation, gene amplification or resistance to the anti-EGFR therapies. The transmembrane protein, Fas (TNFRSF6/CD95), is a member of the tumor necrosis factor receptor superfamily (TNFRSF). It can transmit multiple signals that lead to completely different cell fates. Depending on cellular contexts, Fas either initiates cell death by apoptosis, which is essential for shutting down chronic immune responses and preventing autoimmunity and cancer, or stimulates cell survival, proliferation, and motility, which can promote autoimmunity, cancer growth, and metastasis. With increasing evidence of Fas-mediated pro-survival signaling, the cancer-promoting activities of Fas are now recognized as significant and clinically relevant. While this signaling versatility has been particularly well demonstrated in colon cancer, the molecular mechanisms underlying the survivals pathways are still largely unknown. In this context, the main aim of my Ph.D. project was to study the importance of the crosstalks between Fas and the ErbB family members and more specifically to determine whether the Fas signaling could influence the cancer-promoting signaling of EGFR.More precisely, I describe how the Fas tyrosine phosphorylation status strongly influences the signaling of the EGFR pathway in colorectal cells. My data demonstrate that Fas in its prosurvival state, phosphorylated at Y291 (pY291-Fas), indeed interacts with EGFR and that this interaction significantly intensifies EGFR signaling in anti-EGFR-resistant colorectal cancer cells via the Yes-1/STAT3-mediated pathway. The pY291-Fas accumulates in the nucleus upon EGF treatment and promotes the nuclear localization of phospho-EGFR and phospho-STAT3, the expression of cyclin D1, the activation of STAT3-mediated Akt and MAPK pathways, and finally the cell proliferation and migration. Additionally, I also uncover the potential role that Her3, may play along with Fas, in the colorectal cancer cell escape from anti-EGFR inhibition. All together my Ph.D. studies provide a better understanding of the role of the Fas survival pathways in the ErBb signaling in CRC. Importantly, by demonstrating a connection between the emergence of resistance to anti-ErbB therapies and the Fas pro-survival signal, my work provides a rationale for the development of Fas/phospho-Fas targeted therapy as a new therapeutic option for overcoming anti-EGFR, in patients with secondary anti-EGFR resistance.

Much of the impetus for this research can be attributed to Kenneth W. Hinchcliff, who has studied exercise-induced pulmonary haemorrhage (EIPH) extensively. This thesis focused on EIPH in Thoroughbred racehorses competing in South Africa. Using tracheobronchoscopy, the prevalence and severity of EIPH and the association with racing performance was determined. Thereafter, the prevalence of other respiratory tract disorders and their association with racing performance is reported. This is followed by a study assessing interobserver variability using grading systems in the detection of respiratory tract disorders. Finally, there is a report on the immunopathogenesis of EIPH. Using tracheobronchoscopy after racing, the prevalence and severity of EIPH was assessed in 1,005 racehorses competing at high altitude (> 1,400 meters above sea level) and at sea level in a racing jurisdiction that does not allow the use of furosemide and nasal dilator strips. The prevalence and severity of EIPH was affected by altitude as racing at sea level was associated with a higher prevalence and greater severity of EIPH. Results also suggested that EIPH was associated with superior performance in South African Thoroughbred racehorses. Upper and lower respiratory tract disorders identified following tracheobronchoscopic examination included left arytenoid asymmetry, left laryngeal hemiplegia, epiglottic deformity, epiglottic entrapment, subepiglottic cysts, dorsal displacement of the soft palate, pharyngeal lymphoid hyperplasia (PLH), laryngeal and tracheal dirt, tracheal mucous (TM), tracheal stenosis and tracheal cartilage ring spikes in Thoroughbred racehorses after racing. Overall, there was a low prevalence of grade 2 and 3 arytenoid cartilage asymmetry, left laryngeal hemiplegia, epiglottic entrapment, subepiglottic cysts and epiglottic deformity, while more severe grades of PLH, laryngeal debris, tracheal debris, TM and tracheal cartilage ring spikes had a higher prevalence. An association with sex was identified as tracheal cartilage ring spikes occurred more often in male racehorses. Superior racing performance was identified in racehorses with grade 3 tracheal mucous and tracheal cartilage ring spikes. Endoscopic grading of EIPH, PLH, arytenoid cartilage movement (ACM), and TM was performed by 3 observers that were blinded to each racehorse’s identity and race day performance using previously established grading criteria. Excellent interobserver reliability was seen using the EIPH grading system, while the weighted kappa for PLH, ACM and TM was lower. The study demonstrated sufficient reliability for the use of the EIPH, PLH, ACM and TM grading systems in racehorses competing in South Africa. The study concluded that tracheobronchoscopy seemed to be a practical screening technique that may have prognosticative validity and clinical dependability and that would allow safe and quick assessment of the respiratory tract of a large number of racehorses in field conditions. Venous blood was collected from 10 horses in each EIPH grade classification (grade 0 to 4) following tracheobronchoscopic examinations for the determination of the presence and severity of EIPH. Following RNA isolation and cDNA synthesis, real-time PCR was used to detect equine cytokine-specific mRNA for interleukin (IL) -1, -6, -10, interferon (INF) -ã, and tumor necrosis factor (TNF) -á. Results of this study indicated that increased IL-6, and -10 mRNA production was associated with more severe forms of EIPH. Also, there was greater expression of IL-6 mRNA at sea level and TNF-á mRNA at high altitude. This study concluded that although it was unclear whether the inflammatory response observed in the study was due to pre-existing pulmonary inflammation or as a direct consequence of pulmonary bleeding, the study demonstrated a systemic correlation to pulmonary inflammation. The research reported in this thesis has contributed substantially to the determination of the prevalence, severity and affect on racing performance of respiratory tract disorders in Thoroughbred racehorses competing in South Africa. Also, determination of an association between EIPH and inflammation at a molecular level may assist future researchers in anti-cytokine therapies which may help reduce the prevalence and severity of EIPH. / Thesis (PHD)--University of Pretoria, 2007. / Companion Animal Clinical Studies / unrestricted

Yersinia pestis (Y. pestis), as the causative agent of plague, has caused deaths estimated to more than 200 million people in three historical plague pandemics, including the infamous Black Death in medieval Europe. Although infection with Yersinia pestis can mostly be limited by antibiotics and only 2000-5000 cases are observed worldwide each year, this bacterium is still a concern for bioterrorism and recognized as a category A select agent by the Centers for Disease Control and Prevention (CDC). The investigation into the host-pathogen interactions during Y. pestis infection is important to advance and broaden our knowledge about plague pathogenesis for the development of better vaccines and treatments. Y. pestis is an expert at evading innate immune surveillance through multiple strategies, several mediated by its type three secretion system (T3SS). It is known that the bacterium induces rapid and robust cell death in host macrophages and dendritic cells. Although the T3SS effector YopJ has been determined to be the factor inducing cytotoxicity, the specific host cellular pathways which are targeted by YopJ and responsible for cell death remain poorly defined. This thesis research has established the critical roles of caspase-8 and RIP kinases in Y. pestis-induced macrophage cell death. Y. pestis-induced cytotoxicity is completely inhibited in RIP1-/- or RIP3-/-caspase-8-/- macrophages or by specific chemical inhibitors. Strikingly, this work also indicates that macrophages deficient in either RIP1, or caspase-8 and RIP3, have significantly reduced infection-induced production of IL-1β, IL-18, TNFα and IL-6 cytokines; impaired activation of NF-κB signaling pathway and greatly compromised caspase-1 processing; all of which are critical for innate immune responses and contribute to fight against pathogen infection. Y. pestis infection causes severe and often rapid fatal disease before the development of adaptive immunity to the V bacterium, thus the innate immune responses are critical to control Y. pestis infection. Our group has previously established the important roles of key molecules of the innate immune system: TLR4, MyD88, NLRP12, NLRP3, IL-18 and IL-1β, in host responses against Y. pestis and attenuated strains. Yersinia has proven to be a good model for evaluating the innate immune responses during bacterial infection. Using this model, the role of caspase-8 and RIP3 in counteracting bacterial infection has been determined in this thesis work. Mice deficient in caspase-8 and RIP3 are very susceptible to Y. pestis infection and display reduced levels of pro-inflammatory cytokines in spleen and serum, and decreased myeloid cell death. Thus, both in vitro and in vivo results indicate that caspase-8 and RIP kinases are key regulators of macrophage cell death, NF-κB and caspase-1 activation in Yersinia infection. This thesis work defines novel roles for caspase-8 and RIP kinases as the central components in innate immune responses against Y. pestis infection, and provides further insights to the host-pathogen interaction during bacterial challenge.

The alarming rate of increase in incidence and prevalence of the type 2 diabetes mellitus has prompted intense research on understanding the pathogenesis of the type 2 diabetes. It is observed that the development of type 2 diabetes is preceded by a state of insulin resistance and obesity. Previous studies have suggested that the obesity induced insulin resistance may be mediated by elevated levels of circulating free fatty acids (FFAs). The increase in circulating levels of FFAs may be contributed by the release of FFAs from stored triglycerides (TG) in adipocytes via lipolysis. It is hypothesized that the decrease in levels of circulating FFAs by sequestration and storage of FFAs in adipocytes may prevent deleterious effects of FFAs on insulin sensitivity. Recently our lab and others have shown that the storage of TG in adipocytes is promoted by a novel protein, Fat Specific Protein 27 (FSP27). Although, these studies also revealed FSP27 to be a lipid droplet associated protein that suppresses lipolysis to enhance TG accumulation in adipocytes, the role of FSP27 in lipolysis remains largely undetermined. Therefore, this study investigates the role and regulation of FSP27 in adipocytes in both the basal state, as well as during lipolysis. The studies presented here show FSP27 to be a remarkably short-lived protein (half-life=15 min) due to its rapid ubiquitination and proteasomal degradation. Thus, I tested the hypothesis that lipolytic agents like the cytokine, TNF-α and the catecholamine isoproterenol modulate FSP27 protein levels to regulate FFA release. Consistent with this concept, TNF-α markedly decreased FSP27 mRNA and protein along with lipid droplet size as it increased lipolysis in cultured adipocytes. Similarly, FSP27 depletion using siRNA mimicked the effect of TNF-α to enhance lipolysis, while maintaining stable FSP27 protein levels by expression of HA epitope-tagged FSP27 blocked TNF-α mediated lipolysis. In contrast, the robust lipolytic action of isoproterenol is paradoxically associated with increases in FSP27 protein and a delayed degradation rate that corresponds to decreased ubiquitination. This catecholamine-mediated increase in FSP27 abundance, probably a feedback mechanism to restrain excessive lipolysis by catecholamines, is mimicked by forskolin or 8-Bromo-cAMP treatment, and prevented by Protein Kinase A (PKA) inhibitor KT5720 or PKA depletion using siRNA. These results show that isoproterenol stabililizes FSP27 via the canonical PKA pathway and increased cAMP levels. However, the work presented here also suggests that FSP27 does not get phosphorylated in response to isoproterenol treatment, and the stabilization of FSP27 is independent of isoproterenol mediated lipolysis. The data presented in this thesis not only identifies the regulation of FSP27 as an important intermediate in mechanism of lipolysis in adipocytes in response to TNF-α and isoproterenol, but also suggests that FSP27 may be a possible therapeutic target to modulate lipolysis in adipocytes.

Human obesity is increasing globally at an impressive rate. The rise in obesity has led to an increase in diseases associated with obesity, such as type 2 diabetes. A major prerequisite for this disease is the development of insulin resistance in the muscle and adipose tissues. Interestingly, experiments in rodent models suggest that adipocytes and macrophages can profoundly influence the development of insulin resistance. Accordingly, the number of adipose tissue macrophages increases substantially during the development of obesity. Numerous research models have demonstrated that macrophages promote insulin resistance by secreting cytokines, like TNFα, which impair whole body insulin sensitivity and adipose tissue function. Additionally, enhancements of murine adipose function, particularly glucose disposal, prevent the development of insulin resistance in mice on a high fat diet. Thus, mechanisms which enhance adipose function or attenuate macrophage inflammation are of interest. Our lab previously identified mitogen activated protein kinase kinase kinase kinase 4 (MAP4K4) as a potent negative regulator of adipocyte function. In these studies, TNFα treatment increased the expression of adipocyte MAP4K4. Furthermore, the use of small interfering RNAs (siRNA) to block the increase in MAP4K4 expression protected adipocytes from some of the adverse effects of TNFα. Because MAP4K4 is a potent negative regulator of adipocyte function, an understanding of the mechanisms by which TNFα regulates MAP4K4 expression is of interest. Thus, for the first part of this thesis, I characterized the signaling pathways utilized by TNFα to regulate MAP4K4 expression in cultured adipocytes. Here I show that TNFα increases MAP4K4 expression through a pathway requiring the transcription factors activating transcription factor 2 (ATF2) and the JUN oncogene (cJUN). Through TNFα receptor 1 (TNFR1), but not TNFR2, TNFα increases MAP4K4 expression. This increase is highly specific to TNFα, as the inflammatory agents IL-1β, IL-6 and LPS did not affect MAP4K4 expression. In agreement, the activation of cJUN and ATF2 by TNFα is sustained over a longer period of time than by IL-1β in adipocytes. Finally, MAP4K4 is unique as the expression of other MAP kinases tested fails to change substantially with TNFα treatment. For the second part of this thesis, I assessed the role of MAP4K4 in macrophage inflammation in vitro and in vivo. To accomplish this task, pure β1,3-D-glucan shells were used to encapsulate siRNA. Glucan shells were utilized because they are effectively taken up by macrophages which express the dectin-1 receptor and they survive oral delivery. I demonstrate that these β1,3-D-glucan encapsulated RNAi particles (GeRPs) are efficiently phagocytosed and capable of mediating the silencing of multiple macrophage genes in vitro and in vivo. Importantly, oral treatment of mice with GeRPs fails to increase plasma IFNγ and TNFα or alter serum AST and ALT levels. Orally administered GeRPs are found in macrophages isolated from the spleen, liver, lung and peritoneal cavity and mediate macrophage gene silencing in these tissues. Utilizing this technology, I reveal that MAP4K4 augments the expression of TNFα in macrophages following LPS treatment. Oral delivery of MAP4K4 siRNA in GeRPs silences MAP4K4 expression by 70% and reduces basal TNFα and IL-1β expression significantly. The depletion of MAP4K4 in macrophages protects 40% of mice from death in the LPS/D- galactosamine (D-GalN) model of septicemia, compared to less than 10% in the control groups. This protection associates with significant decreases in serum TNFα concentrations following LPS/D-GalN challenge. Consistent with reduced macrophage inflammation, hepatocytes from mice treated orally with GeRPs targeting MAP4K4 present less apoptosis following LPS/D-GalN treatment. Thus, MAP4K4 is an important regulator of macrophage TNFα production in response to LPS. The results presented here add to the knowledge of MAP4K4 action in adipocyte and macrophage inflammation substantially. Prior to these studies, the mechanism by which TNFα controlled MAP4K4 expression in adipocytes remained unknown. Considering that MAP4K4 is a negative regulator of adipocyte function, identifying the mechanisms that control MAP4K4 expression was of interest. Furthermore, the role of macrophage MAP4K4 in LPS stimulated TNFα production was also unknown. To address this question in vivo, new technology specifically targeting macrophages was needed. Thus, we developed a technology for non toxic and highly specific macrophage gene silencing in vivo. Considering that macrophages mediate numerous diseases, the application of GeRPs to these disease models is an exciting new possibility.

Indiana University-Purdue University Indianapolis (IUPUI). / Tumor necrosis factor-induced protein 6 (TSG-6) has been shown to mitigate inflammation. Its presence in the secretome of adipose stem / stromal cells (ASC) and its role in activities of ASC have been overlooked. This thesis described for the first time the release of TSG-6 from ASC, and its modulation by endothelial cells. It also revealed that protection of endothelial barrier function was a novel mechanism underlying the anti-inflammatory activity of both ASC and TSG-6. Moreover, TSG-6 was found to inhibit mitogen-activated lymphocyte proliferation, extending the understanding of its pleiotropic effects on major cell populations involved in inflammation. Next, enzyme-linked immunosorbent assays (ELISA) were established to quantify secretion of TSG-6 from human and murine ASC. To study the importance of TSG-6 to specific activities of ASC, TSG-6 was knocked down in human ASC by siRNA. Murine ASC from TSG-6-/- mice were isolated and the down-regulation of TSG-6 was verified by ELISA. The subsequent attempt to determine the efficacy of ASC in ameliorating ischemic limb necrosis and the role of TSG-6, however, was hampered by the highly variable ischemic tissue necrosis in the BALB/c mouse strain. Afterwards in a mouse model of cigarette smoking (CS), in which inflammation also plays an important role, it was observed, for the first time, that 3-day CS exposure caused an acute functional exhaustion and cell cycle arrest of hematopoietic progenitor cells; and that 7-week CS exposure led to marked depletion of phenotypic bone marrow stem and progenitor cells (HSPC). Moreover, a dynamic crosstalk between human ASC and murine host inflammatory signals was described, and specifically TSG-6 was identified as a necessary and sufficient mediator accounting for the activity of the ASC secretome to ameliorate CS-induced myelotoxicity. These results implicate TSG-6 as a key mediator for activities of ASC in mitigation of inflammation and protection of HSPC from the myelotoxicity of cigarette smoke. They also prompt the notion that ASC and TSG-6 might potentially play therapeutic roles in other scenarios involving myelotoxicity.