It has been demonstrated that EGCG and caffeine, naturally present in green tea, have thermogenic properties in thermoneutral conditions. The purpose of this study was to quantify the effect of the combined ingestion of EGCG/caffeine on thermogenic responses during a 3h mild cold exposure. Eight healthy males (22± 1 y) were exposed in a randomized, cross over, single blinded fashion to the cold (liquid conditioned suit perfused with 15°C water), after ingesting either a placebo (CON) or an extract of 1600mg of EGCG and 600mg of caffeine (EXP). Thermic, metabolic and electromyographic measurements were monitored at baseline and during cold exposure. After 180min of cold exposure, shivering intensity was significantly reduced by ~32% in EXP condition compared to CON. Area under the curve calculations for total shivering intensity was also reduced by ~21% in EXP (457±99 %MVC.min) compared to CON (361±81 %MVC.min; p=0.007). In contrast, the total area under curve of VO2 was ~25% higher in EXP (33.3±5.5 L O2) compared to CON (25.3±5.1 L O2; p=0.03). Total Heat production (Hprod) also increased by about 11% in the EXP condition (1535±112 kJ) compared to control (1372 ±106 kJ; p=0.002). The decrease in shivering activity combined with an increase in VO2 and Hprod, following the ingestion of EGCG and caffeine in the cold, indicates that nonshivering thermogenesis pathways can be significantly stimulated in adult humans.

The external skeletons, Exoskeletons, are not a new research area in this highly developed world. They are widely used in helping the wearer to enhance human strength, endurance, and speed while walking with them. Most exoskeletons are designed for the whole body and are powered due to their applications and high performance needs. This thesis introduces a novel design of a three-degree of freedom parallel robotic structured hip exoskeleton, which is quite different from these existing exoskeletons. An exoskeleton unit for walking typically is designed as a serial mechanism which is used for the entire leg or entire body. This thesis presents a design as a partial manipulator which is only for the hip. This has better advantages when it comes to marketing the product, these include: light weight, easy to wear, and low cost. Furthermore, most exoskeletons are designed for lower body are serial manipulators, which have large workspace because of their own volume and occupied space. This design introduced in this thesis is a parallel mechanism, which is more stable, stronger and more accurate. These advantages benefit the wearers who choose this product. This thesis focused on the analysis of the structure of this design, and verifies if the design has a reasonable and reliable structure. Therefore, a series of analysis has been done to support it. The mobility analysis and inverse kinematic solution are derived, and the Jacobian matrix was derived analytically. Performance of the CAD model has been checked by the finite element analysis in Ansys, which is based on applied force and moment. The comparison of the results from tests has been illustrated clearly for stability iii and practicability of this design. At the end of this thesis, an optimization of the hip exoskeleton is provided, which offers better structure of this design. / UOIT

The intracellular delivery of molecules to modulate signaling pathways and gene expression is a powerful approach to control stem cell fate decision. For applications in gene therapy and regenerative medicine, the use of genetic material and viral vectors raise concerns because stem cells persist throughout life, and long-term effects of uncontrolled genetic modifications could affect the cellular progeny. An alternative is to deliver directly peptides or proteins using cell-permeable peptides (CPPs) which have the ability of crossing the plasma membrane and carrying cargos into cells. CPPs can therefore be used to deliver factors to direct stem cells proliferation, survival and differentiation. This thesis describes an approach to control stem cell fate based on the delivery of a CPP-conjugated bioactive peptide. A first significant contribution from this work is the development of a flow cytometric assay to accurately quantify the uptake of a panel of CPPs. This study revealed that HIV-transactivator of transcription (TAT) and Antennapedia (Antp) offered the highest level of translocation in different cell types. The uptake was improved by treating the cells with a single, low-voltage electrical pulse that selectively enhances the amount of TAT-conjugated peptides and proteins delivered by at least an order of magnitude, without causing cellular toxicity or apoptosis. Subsequently, flow cytometry, confocal microscopy, capillary electrophoresis and mass spectrometry were used to examine the intracellular fate of TAT-conjugated peptides in order to define the parameters that limit their bioactivity and point to specific sequence modifications that can improve their efficacy. The advances described in this thesis were applied to the development of TAT-eIF2B, a peptide-inhibitor of glycogen synthase kinase-3 (GSK-3). TAT-eIF2B was found to be specific for GSK-3 and had a significant positive effect on the formation of neurospheres in embryonic stem cell cultures and on the survival of myeloid progenitors in cytokine-starved fetal liver cell cultures. On the other hand, GSK-3 inhibition reduced the number of neurospheres generated by human olfactory neuroepithelium cells due to lower proliferation and increased neuronal differentiation. In summary, this work describes the development of a peptide-based technology to deliver bioactive cargoes in cells, and it demonstrates its utility for modulating the activity of a master regulator of stem cell fate decision.

Mastitis is the most prevalent and major cause of economic losses in dairy farms. Bovine mastitis caused by strains of <i>S. aureus</i> is a major economically important disease affecting the dairy industry worldwide. <i>S. aureus</i> is one of the most common udder pathogens that cause either clinical or sub-clinical mammary gland infections. Different treatment regimes have failed to cure <i>S. aureus</i> intramammary infections. Most mastitis vaccination strategies have focused on the enhancement of systemic humoral immunity rather than strengthening local intramammary immunity. Vaccines aimed at enhancing intramammary immunity of dairy cows against <i>S. aureus</i> mastitis have had limited success. Commercially available vaccines show various degrees of success and work in research laboratories with experimental vaccines suggest that in part, the failure of these vaccines lies in the limited antigenic repertoire contained in the vaccine formulations. Moreover, not only does variation in the antigenic composition but also presence of capsular polysaccharide in most pathogenic strains and decreased activity of immune effectors in milk affect the success of vaccines. In addition to these, the ability of <i>S. aureus</i> to attach and internalize into mammary epithelial cells, enables bacteria to escape from the effect of immunity and antibiotics by being hidden in the intracellular niche and thereby causing chronic recurrent intramammary infection. <i>S. aureus</i> also has the ability to become electron-transport-defective and to form slow-growing small colonies that are non haemolytic and less virulent. These small colony variants might hide from the immune surveillance in the intracellular area and revert to the parental strain causing chronic recurrent infections. If immunization targets antigenic molecules that are conserved throughout all pathogenic strains, even the small colony variants can be controlled since the immune system will clear the parental strain which causes lethal infection. Thus, immunization trials should focus on conserved immunogenic antigen molecules among pathogenic strains formulated with an adjuvant and delivered by a route of immunization to induce maximum stimulation of the immune system. Moreover, immunization should focus on inducing Th1 responses, which is protective against <i>S. aureus</i> mastitis. It has been reported that proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity might be used as such antigens to induce protection against parasitic and microbial infections. Previous study in our laboratory on mastitis-causing streptococci indicates that GapC proteins of <i>S. uberis</i> and <i>S. dysgalactiae</i> have potential as vaccine antigens to protect dairy cows against mastitis caused by environmental streptococci. Two conserved cell wall associated proteins with iii glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, GapB and GapC have been identified from <i>S. aureus</i> isolates from bovine intramammary infections. The overall goal of this study was to improve our understanding on intramammary immunity using the GapC and GapB proteins of <i>S. aureus</i> as model antigens for mastitis and to determine the regulation of expression of <i>gapB</i> and <i>gapC</i> genes and their roles in the pathogenesis of bovine <i>S. aureus</i> mastitis. We hypothesized that strengthening local intramammary immunity using GapB and GapC proteins of <i>S. aureus</i> as antigens will protect against bovine <i>S. aureus</i> mastitis. To test this hypothesis we took the approach of using the <i>gapB</i> and <i>gapC</i> genes and constructed plasmids encoding GapB, GapC and GapB::GapC (GapC/B) chimeric proteins. We set six objectives to test our hypothesis using these proteins to enhance the intramammary immunity. In aim 1 we constructed plasmids encoding the GapB, GapC proteins and also constructed a chimeric gene encoding the GapC and GapB proteins as a single entity (GapC/B chimera) as the basis for a multivalent vaccine. In this objective the humoral and cellular immune responses to GapC/B were compared to the responses to the individual proteins alone or in combination in C57 BL/6 mice. Our results showed that the GapC/B protein elicited strong humoral and cellular immune responses as judged by the levels of total IgG, IgG1, IgG2a, IL-4 and IFN-ã secretion and lymphocyte proliferation. These results strongly suggest the potential of this chimeric protein as a target for vaccine production to control mastitis caused by <i>S. aureus</i>. In aim 2 we continued our studies on GapC/B by testing the effects of DNA vaccination with plasmids encoding the individual gapB and gapC genes as well as the gapC/B protein gene with or without a boost with the recombinant proteins. The results showed that DNA vaccination alone was unable to elicit a significant humoral response and barely able to elicit a detectable cell-mediated response to the recombinant antigens but subsequent immunization with the proteins elicited an excellent response. In addition, we found that DNA vaccination using a plasmid encoding the GapC/B chimera followed by a boost with the same protein, although successful, is less effective than priming with plasmids encoding GapB or GapC followed by a boost with the individual antigens. In aim 3 we optimized immune responses in cows by comparing route of vaccination (subcutaneous versus intradermal), site of vaccination (locally at the area drained by the supramammary lymph node versus distantly at area drained by parotid lymph node. Our results showed that both subcutaneous and intradermal immunizations with the GapC/B protein at the area drained by the supramammary and parotid lymph nodes resulted in significantly increased serum and milk titers of total IgG, IgG1, IgG2, iv and IgA in all vaccinated groups as compared to placebo. The anti-GapC/B IgG1 serum and milk titers were significantly higher in all vaccinated group as compared to the placebo group. These results indicated that vaccination at the area drained by the supramammary lymph node resulted in better immune responses. In aim 4 we tested different formulations of the GapC/B antigen with adjuvants such as PCPP, CpG, PCPP + CpG and VSA-3. We found that the VSA-3 formulation induced the best immune responses in cows. In this objective we also monitored immune responses longitudinally over one lactation cycle to determine the duration of immune responses by measuring IgG, IgG1, IgG2, and IgA on monthly blood and milk samples. We found that the duration of immune responses was about four months. In aim 5 we tested the role of GapC in the virulence of <i>S. aureus</i> mastitis using the <i>S. aureus</i> wild type strain RN6390 and its isogenic GapC mutant strain H330. Our results from both in vitro adhesion and invasion assays on MAC- T cells and in vivo infection of ovine mammary glands showed that GapC is an important virulence factor in <i>S. aureus</i> mastitis. In aim 6 we examined the role of sar and agr loci on the expression of <i>gapC</i> and <i>gapB</i> genes by qRT- PCR using <i>S. aureus</i> RN6390 and its isogenic mutants defective in agrA, sarA and sar/agr (double mutant) at exponential and stationary phases of growth. Our results showed that both <i>gapB</i> and <i>gapC</i> expression were down regulated in the mutant strains, indicating that the expression of the <i>gapB</i> and <i>gapC</i> genes is controlled by the universal virulence gene regulators, agr and sar. We also checked the role of environmental factors such as pH, growth media, and oxygen tension on the expression of <i>gapB</i> and <i>gapC</i> using q-RT-PCR. Our results showed that the expression of <i>gapB</i> and <i>gapC</i> genes in different strains of <i>S. aureus</i> was not consistent under the above-mentioned environmental conditions.

Fatty acid synthase (FAS), an enzyme that is over-expressed in many cancers, is necessary for cancer cell proliferation. Previously, we have shown that FAS in cancer cells is regulated at least in part, by Sp1. Indole-3-carbinol (I3C) and its acid condensation product, 3,3’-diindolylmethane (DIM) modulate various transcription factors involved in regulating cellular proliferation and apoptosis. The objective of this study was to determine whether reductions in breast cancer cell proliferation caused by I3C and/or DIM occur as a result of reductions in FAS. DIM and, in some cases, I3C reduced FAS expression in three breast cancer cell lines. However, addition of palmitate or oleate to DIM-treated MCF-7 cells did not restore proliferation. DIM-associated reduction in proliferation of MCF-7 cells also results in a reduction of Sp1 expression, and down-regulation of FAS occurs after inhibition of proliferation. Thus, the anti-proliferative effect of I3C and DIM may be due to their effect on down-regulating Sp1, which in turn could modify several Sp1-associated genes, including FAS.

Fatty acid synthase (FAS), an enzyme that is over-expressed in many cancers, is necessary for cancer cell proliferation. Previously, we have shown that FAS in cancer cells is regulated at least in part, by Sp1. Indole-3-carbinol (I3C) and its acid condensation product, 3,3’-diindolylmethane (DIM) modulate various transcription factors involved in regulating cellular proliferation and apoptosis. The objective of this study was to determine whether reductions in breast cancer cell proliferation caused by I3C and/or DIM occur as a result of reductions in FAS. DIM and, in some cases, I3C reduced FAS expression in three breast cancer cell lines. However, addition of palmitate or oleate to DIM-treated MCF-7 cells did not restore proliferation. DIM-associated reduction in proliferation of MCF-7 cells also results in a reduction of Sp1 expression, and down-regulation of FAS occurs after inhibition of proliferation. Thus, the anti-proliferative effect of I3C and DIM may be due to their effect on down-regulating Sp1, which in turn could modify several Sp1-associated genes, including FAS.

The intracellular delivery of molecules to modulate signaling pathways and gene expression is a powerful approach to control stem cell fate decision. For applications in gene therapy and regenerative medicine, the use of genetic material and viral vectors raise concerns because stem cells persist throughout life, and long-term effects of uncontrolled genetic modifications could affect the cellular progeny. An alternative is to deliver directly peptides or proteins using cell-permeable peptides (CPPs) which have the ability of crossing the plasma membrane and carrying cargos into cells. CPPs can therefore be used to deliver factors to direct stem cells proliferation, survival and differentiation. This thesis describes an approach to control stem cell fate based on the delivery of a CPP-conjugated bioactive peptide. A first significant contribution from this work is the development of a flow cytometric assay to accurately quantify the uptake of a panel of CPPs. This study revealed that HIV-transactivator of transcription (TAT) and Antennapedia (Antp) offered the highest level of translocation in different cell types. The uptake was improved by treating the cells with a single, low-voltage electrical pulse that selectively enhances the amount of TAT-conjugated peptides and proteins delivered by at least an order of magnitude, without causing cellular toxicity or apoptosis. Subsequently, flow cytometry, confocal microscopy, capillary electrophoresis and mass spectrometry were used to examine the intracellular fate of TAT-conjugated peptides in order to define the parameters that limit their bioactivity and point to specific sequence modifications that can improve their efficacy. The advances described in this thesis were applied to the development of TAT-eIF2B, a peptide-inhibitor of glycogen synthase kinase-3 (GSK-3). TAT-eIF2B was found to be specific for GSK-3 and had a significant positive effect on the formation of neurospheres in embryonic stem cell cultures and on the survival of myeloid progenitors in cytokine-starved fetal liver cell cultures. On the other hand, GSK-3 inhibition reduced the number of neurospheres generated by human olfactory neuroepithelium cells due to lower proliferation and increased neuronal differentiation. In summary, this work describes the development of a peptide-based technology to deliver bioactive cargoes in cells, and it demonstrates its utility for modulating the activity of a master regulator of stem cell fate decision.

It has been demonstrated that EGCG and caffeine, naturally present in green tea, have thermogenic properties in thermoneutral conditions. The purpose of this study was to quantify the effect of the combined ingestion of EGCG/caffeine on thermogenic responses during a 3h mild cold exposure. Eight healthy males (22± 1 y) were exposed in a randomized, cross over, single blinded fashion to the cold (liquid conditioned suit perfused with 15°C water), after ingesting either a placebo (CON) or an extract of 1600mg of EGCG and 600mg of caffeine (EXP). Thermic, metabolic and electromyographic measurements were monitored at baseline and during cold exposure. After 180min of cold exposure, shivering intensity was significantly reduced by ~32% in EXP condition compared to CON. Area under the curve calculations for total shivering intensity was also reduced by ~21% in EXP (457±99 %MVC.min) compared to CON (361±81 %MVC.min; p=0.007). In contrast, the total area under curve of VO2 was ~25% higher in EXP (33.3±5.5 L O2) compared to CON (25.3±5.1 L O2; p=0.03). Total Heat production (Hprod) also increased by about 11% in the EXP condition (1535±112 kJ) compared to control (1372 ±106 kJ; p=0.002). The decrease in shivering activity combined with an increase in VO2 and Hprod, following the ingestion of EGCG and caffeine in the cold, indicates that nonshivering thermogenesis pathways can be significantly stimulated in adult humans.

Objectives: The main objective was to assess the effectiveness of early postoperative enteral feeding with an arginine, omega-3 fatty acids and RNA-supplemented formula in improving the wound healing process in patients undergoing surgery for gastric cancer. The assessment was done both measuring local hydroxyproline deposition as an index of reparative collagen synthesis and qualitative indicators of wound healing, including the presence or absence of complications derived from the healing process failure. As a secondary objective, it was assessed the effectiveness of early postoperative administration of the formula in reducing the number of infectious complications. Additional outcome variables were overall morbidity and length of hospital stay.Summary background data:Early postoperative immunonutrition has invariably demonstrated significant improvements in the patients' immunological status , inflammatory response and patient outcome. Even though one of the most frequent complications in patients with neoplastic disease and associated malnutrition is the delay or failure of surgical wound healing, the influence of early immunonutrition on the wound healing process has not yet been evaluated.Methods: Sixty patients of both genders with a preoperative diagnosis of gastric cancer were prospectively randomized in a double-blind fashion to receive early enteral feeding with either an arginine, omega-3 fatty acids, and RNA-supplemented formula or an isocaloric, isonitrogenous control formula, both administered through a jejunostomy tube for 7 days. Six out of them did not complete the study and were excluded from the statistical analysis. Hydroxyproline deposition in subcutaneously placed catheters (Goodson and Hunt method), postoperative wound healing complications and infectious complications were analyzed as main outcome variables.Results: Patients treated with immunonutrition showed higher local hydroxyproline levels (59.7 nmol (5.0 - 201.8), vs. 28.0 nmol (5.8 - 89.6) p: 0.0018), significantly lower episodes of surgical wound healing complications (0 Vs 8 (26.7%) p: 0.005), significantly lower number of infectious complications (2 (6.7%) vs. 9 (30%) p: 0.01) and lower overall morbidity (4 (13.3%) vs. 13 (43.3%) p: 0.01) when compared to patients treated with standard enteral nutrition. Median length of hospital stay was shorter for patients treated with the study formula (13 days vs. 15 days, p: 0.02). Multivariate analysis to evaluate the endpoint 'presence of any postoperative complication' found the feeding group to be a significant predictor after adjusting by patient age and initial weight (OR=2.49 p: 0.001). Conclusions: Early postoperative enteral nutrition with a formula supplemented with arginine, omega 3 fatty acids and RNA increased hydroxyproline synthesis, improved surgical wound healing and significantly reduced the number of postoperative infections and the general morbidity in patients undergoing gastrectomy for gastric cancer.

L'objectiu de la tesi doctoral és estudiar la importància del temps com a factor de benestar quotidià en el context de la societat contemporània. Més concretament, es proposa analitzar la creixent vinculació del temps amb el benestar a partir de les relacions socials que s'estableixen entorn del treball. Quina és l'aportació dels diferents temps de treball al benestar o què es percep socialment com a benestar són dos dels interrogants de partida. La perspectiva teòrica proposada per abordar aquest objecte d'estudi pivota sobre tres eixos. En primer lloc, es proposa un canvi d'enfocament en la concepció del benestar: un canvi que suposa el pas del benestar material al benestar quotidià, el pas d'una aproximació macro a una aproximació micro vers aquest concepte. Aquest canvi es caracteritza per l'ampliació de la perspectiva econòmica i productivista que sovint s'utilitza per definir i mesurar el benestar. En aquest sentit, la tesi defensa l'ús d'un concepte de benestar que permeti considerar tant els aspectes materials com els simbòlics. En segon lloc, es proposa mesurar les necessitats &#8722;individuals i socials&#8722; no només en termes de risc vinculats a l'ocupació, sinó també en termes de promoció del benestar quotidià lligats a la reproducció en general i a la cura en particular. Per això, es parteix de la perspectiva de gènere a l'hora de definir el concepte treball i la seva relació amb el temps, la qual cosa suposa tenir present que el treball no és, estrictament, un sinònim d'ocupació i que el temps no equival, únicament, a la jornada laboral. D'aquesta manera l'objecte d'estudi també inclou els temps i els treballs no reconeguts econòmicament però imprescindibles per a la reproducció i el benestar de les persones. En tercer lloc, s'entén el concepte temps amb tota la seva amplitud, és a dir, no cenyit a les agulles del rellotge, la qual cosa suposa subratllar-ne el caràcter multidimensional i, més específicament, el doble vessant que el caracteritza: l'objectiu i el subjectiu. Concretament, es parteix de la idea que el contingut i la mesura del temps depenen dels contextos socioculturals on es defineixen. Aquesta reflexió no es pot obviar ni per estudiar la importància creixent del temps dins la societat actual, ni per respondre com i per què el temps s'ha situat al centre de les demandes i necessitats socials existents en la societat del benestar. Aquests tres eixos es fan operatius a través de la vida quotidiana, considerada un escenari material i simbòlic. Pel que fa la perspectiva metodològica, en primer lloc, proposa la combinació de les aproximacions quantitativa i qualitativa amb l'objectiu de conèixer els significats subjectius atribuïts al temps dedicat al treball. I, en segon lloc, es proposa la combinació de les anàlisis estructural i biogràfic per tal d'introduir la dimensió longitudinal. Aquest plantejament metodològic es concreta amb l'ús de l'entrevista biogràfica i l'anàlisi estructural del discurs.L'opció per aquestes tècniques d'investigació i anàlisi respon a la doble voluntat de recollir les percepcions subjectives relacionades amb el temps, el treball i el benestar segons les condicions materials d'existència, així com discriminar la influència de les variables curs de vida i generació. Es considera que el material empíric recollit a través d'aquesta tècnica d'investigació permet aprofundir en les raons estructurals i biogràfiques que expliquen la importància del temps com a factor de benestar. / The goal of the PhD is to study the importance of the time as a factor of daily well-being in the context of the western society. More precisely, it proposes to analyze the increasing linking between time, well-being and work. Some questions to answer are: Which is the contribution of social times to well-being? What is perceived socially as a well-being?The theoretical approach included three points. First point proposes change the conception of well-being, that is, from material well-being to daily well-being. This change supposes a step in the use of the concept: from macro and economical approach to a micro and social approach. The investigation defends the use of well-being as a concept which it allows to consider the material characteristics as well as the symbolic ones. The second point proposes to measure the individual and social needs in terms of risk linked to the employment and in terms of promotion of the daily well-being through domestic work and care. Because of that, the research included de gender perspective to use main concepts such as work and time. From this point of view, work is not considering a synonym of employment and time is not considering a synonym of work time. Consequently the object of study also includes times and works not recognized economically but indispensable for human's reproduction and well-being. The third point defends the multidimensional character of time as sociological concept. These three points are made operative through daily life which is considered a material and symbolic framework.The methodological perspective introduces the combination of quantitative and qualitative approaches to knowing the subjective meanings attributed at the time work. And, in order to introduce the longitudinal dimension it proposes the combination of structural and biographical analyses. More over, the empirical work included the biographical interview and the structural analysis of the speech. On the one hand, both techniques approach to the subjective perceptions according to the material conditions. In the other hand, they allow knowing the structural and biographical reasons that explain the importance of the time as a factor of well-being.