Young and ovariectomized animals eating diets rich in omega-3 polyunsaturated fatty acids (n-3 PUFAs) exhibit enhanced bone formation and decrease bone loss, respectively. Eicosapentaenoic acid, an n-3 PUFA found in fish oil, competes with arachidonic acid, an n-6 PUFA, for the cyclooxygenase enzyme, modulating prostaglandin E2, a mediator of bone mechanotransduction. Whether this diet affects bone gains during exercise is not well defined. We hypothesized rats consuming a high n-3 PUFA diet would gain more bone mass with increased bone formation compared to the rats consuming a high n-6 PUFA diet in response to exercise. Virgin Sprague-Dawley rats (5-mo-old, n=18) were assigned to one of two groups: diet rich in corn oil with a n-6:n-3 dietary ratio of 23:1 (O6) or a diet rich in fish oil with an n-6:n-3 dietary ratio of 2:1 (O3). After acclimation, rats completed 9 sessions on alternate days of stimulated muscle contractions at 75% peak isometric strength. Structural and densitometric properties of proximal tibia were measured using in vivo peripheral quantitative CT. Bone formation rate was quantified on the periosteal the surface by standard bone histomorphometry after intraperitoneal injections of calcein. There was a significant main effect due to diet on total volumetric bone mineral density. The diet rich in n-3 PUFAs also allowed for increases in cancellous volumetric bone mineral density at the proximal tibia independent from exercise, as high as 28%. However, proximal tibia metaphysis bone size and shape was not modified due to changes in diet. The training protocol resulted in a robust increase in bone formation, mass, and area at the midshaft tibia. Mineral apposition rate and bone formation rate were significantly greater in the O3 group compared to the O6 group with exercise at the midshaft tibia, ~36% and ~38% respectively. However, the greater bone formation seen in the O3 groups did not translate over to significantly greater bone mass and size as noted by the pQCT results at the same bone site, because there were no detectable differences between groups. In summary, our data demonstrate that a diet high in n-3 PUFAs independently increases bone density at the proximal tibia. In addition, there was enhanced BFR due to a diet high in n-3 PUFAs with exercise, but those increases did not translate over to increased cortical bone mass or size. These data provide evidence that fish oil consumption with and without simulated resistance training exercise can be beneficial to bone outcomes.

Apoptosis is executed by caspase-mediated cleavage of various proteins. Elucidating the consequence of substrate cleavage provides us with insight into cell death and other biological processes. In this study, we applied the diagonal gel approach, a proteomic strategy, to identify substrates of the inflammatory caspase, caspase-1 and the cell death executioner caspase, caspase-3. Our results showed significant overlap between the substrates cleaved by both caspase-1 and -3. Such substrates are implicated in common cellular functions, including maintenance of the cytoskeleton, folding of proteins, translation, glycolysis, bioenergetics, signaling and trafficking. An important finding is that many glycolysis enzymes were targeted specifically by caspase-1. Processing of these glycolysis enzymes by caspase-1 was confirmed by cleaving in vitro transcribed and translated substrates with recombinant caspase-1. We have focused our further analysis on certain glycolysis enzymes. We have characterized the caspase-1 cleavage site in GAPDH. Point mutation of the Aspartic acid at position 189 to Alanine (D189A) in GAPDH blocked its cleavage by caspase-1. In vivo, in a mice model of septic shock, characterized by hyperactivation of caspase-1, we observed depletion of the full-length forms of these glycolysis enzymes in the diaphragm muscle. Further studies in caspase-1 deficient mice will confirm whether this depletion, in caspase-1 proficient mice, was due to caspase-1 processing of the glycolysis enzymes. This provides a direct link between caspase-1 activation and inhibition of glycolysis, which might have important implications on loss of muscle contractility in septic shock.

In many tumors, persistently-active signal transducer and activator of transcription 3 (STAT3) imparts several oncogenic features such as survival, proliferation, angiogenesis, and immune escape. Therefore, STAT3 targeting in cancer and cancer-exposed dendritic cells (DCs) is important for cancer therapy. Our objective is developing delivery modalities of STAT3-targeting small interfering RNA (siRNA) using lipid-modified polyethylenimine (PEI) polyplexes and poly(D,L lactic-co-glycolic) acid (PLGA) nanoparticles (NPs), and evaluating the therapeutic outcomes in vitro and in vivo. Significant increase in siRNA condensation, protection, and cellular uptake by B16.F10 melanoma was seen by stearic-acid-modified PEI (PEI-StA) compared to unmodified PEI. Moreover, PEI-StA increased the STAT3 silencing potency of siRNA compared to PEI. STAT3 knockdown was accompanied with significant induction of interleukin-6 (IL-6) secretion and reduction of vascular endothelial growth factor (VEGF) production and cytotoxicity evidenced by increased Caspase 3 activity in vitro and in vivo, and significant inhibition in tumor growth. Analysis of tumor microenvironment showed CD3+ cells infiltration corresponding to STAT3 knockdown. The levels of CD4+ helper cells, CD8+ cytotoxic cells, and NKT cells significantly increased. DC infiltration and activation significantly increased in tumor mass following STAT3 knockdown as evidenced by high expression of CD86 and CD40. Moreover, IFN-, IL-12, and TNF- significantly increased following STAT3 knockdown by PEI-StA compared to PEI, suggesting Th1-type immunity. Allogenic capacity of DCs isolated from siRNA-treated mice was evidenced by the high T cell proliferation and IL-2 production in mixed lymphocytes reaction (MLR). Then, we explored STAT3 knockdown in DCs exposed to tumor derived factors (TDFs). We investigated encapsulation of siRNA complexes (PEI or PEI-StA) into PLGA NPs (PLGA-P and PLGA-PS). PLGA-P and PLGA-PS had an average diameter of ~ 370 nm and zeta potential of ~ -16 mV. Uptake and endosomal localization was confirmed. After TDFs exposure, DCs showed high STAT3 and low CD86 expression. STAT3 silencing by PLGA-P and PLGA-PS restored DC functionality as evidenced by upregulation of CD86, IL-12, and TNF- and MLR activity. PLGA significantly reduced PEI-associated toxicity. Therefore, STAT3 targeting in B16 cells by siRNA polyplexes of PEI and PEI-StA, or in DCs by PLGA-P and PLGA-PS provide potential strategies for cancer therapy. / Pharmaceutical Sciences

Graduation date: 1991

The aim of this thesis was to examine the role of diet in ankylosing spondylitis (AS). Patients were examined in: i) a postal questionnaire survey of dietary habits and gastrointestinal (GI) symptoms; ii) a study on biomarkers of diet and disease activity; iii) a comparison of cardiovascular risk factors with the general population using data from the Västerbotten Intervention Programme (VIP), and; iv) a 21-week omega-3 fatty acid supplementation study regarding the effects on disease activity. The postal survey (111 respondents) revealed no correlation between dietary habits and disease activity measured by the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). However, GI problems, and in particular GI pain, were prevalent in patients with AS irrespective of NSAID usage.Gastrointestinal pain was predicted by higher BASDAI and a higher consumption of vegetables. Overall, 30 (27%) of the patients experienced an aggravation of gastric symptoms when consuming certain foods. In the study of biomarkers (n=66) no correlation was found between diet and disease activity as assessed by BASDAI. There were, however, positive correlations between BASDAI and the content of arachidonic acid (AA) in plasma phospholipids (rs=0.39, p<0.01) and the estimated activity of the enzyme delta-5-desaturase (rs=0.37, p<0.01). This may reflect a process involved in the inflammation associated with AS that requires further investigation. Comparing data from the VIP for patients (n=89) and controls showed no significant differences regarding diet, physical activity or smoking. Nonetheless, more pronounced correlations between blood lipids and diet were identified among patients than in controls. Furthermore, the levels of cholesterol and triglycerides were lower in patients compared with controls. Lastly, in the supplementation study, a high-dose of long-chain omega-3 fatty acids (4.55 grams/day) was found to lower disease activity, as measured by BASDAI, whereas low-dose treatment (1.95 grams/day) caused no change. In conclusion, within a group of Swedish AS patients we found no correlation between ordinary dietary habits and disease activity. Diet in western populations of patients with AS may, however, be of importance for gastric symptoms and for cardiovascular risk factors. The finding of a lowered disease activity in patients on high-dose supplementation with long-chain omega-3 fatty acids indicates that a radical dietary shift may influence disease activity. The findings of a positive correlation between disease activity and plasma AA, and the decreased levels of blood lipids imply the need for further studies into fatty acid metabolism in AS.

La historia natural de la infección por el virus de la inmunodeficiencia humana (VIH-1) incluye la infección aguda, seguida de una fase asintomática de infección crónica, que desemboca en la fase de crisis o síndrome de inmunodeficiencia adquirida (SIDA). En ausencia de una terapia anti-retroviral potente, la mayoría de los pacientes tarda entre 8 y 10 años en evolucionar hacia la fase SIDA, mientras que una minoría permanecen asintomáticos, denominados "no progresores de alta duración" (LTNP). También se han identificado individuos que, a pesar de permanecer altamente expuestos al virus, permanecen seronegativos (expuestos no identificados o ENI. Justamente los ENI y los LTNP son unos excelentes modelos para el estudio de cuáles son los factores capaces de proporcionar protección al sujeto, lo que abre el camino para desvelar claves esenciales para comprender la patogenia de la infección y, por lo tanto, para desarrolar nuevas estrategias preventivas y terapéuticas.

In adult cardiomyocytes, the established mechanism of excitation-contraction coupling is calcium-induced calcium release (CICR) mediated by L-type Ca2+ channels (Cav1.2). Briefly, membrane depolarization opens voltage-gated Cav1.2 to allow for the influx of extracellular Ca2+ into the cytosol. This small sarcolemmal (SL) Ca2+ influx is necessary for triggering a larger release of Ca2+ from the intracellular Ca2+ storage site, the sarcoplasmic reticulum (SR), through the SR Ca2+ release channel also known as the ryanodine receptor (RyR). RyR-mediated release of SR Ca2+ effectively raises the cytosolic free Ca2+ concentration, allowing for Ca2+ binding to troponin C on the troponin-tropomysin complex, leading to cross-bridge formation and cell contraction. However, previous functional data suggests an additional CICR modality involving reverse mode Na+-Ca2+ exchanger (NCX) activity also exists in neonate cardiomyocytes. To further our understanding of how CICR changes occur during development, we investigated the spatial arrangement of caveolin-3 (cav-3), the principle structural protein of small membrane invaginations named caveolae, and NCX in developing rabbit ventricular myocytes. Using traditional as well as novel image processing and analysis techniques, both qualitative and quantitative findings firmly establish the highly robust and organized nature of NCX and cav-3 distributions during development. Specifically, our results show that NCX and cav-3 are distributed on the peripheral membrane as discrete clusters and are not highly colocalized throughout development. 3D distance analysis revealed that NCX and cav-3 clusters are organized with a distinct longitudinal and transverse periodicity of 1-1.5 μm and that NCX and cav-3 cluster have a pronounced tendency to be mutually exclusive on the cell periphery. Although these findings do not support the original hypothesis that caveolae is the structuring element for a restricted microdomain facilitating NCX-CICR, our results cannot rule out the existence of such microdomain organized by other anchoring proteins. The developmentally stable distributions of NCX and cav-3 imply that the observed developmental CICR changes are achieved by the spatial re-organization of other protein partners of NCX or non-spatial modifications. In addition, the newly developed image processing and analysis techniques can have wide applicability to the investigations on the spatial distribution of other proteins and cellular structures.

Corrections pasted behind title page. / Bibliography: leaves 294-341. / xi, 341, [42] leaves, [42] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Involves the construction of a detailed physical map of the distal band of the long arm of human chromosome 16, 16q24. / Thesis (Ph.D.)--University of Adelaide, Dept. of Cytogenetics and Molecular Genetics, 1997

Thèse (D. en mathématiques)--Université du Québec à Montréal, 2005. / En tête du titre: Université du Québec à Montréal. Bibliogr.: f. [136]-140. Publié aussi en version électronique.

Zugl.: Stuttgart, Univ., Diss., 2006