MMP-8 Activity Profiling In GCF Samples

Background: In healthy tissues a family of enzymes known as matrix metalloproteinases (MMPs) play an important role in regulating turnover and metabolism of connective tissue collagen. MMPs have been implicated in a wide variety of pathological conditions including periodontal disease. MMP-8 has been extensively studied in periodontal health and disease using enzyme-linked immunosorbent assay (ELISA). Although ELISA quantifies the presence of the MMP-8 protein, it is not possible to determine enzyme activity using this method. Furthermore, since members of the MMP family have poor substrate sequence specificity, a peptide substrate alone cannot differentiate the activity of MMP-8 from other MMPs that may be present in biological samples. Objectives: In the present study, a method to specifically measure MMP-8 activity in gingival crevicular fluid (GCF) samples was developed. Methods: GCF was collected from healthy patients and those with periodontal disease using Perio paper strips. Samples were stored frozen until required for analysis. A specific MMP-8 antibody was used to coat 96 well microtitre plates to selectively remove MMP-8 from the GCF samples. Following a washing step, the activity of bound MMP-8 was measured over 70 minutes using a fluorogenic (FRET) substrate. Results: GCF from healthy subjects exhibited basal MMP-8 activity but in diseased samples MMP-8 activity was significantly higher. Minimal binding of other recombinant MMPs to the specific MMP-8 antibody was observed in cross-reactivity studies. Conclusion: We show for the first time that MMP-8 activity was significantly increased in GCF from periodontitis sites compared with activity levels in healthy sites. Further studies of MMP-8 activity in GCF samples should improve our understanding of its destructive role in periodontal disease.

Expressão das proteínas da matriz na discondroplasia da tíbia

A discondroplasia da tíbia (TD) em aves consiste numa anomalia do esqueleto onde existe uma falha nos processos normais da ossificação endocondral. Esta patologia é caracterizada pela formação de uma cartilagem não vascularizada e não mineralizada que se estende até à metáfise. Uma vez que existem várias anomalias do esqueleto em mamíferos com lesões semelhantes às apresentadas pela TD, este trabalho teve como objectivo a caracterização desta patologia em termos das moléculas que podem estar envolvidas no seu desenvolvimento. Assim, foi estudada a expressão das macromoléculas da matriz extracelular, das enzimas degradadoras da matriz (metaloproteinases da matriz: MMPs), bem como das moléculas envolvidas na proliferação e diferenciação celular, na angiogénese e apoptose. A expressão génica foi realizada, por PCR quantitativo em tempo real, em placas de crescimento normais e discondroplásicas obtidas a partir de frangos de carne (broilers) da estirpe Cobb. Os níveis proteicos de algumas MMPs foram analisados por immunoblotting e zimografia de gelatina. No presente estudo não se verificou alteração na expressão dos genes dos colagénios do tipo II, IX, X e XI, bem como do agrecano, nas lesões discondroplásicas. Observou-se uma redução acentuada nos níveis de mRNA da gelatinase-B (MMP-9), da colagenase-3 (MMP-13) e das estromalisinas -2 (MMP-10) e -3 (MMP-11), bem como nos níveis proteicos da gelatinase-A (MMP-2) e da MMP-13. Por outro lado, a MMP-7 aumentou drasticamente a expressão do seu gene. As moléculas envolvidas na proliferação e diferenciação dos condrócitos, tais como a PTHrP, o Ihh, o Cbfa-1 e o Sox-9, mantiveram a sua expressão génica nas lesões. Por outro lado, o TGF-β reduziu a sua expressão. A caspase-3 também dimimuiu a sua expressão na patologia. Em relação aos factores angiogénicos, o FGF manteve a sua expressão e o VEGF aumentou significativamente nas lesões. Este aumento do VEGF juntamente com o aumento da MMP-7 sugere um aumento da hipoxia nas lesões. Os nossos resultados sugerem que a acumulação da cartilagem observada na discondroplasia é devida a uma diminuição da proteólise da matriz, resultado de uma sub-expressão das MMPs, e não de um aumento da produção das macromoléculas da matriz. Desta forma, os nossos resultados sugerem que a falha na expressão e/ou activação das MMPs poderá estar associada ao desenvolvimento da discondroplasia da tíbia em aves. Finalmente, os nossos resultados vêm suportar os resultados anteriores que sugerem uma ligação entre a expressão das MMPs e anomalias no processo de ossificação endocondral.

Salivary proteomics and peptidomics of type 1 diabetes Mellitus

A Diabetes Mellitus (DM) compreende um conjunto de desordens metabólicas comuns caracterizadas por hiperglicemia, que afeta diferentes órgãos do organismo. Ao longo do tempo, ocorrem danos microvasculares no glomérulo renal, retina e nervos periféricos, bem como doença macrovascular nas artérias. A composição da saliva também é afetada pela DM, com consequências na homeostasia oral. No entanto, o proteoma e o peptidoma salivar têm sido pouco explorados na DM tipo 1 e nas suas complicações crónicas. Tendo em conta o crescente interesse na saliva como fluido diagnóstico, o objetivo principal deste trabalho foi avaliar os eventos proteolíticos subjacentes à DM tipo 1 e às suas complicações microvasculares, bem como, caracterizar as alterações induzidas pela DM tipo 1 no proteoma e peptidoma salivar. A DM tipo 1 e particularmente as complicações microvasculares associadas modulam o perfil proteolítico dos fluidos biológicos, com diferenças significativas de atividade observadas na urina e saliva, atribuídas principalmente ao complexo Metaloproteinase da Matriz (MMP)-9/lipocalina associada à gelatinase de neutrófilos, aminopeptidase N, azurocidina e calicreína 1. O aumento da atividade proteolítica observado na saliva total dos diabéticos resultou no aumento da percentagem de péptidos, principalmente de um número acrescido de fragmentos de colagénio do tipo I, refletindo possivelmente um estado inflamatório crónico dos tecidos orais e periodontais. O peptidoma também corrobora uma maior suscetibilidade das proteínas salivares, especificamente, das proteínas ricas em prolina básicas (bPRP) 1, bPRP2 e proteínas ricas em prolina ácidas (aPRP) à proteólise, evidenciando a geração de fragmentos de proteínas associadas à ligação a bactérias. A análise do proteoma salivar baseada em iTRAQ mostrou uma sobre-expressão de L-plastina, fator do adenocarcinoma do pâncreas e das proteínas S100-A8 e S100-A9, enfatizando a importância do sistema imune inato na patogénese da DM tipo 1 e das complicações microvasculares associadas. A análise integrada de todas as proteínas expressas diferencialmente entre os pacientes diabéticos com ou sem complicações microvasculares e indivíduos saudáveis foi realizada com o STRING, onde se observam três conjuntos funcionalmente ligados, um compreende a interação entre o colagénio tipo I, colagénio tipo II e MMP-9, um segundo conjunto envolve a MMP-2 e o colagénio de tipo I e um terceiro conjunto composto por proteínas salivares e inflamatórias. Estes conjuntos estão associados com as vias Kegg de interação recetor-matriz extracelular, de adesão focal e migração transendotelial dos leucócitos. Por outro lado, a análise do proteoma e peptidoma salivar destacou potenciais biomarcadores para o diagnóstico e prognóstico da DM tipo 1 e das suas complicações.

Cinética da transferência de calor em materiais com mudança de fase

É extensa a bibliografia dedicada a potenciais aplicações de materiais com mudança de fase na regulação térmica e no armazenamento de calor ou de frio. No entanto, a baixa condutividade térmica impõe limitações numa grande diversidade de aplicações com exigências críticas em termos de tempo de resposta curto ou com requisitos de elevada potência em ciclos de carga/descarga de calor latente. Foram desenvolvidos códigos numéricos no sentido de obter soluções precisas para descrever a cinética da transferência de calor com mudança de fase, com base em geometrias representativas, i.e. planar e esférica. Foram igualmente propostas soluções aproximadas, sendo identificados correspondentes critérios de validação em função das propriedades dos materiais de mudança de fase e de outros parâmetros relevantes tais como as escalas de tamanho e de tempo, etc. As referidas soluções permitiram identificar com rigor os fatores determinantes daquelas limitações, quantificar os correspondentes efeitos e estabelecer critérios de qualidade adequados para diferentes tipologias de potenciais aplicações. Os referidos critérios foram sistematizados de acordo com metodologias de seleção propostas por Ashby e co-autores, tendo em vista o melhor desempenho dos materiais em aplicações representativas, designadamente com requisitos ao nível de densidade energética, tempo de resposta, potência de carga/descarga e gama de temperaturas de operação. Nesta sistematização foram incluídos alguns dos compósitos desenvolvidos durante o presente trabalho. A avaliação das limitações acima mencionadas deu origem ao desenvolvimento de materiais compósitos para acumulação de calor ou frio, com acentuada melhoria de resposta térmica, mediante incorporação de uma fase com condutividade térmica muito superior à da matriz. Para este efeito, foram desenvolvidos modelos para otimizar a distribuição espacial da fase condutora, de modo a superar os limites de percolação previstos por modelos clássicos de condução em compósitos com distribuição aleatória, visando melhorias de desempenho térmico com reduzidas frações de fase condutora e garantindo que a densidade energética não é significativamente afetada. Os modelos elaborados correspondem a compósitos de tipo core-shell, baseados em microestruturas celulares da fase de elevada condutividade térmica, impregnadas com o material de mudança de fase propriamente dito. Além de visarem a minimização da fração de fase condutora e correspondentes custos, os modelos de compósitos propostos tiveram em conta a adequação a métodos de processamento versáteis, reprodutíveis, preferencialmente com base na emulsificação de líquidos orgânicos em suspensões aquosas ou outros processos de reduzidas complexidade e com base em materiais de baixo custo (material de mudança de fase e fase condutora). O design da distribuição microestrutural também considerou a possibilidade de orientação preferencial de fases condutoras com elevada anisotropia (p.e. grafite), mediante auto-organização. Outros estágios do projeto foram subordinados a esses objetivos de desenvolvimento de compósitos com resposta térmica otimizada, em conformidade com previsões dos modelos de compósitos de tipo core-shell, acima mencionadas. Neste enquadramento, foram preparados 3 tipos de compósitos com organização celular da fase condutora, com as seguintes características e metodologias: i) compósitos celulares parafina-grafite para acumulação de calor, preparados in-situ por emulsificação de uma suspensão de grafite em parafina fundida; ii) compósitos celulares parafina-Al2O3 para acumulação de calor, preparados por impregnação de parafina em esqueleto cerâmico celular de Al2O3; iii) compósitos celulares para acumulação de frio, obtidos mediante impregnação de matrizes celulares de grafite com solução de colagénio, após preparação prévia das matrizes de grafite celular. Os compósitos com esqueleto cerâmico (ii) requereram o desenvolvimento prévio de um método para o seu processamento, baseado na emulsificação de suspensões de Al2O3 em parafina fundida, com adequados aditivos dispersantes, tensioactivos e consolidantes do esqueleto cerâmico, tornando-o auto-suportável durante as fases posteriores de eliminação da parafina, até à queima a alta temperatura, originando cerâmicos celulares com adequada resistência mecânica. Os compósitos desenvolvidos apresentam melhorias significativos de condutividade térmica, atingindo ganhos superiores a 1 ordem de grandeza com frações de fase condutora inferior a 10 % vol. (4 W m-1 K-1), em virtude da organização core-shell e com o contributo adicional da anisotropia da grafite, mediante orientação preferencial. Foram ainda preparados compósitos de armazenamento de frio (iii), com orientação aleatória da fase condutora, obtidos mediante gelificação de suspensões de partículas de grafite em solução aquosa de colagénio. Apesar da estabilidade microestrutural e de forma, conferida por gelificação, estes compósitos confirmaram a esperada limitação dos compósitos com distribuição aleatória, em confronto com os ganhos alcançados com a organização de tipo core-shell.

Cancer-associated fibroblasts in brain metastases

Tese de mestrado. Oncobiologia, Faculdade de Medicina, Universidade de Lisboa, 2015

Platelets induce Pai-1 mRNA expression in monocytes through TGF-BETA

Introduction: Plasminogen activator inhibitor type-1 (PAI-1) is a physiological modulator of fibrinolysis. High plasma PAI-1 is associated with the 4G/5G promoter polymorphism and with increased cardiovascular risk. Here we explored the role of platelets in regulating expression of the PAI-1 gene in monocytes. Methods: Blood from PAI-1 4G/5G genotyped volunteers (n=6) was incubated with the platelet GPVI-specific agonist, cross-linked collagen related peptide (CRP-XL), in the presence or absence of Mab 9E1 that blocks the binding of P-selectin to PSGL1. Monocytes were isolated by +ve selection on CD14 beads and monocyte PAI-1 mRNA expression was measured by real-time PCR. Results: Activation of platelets with CRP-XL resulted in platelets binding to >70% of monocytes and was accompanied by >5000-fold induction of PAI-1 mRNA, peaking at 4hrs. PAI-1 expression was independent of the 4G/5G genotype. Blocking the binding of platelets to monocytes enhanced PAI-1 induction (p<0.05 at 4 hrs). Incubation of isolated monocytes with the releasate from CRP-XL stimulated platelets also led to PAI-1 mRNA expression. The platelet secretome contains >100 different proteins. To identify the soluble factor(s) responsible for induction of PAI-1, neutralizing antibodies to likely candidates were added to monocytes incubated with the platelet releasate. Anti- TGF-beta inhibited platelet releasate-mediated PAI-1 mRNA induction by >80%. Monocyte PAI-1 was also induced by stimulation of PSGL-1 with a P-selectin-Fc chimera, in the absence of platelets, which was also blocked by the TGF-beta antibody. Conclusions: These results suggest that platelets induce PAI-1 mRNA in monocytes predominantly via TGF-beta, released from both platelets, and monocytes via activation by PSGL-1 signalling.This stimulation is independent of 4G/5G genotype

Glucose-dependent insulinotropic polypeptide receptor deficiency leads to modifications of trabecular bone volume and quality in mice.

A role for the gastro-intestinal tract in controlling bone remodeling is suspected since serum levels of bone remodeling markers are affected rapidly after a meal. Glucose-dependent insulinotropic polypeptide (GIP) represents a suitable candidate in mediating this effect. The aim of the present study was to investigate the effect of total inhibition of GIP signaling on trabecular bone volume, microarchitecture and quality. We used GIP receptor (GIPR) knockout mice and investigated trabecular bone volume and microarchitecture by microCT and histomorphometry. GIPR-deficient animals at 16 weeks of age presented with a significant (20%) increase in trabecular bone mass accompanied by an increase (17%) in trabecular number. In addition, the number of osteoclasts and bone formation rate was significantly reduced and augmented, respectively in these animals when compared with wild-type littermates. These modifications of trabecular bone microarchitecture are linked to a remodeling in the expression pattern of adipokines in the GIPR-deficient mice. On the other hand, despite significant enhancement in bone volume, intrinsic mechanical properties of the bone matrix was reduced as well as the distribution of bone mineral density and the ratio of mature/immature collagen cross-links. Taken together, these results indicate an increase in trabecular bone volume in GIPR KO animals associated with a reduction in bone quality.

A-kinase anchoring protein-Lbc promotes pro-fibrotic signaling in cardiac fibroblasts

In response to stress or injury the heart undergoes an adverse remodeling process associated with cardiomyocyte hypertrophy and fibrosis. Transformation of cardiac fibroblasts to myofibroblasts is a crucial event initiating the fibrotic process. Cardiac myofibroblasts invade the myocardium and secrete excess amounts of extracellular matrix proteins, which cause myocardial stiffening, cardiac dysfunctions and progression to heart failure. While several studies indicate that the small GTPase RhoA can promote profibrotic responses, the exchange factors that modulate its activity in cardiac fibroblasts are yet to be identified. In the present study, we show that AKAP-Lbc, an A-kinase anchoring protein (AKAP) with an intrinsic Rho-specific guanine nucleotide exchange factor (GEF) activity, is critical for activating RhoA and transducing profibrotic signals downstream of type I angiotensin II receptors (AT1Rs) in cardiac fibroblasts. In particular, our results indicate that suppression of AKAP-Lbc expression by infecting adult rat ventricular fibroblasts with lentiviruses encoding AKAP-Lbc specific short hairpin (sh) RNAs strongly reduces the ability of angiotensin II to promote RhoA activation, differentiation of cardiac fibroblasts to myofibroblasts, collagen deposition as well as myofibroblast migration. Interestingly, AT1Rs promote AKAP-Lbc activation via a pathway that requires the α subunit of the heterotrimeric G protein G12. These findings identify AKAP-Lbc as a key Rho-guanine nucleotide exchange factor modulating profibrotic responses in cardiac fibroblasts.

Increased numbers of circulating polyfunctional Th17 memory cells in patients with seronegative spondylarthritides

OBJECTIVE: A distinct subset of proinflammatory CD4+ T cells that produce interleukin-17 was recently identified. These cells are implicated in different autoimmune disease models, such as experimental autoimmune encephalomyelitis and collagen-induced arthritis, but their involvement in human autoimmune disease has not yet been clearly established. The purpose of this study was to assess the frequency and functional properties of Th17 cells in healthy donors and in patients with different autoimmune diseases. METHODS: Peripheral blood was obtained from 10 psoriatic arthritis (PsA), 10 ankylosing spondylitis (AS), 10 rheumatoid arthritis (RA), and 5 vitiligo patients, as well as from 25 healthy donors. Synovial tissue samples from a separate group of patients were also evaluated (obtained as paraffin-embedded sections). Peripheral blood cells were analyzed by multiparameter flow cytometry and immunohistochemistry. Cytokine production was examined by enzyme-linked immunosorbent assay and intracellular cytokine staining using specific monoclonal antibodies. Synovial tissue was examined for infiltrating T cells by immunohistochemical analysis. RESULTS: We found increased numbers of circulating Th17 cells in the peripheral blood of patients with seronegative spondylarthritides (PsA and AS), but not in patients with RA or vitiligo. In addition, Th17 cells from the spondylarthritis patients showed advanced differentiation and were polyfunctional in terms of T cell receptor-driven cytokine production. CONCLUSION: These observations suggest a role of Th17 cells in the pathogenesis of certain human autoimmune disorders, in particular the seronegative spondylarthritides.

Endoscopic and surgical treatment of vesico-ureteral reflux in children. Comparative long-term follow-up.

PRINCIPLES: This retrospective study analyzes the long-term results of endoscopic and surgical treatment of vesico-ureteral reflux in children. METHODS: A cohort of 130 patients, 67 girls and 63 boys with a mean age of 30 months were treated either by endoscopic subureteral collagen injection (SCIN) in 92 and by Cohen reimplantation surgery in 123 refluxing ureteral units. Mean follow-up was 4.2 years varying from 1 to 8.7 years. Reflux recurrence, urinary tract infection (UTI) and renal function were evaluated. RESULTS: After SCIN reflux was absent in 64% at 6 months. 20% of the initially 92 refluxing ureters were injected twice. After one or two injections reflux was absent in 71%. In 21% recurrent reflux was of grade I or II, not requiring further treatment. UTI was observed in 27%. After Cohen ureteral reimplantation reflux was absent in 96% at 6 months. UTI was observed in 23%. Renal function at diagnosis and follow-up was compared in children with bilateral grade III reflux only. In patients treated with SCIN it was normal in 77% preoperatively and in 90% at follow-up. In patients treated by open surgery it was normal in 47% preoperatively and in 76% at follow-up. CONCLUSION: For high-grade vesico-ureteral reflux re-implantation surgery remains the gold standard. SCIN is indicated for low and medium grade reflux. Recurrent bacteriuria was observed more often after SCIN and pyelonephritis more often after open surgery. The renal function seems to be preserved with both techniques.

Tgfbi/Bigh3 silencing activates ERK in mouse retina.

BIGH3 is a secreted protein, part of the extracellular matrix where it interacts with collagen and integrins on the cell surface. BIGH3 can play opposing roles in cancer, acting as either tumor suppressor or promoter, and its mutations lead to different forms of corneal dystrophy. Although many studies have been carried out, little is known about the physiological role of BIGH3. Using the cre-loxP system, we generated a mouse model with disruption of the Bigh3 genomic locus. Bigh3 silencing did not result in any apparent phenotype modifications, the mice remained viable and fertile. We were able to determine the presence of BIGH3 in the retinal pigment epithelium (RPE). In the absence of BIGH3, a transient decrease in the apoptotic process involved in retina maturation was observed, leading to a transient increase in the INL thickness at P15. This phenomenon was accompanied by an increased activity of the pro-survival ERK pathway.

Expression and function of endothelin and its receptors in vascular adventitial fibroblasts

Objective: The adventitia has been recognized to play important roles in vascular oxidative stress, remodelling and contraction. We recently demonstrated that adventitial fibroblasts are able to express endothelin-1 (ET-1) in response to angiotensin II (ANG II). However, the mechanisms by which ANG II induces ET-1 expression are unknown. It is also unclear whether the ET-1 receptors are expressed in the adventitia. We therefore examined the role of oxidative stress in the regulation of ET-1. We also investigated the expression of both the ETA and ETB receptors and the roles of these two types of receptors in collagen synthesis and ET-1 clearance in adventitial fibroblasts. Methods and Results: Adventitial fibroblasts were isolated and cultured from the thoracic mouse aorta. Cells were treated with ANG II (lOOnM), ET-1 (lOpM), NADPH oxidase inhibitor apocynin (lOOfiM), the superoxide anion scavenger tempol (lOOfiM), the ANG II receptor antagonists (100[aM), losartan (AT| receptor) and PD 1233 19 (AT2 receptor), the ET-1 receptor antagonists (lOOuM), BQ123 (ETA receptor) and BQ788 (ETB receptor), and the ETB receptor agonist (lOOnM) Sarafotoxin 6C. ET-1 peptide levels were determined by ELISA, while ETA ,ETB and collagen levels were determined by Western blot. ANG II increased ET-1 peptide levels in a time-dependent manner reaching significance when incubated for 24 hours. NAD(P)H oxidase inhibitor, apocynin, as well as the superoxide scanverger, tempol, significantly reduced ANG Il-induced ET-1 peptide levels while over-expression of SOD1 (endogenous antioxidant enzyme) significantly decreased ANG Il-induced collagen I expression, therefore implicating reactive oxygen species in the mediation of ET-1. ANG II increased ETA receptor protein as well as collagen in a similar fashion, reaching significance after 4, 6, and 24 hours treatment. ANG II induced collagen was reduced while in the presence of the ETA receptor antagonist suggesting the role of the ETa receptor in the regulation of the extracellular matrix. ANG II treatment also increased ETB receptor protein levels in a time-dependent manner. ANG II treatment in the presence of the ETB receptor antagonist significantly increased ET-1 peptide levels. On another hand, the ETB receptor agonist, Sarafotoxin 6C, significantly decreased ET-1 peptide levels. These data implicate the role of the ETb receptor in the clearance of the ET-1 peptide. Conclusion: ANG II-induced increases of ET-1 peptide appears to be mediated by reactive oxygen species derived from NAD(P)H oxidase. Both the ETA and ETB receptors are expressed in adventitial fibroblasts. The ETA receptor subtype mediates collagen I expression, while the ETB receptor may play a protective role through increasing the clearance of the ET- 1 peptide.

Effects of dietary restraint and oral contraceptives on bone strength and bone turnover in young women

ABSTRACT Introduction The purpose of this study was to assess specific osteoporosis-related health behaviours and physiological outcomes including daily calcium intake, physical activity levels, bone strength, as assessed by quantitative ultrasound, and bone turnover among women between the ages of 18 and 25. Respective differences on relevant study variables, based on dietary restraint and oral contraceptive use were also examined. Methods One hundred women (20.6 ± 0.2 years of age) volunteered to participate in the study. Informed written consent was obtained by all subjects prior to participation. The study and all related procedures were approved by the Brock University Research Ethics Board. Body mass, height, relative body fat, as well as chest, waist and hip circumferences were measured using standard procedures. The 10-item restrained eating subscale of the Dutch Eating Behaviour Questionnaire (DEBQ) was used to assess dietary restraint (van Strien et al., 1986). Daily calcium intake was assessed by the Rapid Assessment Method (RAM) (Hertzler & Frary 1994). Weekly physical activity was documented by the 4-item Godin Leisure-Time Exercise Questionnaire (Godin & Shephard 1985). Bone strength was determined from the speed of sound (SOS) as measured by QUS (Sunlight 7000S). SOS measurements (m/s) were taken of the dominant and non-dominant sides of the distal one third of the radius and the mid-shaft of the tibia. Resting blood samples were collected from all subjects between 9am and 12pm, in order to evaluate the impact of lifestyle factors on biochemical markers of bone turnover. Blood was collected during the early follicular phase of the menstrual cycle (approximately days 1-5) for all subjects. Samples were centrifliged and the serum or plasma was aliquoted into separate tubes and stored at -80°C until analysis. The bone formation markers measured were Osteocalcin (OC), bone specific alkaline phosphatase (BAP) and 25-OH vitamin D. The bone resorption markers measured were the carboxy (CTx) and amino (NTx) terminal telopeptides of type-I collagen crosslinks. All markers were assessed by ELISA. Subjects were divided into high (HDR) and low dietary restrainers (LDR) based on the median DEBQ score, and also into users (BC) and non-users (nBC) of oral contraceptives. A series of multiple one way ANOVA's were then conducted to identify differences between each set of groups for all relevant variables. A two-way ANOVA analysis was used to explore significant interactions between dietary restraint and use of oral contraceptives while a univariate follow-up analysis was also performed when appropriate. Pearson Product Moment Correlations were used to determine relationships among study variables. Results HDR had significantly higher BMI, %BF and circumference measures but lower daily calcium intake than LDR. There were no significant differences in physical activity levels between HDR and LDR. No significant differences were found between BC and nBC in body composition, calcium intake and physical activity. HDR had significantly lower tibial SOS scores than LDR in both the dominant and non-dominant sites. The post-hoc analysis showed that within the non-birth control group, the HDR had significantly lower tibial SOS scores of bone strength when compared to the LDR but Aere were no significant differences found between the two dietary restraint groups for those currently on birth control. HDR had significantly lower levels of OC than LDR and the BC group had lower levels of BAP than the nBC group. Consistently, the follow-up analysis revealed that within those not on birth control, subjects who were classified as HDR had significantly (f*<0.05) lower levels of OC when compared with LDR but no significant differences were observed in bone turnover between the two dietary restraint groups for those currently on birth control. Physical activity was not correlated with SOS scores and bone turnover markers possibly due to the low physical activity variability in this group of women. Conclusion This is the first study to examine the effects of dietary restraint on bone strength and turnover among this population of women. The most important finding of this study was that bone strength and turnover are negatively influenced by dietary restraint independent of relative body fat. In general, the results of the present thesis suggest that dietary restraint, oral contraceptive use, as well as low daily calcium intake and low physical activity levels were widespread behaviours among this population of college-aged women. The young women who were using dietary restraint as a strategy to lose weight, and thus were in the HDR group, despite their higher relative body fat and weight, had lower scores of bone strength and lower levels of markers of bone turnover compared to the low dietary restrainers. Additionally, bone turnover seemed to be negatively affected by oral contraceptives, while bone strength, as assessed by QUS, seemed unaffected by their use in this population of young women. Physical activity (weekly energy expenditure), on the other hand, was not associated with either bone strength or bone tiimover possibly due to the low variability of this variable in this population of young Canadian women.

The isolation, partial purification and preliminary characterization of a growth factor from chick embryo brains

Chicl( brain growth factor (CBGF) is a mitogen isolated from embryonic chick brains thought to have a potential role as a trophic factor involved in nerve dependent amphibian limb regeneration. In addition, CBGF stimulates 3H-thymidine incorporation in chick embryo brain astrocytes in vitro. In this study, cultured chick embryo brain non-neuronal cells were employed in a bioassay to monitor CBGF activity throughout various stages of its pllrification. Cell culture and assay conditions were optimized. Nonneuronal cells grew best on collagen-coated culture dishes in complete medium, were most responsive to a growth stimulus [10% fetal bovine serum (FBS)] at the second and third subcultures, and were healthiest when rendered "quiescent" in medium supplemented with 1% FBS. The most effective bioassay conditions consisted of a minimum 14.5 hour "quiescence" time (24 hours was used), a 6 hour "prestimulation" time, and a 24 hour 3H-thymidine labeling time. Four-day subconfluent primary non-neuronal cells consisted of 6.63% GFAP positive cells; as a result cultures were thought to be mainly composed of astroblasts. CBGF was purified from 18-day chick embryo brains by ultrafiltration through Amicon PM-30 and YM-2 membranes, size exclusion chromatography through a Biogel P6 column, and analytical reverse-phase high-performance liquid chromatography (rp-HPLC). The greatest activity resided in rp-HPLC fraction #7 (10 ng/ml) which was as effective as 10% FBS at stimulating 3H-thymidine incorporation in chick embryo brain nonneuronal cells. Although other researchers report the isolation of a mitogenic fraction consisting of 5'-GMP from the embryonic chick brain, UV absorbance spectra, rp-HPLC elution profiles, and fast atom bombardment (FAB) mass spectra indicated that CBGF is neither 5'-GMP nor 51-AMP. 2 Moreover, commercially available 5t-GMP was inhibitory to 3H-thymidine incorporation in the chick non-neuronal cells, while Sf-AMP had no effect. Upon treatment with pronase, the biological activity of fraction P6-3 increased; this increase was nearly 30% greater than what would be expected from a simple additive effect of any mitogenic activity of pronase alone together with P6-3 alone. This may suggest the presence of an inhibitor protein. The bioactive component may be a protein protected by a nucleoside/nucleotide or simply a nucleoside/nucleotide acting alone. While the FAB mass spectrum of rp-HPLC fraction #7 did not reveal molecular weight or sequence information, the ion of highest molecular weight was observed at m/z 1610; this is consistent with previous estimations of CBGF's size. 3

The Structural and Functional Changes of Blood Vessels during Aging

The vascular adventitia is recognized as a dynamic mediator of vascular structure and function, yet its role in aging is not understood. The purpose of this thesis was to examine the age-related changes of the vascular adventitia and determine the underlying mediators responsible. Male Sprague-Dawley rats were aged to 15, 30, 50 and 80 weeks before being anesthetised and euthanized by exsanguination. Thoracic aortas, mesenteric and pudental arteries were isolated, formalin fixed, and embedded in paraffin then sectioned at 5μm. Vessels were examined by microscopy and protein expression was determined by indirect immunofluorescence. The thickness of the adventitia increased dramatically with age. Immunofluorescence revealed a robust expression of endothelin system proteins in the adventitia. Additionally, extracellular matrix proteins collagen and fibronectin, and the proliferation marker Ki67 showed strong adventitial origin. The changes observed in the vascular adventitia with aging clearly demonstrate an important role in the process of vascular aging.