Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 161-178).

Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains ix, 144 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 126-144).

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 150-177). Also available on the Internet.

Η HARP είναι ένας αυξητικός παράγοντας με πλειοτροπική δράση που εμπλέκεται στην ανάπτυξη, τη διαφοροποίηση και τη μετανάστευση πολλών ειδών κυττάρων καθώς και στην αγγειογένεση και την καρκινογένεση. Η HARP ασκεί τις δράσεις της μέσω των διαμεμβρανικών της υποδοχέων RPTPβ/ζ, ALK και Ν-συνδεκάνη. Η δομή της HARP αποτελείται από δύο κεντρικές περιοχές ομόλογες με τις επαναλαμβανόμενες περιοχές τύπου I της θρομβοσπονδίνης (Thrombospondin type I Repeat, TSR) και το αμινοτελικό και καρβοξυτελικό της άκρο που έχουν τυχαία διαμόρφωση στο χώρο. Έχει βρεθεί ότι η HARP αποτελεί υπόστρωμα διάφορων πρωτεολυτικών ενζύμων και πεπτίδια της έχουν ανιχνευθεί στο υγρό θρεπτικό μέσο σε καλλιέργειες διαφόρων κυττάρων. Τα πεπτίδια της HARP μπορούν να έχουν διαφορετική ή και αντίθετη δράση από αυτή ολόκληρου του μορίου. Στo πλαίσιo της μελέτης των επιμέρους περιοχών της HARP που συμμετέχουν στις βιολογικές δράσεις της αλλά και της δράσης των πεπτιδίων που προκύπτουν από την πρωτεόλυσή της, σε αυτή την εργασία μελετήθηκε η δράση δύο ανασυνδυασμένων, τροποποιημένων μορφών της HARP που αντιστοιχούν στις κεντρικές TSR περιοχές, των H9-59 και H60-110, στον πολλαπλασιασμό και την προσκόλληση κυττάρων καρκίνου του προστάτη PC3 και στη φωσφορυλίωση των μορίων μεταγωγής σήματος ERK1/2. Τα αποτελέσματα μας έδειξαν ότι τα πολυπεπτίδια H9-59 και H60-110 καταστέλλουν τον πολλαπλασιασμό των κυττάρων PC3, ενώ επάγουν την προσκόλληση των ίδιων κυττάρων. Επιπλέον, τόσο το H9-59 όσο και το H60-110 επάγουν τη φωσφορυλίωση των ERK1/2. / HARP is a heparin-binding growth factor, which plays a key role in cell proliferation, differentiation and migration. HARP is also involved in angiogenesis and tumor growth and progression. HARP mediates its functions through its transmembrane receptors RPTPβ/ζ, ALK and N-syndecan. Structurally, HARP contains two TSR homologous domains and two basic clusters in its N and C-termini. It has been found that HARP is a substrate for several extracellular proteolytic enzymes and HARP peptides have been detected in conditioned media of cells. HARP peptides often have different or even opposite biological activity from the whole molecule. The aim of this project was to study the biological activity of two recombinant, truncated forms of HARP that correspond to the two TSR domains, H9-59 and H60-110, in order to contribute to the study of the HARP domains that mediate its biological activities and also the biological activity of HARP peptides. The results of the study show that both H9-59 and H60-110 inhibit the proliferation of PC3 cells but stimulate their adhesion. Moreover, both H9-59 and H60-110 stimulate the phosphorylation of ERK1/2.

No description available.

No description available.

Magister Chirurgiae Dentium - MChD (Oral Medicine and Periodontics) / In recent years, the development and use of autologous platelet rich concentrates (PC's) has gained traction within the rapidly progressive, multidisciplinary field of regenerative medicine. A PC subtype, marketed as advanced platelet rich fibrin (A- PRF), is a recent advancement of the original PRF protocol and promoted as a "blood concentrate" containing platelets, leukocytes, circulating stem cells and endothelial cells. A-PRF in the form of membranes, plugs, or even shredded particulates are increasingly being used as surgical adjuncts in areas of previous infection or left exposed within the microbial rich oral environment. Although recent literature has noted the biologic benefits of this material within the context of wound healing and regeneration, the antimicrobial potential of APRF has remained unexplored. The aim of this investigation is to determine if A-PRF displays antimicrobial activity against microbes of the oral cavity with a null hypothesis that its activity is no different to a clot of unprocessed venous blood. Methodology: A-PRF and whole blood samples were obtained from consenting individuals and utilised to conduct an in-vitro agar disk diffusion investigation to determine their antimicrobial activity. Standardised samples of A-PRF, unprocessed clotted blood and 0.2% chlorhexidine gluconate (CHX) were tested against organisms cultured from fresh oral rinse samples and pure cultures of candida albicans, streptococcus mutans, staphylococcus aureus and enterococcus faecalis. The antimicrobial activity was assessed in accordance to the established principles of the agar disk diffusion method and measurement of inhibition zones. Results: A-PRF displayed antimicrobial activity against all of the individual organisms tested within this study following a 24 hour incubation period. However, no significant differences were noted between A-PRF and a natural clot of blood when tested against cultures of the oral rinse sample. Finally, the antimicrobial activity of A-PRF is significantly inferior to an equal volume of the CHX preparation. Conclusion: Although A-PRF displays antimicrobial activity; its strength, spectrum and biologic activity within a polymicrobial environment requires further investigation.

Quarenta e duas vacas Holandesas, em gestação e multíparas, e os respectivos bezerros recém-nascidos foram utilizados para determinar se as concentrações de IGF-I no colostro e secreções lácteas podem ser alteradas em resposta à mudanças na concentração sérica de IGF-I pré-parto, avaliar comparativamente a flutuação sérica pré-parto de IGF-I em relação a IgG, bem como as flutuações séricas de IGF-I e IgG nos bezerros na primeira semana de vida e, adicionalmente, alterações na mucosa intestinal dos mesmos. As vacas foram distribuídas ao acaso em dois grupos de 21 animais, o grupo tratado recebeu 500 mg de somatotropina bovina recombinante (rbST) e o grupo controle recebeu injeções de vitamina E. Ambos tratamentos iniciaram 35 dias pré-parto e foram administrados em intervalos de 14 dias. Semanalmente, foi observado o escore corporal e foram coletadas amostras de sangue até a data de parição. Foram coletadas amostras do colostro e das secreções lácteas, diariamente, por sete dias pós-parto. Os bezerros recém-nascidos foram distribuídos ao acaso, em um arranjo fatorial 2X3, correspondendo ao tratamento das mães (controle ou rbST) e às datas de abate (ao nascimento, aos dois dias de idade e aos sete dias de idade). Diariamente, foram coletadas amostras de sangue até a data de abate. Para as análises estereológicas amostras foram coletadas de cinco regiões do intestino delgado. O escore corporal e a concentração sérica de ácidos graxos não-esterificados não diferiram entre os grupos durante todo o período experimental (P>0,05). Os grupos rbST e controle apresentaram diferenças significativas quanto às concentrações séricas de IGF-I na segunda e quarta semanas após o início do período experimental (P<0,05), em resposta às aplicações do rbST, no entanto não foram encontradas diferenças entre os tratamentos no parto (P>0,05). A concentração de IGF-I foi superior no colostro das vacas tratadas com rbST (P<0,05), não diferindo nas secreções subseqüentes. As concentrações séricas de IgG não diferiram entre os tratamentos durante todo o período experimental, bem como as concentrações de IgG do colostro e demais secreções lácteas (P>0,05). Não foram encontradas diferenças entre os tratamentos nas concentrações séricas de IGF-I dos bezerros até o sétimo dia de vida, como também não foram encontradas diferenças entre as concentrações séricas de IgG nos bezerros após 24 horas de vida (P>0,05). O peso dos órgãos e o volume parcial (Vv) da mucosa absortiva não diferiram entre os tratamentos nas três datas de abate, observando-se somente diferenças significativas entre as datas de abate. A porção do jejuno médio apresentou maior Vv em relação aos outros segmentos do intestino delgado ao nascimento e aos sete dias de vida. A aplicação de rbST no período seco influenciou somente a concentração de IGF-I no colostro, não alterando as concentrações séricas nos bezerros até o sétimo dia de vida, como também o Vv da mucosa absortiva do intestino delgado. / Forty-two Holstein cows, in gestation and multiparous, and their newborn calves were used to evaluate if insulin-like growth factor-I (IGF-I) in colostrum and subsequent mammary secretions could be influenced by changes in blood serum IGF-I and compare its temporal changes with the serum immunoglobulin G (IgG) in pre-partum period, as well as the temporal changes in serum IGF-I and IgG in newborn calves during the first week of life, and also, alterations in its intestinal mucosa. Cows were randomly assigned in two groups of twenty-one animals, treated group was injected 500 mg of recombinant bovine somatotropin (rbST), and the control group received vitamin E injection. Both treatments started 35 days pre-partum and were administered every 14 days until partum. Weekly body condition scores were measured and blood was collected until partum. Colostrum and mammary secretions were collected daily for seven days pos-partum. Newborn calves were randomly assigned to a 2X3 factorial arrangement, which the factors depended on mother’s treatment (control and rbST) and slaughter date (just after birth, two days of life and seven days of life). Blood was collected daily until slaughter. For stereological analysis samples were taken from five sites from small intestine. Body condition scores and nonesterified fatty acid concentration didn't differ between the groups during the experimental period (P>0,05). Groups differed about serum IGF-I during pre-partum (P<0,05), which showed differences on second and fourth weeks of experimental period in response to rbST administration. However, no treatment differences were found at partum (P>0,05). IGF-I concentration was higher in colostrum of cows treated with rbST (P<0,05), but it didn't differ in subsequent mammary secretions. IgG serum concentration didn't differ between treatments during the experimental period, neither IgG concentration in colostrum and subsequent mammary secretions (P>0,05). No differences were found either in calves’ IGF-I levels from birth to seven days old or in IgG serum concentration after 24 hours of life (P>0,05). Organ weight and mucosa partial volume (Vv) didn't differ between treatments in the three slaughter dates, only differences among the salughter dates were observed. Segment from the medium jejunum showed higher Vv in relation to others segments at birth and seven days old. The administration of rbST during the dry period influenced only IGF-I concentration in colostrum, however colostrum IGF-I didn’t affect calves’ serum concentration up to seven days old, neither the mucosa Vv of small intestine.

Orientador: José Buratini Júnior / Banca: Paula de Carvalho Papa / Banca: João Carlos Pinheiro Ferreira / Resumo: A compreensão dos mecanismos moleculares controladores do desenvolvimento, função e regressão do CL bovino é necessária para o aprimoramento da manipulação hormonal ovariana. Fortes evidências sugerem o envolvimento de fatores de crescimento fibroblástico (FGFs) na regulação do crescimento e regressão do CL. "Splicing" alternativo de 4 genes formam sete subtipos de FGFRs com afinidade variável por diferentes FGFs. Os membros da subfamília do FGF8 (FGF8, 17 e 18) ativam eficientemente o FGFR3C e 4 e podem atuar em cooperação nos tecidos que expressão estes receptores. O objetivo deste trabalho foi determinar o padrão de expressão dos FGFRs e dos membros da subfamília do FGF8 no CL bovino (CL). Os CLs foram obtidos de ovários de abatedouro e classificados em 4 estádios de desenvolvimento (estádio/1= corpo hemorrágico, estádio/2= CL em desenvolvimento, estádio/3= CL maduro/início da luteólise funcional e estádio/4= luteólise estrutural). O RNAm foi mensurado por PCR semiquantitativo e a proteína localizada por imunohistoquímica. A expressão do RNAm codificante das isoformas 'B' e 'C' de FGFR1 e FGFR2 foi detectada no CL bovino por PCR associado à eletroforese e foi acompanhada pela localização da proteína nas pequenas e grandes células luteínicas. A expressão do RNAm do FGFR1C e 2C não variou durante o desenvolvimento luteínico, distintamente a expressão do FGFR1B aumentou no estádio 3. Embora os FGFRs 3B, 3C e 4 tenham sido detectados de forma inconsistente por PCR associado à eletroforese, o RNAm do FGFR3C e FGFR4 foram detectados por PCR em tempo real em todos os estádios do desenvolvimento luteínico. O RNAm do FGF18 foi detectado por PCR em tempo real em todos os estádios do desenvolvimento luteínico e sua abundancia do RNAm do FGF18 foi maior no estádio 3 comparado com os estádios 1, 2 e 4. Em contraste, os RNAm do FGF8 e 17 ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The molecular mechanisms controlling the development, function and regression of the bovine corpus luteum are necessary for the improvement of reproductive biotechnologies. Strong evidence suggests the involvement of fibroblast growth factors (FGFs) in the regulation of growth, and regression of the corpus luteum (CL). Alternative splicing of 4 genes give rise to seven subtypes of FGFRs with varying affinity for different FGFs. FGF8 subfamily members (FGF8, 17 and 18) efficiently activate FGFR3C and FGFR4 and may act in cooperation in tissues expressing these receptors. The objective of the present study was to determine the pattern of expression of FGF8 subfamily members and FGFRs in the bovine CL. Bovine CLs were obtained from abattoir ovaries and classed into four stages of development (stage 1= corpus hemorragicum, stage 2= developing CL, stage 3= mature/early functional luteolysis CL, and stage 4= structural luteolysis). Expression of mRNA was measured by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) followed by gel analysis (FGFR1-4) and real time RT-PCR (FGF8 subfamily members, FGFR3C and FGFR4) and proteins were localized by immunohistochemistry. Expression of mRNA encoding 'B' and 'C' spliced forms of FGFR1 and FGFR2 was readily detected in the bovine CL and was accompanied by isoform non-specific protein localization. FGFR1C and FGFR2C mRNA expression did not vary throughout CL lifespan, whereas FGFR1B was upregulated in the mature CL (stage III). FGFR3B, FGFR3C and FGFR4 expression was inconsistent in the bovine CL as assessed by PCR associated with gel analysis. FGF18, FGFR3C and FGFR4 mRNA was detected by real time PCR in all four developmental stages, and FGF18 mRNA abundance was higher in stage 3 (2.89  0.05; mean ± SEM) compared with stages 1 (0.3  0.27), 2 (0.56  1.27) and 4 (0.99  0.32). The m RNA expression ... (Complete abstract click electronic access below) / Mestre

Estudo anterior de nosso grupo demonstrou que superfície de titânio (Ti) com nanotopografia obtida por condicionamento com H2SO4/H2O2 e funcionalizada com GDF-5 por simples adsorção promove o aumento da mineralização de culturas primárias de células osteogênicas. O presente estudo teve como objetivos avaliar: 1) os efeitos da pós-adsorção de proteínas principais do plasma- albumina, fibrinogênio e fibronectina, em superfícies de Ti controle e com nanotopografia, funcionalizadas com GDF-5 a 200 ng/mL por simples adsorção, sobre a formação de matriz mineralizada in vitro; 2) parâmetros moleculares e fenotípicos característicos da aquisição do fenótipo osteogênico in vitro sobre superfícies de Ti funcionalizadas com GDF-5 por simples adsorção ou por filmes LbL; 3) parâmetros de formação óssea adjacente a implantes de Ti com nanotopografia funcionalizada com GDF-5 pelos dois métodos, em modelo de tíbia de coelhos. Os resultados mostraram que a pós-adsorção de proteínas plasmáticas não afetou o potencial osteogênico in vitro, com exceção para o efeito inibidor da albumina, quando pós-adsorvida isoladamente. Tanto a superfície de Ti como o método de funcionalização de GDF-5 afetaram, quantitativamente, as formações de matriz mineralizada, com a maior diferenciação osteogênica para Ti com nanotopografia funcionalizada com GDF-5 por simples adsorção e a menor, para os filmes LbL, independentemente das superfícies sobre as quais eles eram montados. A atividade de ALP foi maior em culturas sobre nanotopografia de Ti, incluindo aquelas funcionalizadas com GDF-5, cujos valores, no entanto, não corresponderam, necessariamente, à maior atividade osteogênica. Apesar disso, todos os grupos exibiram expressão de marcadores de diferenciação osteoblástica, com sobre-expressão de osteopontina e osteocalcina para culturas sobre LbL. As análises microtomográfica, histológica e histomorfométrica não revelaram diferenças qualitativas e quantitativas in vivo entre nanotopografias de Ti funcionalizadas ou não com GDF-5, ainda que uma tendência à maior formação óssea tenha sido observada para as superfícies funcionalizadas e, entre essas, para os filmes LbL. Considerados conjuntamente, os resultados do presente estudo contribuem para o melhor entendimento das respostas de osteoblastos e do tecido ósseo quando se propõe a estratégia de funcionalização de superfícies de Ti com GDF-5 visando à otimização da osseointegração. / It has been demonstrated that a nanostructured titanium (Ti) surface obtained by treatment with H2SO4/H2O2 and functionalized with GDF-5 by simple adsorption promotes the enhancement of mineralized matrix formation in osteogenic cell cultures. This study aimed to evaluate: 1) the effects of post-adsorption of major plasma proteins, i.e. albumin, fibrinogen and fibronectin, on control and nanostructured Ti surfaces, functionalized with 200 ng/mL GDF-5 by simple adsorption, on mineralized matrix formation by calvarial osteogenic cell cultures; 2) molecular and phenotypic parameters characteristics of the acquisition of the osteogenic phenotype in vitro on Ti surfaces functionalized with GDF-5 by either simple adsorption or layer by layer (LbL) films; 3) parameters of bone formation adjacent to Ti implants with a nanostructured surface functionalized with GDF-5 by the two methods described in item 2, in a rabbit tibia model. The results showed that the post-adsorption of plasma proteins did not affect the osteogenic potential of cultures, except for the inhibitory effect of albumin when post-adsorbed alone. Either the Ti surface topography or the method for GDF-5 functionalization quantitatively affected mineralized matrix formation, with the higher osteogenic differentiation for nanostructured Ti functionalized with GDF-5 by simple adsorption and the lower one for LbL films, irrespective of the Ti surface topography on which they were mounted. ALP activity was higher for cultures grown on nanostructured Ti, including those functionalized with GDF-5, whose values, however, did not necessarily correspond to the higher osteogenic activity. Despite that, all groups expressed osteoblast differentiation markers, with a remarkable increase in osteopontin and osteocalcin mRNA levels for cultures grown on LbL films. The microtomographic, histologic and histomorphometric analyses revealed no qualitative or quantitative differences in vivo among the nanostructured Ti implants, yet a tendency for enhanced bone formation was observed for the functionalized surfaces and, between them, for the LbL films. Taken together, the results of the present in vitro and in vivo studies contribute to a better understanding of osteoblast and bone tissue responses to the functionalization of Ti surfaces with GDF-5 aiming to optimize osseointegration.