No description available.

The efficiency of a best management practice (BMP) is defined simply as a measure of how well the practice or series of practices removes targeted pollutants. While this concept is relatively simple, mathematical attempts to quantify BMP efficiency are numerous and complex. Intuitively, the pollutant removal capability of a BMP should be fundamental to the BMP selection process. However, as evidenced by the absence of removal efficiency as an influential criterion in many BMP selection procedures, it is typically not at the forefront of the BMP selection and design process. Additionally, of particular interest to any developer or municipal agency is the financial impact of implementing a BMP. Not only does the implementation cost exist, but there are long-term maintenance costs associated with almost any BMP. Much like pollutant removal efficiency, implementation and maintenance costs seem as though they should be integral considerations in the BMP selection process. However, selection flow charts and matrices employed by many localities neglect these considerations. Among the categories of criteria to consider in selecting a BMP for a particular site or objective are site-specific characteristics; local, state, and federal ordinances; and implementation and long-term maintenance costs. A consideration such as long-term maintenance cost may manifest itself in a very subjective fashion during the selection process. For example, a BMPs cost may be of very limited interest to the reviewing locality, whereas cost may be the dominant selection criterion in the eyes of a developer. By contrast, the pollutant removal efficiency of a BMP may be necessarily prioritized in the selection process because of the required adherence to governing legislation. These are merely two possible criteria influencing selection. As more and more selection criteria are considered, the task of objectively and optimally selecting a BMP becomes increasingly complex. One mathematical approach for optimization in the face of multiple influential criteria is the Analytic Hierarchy Process. "The analytic hierarchy process (AHP) provides the objective mathematics to process the inescapably subjective and personal preferences of an individual or a group in making a decision" (Schmoldt, 2001, pg. 15). This paper details the development of two categories of comprehensive BMP selection matrices expressing long-term pollutant removal performance and annual maintenance and operations cost respectively. Additionally, the AHP is applied in multiple scenarios to demonstrate the optimized selection of a single BMP among multiple competing BMP alternatives. Pairwise rankings of competing BMP alternatives are founded on a detailed literature review of the most popular BMPs presently implemented throughout the United States. / Master of Science

No description available.

The Transforming Growth Factor (TGF) superfamily of cytokines and their serine/threonine kinase receptors play an important role in the regulation of cell division, differentiation, adhesion, migration, organization, and death. Smad proteins are the major intracellular signal transducers for the TGF receptor superfamily that mediate the signal from the membrane into the nucleus. Bone Morphogenetic Protein-4 (BMP-4) is a representative of the TGF superfamily, which regulates the formation of teeth, limbs and bone, and also plays a role in fracture repair. Binding of BMP-4 to its receptor stimulates phosphorylation of Smad1, which subsequently recruits Smad4. A hetero-oligomeric complex consisting of Smad1 and Smad4 then translocates into the nucleus and regulates transcription of target genes by interacting with transcription factors. Although the individual steps of the signaling cascade from the receptor to the nucleus have been identified, the exact kinetics and the rate limiting step(s) have remained elusive. Standard biochemical techniques are not suitable for resolving these issues, as they do not offer sufficiently high sensitivity and temporal resolution. In this study, advanced optical techniques were used for direct visualization of Smad signaling in live mammalian cells. Novel fluorescent biosensors were developed by fusing cyan and yellow fluorescent proteins to the signaling molecules Smad1 and Smad4. By measuring Fluorescence Resonance Energy Transfer (FRET) between the two fluorescent proteins, the kinetics of BMP/Smad signaling was unraveled. A rate-limiting delay of 2 - 5 minutes occurred between BMP receptor stimulation and Smad1 activation. A similar delay was observed in the complex formation between Smad1 and Smad4. Further experimentation indicated that the delay is dependent on the Mad homology 1 (MH1) domain of Smad1. These results give new insights into the dynamics of the BMP receptor – Smad1/4 signaling process and provide a new tool for studying Smads and for testing inhibitory drugs. / Die Transforming Growth Factor" (TGF)-Superfamilie der Cytokine und ihrer Serin/Threonin-Kinase-Rezeptoren spielt eine bedeutende Rolle bei der Regulierung der Zellteilung, -differenzierung, -adhäsion, -migration, -organisation, und beim Zelltod. Die Smad-Proteine sind die wichtigsten intrazellulären Signalüberträger für die TGF-Rezeptor-Familie, da sie das Signal von der Zellmembran zum Kern übermitteln. Das ,,Bone Morphogenetic Protein4" (BMP-4) ist ein Vertreter der TGF-Familie, der die Bildung von Zähnen, Gliedmaßen und Knochen reguliert und darüber hinaus eine Rolle bei der Frakturheilung spielt. Das Binden von BMP-4 an seinen Rezeptor stimuliert die Phosphorylierung von Smad1, welches in der Folge Smad4 rekrutiert. Ein hetero-oligomerer Komplex bestehend aus Smad1 und Smad4 verlagert sich dann in den Zellkern, wo er durch Interaktion mit Transkriptionsfaktoren die Transkription von Zielgenen reguliert. Obwohl die einzelnen Schritte der Signalkaskade vom Rezeptor bis in den Zellkern bereits identifiziert wurden, blieben die Kinetik und die geschwindigkeitsbegrenzenden Schritte bisher unbekannt. Gängige biochemische Methoden eignen sich nicht um diese Fragen zu lösen, da sie nicht über ausreichende Empfindlichkeit und zeitliches Auflösungsvermögen verfügen. In der vorliegenden Arbeit wurden hochentwickelte optische Techniken angewandt, um die Smad-vermittelte Signaltransduktion direkt in lebenden Zellen sichtbar zu machen. Neue fluoreszierende Biosensoren wurden konstruiert, indem gelb- und cyan-fluoreszierende Proteine mit den Signalmoleküle Smad1 und Smad4 fusioniert wurden. Durch Messung des "Fluorescent Resonance Energy Transfer" (FRET) zwischen den zwei fluoreszierenden Proteinen konnte die Kinetik der BMP-Smad-Signalkaskade bestimmt werden. Zwischen der Stimulation des Rezeptors und der Aktivierung von Smad1 trat eine geschwindigkeitsbegrenzende Verzögerung von 2-5 Minuten auf. Eine ähnliche Verzögerung wurde bei der Bildung des Komplexes aus Smad1 und Smad4 beobachtet. Weitere Experimente zeigten, dass die Verzögerung von der Mad-Homologie-Domäne 1 (MH1) von Smad1 abhängt. Die Ergebnisse dieser Arbeit geben neue Einblicke in die Dynamik der BMP-Rezeptor-Smad1/4 Signaltransduktion und stellen neue Werkzeuge zur Untersuchung von Smads und zur Austestung inhibitorischer Wirkstoffe zur Verfügung.

Os tecidos ósseos alógenos são utilizados nas cirurgias de reconstrução ortopédicas e odontológicas. O número crescente de transplantes de ossos na última década beneficia diversos pacientes. Os profissionais dos Bancos de Tecidos utilizam protocolos que reduzem os riscos de transmissão de doenças infectocontagiosas, entretanto, não eliminam esta possibilidade. Portanto, é importante que tecidos sejam esterilizados por um método eficaz, sendo usualmente aplicada a radiação ionizante. Porém, a radiação ionizante pode acarretar alterações estruturais e biológicas nos ossos em relação à dose. O objetivo deste trabalho foi estudar os efeitos da aplicação da radiação gama e de feixe de elétrons, nas doses de 15 kGy, 25 kGy e 50 kGy nos tecidos ósseos desmineralizados preservados liofilizados ou congelados e avaliar as possíveis alterações no colágeno, na concentração de proteínas totais, BMP-2 e BMP-7. Para tanto foram utilizadas as técnicas de espectroscopia Raman, Bradford e ELISA. Foram utilizadas 5 diáfises de femur humano e parte das amostras foram desmineralizadas. As proteínas ósseas foram extraídas e quantificadas. Com os resultados da espectroscopia Raman, observamos que a eficiência da desmineralização foi de 85 a 90% porém com alterações na estrutura do colágeno. Alterações também foram observadas nos ossos congelados e irradiados nas doses de 25 kGy e 50 kGy. Nos resultados da quantificação de proteínas totais e específicas, ocorreram diminuições gradativas das concentrações médias das proteínas em relação à dose de radiação nos grupos estudados. Nas doses de radiação usualmente aplicadas aos tecidos ósseos, 15 kGy ou 25 kGy, as reduções nas concentrações das BMP-2 e BMP-7, foram menores que 20%. As reduções na dose de 50 kGy foram entre 27% a 53%, sendo influenciadas pelo tipo de radiação e pelo tipo de preservação dos ossos. / Allogenic bone tissues are used in orthopedic and dental reconstructive surgery. The growing number of bone transplants in the last decade benefits many patients. The professionals of Tissue Banks use protocols that reduce the risk of transmission of infectious diseases, however do not eliminate this possibility. Therefore, it is important that tissues are sterilized by an effective method, usually being applied ionizing radiation. However, ionizing radiation can cause structural and biological changes in the bones relative to the dose. The objective of this work was to study the effects of the application of gamma radiation and electron beam at doses of 15 kGy, 25 kGy and 50 kGy in the bone tissues demineralized preserved freeze-dried or frozen and to evaluate possible changes in collagen, the protein concentration total, BMP-2 and BMP-7. For both Raman spectroscopy techniques, Bradford and ELISA were used. They used 5 diaphyses human femur and part of the samples was demineralized. Bone proteins were extracted and quantified. With the results of Raman spectroscopy, we found that the efficiency of demineralization was 85-90% but with changes in the structure of collagen. Changes were also observed in frozen and irradiated bone at doses of 25 kGy and 50 kGy. The results of quantification of total and specific proteins, there were gradual decreases in average concentrations of proteins in relation to radiation dose in both groups. In the radiation doses usually applied to the bone tissue (15 kGy and 25 kGy) reductions in concentrations of BMP- 2 and BMP-7, were lower than 20%. The reductions at a dose of 50 kGy were between 27% to 53%, being influenced by the type of radiation and the kind of preservation of the bones.

Stormwater runoff from urbanized areas can have detrimental impacts on groundwater and surface water supplies by mobilizing contaminants such as bacteria and nutrients from surrounding areas. Best Management Practices (BMPs) are commonly designed to mitigate these impacts, but the processes governing the effectiveness of these BMPs are often not well understood. Biofiltration BMPs, which include storage, sediment removal, and infiltration processes, are particularly challenging to quantify. This research involved an investigation of the processes associated with a biofiltration BMP located in West Boylston, MA adjacent to the Wachusett Reservoir. The basin treats runoff from an 8-acre watershed with two roadways (Routes 12 and 110) and surrounding residential and commercial land uses. Water exits the basin by either seepage directly to groundwater or by seepage through a two-foot filtration bed to an outfall pipe on one side of the basin. A field sampling program was conducted in collaboration with the Massachusetts Department of Conservation and Recreation to characterize the various flow paths of contaminants upstream, within, and downstream of the biofiltration facility. The program included collection of volumetric flow information, field parameters (dissolved oxygen, specific conductance, pH, and temperature), and water quality samples. Samples were tested for alkalinity, bacteria, dissolved organic carbon, nutrients, additional anions and cations, and suspended sediments. Stormwater samples were collected for storm events that included substantial rainfall and illustrated seasonal variability. A set of seven monitoring wells installed for this project provided information on groundwater flow and quality at the site. The field program provided quantitative data on the flows and transformations that occur within and in the groundwater downstream of the biofiltration basin. The results demonstrated that stormwater infiltration to groundwater is an important component to consider for BMP design. The flow path through the outfall was effective in removing sediments, but was found to have limited capacity for water quality treatment, since only small changes in stormwater quality occurred between the culvert inflow, basin, and outfall samples. However, analysis of the flow data showed that infiltration to groundwater was comparable to discharge through the outfall. Furthermore, the signatures of stormwater infiltration could still be seen in the wells, indicating that the infiltration from the stormwater basin can impact groundwater quality. The groundwater pathway was found to impact the chemistry of the constituents, and was particularly effective in removing bacteria and phosphorus. The results demonstrate the value of groundwater recharge as a component of BMP design, and provide a basis for a number of specific design recommendations related to biofiltration basins.

FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / Este trabalho tem como objetivo avaliar a estabilidade de genes de referÃncia e a expressÃo de fatores de crescimento e de receptores de hormÃnios em folÃculos secundÃrios de caprinos. AlÃm disso, visa quantificar a expressÃo dos RNA mensageiros para BMP-6 e BMP-7 em folÃculos prÃ-antrais e antrais caprinos e avaliar os efeitos de FSH, BMP-6 e BMP-7 sobre o crescimento e expressÃo gÃnica em folÃculos secundÃrios caprinos durante 6 dias de cultivo. Para avaliar a estabilidade dos genes de referÃncia, folÃculos secundÃrios (150-200 Âm) foram isolados mecanicamente de ovÃrios caprinos. ApÃs a extraÃÃo do RNA total e sÃntese de DNA complementar, realizou a quantificaÃÃo dos mRNA, por PCR em tempo real, utilizando-se primers especÃficos para genes de referÃncia (GAPDH, β-tubulina, β-actina, PGK, UBQ, RPL-19, rRNA18S), fatores de crescimento (GDF-9, BMP-15, BMP-6, FGF-2, VEGF, KL e IGF-1) e receptores de hormÃnio (FSH-R, LH-R e GH-R). Para avaliar a expressÃo de BMP-6 e BMP-7, folÃculos primordiais, primÃrios e secundÃrios, bem como pequenos e grandes folÃculos antrais foram obtidos e os nÃveis de mRNA de BMP-6 e 7 foram quantificados. Nos estudos in vitro, os efeitos da BMP-6 (50 ng/mL) e BMP-7 (50 ng/mL) na presenÃa ou ausÃncia de FSH (50 ng/mL) sobre o desenvolvimento in vitro de folÃculos secundÃrios e sobre a expressÃo de mRNA para BMP-6 e 7 e FSH-R foi avaliada apÃs 6 dias de cultivo. Os resultados mostraram que UBQ e β-actina sÃo os genes de referÃncia mais estÃveis em folÃculos prÃ-antrais caprinos fresco cultivados por 12 dias. Os RNAs mensageiros para os fatores de crescimento (EGF, GDF-9, BMP-15, VEGF, FGF-2, BMP-6, IGF-1 e KL) e os receptores de FSH, LH e GH sÃo expressos em diferentes nÃveis em folÃculos prÃ-antrais de caprinos, sendo que o IGF-1 e o EGF apresentaram, respectivamente, o maior e o menor nÃvel de mRNA. O nÃvel de mRNA para BMP-6 em folÃculos primÃrios e secundÃrios foi significativamente maior do que aqueles em primordial, enquanto que os nÃveis de mRNA para BMP-7 foi maior nas cÃlulas da granulosa/teca de grandes do que nos pequenos folÃculos antrais. ApÃs o cultivo de folÃculos secundÃrios durante 6 dias, FSH aumentou o diÃmetro folicular e FSH e BMP-7 aumentou significativamente os nÃveis de mRNA para BMP-7 e FSH-R. Jà a BMP-6 na presenÃa ou ausÃncia de FSH aumentou o diÃmetro dos folÃculos secundÃrios. AlÃm disso, FSH aumentou os nÃveis de mRNA para BMP-6, enquanto ambos BMP-6 e FSH e aumentaram os nÃveis de mRNA para FSH-R, apÃs o perÃodo de cultivo. Em conclusÃo, UBQ e β-actina sÃo os dois genes mais estÃveis para folÃculos secundÃrios caprinos e o FSH e as BMPs dos tipos 6 e 7 estimulam o crescimento de folÃculos prÃ-antrais in vitro durante 6 dias de cultivo. / This study aims to evaluate the stability of reference genes and the expression of growth factors and hormone receptors in goat secondary follicles. It also seeks to quantify the expression of messenger RNA for BMP-6 and BMP-7 in goat preantral follicles and to evaluate the effects of FSH, BMP-6 and BMP-7 on growth and gene expression in secondary follicles goats after 6 days of culture. To evaluate the stability of reference genes, secondary follicles (150-200 μm) were mechanically isolated from goat ovaries. After extraction of total RNA and synthesis of complementary DNA, the quantification of mRNA was carried out by real-time PCR using specific primers for genes of reference (GAPDH, β-tubulin, β-actin, PGK, UBQ, RPL - 19, rRNA18S), growth factors (GDF-9, BMP-15, BMP-6, FGF-2, VEGF, KL and IGF-1) and hormone receptor (FSH-R, LH-R and GH-R). To evaluate expression of BMP-6 and BMP-7, primordial follicles, primary and secondary as well as small and large antral follicles were obtained and the mRNA levels of BMP-6 and 7 were measured. For in vitro studies, the effects of BMP-6 (50 ng / mL) and BMP-7 (50 ng / mL) in the presence or absence of FSH (50 ng / mL) on the development of secondary follicles and on the expression of mRNA for BMP-6 and 7 and FSH-R was evaluated after 6 days of culture. Results showed that mRNA for growth factors (EGF, GDF-9, BMP-15, VEGF, FGF-2, BMP-6, IGF-1 and KL) and the receptors for FSH, LH and GH are expressed in at different levels in preantral follicles of goats. Moreover, among the growth factors studied, IGF-1 and EGF had higher and lower levels or RNA, respectively. UBQ and β-actin genes were the mostr stable reference in fresh and cultured preantral follicles. The level of mRNA for BMP-6 in primary and secondary follicles was significantly higher than those in primordial follicles, while the levels of mRNA for BMP-7 was higher in granulosa cells / theca in large antral follicles than in small antral follicles. After culture of secondary follicles for 6 days, FSH increased follicular diameter and FSH and BMP-7 significantly increased the levels of mRNA for BMP- 7 and FSH-R. In addition, BMP-6 in the presence or absence of FSH increased the diameter of secondary follicles after 6 days of culture. Real-time PCR shoed that FSH increased the levels of mRNA for BMP-6, while both BMP-6 and FSH and increased levels of mRNA for FSH-R, after a period of 6 days of culture. In conclusion, UBQ and β-actin are the two most stable genes for secondary follicles goats and FSH and BMPs of types 6 and 7 stimulate the growth of preantral follicles after 6 days of in vitro culture.

The transforming growth factor-ß (TGFß) pathway, including the bone morphogenetic protein (BMP), plays critical roles during embryogenesis and in adult tissue homeostasis. Hence, malfunctions in TGFß/BMP signalling result in several diseases. Signalling is initiated by ligand binding to cell surface receptor kinases, which phosphorylate and activate the R-SMAD transcription factors. R-SMADs translocate to the nucleus and regulate the transcription of hundreds of genes. The cellular responses to TGFß/BMP signals are tightly controlled and highly regulated. TGFß/BMP receptors and R-SMADs, as the intracellular mediators of TGFß/BMP ligands, are key targets for regulation to control duration and potency of signalling. Reversible ubiquitylation of R-SMADs and TGFß/BMP receptors is a key mechanism to control TGFß/BMP signalling. Several E3 ubiquitin ligases have been reported to regulate the turnover and activity of TGFß/BMP receptors and R-SMADs, however little is known about their cognate deubiquitylating enzymes (DUBs). A proteomic screen identified the DUBs OTUB1 and USP15 as potential novel regulators of the TGFß and BMP pathways respectively. Endogenous OTUB1 was recruited to the active phospho-SMAD2/3 complex only upon TGFß induction and OTUB1 had a crucial role in TGFß-mediated gene transcription and cellular migration. OTUB1 inhibited the ubiquitylation of phospho-SMAD2/3 by binding to and inhibiting the E2 ubiquitin-conjugating enzymes independently of its catalytic activity. Consequently, the depletion of OTUB1 in cells caused a rapid loss in levels of TGFß-induced phospho-SMAD2/3, which was rescued by the proteasomal inhibitor Bortezomib. These findings demonstrated a novel signal-induced phosphorylation-dependent recruitment of OTUB1 to its target. Hence, OTUB1 could be exploited as a target to intervene against diseases that are provoked by an imbalance in TGFß signalling. DUBs are highly regulated enzymes and recent reports have shed light into the molecular regulation OTUB1. The N-terminal region of OTUB1 harbours an ubiquitin binding domain, which is critical for its function to inhibit ubiquitylation. While investigating the role of OTUB1 in TGFß signalling, it became apparent that OTUB1 itself could be post-translationally modified by phosphorylation. Two phosphorylation sites at the OTUB1 N-terminal region have been identified by mass spectrometry. S18 of OTUB1 was phosphorylated in vitro by the type I TGFß receptor (ALK5), whereas S16 was phosphorylated by the constitutively active kinase CK2 in vitro and in vivo. Phosphorylation of the OTUB1 N-terminal region could affect its physiological function and requires further investigation. Although much is known about DUBs that target the type I TGFß receptor, no DUBs that target the type I BMP receptors had been identified. USP15 was identified in a proteomic screen as an interactor of SMAD6, which is a negative regulator of the BMP pathway. USP15 also binds to and deubiquitylates the type I BMP receptor (ALK3), thereby enhancing BMP signalling. Consequently, USP15 impacts BMP-induced SMAD1 phosphorylation, mouse osteoblastic differentiation and Xenopus embryogenesis. A proteomic approach identified O-GlcNAc transferase (OGT) as an interactor of SMAD2. SMADs have not been associated with O-GlcNAc modifications and the regulation of TGFß/BMP signalling by O-GlcNAcylation has not been investigated. Endogenous SMADs1-3 bound OGT and pulled down potential O-GlcNAc modified proteins. Furthermore, SMAD4 was possibly O-GlcNAcylated, which implies that O-GlcNAc modification could regulate TGFß/BMP signalling. Further investigation is needed to decipher the precise molecular mechanisms of this potential regulation.

Cell signalling determines physiological responses to many cellular stimuli and environmental changes. The transforming growth factor-beta (TGFβ)/bone morphogenetic protein (BMP) signalling pathways begin by binding of ligand to the heterodimeric receptor complex, followed by activation of Smads that translocate to the nucleus to regulate transcription of genes that further mediate cellular physiology. The TGFβ/BMP pathways are very important for proper tissue development and homeostasis, thus precise spatial and temporal regulation of the signalling pathway is required and achieved by many positive and negative signalling regulators. This thesis work identified the liver kinase B1 (LKB1) pathway as a negative regulator of TGFβ/BMP signalling pathways. In the first paper, we established LKB1 as a negative regulator of TGFβ signalling and TGFβ-induced epithelial to mesenchymal transition (EMT). LKB1 impairs Smad4 binding capacity to DNA leading to suppressed TGFβ-activated gene transcription. The second paper describes further the mechanism of LKB1 negative regulation on BMP signalling, by mediating BMP type I receptor degradation resulting in inhibition of BMP-induced cell differentiation. Downstream of LKB1, salt inducible kinase 1 (SIK1) is a TGFβ target gene and its expression is up-regulated by Smad2/3/4-mediated gene transcription. The third paper elucidates the mechanism of SIK1 transcriptional induction via an enhancer element located 3’ of the gene and SIK1-mediated type I TGFβ receptor degradation, which requires the activity of Smad7 and of the Smurf2 ubiquitin ligase. The fourth manuscript finds sucrose non-fermenting (SNF) 1-like kinase 2 (NUAK2) as another TGFβ target gene and its up-regulation results in modification of the mammalian target of rapamycin (mTOR) pathway that controls protein synthesis. NUAK2 cooperates with LKB1 leading to Raptor phosphorylation and inhibition of mTOR-mediated protein synthesis. Collectively, this thesis work has provided a functional link between two important signalling pathways, the metabolic LKB1 pathway and TGFβ/BMP pathway.

Introduction: Mammalian cell recombinant bone morphogenetic protein (rBMP) synthesis is reported to be poor. The BMP pro-domain may be involved in folding, stability and secretion. Objectives: Investigate the effect of inhibition of pro-domain cleavage on rhBMP-2 production. Methods: CHO cells transfected with human BMP-2 (hBMP-2) were cultured in the presence of the proprotein convertase inhibitor 9DR in short (multi-well) and long-term (bioreactor) cultures. Mature and proBMP secretion was measured by ELISA and characterized by Western blot. BMP activity was determined by C2C12 bioassay. Results: 9DR significantly enhanced the yields of both pro- and mature hBMP-2 in short and long-term cultures, without any negative effects on cell growth or viability. The rhBMP-2 was biologically active. ProBMP-2 could be converted by exogenous furin treatment into mature BMP-2 as shown by Western blot. Conclusions: 9DR increases rhBMP-2 production and is a simple, but effective way to enhance the yield of active, mature BMP-2 in CHO cells.