Thesis (PhD (Med))--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The increase in the prescription and use of glucocorticoids (GCs) to treat various diseases and resulting decrease in bone density and development of osteoporosis is of growing concern. Glucocorticoid-induced osteoporosis (GCIO) is a relatively under-researched disease with the mechanism by which GCs affect bone metabolism not yet fully delineated. This holds especially true for the early events in bone development. The negative effects of GCs are predominantly seen in osteoblasts, the cells responsible for bone formation, in that GCs diminish both the numbers and function of osteoblastic cells. Osteoblast precursor cell proliferation is crucial to ensure the existence of a healthy pool of osteoblastic cells needed to form new bone after bone resorption by osteoclasts. Previously, it was shown that GCs reduce the proliferation of immortalised osteoblastic cell lines. In addition, early immortalised preosteoblasts were more sensitive to GCs than their mature counterparts. However, these cells have corrupted cell cycles; therefore, primitive primary mesenchymal stromal cells (MSCs) were used in this study to examine the effect of GCs on the mitogen-induced proliferation of early osteoblast precursor cells (naïve MSCs and preosteoblasts) using the synthetic GC, dexamethasone (Dex). Mitogenic conditions established for naïve rat mesenchymal stromal cells (rMSCs) indicated that mild (5% FBS) stimulation is sufficient to induce proliferation, whereas a higher FBS concentration (20% FBS) was mitogenic in primary preosteoblasts. It was also found that pharmacological doses of Dex drastically decreased the mitogen-induced proliferation of both naïve rat MSCs (rMSCs) and preosteoblasts. Mitogen-activated protein kinase (MAPK) signalling pathways, such as ERK1/2, govern cell proliferation. GCs have been shown to decrease the activity of ERK1/2, which is associated with decreased proliferation in osteoblastic cells. In the present study, western blot analysis showed that Dex reduced the proliferation-associated shoulder of the ERK1/2 activity profile in both naïve rMSCs and preosteoblasts. Moreover, the ERK1/2 signalling pathway was shown to be essential for mitogen-stimulated growth of naïve rMSCs and preosteoblasts as the MEK1/2 inhibitor, U0126, inhibited mitogen-induced proliferation. Using western blot analysis, it was shown that, after mitogen administration, ERK1/2 activity exhibited a typical proliferation profile, which was blocked by U0126. Protein tyrosine phosphatases (PTPs) dephosphorylate and inactivate ERK1/2. Utilising sodium vanadate, an inhibitor of PTPs, in vitro phosphatase assays revealed that PTP activity was the predominant phosphatase activity present in naïve rMSCs and preosteoblast lysates after concomitant mitogen and Dex stimulation. The mRNA of the dual specificity phosphatase, MKP-1, was rapidly (within 30 minutes) upregulated after mitogen and Dex administration in both naïve rMSCs and preosteoblasts. However, the protein expression pattern of MKP-1 did not correspond to the mRNA induction, suggesting that the MKP-1 protein could be subjected to rapid degradation. These findings suggest that MKP-1 could possibly be involved in the GC regulation of mitogen-induced proliferation of early osteoblast precursor cells, but closer investigation is needed to fully elucidate this role. In addition, the involvement of other PTPs should not be excluded and warrants further investigation. During the course of the present study, it was found that strong mitogenic stimulation with 20% FBS led to oncogene-induced senescence (OIS). Flow cytometry analysis revealed the presence of two populations in naïve rMSCs preparations and DNA content analysis was consistent with that of cells undergoing OIS. These results indicated that the more primitive osteoblast precursor cells (naïve rMSCs) are more responsive to mitogens than their mature counterparts (preosteoblasts). In addition, it was found that the magnitude of ERK1/2 activation was increased in naïve rMSC after strong mitogenic stimulation, indicating that naïve rMSCs are still highly sensitive to stimulation with strong mitogens. In summary, these findings show that Dex decreased the proliferation of naïve rMSCs and preosteoblasts concomitantly with a decrease in ERK1/2 activity. In addition, Dex upregulated MKP-1 mRNA, but the same effect was not seen on the MKP-1 protein levels. Therefore, this suggests that PTP/s other than MKP-1 could be responsible for the inactivation of ERK1/2 by Dex, leading to decreased proliferation in naïve rMSCs and preosteoblasts. Further identification of PTPs that regulate osteoblast precursor cell numbers and function could lead to the elucidation of the mechanism through which GCs act to negatively influence bone density. This will improve our insights into the pathogenesis of GCIO and aid in the identification of therapeutic targets which can be exploited to develop new agents to treat osteoporosis. / AFRIKAANSE OPSOMMING: Die toename in voorskrifte en gebruik van glukokortikoïede (GKs) om verskillende siektes te behandel en die gevolglike afname in been digtheid, is kommerwekkend. Glukokortikoïed geïnduseerde osteoporosis (GKIO) is 'n relatief min genavorste siekte waarvan die meganisme waardeur GKs been-metabolisme affekteer nog nie ten volle ontrafel is nie. Dit is veral waar ten opsigte van die vroeë stadia in beenontwikkeling. Die negatiewe uitwerking van GK's word oorwegend in osteoblaste, die selle wat verantwoordelik is vir beenformasie, waargeneem, waar GKs beide die getalle en funksie van osteoblaste verminder. Osteoblast voorloper-sel proliferasie is belangrik vir die handhawing van 'n gesonde poel osteoblastiese selle wat benodig word om nuwe been te vorm na beenresorpsie deur osteoklaste. Daar is gevind dat GKs proliferasie van verewigde preosteoblastiese sellyne verminder en dat jong verewigde preosteoblaste meer sensitief is vir GKs as hul meer volwasse ekwivalent. Die selle se selsiklusse is egter gekorrupteer en daarom was primitiewe primêre rot mesenkiem stromaselle (rMSCs) in hierde studie gebruik om die effek van GKs op mitogeen-geïnduseerde proliferasie van vroeë osteoblasvoorloperselle (naïwe MSC en preosteoblaste) deur die sintetiese GK, deksametasoon (Dex), te bestudeer. Mitogeniese kondisies vir naïwe rMSCs het getoon dat matige (5% FBS) stimulasie voldoende is om proliferasie te induseer, terwyl 'n hoë FBS konsentrasie (20% FBS) mitogenies was in primêre preosteoblaste. Daar is ook gevind dat farmokolgiese dosisse Dex die mitogeen-geïnduseerde proliferasie van beide naïwe rMSCs en preosteoblaste verminder. Die mitogeen-geïnduseerde protein kinase (MAPK) pad beheer selproliferasie. Die ekstrasellulêre gereguleerde kinase pad (ERK1/2) is voorheen as die hoofpad wat MBA 15.4 and MG 63 proliferasie beheer geïdentifiseer. Daar is gewys dat GKs die aktiwiteit van ERK1/2 verlaag en proliferasie van die selle verminder. In die huidige studie het western blot analise gewys dat Dex die proliferasie geassosieerde skoueraktiwiteit van die ERK1/2 aktiwiteitsprofiel in beide naïwe rMSCs en preosteoblaste verminder. Die noodsaaklike rol van ERK1/2 pad in mitogeen-gestimuleerde groei van die selle is bevestig deur die MEK1/2 inhibitor, U0126, wat die mitogeen-geïnduseerde proliferasie geïnhibeer het. Western blot analise het gewys dat die ERK1/2 aktiwiteit na mitogeen toediening 'n tipiese proliferasie profile toon wat deur U0126 geblokkeer word. Protein tirosien fosfatases (PTPs) defosforileer and inaktiveer ERK1/2. In vitro fosfatase bepalings met natrium vanadaat, 'n inhibitor van PTPs, het bevestig dat PTP die predominante fosfatase akitiwiteit is in naïwe rMSCs en preosteoblaste lisate is na gelyktydige mitogeen en Dex stimulasie. Die mRNA van die dubbele spesifisiteits fosfatase, MKP-1, is vinnig (binne 30 minute) opgereguleer is na mitogeen en Dex toediening in beide naïwe rMSCs en preosteoblaste. Die proteinekspressie van MKP-1 het egter nie met die mRNA ekspressie ooreengestem nie, wat suggereer dat die MKP-1protein blootgestel is aan vinnige degradasie. Hierdie bevindings stel voor dat MKP-1 moontlik 'n rol speel in die GC-regulering van mitogeen-geïnduseerde proliferasie van vroeë osteoblast voorloperselle maar verdere ondersoek is nodig om die rol ten volle te verklaar. Die betrokkenheid van ander PTPs moet egter nie uitgesluit word nie en regverdig verdere studie. Die huidige studie het bevind dat sterk mitogeniese-stimulasie met 20% FBS tot onkogene- geïnduseerde selgroeistilte (senescence) (OIS) lei. Vloeisitometriese analise het die teenwoordigheid van twee afsonderlike populasies getoon in die naïwe rMSCs preparate en die DNA inhoud was verenigbaar met die van selle wat OIS ondergaan. Die bevindinge stel voor dat die meer primatiewe osteoblast voorloperselle (naïwe rMSCs) is meer vatbaar vir mitogene-stimulasie as hul volwasse ekwivalente (preosteoblaste). Ook is gevind dat die mate van ERK1/2 aktivering hoër was in naïwe rMSCs, selfs na sterk mitogeniese stimulasie wat daarop dui dat naïwe rMSCs steeds hoogs sensitifief is vir stimulasie met sterk mitogene. In opsomming, dui die bevindinge dat Dex die proliferasie van naïwe rMSCs en preosteoblaste onderdruk wat met 'n verlaging van ERK1/2 aktiwiteit gepaard gaan. Verder, het Dex, MKP-1 mRNA opgereguleer maar die effek is nie op die proteinvlak waargeneem nie. Dit suggereer dat PTP/s anders as MKP-1 verantwoordelik kan wees vir die Dex inaktivering van ERK1/2 wat die proliferasie van naïwe rMSCs en preosteoblaste onderdruk.

Abstract This research was conducted to understand whether phosphorus limits the phytoplankton production in the South China Sea (SCS). In the nutrient enrichment experiments nitrate and phosphate were supplemented to surface sea water and the enhancement of chlorophyll a concentration during incubation was observed. Seasonal field survey was conducted to measure ambient abundance of phosphorus including phosphate (SRP) and dissolve organic phosphorus (DOP), as well as alkaline phosphatase activity (APA) in the nautral sea water in the contiential shelf and basin of the SCS. Except at the contiential shelf in summer and the mouth of Zhu Jiang River in fall, the nutrient concentration of surface water was low in the SCS. The average¡£NO3+NO2¡¤was 20 nM (fall) - 360 nM (winter ). The average SRP concentration was 16 nM (fall) - 87 nM (winter). The average DOP concentration was 0.08 £gM (summer)- 0.25 £gM (winter). The ¡£NO3+NO2¡¤/ SRP ratio was smaller than the Redfield N/P ratio of 16. The average chlorophyll a concentration (Chl a) was 0.13 £gg l-1 (summer) - 0.48 £gg l-1 (winter). The average concentration of the particlulate organic carbon (POC) was 4.58 £gM (spring) - 8.11 £gM (winter). The average APA was 16 n mol l-1 h-1 (fall) - 87 n mol l-1h-1 (winter). The average of APA/Chl a was 33.94 n mol £gg -1 h-1 (winter) - 97.22 n mol £gg -1 h-1 (spring). The results of the enrichment experiment show that the phosphorus deficiency was observed on the contiential shelf in the summer of 2001 and at the mouth of Zhu Jiang in the fall of 2002. The common characteristics of the phosphorus deficient regions were low salinity (29.90- 30.87 psu ), high¡£NO3+NO2¡¤(1.31 - 3.01 £gM) , and a ¡£NO3+NO2¡¤/ SRP ratio higher than 16. Chl a increased significantly (p<0.05) by the enrichments of phosphorus. In spring and winter when all regions were N-limited, N enrichment significantly (p<0.05) increased Chl a. In fall, all the contiential shelf region except the mouth of Zhu Jiang River, the slope and basin regions were NP co-limited. The Bashi Strait in summer was also NP co-limited. P-limitation that was seen in the contiential shelf SCS and at Zhu Jiang River mouth, was probably caused by the influence of river discharge. N/P, SRP or APA were not effective parameters to assess whether marine phytoplankton growth was limited phosphorus, nitrogen or both. In the SCS, the P-limited water masses were, in general, low in salinity, high in¡£NO3+NO2¡¤,Chl a,¡£NO3+NO2¡¤ / Chl a, APA and a N/P ratio higher than 16. The water masses that were N-limited was high in salinity, and low in¡£NO3+NO2¡¤, Chl a,¡£NO3+NO2¡¤/ Chl a and APA, as well as a N/P ratio smaller than 16. The water masses that were nitrogen and phosphorus co-limited were different from the N-limited ones in that they were low in SRP, SRP/Chl a, and DOP. The SRP and DOP concentration in the NP co-limited region were 11 - 28 nM and 0.09 - 0.23

Made available in DSpace on 2015-03-26T13:51:44Z (GMT). No. of bitstreams: 1 01 - capa_abstract.pdf: 76671 bytes, checksum: 6479c7684e1e1f9436c94cf7407a6fac (MD5) Previous issue date: 2007-08-17 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The objective of this work was to evaluate the role of soil microorganisms in the availability of insoluble phosphate sources and to study the sensibility of phosphate solubilizing bacteria to commercial formulations of glyphosate. Rhizosphere soil samples were collected from Eucalyptus grandis x Eucalyptus urophylla hybrids with different diameters at breast height, planted at distinct topographical regions, namely top, slope, and lowland. Acid phosphatase activity varied from 24.40 to 190.07 &#956;g p-nitrophenol g-1 soil h-1, while alkaline phosphatase showed values ranging from 0.70 to 20.55 &#956;g p-nitrophenol g-1 soil h-1. The highest phosphatase activity was observed for the rizhosphere soli from plants located at the top region. The solubilization potential of Ca, Fe and Al phosphates by the soil microorganisms varied from 17.38 to 7949.71 &#956;g P g-1 dry soil. Calcium phosphate promoted the highest values for phosphate solubilization. The solubilization potential for Catalão and Araxá rock phosphates varied from 27.08 to 1209.71 &#956;g P g-1 dry soil, and Catalão phosphate was the most soluble phosphate source. The final pH of the culture medium was negatively correlated to phosphate solubilization. Rhizosphere bacteria were isolated and characterized as to their phosphate solubilization capacity. The solubilization index (SI) for calcium phosphate by the bacterial isolates grown in solid medium varied from 0 to 4.07. The amount of solubilized phosphate in liquid medium varied from 80.17 to 22,259.33 &#956;g P. The growth and the phosphate solubilization capacity of the isolates were also evaluated in the presence of the commercial formulations Roundup Transorb®, Roundup NA®, Zapp QI®, and Scout®. The presence of the herbicides in the culture medium caused a significant reduction in the growth of the isolates tested. For the isolate To 66, the presence of Roundup Transorb® and Zapp QI® significantly reduced the phosphate solubilization potential of this isolate, while for Scout the reverse was observed. The tested formulations affected the growth and solubilization capacity of some of the isolates in vitro. It is hypothesized that such effect resulted from specific components of the tested formulations and not from glyphosate itself, once the herbicide was present at the same concentration in each of the commercial formulations tested. Further investigations on the action of these substances on the soil microbiota must be conducted in situ for a better understanding of the potential deleterious effects of such formulations on key processes that take place in the soil. / Objetivou-se neste trabalho avaliar o papel dos microrganismos do solo na solubilização de fontes insolúveis de fósforo, bem como a sensibilidade de bactérias solubilizadoras de fosfato a formulações comerciais de glyphosate. A atividade de fosfatases e o potencial de solubilização, em meio NBRIP líquido, dos fosfatos de cálcio, ferro e alumínio, e dos fosfatos naturais de Araxá e Catalão pela microbiota total do solo rizosférico de plantas do híbrido Eucalyptus grandis x Eucalyptus urophylla com diferentes diâmetros à altura do peito, oriundas de posições topográficas distintas, a saber, topo, encosta e baixada, foram avaliados in vitro. Procedeu-se também o isolamento de bactérias da rizosfera destas plantas e a sua caracterização quanto à capacidade de solubilização de fosfato de cálcio, por meio da determinação do índice de solubilização em meio sólido. O crescimento e a capacidade de solubilização de fosfato dessas bactérias foram também avaliados em meio líquido, na presença das formulações comerciais Roundup Transorb®, Roundup NA®, Zapp QI® e Scout®. Os solos coletados de plantas do topo e da baixada apresentaram maior solubilização de fosfato de cálcio pela microbiota, enquanto o solo da encosta não apresentou diferenças entre as fontes inorgânicas testadas. O fosfato de Catalão foi a fonte natural mais solubilizada pela microbiota do solo. O pH final do meio de cultura correlacionou-se negativamente com os valores de fósforo solubilizado. A atividade das fosfatases ácida e alcalina foi maior nos solos rizosféricos de plantas do topo. Os isolados obtidos apresentaram diferenças quanto ao índice de solubilização (IS) de fosfato de cálcio, apresentando valores entre zero, relativo aos isolados que perderam a capacidade de solubilização, e 4,07. Também foram observadas diferenças entre os isolados quanto à sua capacidade de solubilização de fosfato em meio líquido. A presença de herbicidas no meio de cultura reduziu significativamente o crescimento de todos os isolados testados. Os isolados To 66 e To 3 foram os mais sensíveis à presença do herbicida Roundup Transorb®. Para o isolado To 66, a adição ao meio de cultura de Roundup Transorb® e Zapp QI® reduziu significativamente o potencial de solubilização deste isolado, enquanto o herbicida Scout® teve efeito oposto. O potencial de solubilização do isolado To 11 não foi alterado na presença dos herbicidas. Os herbicidas testados afetam o crescimento e a capacidade de solubilização de alguns isolados in vitro, sendo necessário investigar se esse efeito também ocorre no solo.

<p dir="ltr">Phosphatidylinositol phosphates (PIPs) and soluble inositol phosphates (IPs) serve as critical secondary messenger molecules that regulate cellular processes. The INPP5 family of phosphatases play an essential role in regulating levels of PIP-5’ and IP-5’ molecules. Src homology 2-containing-inositol phosphatases (SHIP), are a subgroup of the INPP5 family that consists of two members, SHIP1 and SHIP2. Both SHIP proteins have been identified to hydrolyze PI(3,4,5)P3 into PI(3,4)P2. Interestingly, the dysregulation of PI(3,4,5)P3 and SHIP proteins have been observed in multiple diseases, such as cancer, diabetes, and neurodegenerative disease. Recently, SHIP1 was identified as a potential risk factor for the development of Late-onset Alzheimer’s Disease (LOAD). Furthermore, knockdown and inhibition of SHIP1 using small-molecule inhibitors were shown to reduce phenotypes associated with LOAD. Taking these studies together suggests SHIP1 to be a potential therapeutic target for the treatment of LOAD.</p><p dir="ltr"><br></p><p dir="ltr">Despite SHIP1’s therapeutic potential, the development of specific small-molecule inhibitors that target SHIP1 has been challenging. One explanation for this challenge is that very little is known about the overall structure and function of SHIP1. In this thesis I will discuss in detail how we generated multiple SHIP1 constructs to improve our understanding of SHIP1’s overall structure and function in an <i>in vitro </i>setting.</p><p><br></p><p dir="ltr">Efficient protein production is essential for studying enzyme structure and function. The choice of expression system can impact protein yield and stability. The E. coli (BL21) and Baculovirus expression systems are two commonly used systems for protein production. While E. coli is cost-effective and can yield a large amount of protein, the Baculovirus system offers advantages in terms of protein folding and post-translational modifications. Using both systems to generate SHIP1 protein, we demonstrate that the Baculovirus system significantly enhances SHIP1 solubility for all generated constructs, making it the preferable choice for investigating the structure and function of SHIP1.</p><p><br></p><p dir="ltr">SHIP1, a 133 kDa protein, which comprises five established domains: an N-terminal Src Homolgy 2 (SH2) domain, 2.) a pleckstrin homology-related (PH) domain, 3.) an inositol phosphatase catalytic (Ptase) domain, 4.) a C2 domain, and 5.) a C-terminal domain containing proline-rich regions (PXXP) and tyrosine phosphorylated (NPXY) motifs. Despite their regulatory roles in phosphatase activity, protein-protein interactions, and membrane association, limited information is available about their structures and how they contribute SHIP1’s biochemical functions. In this study, we utilized baculovirus-expressed SHIP1 constructs to investigate the impact of each domain on macromolecular structure. Interestingly, a previously unrecognized domain within SHIP1 that directly impacts the enzyme's oligomeric state was identified. This work highlights that SHIP1's individual domains can significantly impact its overall structure and function, providing valuable insights for the development of potential therapeutics in the treatment of LOAD.</p><p><br></p><p dir="ltr">Accurate determination of phosphatase kinetics is vital for understanding the enzymatic activity and its potential involvement in disease. Using our baculovirus generated SHIP1 constructs, we employed in-vitro assays, including the malachite green (MG) and the 2-amino-6-mercapto-7-methylpurine riboside (MESG) coupled enzyme assays, to gain insight into SHIP1 kinetics. Results from the MG assay shows that SHIP1 can hydrolyze the PI(3,4,5)P3 diC8 substrate more efficiently than I(1,3,4,5)P4. Additionally, SHIP1’s PH domain was observed to increase the turnover of PI(3,4,5)P3 diC8. Furthermore, dimerization of SHIP1 was not observed to alter SHIP1 kinetics in any way. Lastly, no major differences in I(1,3,4,5)P4 kinetics were observed with the addition of SHIP1’s N-terminus. These results offer the first comprehensive biochemical characterization of SHIP1 across its substrates and N-terminal domains.</p><p><br></p><p dir="ltr">The development of potent and specific small-molecule inhibitors that target SHIP1 remains challenging. One potential cause for this challenge is that no structures of SHIP1 have been solved in complex with active compounds, making structure-based drug design impossible. In this study, we developed a covalent compound, <b>TAD-58547</b>, from a previously published fragment-based screen that was conducted on SHIP1’s Ptase and C2 domain. <b>TAD-58547 </b>was shown to effectively inhibited SHIP1's Ptase and C2 domains at modest potency. Using X-ray crystallography, this compound was observed to form a covalent interaction with a cysteine residue near the Phosphatase-C2 domain interface. Intriguingly, the inhibitor's potency was observed to be reduced in the presence of the SH2 domain. In addition to testing <b>TAD-58547</b> against our SHIP1 constructs, we investigated the effect of SHIP1’s N-terminus on the potency of a literature compound, <b>TAD-58616</b>. This compound was shown to inhibit all our tested constructs at low µM concentrations. Furthermore, using x-ray crystallography <b>TAD-58616 </b>was solved in complex with SHIP1’s Ptase and C2 domain. Intriguingly, density for <b>TAD-58616 </b>was shown to interact with a site previously identified from the fragment-based screen. While we initially determined this site to be a result of crystal packing, fragments bound to this site may have the potential to inhibit SHIP1. The work presented in this study reinforced the importance of testing inhibitors against physiological relevant forms of SHIP1, when developing potential therapeutics.</p><p><br></p><p dir="ltr">Lastly, new evidence has suggested that the binding of phosphorylated immunoreceptor tyrosine-based activation motifs (p-ITAM) and immunoreceptor tyrosine-based inhibitory motifs (p-ITIM) to SHIP1’s N-terminal SH2 domain is essential for its “Anchorage and Activation” at the plasma membrane (PM). With this model it is believed that SHIP1’s SH2 domain, places the phosphatase into an auto-inhibited state. Upon binding to immune receptor proteins and adaptor proteins that contain ITAM/ITIM sequences, SHIP1 becomes un-auto-inhibited, allowing it to efficiently hydrolyze PI(3,4,5)P3 embedded in the PM. While this model does support the notion that SHIP1 activity is mediated by its PM localization, our biophysical and biochemical characterization add another level of complexity to this regulatory event. Taking all these results together, we propose a novel model for SHIP1 called “Anchorage and Assist” and suggest innovative therapeutic strategies for targeting SHIP1.</p><p><br></p><p dir="ltr">In conclusion, this thesis highlights the importance of choosing suitable expression systems for efficient protein production. Additionally, it offers insight into SHIP1's regulatory mechanisms through the discovery of a novel domain impacting its oligomeric state. Furthermore, the accurate determination of SHIP1 kinetics enhances our understanding of this phosphatase and its potential implications in disease. Also, the identification and crystallization of a novel and previously determined inhibitor scaffolds in complex with SHIP1 increases our ongoing efforts to develop a small-molecule inhibitor that specifically targets SHIP1. Lastly, using recently published data, detailing SHIP1 PM localization and activation, we proposed a new model for SHIP1 activity and suggest novel therapeutic strategies for targeting SHIP1.</p>

No description available.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Arbuscular Mycorrhizal Fungi (AMF) can increase the tolerance of plants to heavy metals, as well as their ability to enhance the acquisition of phosphorus (P). The aim of this study was to evaluate how the inoculation with AMF and P application can remedy the effects of high levels of copper (Cu) in the soil on Crotalaria juncea. The effects of AMF and P on the growth of plants, the enzymatic activity of acid phosphatase (APases) in plant and soil as well as the presence of glomalina. The experiment was conducted in a greenhouse in a 3 x 2 factorial arrangement (natural content of P, 40 and 100 mg kg-1 of P, with and without inoculation Rhizophagus clarus) with three replications in a soil with high levels of Cu (60 mg kg -1). Besides the treatments of P and AMF in soil with high levels of Cu, were evaluated two additional treatments in soil with natural levels of Cu (0.55 mg kg-1) containing 40 mg kg-1 of P, with and without AMF inoculation. The results showed that the combination of P and AMF (Rhizophagus clarus) may be an interesting strategy for the reduction of Cu phytotoxicity in Crotalaria juncea, as provided increments in dry matter production of plants and a decrease in the activity of acidic enzyme APases in soil and plants. Furthermore, it was showed that Glomalin produced by AMF can decrease Cu availability to the plants with phytoprotector consequent effect. / Os Fungos Micorrízicos Arbusculares (FMA) podem aumentar a tolerância das plantas a metais pesados, bem com sua capacidade de melhorar a aquisição de fósforo (P). O estudo objetivou avaliar como a inoculação com FMA e a aplicação de P podem remediar os efeitos de altos teores de Cu no solo sobre Crotalaria juncea. Foram avaliados os efeitos de FMA e P sobre o crescimento de plantas, a atividade enzimática de fosfatase ácida (APases) na planta e no solo, bem como a presença de glomalina. O experimento foi montado em casa de vegetação em esquema fatorial 3 x 2 (teor natural de P, 40 e 100 mg kg-1 de P, com e sem inoculação de Rhizophagus clarus com três repetições em um solo com altos teores de Cu (60 mg kg-1). Além destes tratamentos de P e FMA em solo com altos teores de Cu foram avaliados dois tratamentos adicionais em solo com teores naturais de Cu (0.55 mg kg-1) contendo 40 mg kg-1 P, com e sem inoculação de FMA. Os resultados demonstram que a combinação entre fósforo e o FMA (Rhizophagus clarus) pode ser uma estratégia interessante para a redução da fitotoxidez de Cu em Crotalaria juncea, pois proporcionaram incrementos na produção de matéria seca das plantas e uma diminuição na atividade das enzimas APases ácida no solo e nas plantas. Além disso, foi demonstrado que o aumento nos teores de glomalina produzida pelos FMA pode diminuir a disponibilidade do Cu para as plantas com consequente efeito fitoprotetor.

Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-02-20T17:57:53Z No. of bitstreams: 2 Tese - Carla Eloiza B. C. Seabra.pdf: 1211089 bytes, checksum: 1125d30550099fb430808207eb415619 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-02-20T17:58:36Z (GMT) No. of bitstreams: 2 Tese - Carla Eloiza B. C. Seabra.pdf: 1211089 bytes, checksum: 1125d30550099fb430808207eb415619 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-02-20T17:59:00Z (GMT) No. of bitstreams: 2 Tese - Carla Eloiza B. C. Seabra.pdf: 1211089 bytes, checksum: 1125d30550099fb430808207eb415619 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-02-20T17:59:00Z (GMT). No. of bitstreams: 2 Tese - Carla Eloiza B. C. Seabra.pdf: 1211089 bytes, checksum: 1125d30550099fb430808207eb415619 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-07-29 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Mahogany (Swietenia macrophylla King) is in high demand world-wide, and is one of Brazil’s most highly valued forestry species. Balsa (Ochroma pyramidale Cav. ex. Lamb.) also has broad usage and great market potential owing to extremely rapid growth rates. Rather than harvesting these increasingly rare timber species from native forests, high density commercial plantings have been established. However, there is little information concerning the nutritional requirements of both species, especially regarding the response to soil phosphorous (P). Phosphorous is expensive in Brazil and over use is associated with adverse environmental consequences. In this study, we examined the effects of four levels of P fertilization (0, 1, 10 and 100 kg ha-1) on biometric and physiological parameters of mahogany and balsa wood seedlings grown in the greenhouse. The response to P was markedly different for the two species. For mahogany, seedling height, seedling diameter, leaf area, leaf weight, stem dry weight, and total dry weight increased at the 100 kg/ha-1 P level. The greatest increase (3- to 5- fold) occurred for leaf area and leaf weight, whereas root dry mass was not influenced by P. Similarly, %N and %P (but not %C) in leaf, stem and root tissue increased with increasing P level, but increased drastically at the 100 kg/ha-1 P treatment. The divalent cations (Ca and Mg) increased in foliar tissue, yet the concentrations of organic acids in xylem fluid declined with increasing P. Leaf macro- and micro-nutrients concentrations were influenced by P treatments, with the exception of Zn and Fe. Leaf, stem and root P utilization efficiency was highest at the 0, 1 or 10 level. Root phosphatase activity was unaffected by P level. By contrast, balsa responded greatly to increasing P concentration, although the most dramatic increase was at 100 kg/ha-1. For example, there was a 9-, 4-, 66-, 25-, 65-, 25- and 32-fold increase for seedling height, seedling diameter, leaf area, leaf dry mass, stem dry mass, root dry mass, and total dry mass, respectively, for the 0 compared to the 100 kg/ha-1 treatment. Leaf, stem and root %N decreased and %P increased with increasing P levels. Macro and micro nutrient levels wereinfluenced by P treatment, with the exception of Mn. The concentrations of organic acids in xylem fluid were altered by P fertilization; and malic, succinic, and lactic acids were highest at the 100 kg/ha-1 treatment. Leaf, stem and root P utilization efficiency increased greatly with increasing P level; however, root phosphatase activity decreased with increasing P levels. The implications of these physiologic data are discussed in relation to management strategies for the culture of mahogany and balsa. / Plantios comerciais de mogno e pau-de-balsa, com maiores relações custo/benefício, podem alavancar o mercado de madeira de espécies nativas, tanto interno quanto externo. Mogno e pau-de-balsa estãoentre as mais valiosas espécies de madeira com diversos usos, sendo ambas economicamente promissoras. Todavia, informações sobre a influência do fósforo (P) na nutrição e desenvolvimento das mesmas são incipientes. Em condições de casa de vegetação, avaliou-se os efeitos de quatro doses de P (0; 1; 10 e 100 kg ha-1) sobre parâmetros biométricos e fisiológicos dessas espécies. Para mogno, as doses crescentes de P elevaram o crescimento da parte aérea, os conteúdos de carbono, nitrogênio, P e, as concentrações foliares de macronutrientes e manganês, e diminuíram a eficiência de utilização de P, as concentrações foliares de boro e cobre e, a concentração dos ácidos orgânicos no fluido do xilema. Não foi verificado efeito sobre o crescimento de raiz, as concentrações foliares de Zn e Fe, e a atividade de fosfatase de raiz. Destaca-se que houve correlação inversa entre a atividade de fosfatase ácida de raiz e o crescimento da parte aérea e, P e N acumulados na planta. Para pau-de-balsa, o aumento das doses de P elevou o crescimento da parte aérea, a eficiência de utilização de P e a concentração de ácidos orgânicos no fluido do xilema. As concentrações foliares de nutrientes foram afetadas de forma variada pela aplicação de doses de P: P, magnésio e cálcio aumentaram e, nitrogênio, potássio, enxofre, boro, zinco e cobre diminuíram. Doses crescentes de P diminuíram a atividade de fosfatase ácida de raiz. Não foi verificado efeito sobre as concentrações foliares de manganês, ferro e do ácido oxálico no fluido no xilema. Em geral, doses crescentes de P melhoram a formação das mudas dessas espécies. Porém, uma adubação equivalente a 100 kg P ha-1 pode causar desequilíbrio de crescimento em mudas de pau-debalsa.

The purpose of the study was to elucidate the mechanisms by which polyunsaturated fatty acids (PUFAs) prevent bone loss. MG-63 human osteoblasts and MC3T3-E1 murine osteoblasts were exposed to the n-6 PUFA arachidonic acid (AA) and the n-3 PUFA docosahexaenoic acid (DHA) as well as oestrogen (E2) and parathyroid hormone (PTH) and the effects thereof tested on a variety of biological parameters characteristic of osteoblasts. These parameters included prostaglandin E2 (PGE2) synthesis, proliferation, differentiation to mature mineralising osteoblasts as well as osteoprotegerin (OPG) and receptor activator of nuclear factor êB ligand (RANKL) secretion. Results showed that AA stimulates PGE2 production significantly in both cell lines. Stimulated PGE2 production by MC3T3-E1 cells however, was significantly higher, which might be attributed to auto-amplification by PGE2 itself in this cell line. Pre-incubation of the MG-63 cells with cyclo-oxygenase (COX)-blockers inhibited PGE2 production significantly, suggesting that both COX enzymes were involved in PGE2 synthesis. The number of functional osteoblasts is important for bone formation therefore in vitro osteoblastic cell proliferation was investigated. In contrast to the hormones E2 and PTH, both AA and DHA inhibited proliferation significantly. The AA-mediated anti-proliferative effect is possibly independent of PGE2 production, as PGE2 per se had little effect on proliferation. DHA inhibited proliferation of MG-63 cells more severely, which might be attributed to the osteosarcoma nature of the MG-63 cells. The anti-proliferative effect of these PUFAs might be attributed to modulation of cell cycle progression or anti-mitotic effects of PUFA peroxidation products. Morphological studies showed apoptotic cells after DHA exposure in MG-63 cells. There is a reciprocal relationship between reduced proliferation and the subsequent induction of cell differentiation in vitro. High basal levels of alkaline phosphatase (ALP) activity, a marker of the mature mineralising osteoblastic phenotype, were detected in MC3T3-E1 cells. Long-term exposure to AA inhibited ALP activity in these cells. This process might be PGE2-mediated. Exposure to PUFAs, however, did not compromise the ability of the MC3T3-E1 cells to differentiate to mature mineralising osteoblasts. In contrast with MC3T3-E1 cells, MG-63 cells demonstrated low basal ALP activity and were unable to differentiate to mature mineralising osteoblasts. In the absence of osteogenic-inducing supplements, PUFAs induced adipocyte-like features that might be due to the expression of high levels of PPARã in this cell line. Lipid-filled vacuoles were absent in the MC3T3-E1 cells suggesting that the MC3T3-E1 cell line may not express PPARã mRNA. The study furthermore demonstrated that PUFAs are able to modulate OPG and RANKL secretion in osteoblasts. AA inhibited OPG secretion dose-dependently in both cell lines, this could be PGE2-mediated. AA dose-dependently stimulated soluble RANKL (sRANKL) secretion in MC3T3-E1 cells thereby affecting the OPG/RANKL ratio in a negative way, supporting various reports that AA and PGE2 do cause bone resorption. No sRANKL could be detected after exposing the MC3T3-E1 cells to DHA suggesting that DHA could be protective to bone. In conclusion, contrary to in vivo evidence, this in vitro study could not indisputably demonstrate protective effects of PUFAs on the osteoblastic cell lines tested. / Thesis (PhD (Physiology))--University of Pretoria, 2006. / Physiology / unrestricted

La myopathie myotubulaire (XLMTM, OMIM 310400) est causée par des mutations dans le gêne MTM1 situé sur le chromosome X et apparaît à une fréquence de 1/50 000 naissances mâles. Les patients présentent une hypotonie et une faiblesse musculaire généralisées et de graves problèmes respiratoires à la naissance. En absence de traitement, les patients sont nombreux à décéder durant la première année de vie. Mon travail de thèse consiste à développer un traitement de thérapie génique pour la myopathie myotubulaire. L'injection intraveineuse du vecteur thérapeutique rAAV9-DES-Mtm1 chez le modèle murin de la myopathie myotubulaire permet d'obtenir de la protéine transgénique dans l'ensemble des muscles squelettiques de l'organisme. Un an après le traitement, les muscles malades retrouvent une morphologie normale et les souris sont en vie. La seconde partie porte sur l'évaluation des effets de la protéine MTMR2, un homologue de la myotubularine (MTM1), sur le muscle déficient en myotubularine, comme alternative à la thérapie génique avec MTM1. L'administration d'un vecteur AAV comportant le transgène MTMR2 dans le muscle malade améliore, partiellement, le phénotype musculaire. Ces résultats suggèrent que l'augmentation du niveau d'expression de MTMR2 dans le muscle malade pourrait être un traitement efficace. Enfin j'ai étudié le rôle de l'activité phosphatase de la myotubularine dans son action thérapeutique. J'ai comparé l'effet d'une forme inactive de myotubularine (MTMIC375S) à celui du transgène thérapeutique sur le muscle atteint de myopathie myotubulaire. Les résultats montrent que l'activité phosphatase de la myotubularine est nécessaire pour son activité thérapeutique. / Myotubular myopathy (XLMTM, OMlM 310400) is caused by mutations in the MTM1 gene located on the X chromosome and appeared at a frequency of 1/50 000 male births. Most of the patients arc affected by hypotonia and generalized muscular weakness as weIl as grave respiratory problems in the birth. In absence of an effective therapeutic treatment they are many to die during the first year of their life. My thesis work consists in developing a treatment of gene therapy for myotubular myopathy. The intravenous injection of the therapeutic vector rAAV9-DES-MTM1 into the murin model of the myotubular myopathy allows transgenic protein in the whole skeletal muscle of the body. The therapeutic protein cures the muscular phenotype rapidly after injection. At one year after treatment, the thick muscles still be cured, mice are alive and recovered a normal development. Then, I estimated the effects of the protein MTMR2, a counterpart of the myotubularine (MTM1), on muscles from mice devoid of myotubularin, as alternative at the gene therapy with MTM1. The administration of a vector AAV containing the transgene MTMR2 in the thick muscles improves, partially, the muscular phenotype. These results suggest that the increase of MTMR2 protein level MTMR2 thick muscle could be an effective treatment. Finally, I studied the role of myotubularin phosphatase activity on its therapeutic action, I compared the effect of an inactive shape of myotubularin (MTM1C375S) on muscle affected by myotubular myopathy as the effect of therapeutic transgene on the same muscle. The results show that the phosphatase activity of the myotubularin is necessary for its therapeutic activity.

La Myotubularine (MTM1) est une 3-phosphatase à phosphoinositides (PI) mutée dans la myopathie centronucléaire liée au chromosome X (XLCNM), caractérisée par une faiblesse musculaire et un positionnement anormal des noyaux dans les fibres musculaires. MTM1 définit une grande famille de phosphatases, exprimées dans tous les tissus, et qui englobent des phosphatases catalytiquement actives et inactives. Les myotubularines actives dephosphorylent le phosphatidylinositol 3 monophosphate [PtdIns3P] et le 3,5-bisphosphate [PtdIns(3,5)P2] en PtdIns et PtdIns5P, respectivement. Le rôle de MTM1 et son activité phosphatase à lipide dans le muscle restaient peu connus. L’étude approfondie de la protéine a révélé une association de MTM1 au réticulum sarcoplasmique des triades, un sous-compartiment impliqué dans la régulation calcique. La caractérisation de la souris Mtm1 KO, qui reproduit la XLCNM, a témoigné d’une anomalie de l’organisation et de la forme du réticulum sarcoplasmique. Afin d’explorer l’implicationde l’activité phosphatase de MTM1 dans l’organisation de réticulum sarcoplasmique, j’ai utilisé une approche in vivo avec des virus adéno-associé (AAV) pour moduler l’activité phosphatase en sur-exprimant MTM1 et sa forme phosphatase-inactive (MTM1-C375S) dans un muscle sauvage. L’observation des muscles transduits a dévoilé une implication de MTM1 dans le remodelage du réticulum sarcoplasmique et un rôle potentiel de PtdIns3P avec MTM1 dans la courbure des membranes du réticulum sarcoplasmique. Afin de comprendre l’importance de l’activité phosphatase dans le maintien du phénotype XLCNM, les muscles de souris Mtm1 KO ont été injectés avec ces AAVs contenant la forme active et inactive de MTM1 au moment de l’apparition des premiers signes de XLCNM. Étonnamment, la forme phosphatase-inactive(MTM1-C375S) a sauvé le phénotype de la souris Mtm1 KO de la même façon que la forme active, suggérant que l'activité de phosphatase de MTM1 n’est pas nécessaire pour le maintien de la structure intracellulaire des fibres du muscle adulte. Ces données suggèrent que MTM1 exerce une fonction phosphatase-indépendante dans le maintien de la structure musculaire, certainement via des interactions protéine-protéine, et une fonction phosphatase-dépendente dans le remodelage de la forme du réticulum sarcoplasmique dans le muscle squelettique. / Myotubularin (MTM1) is a phosphoinositide (PI) 3-phosphatase mutated in X-linked centronuclear myopathy (XLCNM), a rare congenital myopathy characterized by muscle weakness and abnormal positioning of nuclei in muscle fibers. MTM1 defines a large family of ubiquitously expressed catalytically active and inactive phosphatases. Active myotubularins dephosphorylate both phosphatidylinositol 3-phosphate [PtdIns3P] and 3,5-bisphosphate [PtdIns(3,5)P2] to PtdIns andPtdIns5P, respectively. The specific role of MTM1 and its PI phosphatase activity in muscle remains unknown. Comprehensive analysis of the protein unveiled the association of MTM1 with the sarcoplasmic reticulum (SR) at the triads. Characterization of Mtm1-KO mouse, which reproduce the XLCNM phenotype, revealed a defect of SR organization and shape. In order to gain insight into the involvement of MTM1 phosphatase activity on SR shape and organization, we employed an in vivo approach using Adeno-Associated Virus (AAV) to modulate the phosphatase activity by overexpressingMTM1 and its phosphatase inactive mutant in wild type muscle. The analysis of transduced muscle revealed the involvement of MTM1 in the SR remodeling and its potential role together with PtdIns3P in modulating membrane curvature. In order to understand the importance of the phosphatase activity in the generation of the XLCNM phenotype, Mtm1 KO mice were injected with AAV expressing the active form and the phosphatase inactive form. Surprisingly, both, the phosphatase active and the phosphatase inactive mutant corrected the Mtm1-KO mouse phenotype to a similar extent, thus suggesting that the PI-phosphatase activity of MTM1 is not essential for adult skeletal muscle maintenance. Our data indicates that MTM1 has a phosphatase-independent function in adult muscle structure maintenance and a phosphatase-dependent function in sarcoplasmic reticulum remodeling and shape in skeletal muscle.