The Effects of Estradiol, Androgens and the Selective Estrogen Modulators: Ospemifene, Raloxifene and Tamoxifen on Explant Cultures of Human Breast Tissue

The impact of menopausal hormone therapy (MHT) on increasing the risk for breast cancer (BC) remains controversial. To understand MHT-elicited cellular breast effects and the potential risks, included with using this therapy, a further investigation into this controversy is the subject of this thesis. In this thesis, to study the effects of estrogen, progestin, androgens and selective estrogen receptor modulators (SERMs), a modified tissue explant culture system was used. The different types of human breast tissues (HBTs) used in this study were normal HBTs, obtained from reduction mammoplasties of premenopausal women (prem-HBTs) or postmenopausal (postm-HBTs) women and peritumoral HBTs (peritum-HBTs) which were obtained from surgeries on postmenopausal BC patients. The explants were cultured up to three weeks in the presence or absence of estradiol (E2), medroxyprogesterone acetate (MPA), testosterone (T), dihydrotestosterone (DHT) and SERMs - ospemifene (OSP), raloxifene (RAL) and tamoxifen (TAM). The cultured HBTs maintained morphological integrity and responded to hormonal treatment in vitro. E2, MPA or E2/MPA increased proliferative activity and was associated with increased cyclin-D1 and caused changes in the cell cycle inhibitors p21 and p27, whereas the androgens T and DHT inhibited proliferation and increased apoptosis in HBT epithelia and opposed E2-stimulated proliferation and cell survival. The postm-HBTs were more sensitive to E2 than prem-HBTs. The effects of OSP, RAL and TAM on HBT epithelium were antiproliferative. E2, androgens and SERMs were associated with marked changes in the proportions of epithelial cells expressing steroid hormone receptors: E2 increased ER�� expressing cells and decreased androgen receptor (AR) positive cells, whereas T and DHT had opposite effects. The OSP, RAL and TAM, also decreased a proportion of ER�� positive cells in HBT epithelium. At 100 nM, these compounds maintained the relative number of AR positive cells, present at control level, which may partly explain proliferative inhibition. In conclusion, the proliferative activity of E2, in the epithelium of postm-HBTs, is opposed by T and DHT, which suggests that the inclusion of androgens in MHT may decrease the risk for developing BC.

Multidata inverse problems and Bayesian solution methods in astronomy

Statistical analyses of measurements that can be described by statistical models are of essence in astronomy and in scientific inquiry in general. The sensitivity of such analyses, modelling approaches, and the consequent predictions, is sometimes highly dependent on the exact techniques applied, and improvements therein can result in significantly better understanding of the observed system of interest. Particularly, optimising the sensitivity of statistical techniques in detecting the faint signatures of low-mass planets orbiting the nearby stars is, together with improvements in instrumentation, essential in estimating the properties of the population of such planets, and in the race to detect Earth-analogs, i.e. planets that could support liquid water and, perhaps, life on their surfaces. We review the developments in Bayesian statistical techniques applicable to detections planets orbiting nearby stars and astronomical data analysis problems in general. We also discuss these techniques and demonstrate their usefulness by using various examples and detailed descriptions of the respective mathematics involved. We demonstrate the practical aspects of Bayesian statistical techniques by describing several algorithms and numerical techniques, as well as theoretical constructions, in the estimation of model parameters and in hypothesis testing. We also apply these algorithms to Doppler measurements of nearby stars to show how they can be used in practice to obtain as much information from the noisy data as possible. Bayesian statistical techniques are powerful tools in analysing and interpreting noisy data and should be preferred in practice whenever computational limitations are not too restrictive.

Studies on ATP-diphosphohydrolase nucleotide-binding sites by intrinsic fluorescence

Potato apyrase, a soluble ATP-diphosphohydrolase, was purified to homogeneity from several clonal varieties of Solanum tuberosum. Depending on the source of the enzyme, differences in kinetic and physicochemical properties have been described, which cannot be explained by the amino acid residues present in the active site. In order to understand the different kinetic behavior of the Pimpernel (ATPase/ADPase = 10) and Desir��e (ATPase/ADPase = 1) isoenzymes, the nucleotide-binding site of these apyrases was explored using the intrinsic fluorescence of tryptophan. The intrinsic fluorescence of the two apyrases was slightly different. The maximum emission wavelengths of the Desir��e and Pimpernel enzymes were 336 and 340 nm, respectively, suggesting small differences in the microenvironment of Trp residues. The Pimpernel enzyme emitted more fluorescence than the Desir��e apyrase at the same concentration although both enzymes have the same number of Trp residues. The binding of the nonhydrolyzable substrate analogs decreased the fluorescence emission of both apyrases, indicating the presence of conformational changes in the neighborhood of Trp residues. Experiments with quenchers of different polarities, such as acrylamide, Cs+ and I- indicated the existence of differences in the nucleotide-binding site, as further shown by quenching experiments in the presence of nonhydrolyzable substrate analogs. Differences in the nucleotide-binding site may explain, at least in part, the kinetic differences of the Pimpernel and Desir��e isoapyrases.

Conventional and novel strategies in the treatment of adrenocortical cancer

Adrenocortical carcinoma is a highly malignant neoplasm with an incidence of two per million people per year. Several treatment strategies have resulted in temporary or partial tumor regression but very few cases have attained long survival. Surgical resection of the primary tumor and metastases is most effective. Several chemotherapeutic protocols have been employed with variable success. Mitotane (o,p'-DDD) is an adrenalytic drug effective in inducing a tumor response in 33% of patients treated. Mitotane requires metabolic transformation for therapeutic action. Tumors may vary in their ability to metabolize mitotane and the ability of tumors to transform mitotane may predict the clinical response to the drug. Preliminary data show a possible correlation between metabolic activity of neoplastic adrenocortical tissue and response to mitotane. We have attempted to develop mitotane analogs with enhanced adrenalytic effect. Compared to mitotane, a di-chloro compound, the bromo-chloro and di-bromo analogs appear to have a greater effect. Future approaches to the treatment of adrenocortical carcinoma are likely to be based on blocking or reversing the biological mechanisms of tumorigenesis. Angiogenic and chemotactic mechanisms may play a role in adrenal tumor growth and inhibition of these mechanisms may result in inhibition of tumor growth. New mitotane analogs with greater adrenalytic potential could be a promising approach to developing more effective and selective therapies for adrenal cancer. Alternative approaches should attempt to suppress tumor growth by means of compounds with anti-angiogenic and anti-chemotactic activity.

Antiproliferative effects of 1,25-dihydroxyvitamin D3 on breast cells: a mini review

The hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), the active form of vitamin D3, is an important regulator of calcium homeostasis, exerts antiproliferative effects on various cell systems and can induce differentiation in some kinds of hematopoietic cells. These effects are triggered by its receptor, vitamin D receptor (VDR), a phosphoprotein member of the nuclear receptor superfamily, which functions as a transcriptional factor. VDR binds as a heterodimer with retinoid X receptor (R X R) to hexameric repeats, characterized as vitamin D-responsive elements present in the regulatory region of target genes such as osteocalcin, osteopontin, calbindin-D28K, calbindin-D9K, p21WAF1/CIP1, TGF-��2 and vitamin D 24-hydroxylase. Many factors such as glucocorticoids, estrogens, retinoids, proliferation rate and cell transformation can modulate VDR levels. VDR is expressed in mammary tissue and breast cancer cells, which are potential targets to hormone action. Besides having antiproliferative properties, vitamin D might also reduce the invasiveness of cancer cells and act as an anti-angiogenesis agent. All of these antitumoral features suggest that the properties of vitamin D could be explored for chemopreventive and therapeutic purposes in cancer. However, hypercalcemia is an undesirable side effect associated with pharmacological doses of 1,25-(OH)2D3. Some promising 1,25-(OH)2D3 analogs have been developed, which are less hypercalcemic in spite of being potent antiproliferative agents. They represent a new field of investigation.

Design and applications of modified oligonucleotides

Oligonucleotides have a wide range of applications in fields such as biotechnology, molecular biology, diagnosis and therapy. However, the spectrum of uses can be broadened by introducing chemical modifications into their structures. The most prolific field in the search for new oligonucleotide analogs is the antisense strategy, where chemical modifications confer appropriate characteristics such as hybridization, resistance to nucleases, cellular uptake, selectivity and, basically, good pharmacokinetic and pharmacodynamic properties. Combinatorial technology is another research area where oligonucleotides and their analogs are extensively employed. Aptamers, new catalytic ribozymes and deoxyribozymes are RNA or DNA molecules individualized from a randomly synthesized library on the basis of a particular property. They are identified by repeated cycles of selection and amplification, using PCR technologies. Modified nucleotides can be introduced either during the amplification procedure or after selection.

Recognition of nucleic acids by metal ion complexes

Metal-ion-mediated base-pairing of nucleic acids has attracted considerable attention during the past decade, since it offers means to expand the genetic code by artificial base-pairs, to create predesigned molecular architecture by metal-ion-mediated inter- or intra-strand cross-links, or to convert double stranded DNA to a nano-scale wire. Such applications largely depend on the presence of a modified nucleobase in both strands engaged in the duplex formation. Hybridization of metal-ion-binding oligonucleotide analogs with natural nucleic acid sequences has received much less attention in spite of obvious applications. While the natural oligonucleotides hybridize with high selectivity, their affinity for complementary sequences is inadequate for a number of applications. In the case of DNA, for example, more than 10 consecutive Watson-Crick base pairs are required for a stable duplex at room temperature, making targeting of sequences shorter than this challenging. For example, many types of cancer exhibit distinctive profiles of oncogenic miRNA, the diagnostics of which is, however, difficult owing to the presence of only short single stranded loop structures. Metallo-oligonucleotides, with their superior affinity towards their natural complements, would offer a way to overcome the low stability of short duplexes. In this study a number of metal-ion-binding surrogate nucleosides were prepared and their interaction with nucleoside 5��-monophosphates (NMPs) has been investigated by 1H NMR spectroscopy. To find metal ion complexes that could discriminate between natural nucleobases upon double helix formation, glycol nucleic acid (GNA) sequences carrying a PdII ion with vacant coordination sites at a predetermined position were synthesized and their affinity to complementary as well as mismatched counterparts quantified by UV-melting measurements.

The study of ligand binding specificities of the lipid binding proteins : recombinant human a-tocopherol transport protein (a-ttp), supernatant protein factor (spf) and S. cerevisiae Sec 14p for vitamin e (rrr-a-tocopherol) and other hydrophobic ligands.

One of the various functions of proteins in biological systems is the transport of small molecules, for this purpose proteins have naturally evolved special mechanisms to allow both ligand binding and its subsequent release to a target site; a process fundamental to many biological processes. Transport of Vitamin E (a-tocopherol), a lipid soluble antioxidant, to membranes helps in the protection of polyunsaturated fatty acids against peroxidative damage. In this research, the ligand binding characteristics of several members of the CRALTRIO family of lipid binding proteins was examined; the recombinant human a-Tocopherol Transfer Protein (a-TIP), Supernatant Protein Factor (SPF)ffocopherol Associated Protein (TAP), Cellular Retinaldehyde Binding Protein (CRALBP) and the phosphatidylinositol transfer protein from S. cerevisiae Sec 14p. Recombinant Sec 14p was expressed and purified from E. coli for comparison of tocopherol binding to the two other recombinant proteins postulated to traffic a-tocopherol. Competitive binding assays using [3H]-a-tocopherol and Lipidex-l000 resin allowed determination of the dissociation constants ~) of the CRAL-TRIO proteins for a-tocopherol and - 20 hydrophobic ligands for evaluation of the possible biological relevance of the binding interactions observed. The KIs (nM) for RRR-a-tocopherol are: a-TIP: 25.0, Sec 14p: 373, CRALBP: 528 and SPFffAP: 615. This indicates that all proteins recognize tocopherol but not with the same affinity. Sec 14p bound its native ligand PI with a KI of381 whereas SPFffAP bound PI (216) and y-tocopherol (268) similarly in contrast to the preferential binding ofRRR-a-tocopherol by a-TIP. Efforts to adequately represent biologically active SPFff AP involved investigation of tocopherol binding for several different recombinant proteins derived from different constructs and in the presence of different potential modulators (Ca+2, Mg+2, GTP and GDP); none of these conditions enhanced or inhibited a-tocopherol binding to SPF. This work suggests that only aTTP serves as the physiological mediator of a-tocopherol, yet structural homology between proteins allows common recognition of similar ligand features. In addition, several photo-affmity analogs of a-tocopherol were evaluated for their potential utility in further elucidation of a-TTP function or identification of novel tocopherol binding proteins.

Towards the enantioselective synthesis of lycoricidine alkaloids /

Two efficient, regio- and stereo controlled synthetic approaches to the synthesis of racemic analogs of pancratistatin have been accomplished and they serve as the model systems for the total synthesis of optically active 7-deoxy-pancratistatin. In the Diels-Alder approach, an efficient [4+2] cycloaddition of 3,4-methylenedioxyco- nitrostyrene with Danishefsky's diene to selectively form an exo-nitro adduct has been developed as the key step in the construction of the C-ring of the target molecule. In the Michael addition approach, the key step was a conjugate addition of an organic zinc-cuprate to the 3,4-methylenedioxy-(B-nitrostyrene, followed by a diastereocontroUed closure to form the cyclohexane C-ring of the target molecule via an intramolecular nitro-aldol cyclization on a neutral alumina surface. A chair-like transition state for such a cyclization has been established and such a chelation controlled transition state can be useful in the prediction of diastereoselectivity in other related 6-exo-trig nitroaldol reactions. Cyclization of the above products fi^om both approaches by using a Bischler-Napieralski type reaction afforded two lycoricidine derivatives 38 and 50 in good yields. The initial results from the above modeling studies as well as the analysis of the synthetic strategy were directed to a chiral pool approach to the total synthesis of optically active 7-deoxy-pancratistatin. Selective monsilylation and iodination of Ltartaric acid provided a chiral precursor for the proposed key Michael transformation. The outlook for the total synthesis of 7-deoxy-pancratistatin by this approach is very promising.A concise synthesis of novel designed, optically pure, Cz-symmetrical disulfonylamide chiral ligands starting from L-tartaric acid has also been achieved. This sequence employs the metallation of indole followed by Sfj2 replacement of a dimesylate as the key step. The activity for this Cz-symmetric chiral disulfonamide ligand in the catalytic enantioselective reaction has been confirmed by nucleophilic addition to benzaldehyde in the disulfonamide-Ti (0-i-Pr)4-diethylzinc system with a 48% yield and a 33% e.e. value. Such a ligand tethered with a suitable metal complex should be also applicable towards the total synthesis of 7-deoxy-pancratistatin.

Amino acid transport : the special case of a H/L-glutamate cotransport system in Asparagus sprengeri mesophyll cells /

The addition of L-Glutamate (L-GLU) and L-Hethionine ~ulfoximine (L-HSO) to mechanically isolated. photosynthetically competent, Asparagus sprengeri mesophyll cells ~u~pended in 1mM CaS04 cau~ed an immediate transient alkalinization of the cell su~pension medium in both the light and dark. The alkalinization response was specific and stereospecific as none of the L-isomers of the other 19 protein amino acids tested or D-GLU gave this response. Uptake of 14C-L-GLU was stimulated by the light. The addition of non-radioactive L-GLU. or L-GLU analogs together with 14C-L-GLU showed that only L-GLU and L-HSO stimulated alkalinization whilst inhibiting the uptake of 14C-L-GLU. Both the L-GLU dependent alkalinization and the upt~ke of 14C-L-GLU were stimulated when the external pH was decreased from 6.5 to 5.5. Increasing external K+ concentrations inhibited the uptake of 14C-L-GLU. Fusicoccin (FC) stimulated uptake. The L-GLU dependent alkalinization re~ponse exhibited monophasic saturation kinetics while the uptake of 14C-L-GLU exhibited biphasic saturation kinetics. In addition to a saturable component. the uptake kinetics also showed a linear component of uptake. Addition of L-GLU and L-MSO caused internal acidification of the cell as measured by a change in the distribution of 14C-DMO. There was no change in K+ efflux when L-GLU was added. A H+ to L-GLUinflux stoichiometry of 3:1 wa~ mea~ured at an external I.-GLU concentration of O.5mM and increased with increasing external 13 L-QLU concentration. Metabolism of L-GLU was detected manometrlcally by observing an increase in COa evolution upon the addition of L-QLU and by detection of i*C02 evolution upon the addition of ��*C-L-GLU. ��*C02 evolution was higher in the dark than in the light. The data are consistent with the operation of a H+/L-QLO cotransport system. The data also show that attempts to quantify the stoichlometry of the process were complicated by the metabolism of L-GLU.

Two enantiodivergent syntheses of balanol and the chemoenzymatic synthesis of oseltamivir

The present thesis outlines our latest findings on the reactivity of the Burgess reagent with oxiranes. Structural, mechanistic, and computational studies are presented. Included is the development of a (-)-menthyl version of the Burgess reagent and its application to the synthesis of enantiomerically pure ~-amino alcohols. This methodology has been exploited in the formal enantiodivergent synthesis of the (+)- and (-)-isomers of balanol. Also described is a second generation approach to both balanol enantiomers; each commencmg with the chemoenzymatic dihydroxylation of bromobenzene. This study also describes the steric and functional limitations of the toluene dioxygenase-mediated oxidation of benzoate esters. The metabolite derived from ethyl benzoate was employed in a formal synthesis of oseltamivir. Finally, several synthetic approaches to oseltamivir and its analogs are presented, each proceeding through a different vinyl aziridine derived from bromobenzene and ethyl benzoate.

Novel glutathione disulfide transferase function of CC-glutaredoxins involved in disease resistance and flower development

Glutaredoxins are oxidoreductases capable of reducing protein disulfide bridges and glutathione mixed disulfides through the process of deglutathionylation and glutathionylation. Lately, redox-mediated modifications of functional cysteine residues of TGA1 and TGA8 transcription factors have been postulated. Namely, GRX480 and ROXY1 glutaredoxins have been previously shown to interact with TGA proteins and have been suggested to regulate redox state of these proteins. TGA1, together with TGA2, is involved in systemic acquired resistance (SAR) establishment in the plant Arabidopsis thaliana through PR1 (Pathogenesis related 1) gene activation. They both form an enhanceosome complex with the NPR1 protein (non-expressor of pathogenesis related gene 1) which leads to PR1 transcription. Although TGA1 is capable of activating PR1 transcription, the ability of the TGA1 NPR1 enhanceosome complex to assembly is based on the redox status of TGA1. We identified GRX480 as a glutathionylating enzyme that catalyzes the TGA1 glutathione disulfide transferase reaction with a Km of around 20��M GSSG (oxidized glutathione). Out of four cysteine residues found within TGA1, C172 and C266 were found to be glutathionylated by this enzyme. We also confirmed TGA1 glutathionylation in vivo and showed that this modification takes place while TGA1 is associated with the PR1 promoter enzymatically via GRX480. Furthermore, we show that glutathionylation via GRX480 abolishes TGA1's interaction with NPR1 and consequently prevents the TGA1-NPR1 transcription activation of PR1. When glutathionylated, TGA1 is recruited to the PR1 promoter and acts as a repressor. Therefore, glutathionylation is a mechanism that prevents TGA1 NPR1 interaction, allowing TGA1 to function as a repressor of PR1 transcription. Surprisingly, GRX480 was not able to deglutathionylate proteins demonstrating the irreversible nature of the reaction. Moreover, we demonstrate that other members of CC-class glutaredoxins, namely ROXY1 and ROXY2, can also catalyze protein glutathionylation. The TGA8 protein was previously shown to interact with NPR1 analogs, BOP1 and BOP2 proteins. However, unlike the case of TGA1 NPR1 interaction, here we demonstrate that TGA8-BOP1 interaction is not redox regulated and that TGA8 glutathionylation by ROXY1 and ROXY2 enzymes does not abolish this interaction in vitro. However, TGA8 glutathionylation results in TGA8 oligomer disassembly into smaller complexes and monomers. Our results suggest that CC-Grxs are unable to reduce mixed disulfides, instead they efficiently catalyze the opposite reaction which distinguishes them from traditional glutaredoxins. Therefore, they should not be classified as glutaredoxins but as protein glutathione disulfide transferases.

Le diab��te maternel influence la morphogen��se r��nale et la programmation p��rinatale

Le diab��te maternel est un facteur de risque majeur pour le d��veloppement de malformations cong��nitales. Dans le syndrome de l���embryopathie diab��tique, l���exposition prolong��e du f��tus �� de hautes concentrations ambientes de glucose induit des dommages qui peuvent affecter plusieurs organes, dont les reins. Les malformations r��nales sont la cause de pr��s de 40 pourcent des cas d���insuffisance r��nale infantile. L���hyperglyc��mie constitue un environnement ut��rin adverse qui nuit �� la n��phrogen��se et peut causer l���agen��se, la dysplasie (aplasie) ou l���hypoplasie r��nale. Les m��canismes mol��culaires par lesquels les hautes concentrations ambientes de glucose m��nent �� la dysmorphogen��se et aux malformations demeurent toutefois mal d��finis. Le diab��te maternel pr��dispose aussi la prog��niture au d��veloppement d���autres probl��mes �� l�����ge adulte, tels l���hypertension, l���ob��sit�� et le diab��te de type 2. Ce ph��nom��ne appel�� ���programmation p��rinatale��� a suscit�� l���int��r��t au cours des derni��res d��cennies, mais les m��canismes responsables demeurent mal compris. Mes ��tudes doctorales visaient �� ��lucider les m��canismes mol��culaires par lesquels le diab��te maternel ou un environnement in utero hyperglyc��mique affecte la n��phrogen��se et programme par la suite la prog��niture a d��velopper de l���hypertension par des observations in vitro, ex vivo et in vivo. Nous avons utilis�� les cellules MK4, des cellules embryonnaires du m��senchyme m��tan��phrique de souris, pour nos ��tudes in vitro et deux lign��es de souris transg��niques (Tg) pour nos ��tudes ex vivo et in vivo, soient les souris HoxB7-GFP-Tg et Nephrin-CFP-Tg. Les souris HoxB7-GFP-Tg expriment la prot��ine fluorescente verte (GFP) dans le bourgeon ur��t��rique (UB), sous le contr��le du promoteur HoxB7. Les souris Nephrin-CFP expriment la prot��ine fluorescente cyan (CFP) dans les glom��rules, sous le contr��le du promoteur nephrin sp��cifique aux podocytes. Nos ��tudes in vitro visaient �� d��terminer si les hautes concentrations de glucose modulent l���expression du g��ne Pax2 dans les cellules MK4. Les cellules MK4 ont ��t�� trait��es pendant 24h avec du milieu contenant soit 5mM D-glucose et 20mM D-mannitol ou 25mM D-glucose et avec ou sans antioxydants ou inhibiteurs de p38 MAPK, p44/42 MAPK, PKC et NF-kB. Nos r��sultats ont d��montr�� que le D-glucose ��lev�� (25mM) augmente la g��n��ration des esp��ces r��actives de l���oxyg��ne (ROS) dans les cellules MK4 et induit sp��cifiquement l���expression du g��ne Pax2. Des analogues du glucose tels le D-mannitol, L-glucose ou le 2-Deoxy-D-glucose n���induisent pas cette augmentation dans les cellules MK4. La stimulation de l���expression du g��ne Pax2 par le D-glucose dans les cellules MK4 peut ��tre bloqu��e par des inhibiteurs des ROS et de NF-kB, mais pas par des inhibiteurs de p38 MAPK, p44/42 MAPK ou PKC. Ces r��sultats indiquent que la stimulation de l���expression du g��ne Pax2 par les concentrations ��lev��es de glucose est due, au moins en partie, �� la g��n��ration des ROS et l���activation de la voie de signalisation NF-kB, et non pas via les voies PKC, p38 MAPK et p44/42 MAPK. Nos ��tudes ex vivo s���int��ressaient aux effets d���un milieu hyperglyc��mique sur la morphogen��se de la ramification du bourgeon ur��t��rique (UB). Des explants de reins embryonnaires (E12 �� E18) ont ��t�� pr��lev��s par micro-dissection de femelles HoxB7-GFP gestantes. Les explants ont ensuite ��t�� cultiv��s dans un milieu contenant soit 5mM D-glucose et 20mM D-mannitol ou 25mM D-glucose et avec ou sans antioxydants, catalase ou inhibiteur de PI3K/AKT pour diverses dur��es. Nos r��sultats ont d��montr�� que le D-glucose stimule la ramification du UB de mani��re sp��cifique, et ce via l���expression du g��ne Pax2. Cette augmentation de la ramification et de l���expression du g��ne Pax2 peut ��tre bloqu��e par des inhibiteurs des ROS et de PI3K/AKT. Ces ��tudes ont d��montr�� que les hautes concentrations de glucose alt��rent la morphogen��se de la ramification du UB via l���expression de Pax2. L���effet stimulant du glucose semble s���effectuer via la g��n��ration des ROS et l���activation de la voie de signalisation Akt. Nos ��tudes in vivo visaient �� d��terminer le r��le fondamental du diab��te maternel sur les d��fauts de morphogen��se r��nale chez la prog��niture. Dans notre mod��le animal, le diab��te maternel est induit par le streptozotocin (STZ) chez des femelles HoxB7-GFP gestantes (E13). Les souriceaux ont ��t�� ��tudi��s �� diff��rents ��ges (naissants et ��g��s de une, deux ou trois semaines). Nous avons examin�� leurs morphologie r��nale, nombre de n��phrons, expression g��nique et les ��v��nements apoptotiques lors de cette ��tude �� court terme. La prog��niture des m��res diab��tiques avait un plus faible poids, taille et poids des reins, et poss��dait des glom��rules plus petits et moins de n��phrons par rapport �� la prog��niture des m��res contr��les. La dysmorphogen��se r��nale observ��e est peut-��tre caus��e par l���augmentation de l���apoptose des cellules dans la r��gion du glom��rule. Nos r��sultats ont montr�� que les souriceaux n��s de m��res diab��tiques poss��dent plus de podocytes apoptotiques et plus de marquage contre la caspase-3 active dans leurs tubules r��naux que la prog��niture des m��res contr��les. Les souriceaux des m��res diab��tiques montrent une augmentation de l���expression des composants du syst��me r��nine angiotensine (RAS) intrar��nal comme l���angiotensinog��ne et la r��nine, ainsi qu���une augmentation des isoformes p50 et p65 de NF-kB. Ces r��sultats indiquent que le diab��te maternel active le RAS intrar��nal et induit l���apoptose des glom��rules, menant �� une alt��ration de la morphogen��se r��nale de la prog��niture. En conclusion, nos ��tudes ont permis de d��montrer que le glucose ��lev�� ou l���environnement in utero diab��tique alt��re la morphogen��se du UB, qui r��sulte en un retard dans la n��phrogen��se et produit des reins plus petits. Cet effet est d��, au moins en partie, �� la g��n��ration des ROS, �� l���activation du RAS intrar��nal et �� la voie NF-kB. Nos ��tudes futures se concentreront sur les m��canismes par lesquels le diab��te maternel induit la programmation p��rinatale de l���hypertension chez la prog��niture adulte. Cette ��tude �� long terme porte sur trois types de prog��nitures : adultes n��s de m��res contr��les, diab��tiques ou diab��tiques trait��es avec insuline pendant la gestation. Nous observerons la pression systolique, la morphologie r��nale et l���expression de divers g��nes et prot��ines. Nous voulons de plus d��terminer si la pr��sence d���un syst��me antioxydant (catalase) peut prot��ger la prog��niture des effets n��fastes des ROS caus��s par l���environnement in utero hyperglyc��mique. Les souris Catalase-Tg expriment la catalase sp��cifiquement dans les tubules proximaux et nous permettrons d���explorer notre hypoth��se sur le r��le des ROS dans notre mod��le exp��rimental de diab��te maternel.

Caract��risation de nouveaux analogues des cannabinoides

Ce m��moire pr��sente l�����tude de certains effets pharmacologiques de nouveaux analogues synth��tiques des cannabino��des. Un des objectifs de longue date des recherches sur les cannabino��des a ��t�� la d��couverte de puissants analogues synth��tiques de substances naturelles, qui pourraient ��tre d��velopp��s comme m��dicaments. Cela n��cessite, entre autres, qu���ils soient exempts d���effets psychotropes qui caract��risent l���usage r��cr��atif du Cannabis. Le moteur derri��re cet objectif a ��t�� la longue histoire de l���usage du Cannabis comme substance m��dicale, en particulier dans le traitement de la douleur et de l���inflammation. Parmi les nombreux effets pharmacologiques des cannabino��des, deux sont d���un grand int��r��t th��rapeutique : l���effet antiprolif��ratif sur les cellules tumorales et l���effet anti-angiog��ne. Dans ce m��moire, l�����tude de ces deux effets a ��t�� r��alis��e sur des cultures cellulaires tumorales et endoth��liales humaines. Les tests de prolif��ration sur les deux types de cellules n���ont pas montr�� de cytotoxicit��. Ce qui a permis de poursuivre l�����tude de l���effet anti-angiog��ne, et qui a mis fin �� l�����tude de l���effet anti-tumoral. L���inhibition de l���angiog��n��se a ��t�� investigu��e en r��alisant des tests de recolonisation d���une zone d��nud��e dans une monocouche de cellules endoth��liales et des tests de formation de microtubules sur matrice g��lifi��e. L���effet anti-angiog��ne n���a pas pu ��tre ��valu�� �� cause de probl��mes de contamination, n��anmoins certaines mol��cules montrent un effet anti-migratoire. L���absence de cytotoxicit�� et l���analogie structurale avec le ������-t��trahydrocannabinol encourage �� continuer l���investigation des effets pharmacologiques de ces nouvelles mol��cules synth��tiques. Il serait aussi pertinent d�����tudier, dans une th��se de doctorat, les m��canismes d���actions mol��culaires par lesquels agissent ces mol��cules.

Addition st��r��os��lective de nucl��ophiles sur un centre ac��tal : synth��se de nucl��osides 1���,2���-cis

Plusieurs analogues de nucl��osides th��rapeutiques (Ara-C, Clofarabine), utilis��s pour le traitement de leuc��mies, pr��sentent un arrangement 1���,2���-cis entre la nucl��obase reli��e au centre anom��re et le substituant (��lectroattracteur) en C-2���. R��cemment, notre laboratoire a d��velopp�� une approche synth��tique pour former s��lectivement des analogues de nucl��osides et de thionucl��osides 1���,2���-trans et 1���,2���-cis �� partir de pr��curseurs acycliques. Ce m��moire pr��sente une nouvelle m��thodologie pour acc��der efficacement aux analogues de nucl��osides 1���,2���-cis �� partir de furanosides. Diff��rents groupements en position anom��rique ont ��t�� examin��s, sous conditions cin��tiques en utilisant le bromure de dim��thylbore pour g��n��rer s��lectivement des produits acycliques ou cycliques. Les interm��diaires cin��tiques de diff��rents furanosides de m��thyle form��s en pr��sence de Me2BBr ont ��t�� pi��g��s in situ par un thiol pour g��n��rer des thioac��tals acycliques avec de bonnes voire d���excellentes diast��r��os��lectivit��s. Les produits g��n��r��s sont en accord avec une r��tention globale de l���information st��r��ochimique du centre ac��tal et deux d��placements SN2 cons��cutifs ont ��t�� sugg��r��s pour rationaliser ces r��sultats. Toutefois, l���objectif de synth��tiser des analogues de nucl��osides �� partir de furanosides de m��thyle a ��chou��. Tel que d��montr�� par le Dr Michel Pr��vost, l���activation par Me2BBr des lactols des quatres diff��rents furanosides suivie d���une addition in situ d���une base silyl��e a permis de former diast��r��os��lectivement les analogues de nucl��osides 1���,2���-cis correspondants avec d���excellents rendements. Nous avons d��montr�� que d���autres substrats peuvent ��tre employ��s et que l���induction st��r��ochimique est sous contr��le du substituant ��lectroattracteur en C-2. D���autres acides de Lewis, tel que TMSBr, peuvent ��galement ��tre utilis��s. Cette m��thodologie a ��galement ��t�� ��tendue �� d���autres nucl��ophiles tels que des Grignards ou des ��thers d�����nols silyl��s, conduisant �� de bonnes s��lectivit��s.