Th.--Sci. nat.--Brest, 1977. N°: 43. / Rés. en angl.

Breast cancer is the most commonly diagnosed cancer amongst Canadian women. Though numerous treatments are available, in many instances tumours become refractory or recur. Therefore, understanding the biological events that lead to the progression and therapeutic resistance of breast cancer is essential for the development of novel treatment options for this disease. Numerous members of the protein arginine methyltransferase (PRMT) family, which are the enzymes responsible for catalyzing methylation on arginine residues are aberrantly regulated in breast cancer. Hence, understanding the precise contribution of PRMTs to the development and progression of breast cancer is important. This Thesis will present my findings on the alternatively spliced PRMT1 isoform, PRMT1v2, previously identified to be overexpressed in breast cancer cell lines and here shown to promote breast cancer cell survival and invasion. Second, a novel role is ascribed to PRMT6, another PRMT aberrantly expressed in breast cancer. PRMT6 promotes chemoresistance to the drug bortezomib by mediating stress granule formation through down-regulation of eIF4E. Increased stress granule formation in bortezomib-resistant cancer cells promotes cell survival. Third, DDX3, a prototypical PRMT substrate which is overexpressed in breast cancer cell lines and stimulates transformation of mammary epithelial cells is a novel substrate of PRMT1, CARM1, and PRMT6. Lastly, TDRD3, a reader/effector of arginine methylation also overexpressed in breast tumours regulates breast cancer cell proliferation, anchorage-independent growth and cell motility and invasion.

Three major systems participate in the control of the peripheral circulation: the renin-angiotensin, the arginine vasopressin (AVP) and the sympathetic nervous systems. These studies examined the roles of the AVP and the sympathetic nervous systems in the regulation of blood pressure at both the central and the peripheral level. Anatomical studies have revealed that hypothalamic neurons containing AVP extend to the nucleus tractus solitarius (NTS) in the medulla. Since the NTS is the primary site of termination of the afferent neurons of the baroreceptor reflex arc, it suggests that AVP may be involved in central cardiovascular regulation. The effect of central AVP on mean arterial pressure (MAP) and sympathetic nerve activity, estimated from plasma catecholamine levels, was investigated. The injection of AVP into the fourth cerebroventricle and NTS of conscious, unrestrained rats increased MAP and plasma noradrenaline and adrenaline levels, suggesting that AVP may act centrally at the NTS to modulate sympathoadrenal outflow. However, the injection of a selective vascular antagonist of AVP, d(CH₂)₅Tyr(Me)AVP, into the fourth ventricle or NTS did not affect MAP or plasma catecholamine levels, either in normotensive rats, in rats subjected to hypotensive stress, or in neurogenically-stressed rats. This suggests that endogenously-released AVP may not have a tonic influence on central cardiovascular regulation. The role of AVP in the control of MAP, cardiac output (CO) and its distribution was investigated in anesthetized, surgically-stressed rats. The i.v. injection of d(CH₂)₅Tyr(Me)AVP decreased MAP and total peripheral resistance (TPR), did not alter CO, and increased the distribution of blood flow (BF) to the stomach and skin. The vascular role of AVP was found to be greater in the absence of influence from the renin-angiotensin and the sympathetic nervous systems. After blockade of the renin-angiotensin system by the infusion of saralasin the AVP antagonist increased BF to the skin and muscle, while after blockade of the α-adrenergic system with the infusion of phentolamine, the AVP antagonist markedly increased BF to the muscle. Thus, the amount of vasoconstriction produced by AVP in different vascular beds was found to depend on the endogenous vasomotor tone from the renin-angiotensin and α-adrenergic systems. Cross-circulation studies were conducted to concurrently observe the peripheral and central effects of α-agonists in two anesthetized rats, designated rat A and B, respectively. The i.v. injection of clonidine into rat A was found to increase MAP and decrease HR in rat A, and reduce MAP and HR in rat B. Since the stimulation of peripheral α-adrenoceptors in rat A by clonidine increased MAP, it suggests that the effects of peripheral post-junctional α₂-adrenoceptors predominate over those of peripheral pre-junctional α₂-adrenoceptors. In contrast, the i.v. injection of the α₁-agonist, methoxamine, in rat A increased MAP and decreased HR in rat A, and increased both MAP and HR in rat B. This suggests that central α₁-adrenoceptors may mediate responses in the opposite direction to those produced by α₂-adrenoceptors. To verify the results of the cross-circulation studies in animals free of the influence of surgery and anesthesia, and to determine whether the responses to a-agonists were mediated by changes in sympathoadrenal outflow, clonidine and a more selective α₂-agonist, B-HT 920, were injected centrally in conscious rats. The i.e.v. injection of clonidine (1 µg) significantly decreased MAP and HR and slightly decreased plasma noradrenaline and adrenaline levels; however, contrary to expectations, the i.c.v. injection of B-HT 920 (1, 10 µg) increased MAP, decreased HR and slightly increased plasma noradrenaline and adrenaline levels. To determine whether the responses to central injection of clonidine or B-HT 920 were due to the stimulation of α₂-adrenoceptors, i.c.v. injections of these drugs were given after pretreatment with rauwolseine, a selective α₂-antagonist. The i.c.v. injection of rauwolscine in conscious rats increased MAP and plasma noradrenaline and adrenaline levels, suggesting that central α₂-adrenoceptors may mediate tonic inhibition of the cardiovascular system. However, i.c.v. injections of clonidine or B-HT 920 produced the same responses in the absence or presence of rauwolscine. Further studies with different α-adrenergic agonists and antagonists with various selectivities are necessary before we can explain the differential effects of central clonidine and B-HT 920. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

No description available.

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1990. / Extra t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1990. / Extra t.p. with thesis statement inserted. Includes bibliographical references.

The twin-arginine protein transport (Tat) system is one of two general protein transport systems in the cytoplasmic membranes of bacteria, and is conserved in the thylakoid membranes of plants. The Tat system transports fully folded proteins of varying sizes across the membrane. This feat is achieved by the use of just three conserved integral membrane proteins, TatA, TatB and TatC. Passenger proteins are designated for transport by the system by the use of an N-terminal extension, termed a signal peptide which contains an invariant twin arginine motif. The TatC component of the transport system is responsible for recognising the signal peptide, as well as binding to TatB and the TatA components. TatC therefore emerges as the functional and organisational ‘core’ of the Tat system. There is a wealth of functional data relating to the TatC protein, but no high resolution structural information on TatC was available to interpret this data. In order to gain an understanding of the Tat system at the molecular level, a structure was required of TatC. Preliminary expression screening using green fluorescent protein had previously identified the TatC protein from the thermophilic bacterium Aquifex aeolicus as being amenable for structural work. This study purified the A. aeolicus TatC construct in a variety of detergents, used native mass spectrometry and light scattering techniques to assay the sample homogeneity and set up extensive crystallisation trials. Following optimisation of a selenomethionine minimal media growth protocol, crystals grown from selenomethionine-substituted TatC purified in lauryl maltose neopentyl glycol diffracted to 3.5Å resolution, and allowed the structure of TatC to be solved by single wavelength anomalous dispersion. Analysis of the novel structure of TatC, in combination with an isothermal titration calorimetric binding assay, gave information on how TatC binds to signal peptides. Further structural analysis combined with additional experimental data from collaborators allowed a model to be proposed for how TatC binds to the two additional integral membrane components of the system, TatB and TatA, as part of the transport mechanism. Subsequent work was undertaken in this study to attempt to gain a co-crystal structure of TatC with the signal peptide. Construct and detergent screening was carried out in order to aim towards high resolution structural characterisation of the TatBC complex.

Arginine uptake and degradation systems are common throughout bacteria and archaea. The genome of human pathogen Chlamydia pneumoniae encodes three proteins now called AaxA, AaxB, and AaxC which function together to take up arginine, decarboxylate it, and expel the decarboxylation product, agmatine. AaxB is the previously characterized pyruvoyl-dependent arginine decarboxylase, AaxC is an inner membrane amino acid transport protein that functions as an arginine-agmatine antiporter, and AaxA is an outer membrane porin, which facilitates the uptake of arginine and also functions as a general porin with broad specificity. C. pneumoniae is a non-typical Gram negative bacteria and an obligate intracellular parasite with a unique 2-phase life cycle. The role of this system for arginine-agmatine exchange has yet to be determined but it may function to deplete host cell arginine as a means of inactivating host inducible nitric oxide synthase (iNOS), a molecule used in the innate immune response that has been shown to have an inhibitory affect on the growth of C. pneumoniae in cell culture. AaxB and AaxC are able to complement the loss of extreme acid-resistance in E. coli mutants that lack their own system for arginine-agmatine exchange, making pH homeostasis another possible role for this system. The porin AaxA is able to enhance arginine-agmatine exchange by AaxB and AaxC in E. coli mutants as well as by the native arginine decarboxylase AdiA and the native arginine-agmatine antiporter AdiC in wild type E. coli. AaxA is not an arginine-specific porin and instead acts as a general porin with a broad specificity. AaxA discriminates only against large and negatively charged solute molecules, and therefore it may have a broad role in the uptake of various biomolecules essential for chlamydial growth in addition to its role as part of a system for arginine-agmatine exchange. / text

No description available.

No description available.