Five studies were designed to characterize the effects of a non-steroidal aromatase inhibitor, letrozole, on ovarian function in cattle. The general hypothesis was that non-steroidal aromatase inhibitors have potential as a steroid-free option for the control of ovarian function for the purposes of fixed-time artificial insemination and embryo production. The specific objectives were to determine the effect of route and vehicle, type of aromatase inhibitor, and duration of aromatase inhibitor treatment (short vs prolonged) on ovarian follicles in cattle, and to test the efficacy of an aromatase inhibitor-based protocol to synchronize ovulation in cattle. In the first experiment, heifers were treated with letrozole intravenously (n=10) or intramuscularly (n=10) or allocated in iv and im control groups (n=5/group). During the second experiment, heifers were divided randomly into two groups (n=15/group) and an intravaginal device containing 1 g of letrozole or a blank device (control) was inserted. The third experiment was designed with the goal of formulating and testing an intravaginal device that provides biologically active circulating concentrations of an aromatase inhibitor for a minimum of 4 days. The biological significance of the pharmacokinetic differences between the letrozole intravaginal devices resulting from the third study was evaluated during the fourth study. A final study was designed to determine the effect of stage of the estrous cycle on the proportion of animals that ovulated and the synchrony of ovulation of heifers treated with an aromatase inhibitor-based ovulation-synchronization protocol and to determine subsequent pregnancy outcomes. In all the studies, the effects of aromatase inhibitor on ovarian function were assessed by transrectal ultrasound examination of the ovaries, and blood samples were collected for hormone concentration determination. Results demonstrated that route of administration, or more precisely, the nature of iii the vehicle used for the administration of letrozole (intravenous, intramuscular depot, short release intravaginal or prolonged release intravaginal) has an impact on the effects of letrozole on hormonal profiles and ovarian dynamics. The intramuscular route appeared to provide a prolonged release of letrozole from the injection site which had a marked effect on estradiol production, dominant follicle lifespan, and CL form and function. Letrozole treatment during the ovulatory follicle wave by means of a gel-based intravaginal releasing device during the second study resulted in more rapidly growing dominant follicles and larger ovulatory follicles, delayed ovulation (by 24 h) of a single follicle and formation of a CL that secreted higher levels of progesterone. A wax-based vehicle allowed for a steady and continuous delivery of the active compound over the treatment period. During the third study, the addition of a letrozole-containing gel coating increased the rate of initial absorption and hastened the increase on plasma concentrations of the active ingredient, while the letrozole-containing wax-based vehicle prolonged drug-delivery from the intravaginal device. When tested in vivo during the fourth study, we confirmed that letrozole-impregnated intravaginal devices formulated with a wax base plus a gel coat vehicle was most suitable for the application of a letrozole-based protocol for the synchronization of ovulation in cattle, since it effectively delivered elevated concentrations of letrozole, and reduced estradiol production resulting in increased follicular growth and lifespan, without adversely affecting progesterone production. The application of a letrozole-impregnated intravaginal device for 4 days, combined with PGF treatment at device removal and GnRH 24 h post-device removal increased the percentage of ovulations and synchrony of ovulation in cattle, regardless the stage of the estrous cycle at initiation of treatment. As observed in previous studies, the effects observed could be associated with an increase in circulating LH iv concentrations. However, the effects of treatment on gonadotropin concentrations are inconclusive, possibly due to inadequate sampling frequency. The impact of letrozole treatment of oocyte fertility remains unknown. The results of the five experiments support our general hypothesis that non-steroidal aromatase inhibitors have potential as a steroid-free option for the control of ovarian function in cattle. However, further research is needed in order to elucidate the effects of letrozole treatment during the proestrous on oocyte competence and fertility of the resulting ovulations in cattle.

The cellular inhibitor of apoptosis protein (cIAP)-2 plays an important role in the protection against apoptosis by inhibiting the endogenous IAP inhibitor Smac, thus allowing other members of the IAP family, such as XIAP to block caspases. Additionally, cIAP2 functions as a ubiquitin ligase and mediates survival/proliferative signaling through NF-κB. cIAP2 is overexpressed in many human cancers and is believed to play an oncogenic role. This led to the development of small molecule IAP antagonists aimed at eliciting apoptosis in cancer cells. However, the loss of cIAP2 is also associated with multiple myeloma, in which constitutively active NF-κB signaling contributes to pathogenesis of the disease and suggests that cIAP2 may also perform a tumour suppressive function. We demonstrate a novel role for cIAP2 in maintaining p53 levels in mammary epithelial cells that express wildtype p53. Downregulation of cIAP2 resulted in activation of IKKs, which led to increased Mdm2-mediated degradation of p53. cIAP2 depletion also led to increased phosphorylation of ERK1/2. Reduction of p53 levels, in combination with survival signaling provided by NF-κB and MEK-ERK pathways were associated with increased colony formation in vitro and increased DMBA-induced adenocarcinomas in cIAP2-null mice. Treatment of cells with IAP antagonists resulted in significant cytotoxicity only in p53-mutant MDA-MB-231 cells, which was associated with autocrine production of TNF-α. We propose that the transcription of TNF-α is potentiated by gain-of-function mutation in p53 since downregulation of mutant p53 in MDA-MB-231 cells decreased TNF-α mRNA. Downregulation of cIAPs in p53-mutant cells resulted in a decrease in nuclear IKK-α, which may result in decreased IKK-α-mediated survival signaling. In contrast, cIAP downregulation in p53-wildtype cells resulted in no change in nuclear IKK-α, degradation of the corepressor SMRT and cell survival. We show that the effects of cIAP2 downregulation are context-dependent. Downregulation of cIAP2 in p53-wildtype cells results in a decrease in p53 and an increase in survival and proliferative signaling. These results suggest a tumour suppressor function for cIAPs that may account for cIAP mutation-associated cancers such as multiple myeloma. Moreover, our data also defines gain-of-function p53 mutation as a possible contributor to IAP antagonist sensitivity.

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Prostate cancer is the second leading cause of death from cancer in Australian men. As prostate cancer cells are reliant on androgens for growth and survival, the standard therapy for metastatic disease is androgen ablation therapy (AAT). AAT inhibits androgen signalling by altering androgen synthesis or prevent binding of androgens to their intracellular mediator, the androgen receptor (AR). Although initially effective, virtually all patients relapse, beyond which there are limited treatment options. The failure of AAT is not necessarily due to a decreased requirement for androgen signalling, but rather the AR is able to maintain signalling and tumour growth in an androgen-depleted environment. Therefore novel strategies that directly target the AR may provide a more effective therapeutic approach. We have endeavoured to suppress AR activity in prostate cancer cells by utilising a dominant negative AR. The most effective dominant negative construct developed, ARi41O, lacks amino acids 39-410 in the AR amino terminal transactivation domain. In studies of transcriptional activity, ARi410 has no intrinsic activity but inhibits the activity of wild type AR (wtAR) and also clinically relevant AR variants, by up to 95%. The objective of this thesis was to characterise the mechanisms of action of ARi410 and assess the functional effects of introducing this dominant negative receptor into prostate cancer cells. To investigate the mechanism by which ARi410 suppresses AR activity, a robust and sensitive AR inhibition assay was developed. This assay revealed that ARi410 is a potent inhibitor of AR activity on three independent AR-regulated promoters, regardless of the level of AR expression. Furthermore, while ARi410 can inhibit AR activity, it does not alter AR protein levels. By using ARi410 variants with mutations and/or deletions in regions of functional importance, the AR inhibition assay was also used to identify the critical regions of ARi410 required for its dominant negative activity. These studies demonstrate that the dominant negative activity of ARi41 0 is ligand-dependent, requires dimerisation through the ligand binding domain (LBD) and an intact DNA-binding domain (DBD). Further investigation into the mechanism of dominant negative activity revealed that ARi410 does not alter the subcellular localisation of AR, as both receptors are predominantly cytoplasmic in the absence of ligand and rapidly co-localise to the nucleus in response to androgens. Furthermore, an interaction between AR and ARi410 was observed in the presence and absence of ligand, and electrophoretic mobility shift assays demonstrated that AR and ARi410 form heterodimers on DNA. These studies led to the conclusion that the mechanism of dominant negative activity by ARi4I0 involves the formation of inactive receptor heterodimers that assemble on DNA and suppress AR activity. To determine the functional consequence of expressing the dominant negative androgen receptor in prostate cancer cells, an adenoviral method of gene delivery was developed. Adenoviral expression of ARi410 in LNCaP prostate cancer cells did not allow assessment of cell viability due to cell-specific toxicity of the viral vectors when expressed long-term. However, short-term expression of ARi410 in LNCaP cells resulted in inhibition of AR signalling, as determined by reduced expression of the androgen regulated genes apolipoprotein D and kallikrein 2. Importantly, this finding is consistent with the inhibitory activity of ARi410 observed using synthetic AR-regulated reporter genes in the AR inhibition assay, and demonstrates that ARi410 can effectively suppress endogenous AR signalling. The results of this thesis indicate that heterodimerisation between AR and ARi410 is the most likely mechanism of dominant negative inhibition of AR function by ARi410, and that the DBD and dimerisation through the LBD are required for optimal dominant negative activity. Furthermore, this thesis has demonstrated that ARi410 is an effective inhibitor of AR signalling and provides a basis for further functional studies and evaluation of the dominant negative androgen receptor in vitro and in vivo. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1338478 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

This thesis presents the first example of reversible photoregulation of the binding of a protease, α-chymotrypsin, to a surface. A modular approach is used involving the azobenzene photoswitch group, a surface linker and an enzyme binding group. This approach is designed to be easily extended to the photoregulation of binding of other proteases to surfaces by use of enzyme binding groups selective to these proteases. Chapter one gives a brief outline of some of the important areas involved in to this work, including molecular switches, proteases and surface modification. Chapter two describes the synthesis of azobenzene-containing boronate esters designed as photoswitch inhibitors of α-chymotrypsin. Boronate esters were prepared containing the aminophenylboronate group or the peptidomimetic borophenylalanine group for enzyme binding and a range of substituents designed for enzyme affinity and/or surface attachment. Syntheses primarily involved peptide coupling reactions and azobenzene formation by condensation of nitrosobenzenes and anilines. Coupling reactions were successfully carried out using EDCI or isobutyl chlorofomate in several cases where other reagents gave unacceptable decomposition. Chapter three describes the syntheses and HPLC stability studies of derivatives of a noncovalent α-chymotrypsin inhibitor. Several dipeptide-based compounds containing either an amide group for surface attachment or an azobenzene group for photoswitching were prepared, primarily using peptide coupling reactions. Each compound was incubated with α-chymotrypsin to assess its stability, and all were found by HPLC monitoring to be stable to α-chymotrypsin catalysed hydrolysis. Chapter four describes syntheses of azobenzene-containing trifluoromethylketones and α-ketoesters designed as photoswitch inhibitors of α-chymotrypsin. Trifluoromethylketones/α-ketoesters containing amine groups for surface attachment were prepared, primarily using peptide coupling reactions, but could not be isolated due to the incompatibility of the electrophilic ketone and primary amine groups. Trifluoromethylketones/α-ketoesters containing terminal alkynes for surface attachment were prepared either by the attachment of an alkyne substituent group to a symmetrical azobenzene core or by Pd-catalysed reaction of a protected alkyne with an azobenzene having a halide substitutent. Chapter five describes syntheses of sulfur-containing surface linkers for use in surface attachment of the photoswitch inhibitors described in chapters 2-4. A range of compounds containing disulfide or protected thiol groups for surface attachment and azide or carboxylic acid groups for inhibitor attachment were prepared. Syntheses primarily involved coupling of functionalised alcohols/amides to carboxylic acid-containing disulfides/thioacetates. Selected linkers were attached to azobenzenes by amide coupling or azide-alkyne cycloaddition for surface attachment, photoswitching and/or enzyme assay. Azide-alkyne cycloaddition yields were initially poor, but were improved by use of stoichiometric amounts of copper catalyst. Chapter six describes UV/vis photoisomerisation studies and enzyme assays carried out to assess enzyme photoswitching of the compounds described in chapters 2-5. The trifluoromethylketones and α-ketoesters described in chapter 4 gave the best results, with moderate inhibition of α-chymotrypsin (µM affinity constants) and up to 5.3 fold changes in inhibition on UV/vis irradiation. Many of the boronate esters described in chapter 2 were found to inhibit α-chymotrypsin, but were somewhat unstable to irradiation. The dipeptide-based compounds described in chapter 3 were inactive against α-chymotrypsin. Good photoisomerisation was obtained for an azobenzene containing a symmetrical disulfide surface linker and poor photoisomerisation was obtained for an azobenzene containing a lipoic acid surface linker. Chapter seven describes surface attachment of selected photoswitch inhibitors and studies of photoregulated enzyme binding to the resultant functional surfaces. Self assembled monolayers (SAMs) of disulfides were formed on gold surfaces and characterised by electrochemistry and contact angle measurements. Binding of α-chymotrypsin to SAMs containing a photoswitch inhibitor was detected by quartz crystal microbalance (QCM), but was found to be largely irreversible. An alkyne-containing photoswitch inhibitor was attached to a surface plasmon resonance (SPR) chip in a two step procedure involving generation of an azide modified surface followed by azide-alkyne cycloaddition. Binding of α-chymotrypsin to the resultant modified surface was detected by SPR and successfully regulated by UV/vis irradiation. Chapter eight provides conclusions for the work described in this thesis and suggests future directions. Chapter nine gives experimental details for the work described in this thesis.

Universiẗat, Diss., 2007--Giessen.

Universiẗat, Diss., 2007--Giessen.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2006. / Härtill 4 uppsatser.

Univ., Diss.--Münster, 2004.