published_or_final_version / Pharmacology and Pharmacy / Master / Master of Philosophy

Doctor of Philosophy / Department of Chemistry / Stefan H. Bossmann / This paper describes two methods for utilizing cancer associated proteases for targeting cancer therapy to the tumor. The first method is designing a drug delivery system based on liposomes that are sensitive to cancer associated proteases. Upon contact with the protease, the liposome releases its contents. The second method is designing a prodrug that is based on a porin isolated from Mycobacterium smegmatis. The porin is modified with protease consensus sequences, inhibiting its toxicity. Upon contact with the protease, the drug is activated. Protease sensitive liposomes were synthesized that were sensitive to urokinase plasminogen activator. This was done by synthesizing a cholesterol-anchored, uPA consensus – sequence-containing, acrylic acid block copolymer and using it to form a covalently bound polymer cage around the outside of a hypertonic liposome. Liposomes were synthesized that had a diameter of 136 nm. Upon addition of the polymer the diameter increased by 2.69 nm, indicating it had successfully embedded into the liposome membrane. After crosslinking with either a short peptide containing a lysine (so that it is a diamine) or ethylenediamine, the diameter increased between 5.33 nm and 14.1 nm (depending on the type and amount of the crosslinked). Fluorescence release assays showed that the polymer cage could add in excess of thirty atmospheres of osmotic pressure resistance, and, under isobaric conditions, would prevent release of much of the liposomal contents. Upon treatment with uPA, the polymer caged liposomes released a significantly larger amount of their contents making the liposomes protease sensitive. MspA was shown to be a very stable protein able to be imaged by AFM. AFM imaging demonstrated that MspA is able to form native pore structures in membranes making it a good imitator of the membrane attack complex. MspA was demonstrated to be highly cytotoxic, but poor at distinguishing between cells. Pro-MspA was synthesized by adding a hydrophilic peptide to MspA that prevents insertion. A uPA cleavage sequence embedded causes the MspA to become activated at the cancer site. This was demonstrated in tests against uPA and non-uPA producing cell lines.

Neste estudo retrospectivo, controlado, de 94 adolescentes consecutivos com maloclusão de Classe II, divisão 1ª de Angle e retrognatismo mandibular, no surto de crescimento puberal, foram avaliadas as mudanças dentoesqueléticas decorrentes do tratamento com aparelho de Herbst (Grupo A coroas de aço e B splints acrílicos) e de acompanhamento (Grupo C - controle), em um período de 12 meses. As telerradiografias laterais foram obtidas em T1 (inicial) e T2 (no final do período de observação). As variáveis cefalométricas foram analisadas com testes paramétricos. Os resultados mostraram que os dois grupos tratados caracterizaram-se diferencialmente do grupo controle, com restrição do crescimento maxilar e maior crescimento mandibular, o que permitiu melhorar a relação sagital. Da mesma forma, no aspecto dentoalveolar houve melhora da sobressaliência pela retroinclinação dos incisivos superiores e vestibularização dos incisivos inferiores, distalização com controle vertical dos molares superiores e mesialização dos molares inferiores. Os três grupos mostraram semelhança na extrusão dos molares inferiores, na preservação da morfologia mandibular e do padrão facial. O plano oclusal rotacionou no sentido horário nos grupos tratados e anti-horário no grupo controle. Pode-se concluir que no tratamento com dois tipos de aparelho de Herbst da maloclusão de classe II, em adolescentes no pico máximo de crescimento, observaram-se um conjunto de mudanças que melhoraram as relações dentoesqueléticas sagitais, independentemente do crescimento. No aspecto vertical, apesar das pequenas mudanças observadas houve preservação do padrão facial assim como no grupo controle. / This retrospective controlled study enrolled 94 consecutive adolescents during growth spurt with Angle class II division 1 malocclusion and mandibular retrognathism and evaluated skeletal and dental changes resulting from treatment with a Herbst appliance (Group A: steel crowns; Group B: acrylic splints) and compared them with results of a follow-up group (Group C: control) for 12 months. Lateral radiographs were obtained in the beginning of the treatment (T1) and at the end of the observation period (T2). Cephalometric variables were analyzed using parametric tests. The results of the two treatment groups were different from those found for the control group. Maxillary growth was restricted, whereas mandibular growth was greater, which resulted in a better sagittal relation. At the same time, the analysis of dentoalveolar aspects revealed that overjet was reduced due to distal tipping of maxillary incisors and buccal inclination of mandibular incisors, vertical control of maxillary molars and mesial movement of mandibular molars. The three groups had similar extrusion of mandibular molars, preservation of mandibular morphology and facial pattern. The occlusal plane rotated clockwise in the treated groups and counterclockwise in the control group. It can be concluded that the treatment of class II malocclusion in adolescents during growth spurt with two types of Herbst appliance resulted in a set of changes that improved sagittal skeletal and dental relations, regardless of growth. Vertically, facial pattern was preserved in treatment and control groups, despite some small differences.

Peroxisome proliferator-activated receptor alpha (PPARa) and delta (PPARd) belong to the nuclear receptor superfamily. PPARa is a target of lipid-lowering drugs and PPARd promotes fatty acid utilization and is a promising anti-diabetic drug target. However, evidence is growing that PPARd-agonism can stimulate fat accumulation in liver, which may aggravate the toxic situation in diabetics. The aim of the study was to characterise the hepatic transcriptional and lipid response of humanized mouse models to PPARd-agonists. In our studies of mice conditionally-expressing human PPARd (hPPARd), or the dominant-negative derivative of hPPARd (hPPARd?AF2) or wild-type animals, we demonstrated that GW501516, a potent PPARd activator, promoted up-regulation of the genes involved in lipid turnover, stimulated significant weight loss and promoted hepatic steatosis in these mouse models. There was time-dependent accumulation of hepatic triglycerides observed in wild-type and in conditionally-expressing hPPARd mice fed a diet containing PPARd synthetic ligand. This was not seen in animals conditionally-expressing hPPARd?AF2, neither in PPARa-KO or PPARd-KO animals. Concurrently, activation of PPARd in humanised animals caused significant depletion, as compared with controls, of adipose tissue deposits when fed normal or high fat diet. This effect was completely absent in PPARa-KO or PPARd-KO mice, fed diet containing GW501516. Genome-wide transcriptional profiling of GW501516 effects in the livers of these different mouse strains was performed. In PPARa-KO mice fed PPARd-agonist, some direct PPARd target genes were still up-regulated, demonstrating that they are not sufficient for the observed phenotype. In addition the blood HDL-raising effects of GW501516 were preserved in the PPARa-KO mice. This suggests a novel finding that both PPARd and PPARa receptors are essential for GW501516-driven weight loss and hepatic steatosis, with PPARa working downstream of PPARd.

The steroid receptor RNA activator gene (SRA1) generates two distinct entities. SRA RNA coactivates several NRs whereas SRA protein (SRAP) is suspected to regulate the activity of several transcription factors, including estrogen receptors (ER). Splicing of SRA intron-1 is the major event defining SRAP coding frame. Fully spliced, coding SRA and intron-1 retained, non-coding SRA coexist in breast cancer cells. The relative proportion between the two types of SRA RNA maintains a balance between two genetically linked entities, SRA and SRAP. In this study, a minigene model was used to demonstrate that the primary sequence of SRA exon-1-intron-1-exon-2 is sufficient for alternative splicing of SRA intron-1. In addition, a modified oligoribonucleotidic construct promotes SRA intron-1 retention in breast cancer cells. This oligoribonucleotide differentially alters estradiol-induced transcription of ER regulated genes. Together, results presented herein demonstrate that the SRA-SRAP balance, which can be artificially modified by targeting alternative splicing of SRA intron-1, might be a new critical target to treat breast cancer patients.

Atherosclerotic cardiovascular disease is the leading cause of death of adults in North America. Diabetes is a classical risk factor for atherosclerotic cardiovascular disease. Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of fibrinolysis. Elevated levels of PAI-1, oxidized low-density lipoprotein (oxLDL) and glycated LDL (glyLDL) were detected in patients with diabetes. Increased oxidative stress is associated with diabetic cardiovascular complications. Previous studies in our laboratory demonstrated that oxLDL or glyLDL increased the production of PAI-1 or reactive oxygen species (ROS) in vascular endothelial cells (EC). This study was undertaken to investigate transmembrane signaling mechanisms involved in oxLDL or glyLDL-induced upregulation of PAI-1 in cultured vascular EC. Further, we examined the mechanism for oxLDL or glyLDL-induced oxidative stress in EC. The results of the present studies demonstrated novel transmembrane signaling pathway for oxLDL-induced PAI-1 production in vascular EC. We demonstrated that lectin-like oxLDL receptor-1, H-Ras, a small G-protein and Raf-1/ERK-1/2 mediate oxLDL-induced PAI-1 expression in cultured EC. GlyLDL may activate EC via a distinct transmembrane signaling pathway. The results of the present study demonstrated that receptor for advanced glycation end products, NADPH oxidase and H-Ras/Raf-1 are implicated in the upregulation of heat shock factor-1 or PAI-1 in vascular EC under diabetes-associated metabolic stress. We investigated the effects of oxLDL or glyLDL on mitochondrial function in EC. Treatment with oxLDL or glyLDL significantly impaired the activities of electron transport chain (ETC) enzymes and also increased mitochondria-associated ROS in EC. The findings suggest that oxLDL or glyLDL attenuated activity of ETC and increased ROS generation in EC, which potentially contributes to oxidative stress in vasculature. In conclusion, diabetes-associated lipoproteins may upregulate stress response mediators and PAI-1 production via distinct transmembrane signaling pathways. OxLDL or glyLDL may increase ROS production via NOX activation and the impairment of mitochondrial ETC enzyme activity in EC. The understanding and identification of the regulatory mechanisms involved in diabetes-associated lipoprotein-induced signaling may help pharmacological design for the management of diabetic cardiovascular complications.

Objective : Low back pain (LBP), and in particular sacroiliac joint syndrome, is a significant health concern for both patient and their chiropractor with regards to quality of life and work related musculoskeletal disorders. Therefore, chiropractors often utilise mechanical aids to reduce the impact on the chiropractor’s health. It is, however, important to establish whether these mechanical aids are indeed clinically effective, therefore, this study evaluated the Activator Adjusting Instrument (AAI) against an AAI placebo to determine whether this adjusting instrument is an effective aid for both the chiropractor and the patient. Method : This randomised, placebo controlled clinical trial consisted of 40 patients (20 per group), screened by stringent inclusion criteria assessed through a telephonic and clinical assessment screen. Post receipt of informed consent from the patients, measurements (NRS, Revised Oswestry Disability Questionnaire, algometer) were taken at baseline, prior to consultation three and at the follow consultation. This procedure occurred with four interventions over a two week period. Results: The AAI group showed clinical significance for all clinical measures as compared to the AAI placebo group which attained clinical significance only for the Revised Oswestry Disability Questionnaire. By comparison there was only a statistically significant difference between the groups in terms of the algometer readings (p= 0.037). Conclusion : Therefore, it is evident that the AAI seems to have clinical benefit beyond a placebo. However this is not reflected in the statistical analysis. It is, therefore, suggested that this study be repeated with a larger sample size in order to verify the effect on the statistical analysis outcomes.

The steroid receptor RNA activator gene (SRA1) generates two distinct entities. SRA RNA coactivates several NRs whereas SRA protein (SRAP) is suspected to regulate the activity of several transcription factors, including estrogen receptors (ER). Splicing of SRA intron-1 is the major event defining SRAP coding frame. Fully spliced, coding SRA and intron-1 retained, non-coding SRA coexist in breast cancer cells. The relative proportion between the two types of SRA RNA maintains a balance between two genetically linked entities, SRA and SRAP. In this study, a minigene model was used to demonstrate that the primary sequence of SRA exon-1-intron-1-exon-2 is sufficient for alternative splicing of SRA intron-1. In addition, a modified oligoribonucleotidic construct promotes SRA intron-1 retention in breast cancer cells. This oligoribonucleotide differentially alters estradiol-induced transcription of ER regulated genes. Together, results presented herein demonstrate that the SRA-SRAP balance, which can be artificially modified by targeting alternative splicing of SRA intron-1, might be a new critical target to treat breast cancer patients.

Atherosclerotic cardiovascular disease is the leading cause of death of adults in North America. Diabetes is a classical risk factor for atherosclerotic cardiovascular disease. Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of fibrinolysis. Elevated levels of PAI-1, oxidized low-density lipoprotein (oxLDL) and glycated LDL (glyLDL) were detected in patients with diabetes. Increased oxidative stress is associated with diabetic cardiovascular complications. Previous studies in our laboratory demonstrated that oxLDL or glyLDL increased the production of PAI-1 or reactive oxygen species (ROS) in vascular endothelial cells (EC). This study was undertaken to investigate transmembrane signaling mechanisms involved in oxLDL or glyLDL-induced upregulation of PAI-1 in cultured vascular EC. Further, we examined the mechanism for oxLDL or glyLDL-induced oxidative stress in EC. The results of the present studies demonstrated novel transmembrane signaling pathway for oxLDL-induced PAI-1 production in vascular EC. We demonstrated that lectin-like oxLDL receptor-1, H-Ras, a small G-protein and Raf-1/ERK-1/2 mediate oxLDL-induced PAI-1 expression in cultured EC. GlyLDL may activate EC via a distinct transmembrane signaling pathway. The results of the present study demonstrated that receptor for advanced glycation end products, NADPH oxidase and H-Ras/Raf-1 are implicated in the upregulation of heat shock factor-1 or PAI-1 in vascular EC under diabetes-associated metabolic stress. We investigated the effects of oxLDL or glyLDL on mitochondrial function in EC. Treatment with oxLDL or glyLDL significantly impaired the activities of electron transport chain (ETC) enzymes and also increased mitochondria-associated ROS in EC. The findings suggest that oxLDL or glyLDL attenuated activity of ETC and increased ROS generation in EC, which potentially contributes to oxidative stress in vasculature. In conclusion, diabetes-associated lipoproteins may upregulate stress response mediators and PAI-1 production via distinct transmembrane signaling pathways. OxLDL or glyLDL may increase ROS production via NOX activation and the impairment of mitochondrial ETC enzyme activity in EC. The understanding and identification of the regulatory mechanisms involved in diabetes-associated lipoprotein-induced signaling may help pharmacological design for the management of diabetic cardiovascular complications.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [137]-148).