Made available in DSpace on 2015-07-22T20:50:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-05-25. Added 1 bitstream(s) on 2015-08-11T03:26:12Z : No. of bitstreams: 1 Publico-12837a.pdf: 1258726 bytes, checksum: a7e0f280ec3b88f2f775c2b325704cd7 (MD5). Added 1 bitstream(s) on 2015-08-11T03:26:12Z : No. of bitstreams: 2 Publico-12837a.pdf: 1258726 bytes, checksum: a7e0f280ec3b88f2f775c2b325704cd7 (MD5) Publico-12837b.pdf: 2043061 bytes, checksum: 4edc37b57c73112ae7353868a78233ca (MD5). Added 1 bitstream(s) on 2015-08-11T03:26:12Z : No. of bitstreams: 3 Publico-12837a.pdf: 1258726 bytes, checksum: a7e0f280ec3b88f2f775c2b325704cd7 (MD5) Publico-12837b.pdf: 2043061 bytes, checksum: 4edc37b57c73112ae7353868a78233ca (MD5) Publico-12837c.pdf: 1773766 bytes, checksum: 56addbd5601cb4e7336afb640ed56fe8 (MD5) / Objetivo: O prolapso de órgãos pélvicos (POP) resulta da falha na sustentação do assoalho pélvico, e anormalidades do tecido conjuntivo podem estar envolvidas na etiologia e/ou na progressão da disfunção. Analisar-se-á a expressão diferencial de genes e de proteínas que participam da biossíntese do colágeno e da elastina: lisil oxidases (LOXs), fibulina-5, fibrilinas-1 e -2 e pró-colágeno C proteinase (PCP/BMP1), no tecido vaginal de mulheres sem e com POP acentuado consoante seu estado hormonal. Casuística e Métodos: Durante a histerectomia total, biópsias de parede vaginal anterior foram obtidas de mulheres caucasianas na pré-menopausa (fase proliferativa do ciclo menstrual) e na pós-menopausa com POP acentuado (POPQ estadio III e IV), e de controles assintomáticas (POPQ 0). RNAm e proteínas totais foram extraídos usando Trizol e RIPA Buffer, e os genes e proteínas de interesse quantificados por RT-PCR em tempo real e Imunobloting, respectivamente. As seguintes análises comparativas foram realizadas: (1) expressão dos genes e das proteínas da família LOX (LOX e LOXL1-4), fibulina-5 e fibrilinas- 1 e -2 em pacientes na pré-menopausa com e sem POP; (2) expressão do gene e da proteína PCP/BMP1 em pacientes na pré- e pós-menopausa com POP, e respectivos controles. Os testes de Wilcoxon signed-rank e Fisher foram usados para as análises estatísticas (p<0.05). Resultados: Obtivemos amostras de 15 pacientes e 11 controles na pré-menopausa para o estudo (1), e 39 pacientes na pré-menopausa (POP=23 e Controle=16) e 18 na pósmenopausa (POP=13 e Controle=5) para o estudo (2). A partir das análises, observamos (1) diminuição significativa na expressão dos genes LOX, LOXL1 e LOXL3, bem como nas proteínas LOX e LOXL3 no tecido vaginal de pacientes POP na pré-menopausa comparadas com mulheres assintomáticas (p<0.05); (2) hipoexpressão do gene PCP/BMP1 nos tecidos vaginais de mulheres com POP acentuado comparadas com controles, tanto na prémenopausa como na pós-menopausa (ambos p=0.01); redução significativa das isoformas 130kDa, 92,5kDa e 82,5kDa da PCP/BMP1 no tecido vaginal de pacientes na pósmenopausa (p=0.01), bem como hiperexpressão da isoforma 130kDa nas mulheres com POP acentuado na pré-menopausa (p=0.009), comparadas com as respectivas controles. Conclusão: As expressões das enzimas LOXs e pró-colágeno C proteinase estão alteradas no tecido vaginal de mulheres com POP, e são moduladas pelo estado hormonal. A alteração na regulação destas enzimas, envolvidas na biossíntese da matriz extracelular, pode contribuir para deficiente síntese do tecido conjuntivo e do suporte vaginal, e estar envolvida no desenvolvimento do POP. / Objective: Pelvic organ prolapse (POP) results from the failure of pelvic floor support, and connective tissue abnormalities may be involved in the etiology and/or progression of the dysfunction. We aimed to analyze the differential expression of genes and proteins related to the collagen and elastin biogenesis: lysyl oxidases (LOXs), fibulin-5, fibrillin -1 and -2, and procollagen C proteinase (PCP/BMP1) in vaginal tissue of women without and with advanced POP controlled by hormonal status. Materials and Methods: During total hysterectomy, anterior vaginal wall biopsies were obtained from Caucasian premenopausal women (proliferative phase of menstrual cycle) and postmenopausal women with severe POP (POPQ stage III and IV) and asymptomatic controls (POPQ 0). Total mRNA and protein were extracted using Trizol and RIPA buffer, and the genes and proteins of interest were quantified by real-time RT-PCR and Immunoblotting, respectively. The following analysis were performed: (1) expression of LOX family genes and proteins (LOX and LOXL1-4), fibulin-5, fibrillin-1 and -2 in premenopausal women with and without POP; (2) PCP/BMP1 gene and protein expression in vaginal tissue of pre- and postmenopausal POP women, and respective controls. Wilcoxon signed-rank and Fisher tests were used for statistical analysis (p<0.05). Results: Samples from 15 premenopausal patients and 11 controls were obtained for study (1); 39 premenopausal (POP=23 and Control=16) and 18 postmenopausal women samples (POP=13 and Control=5) for study (2). We observed: (1) significant decrease in expression of LOX, LOXL1 and LOXL3 genes, as well as LOX and LOXL3 proteins in vaginal tissue of premenopausal POP patients compared with asymptomatic women (p<0.05); (2) PCP/BMP1 gene downregulation in the vagina of women with severe POP compared with controls, in both premenopausal and postmenopausal phase (both p=0.01); significant reduction of 130 kDa, 92.5 kDa and 82.5 kDa PCP/BMP1 isoforms in vaginal tissue of postmenopausal patients (p=0.01), and 130 kDa isoform upregulation in premenopausal women with severe POP (p=0.009), compared with their respective controls. Conclusion: The expression of LOXs enzymes and PCP/BMP1 are altered in vaginal tissue of women with severe POP, and are modulated by hormonal status. Dysregulation of these enzymes involved in the extracellular matrix biogenesis may contribute to impaired tissue and vaginal support, and may be involved in POP development. / TEDE / BV UNIFESP: Teses e dissertações

<p> </p> <p>Learning about ‘behavior’ has always been at the heart of my research endeavors. While my undergraduate work in evolution and ecology exposed me to the science behind why a behavior exists, in my graduate work, I intended to explore how to use something’s behavior to widen its applicability. In this thesis, <em>I will present three works that utilize some of the fundamental</em></p> <p><em>behaviors (i.e., properties) of elastin-like polypeptides (ELP) to improve existing protein purification methods or explore their applicability in bladder cancer imaging and immunotherapy. </em></p> <p>Bladder cancer has high recurrence rates (60-70 % annually) that necessitate multiple follow-up therapies making it one of the costliest cancers per patient. In this work, we have attempted to address two leading causes of the recurrence. First is a low sensitivity (62-84 %) and variable specificity (43-95 %) of white light cystoscopy used to diagnose and remove tumors. We aimed to address the heart of this problem, i.e., the non-specific mode of detection using white light. Only the trained eyes can discern abnormal from normal-appearing tissues even then, leaving up to 45% of tumors unresected to colonize and spread. <em>We developed and characterized near infrared dye-peptide-ligand conjugates (NIR-ELP-ligand) that undergo receptor-mediated binding and internalization to human bladder cancer cells in vitro and tissues ex vivo.</em> By using a molecular target-based probe in combination with NIR imaging, we can aid in improving the detection limit via selective binding to the tumor and reduction in background autofluorescence.</p> <p>Bacillus-Calmette Guérin (BCG) instillation in the bladder is the gold-standard</p> <p>immunotherapy used after surgical removal of bladder tumors. This was approved as a response to the inefficiency of surgery alone in improving cancer status. It has succeeded by reducing the recurrence rate to 30-50 %. But it comes with the complications of putting a live mycobacterium</p> <p>in the human body and giving a patient a urinary tract infection right after surgical tumor resection. <em>Thus, we aimed to deliver nucleic acid as immunotherapeutic cargo in a selective manner to elicit robust anti-tumor immune responses while minimizing the side effects due to its carrier.</em> Towards</p> <p>this goal, we have developed a highly modular and adaptable ELP-ligand fusion protein-based nucleic acid delivery carrier targeted toward bladder cancer. Before developing targeted peptide-based cancer imaging and nucleic acid delivery modalities, we addressed the Achilles heel of peptide-based approaches. The peptide and protein industry suffers</p> <p>through complex, time-consuming, inconsistent, and low-yielding purification methods. <em>We have developed a scalable, facile, and reproducible protein purification method that delivers ELP and ELP fusion proteins free of host cell proteins and nucleic acids and has low lipopolysaccharide</em></p> <p><em>content in just 3 h starting from a bacterial pellet. </em>Thus, for a coherent narrative, the thesis is structured as follows:</p> <p>1. Introduction</p> <p>2. ELP as a protein purification tag: Development of a rapid purification method for ELPs and ELP fusion proteins.</p> <p>3. ELP as a cancer imaging agent: Development of NIR-ELP-Ligand imaging probe targeting bladder cancer.</p> <p>4. ELP as a drug delivery agent: Utilizing ELP-ligand fusion protein in the formulation of targeted nucleic acid delivery carrier to bladder cancer.</p>

Indiana University-Purdue University Indianapolis (IUPUI) / The passive properties of the venous wall are important in the development of venous pathology. Increase in venous pressure due to retrograde flow (reflux) and obstruction of venous flow by intrinsic and extrinsic means are the two possible mechanisms for venous hypertension. Reflux is the prevailing theory in the etiology of venous insufficiency. The objective of this thesis is to quantify the passive biomechanical response and structural remodeling of veins subjected to chronic venous reflux and hypertension. To investigate the effects of venous reflux on venous mechanics, the tricuspid valve was injured chronically in canines by disrupting the chordae tendineae. The conventional inflation-extension protocol in conjunction with intravascular ultrasound (IVUS) was utilized to investigate the passive biomechanical response of both control common iliac veins (from 9 dogs) and common iliac veins subjected to chronic venous reflux and hypertension (from 9 dogs). The change in thickness and constituent composition as a result of chronic venous reflux and hypertension was quantified using multiphoton microscopy (MPM) and histological evaluation. Biomechanical results indicate that the veins stiffened and became less compliant when exposed to eight weeks of chronic venous reflux and hypertension. The mechanical stiffening was found to be a result of a significant increase in wall thickness (p < 0.05) and a significant increase in the collagen to elastin ratio (p < 0.05). After eight weeks of chronic reflux, the circumferential Cauchy stress significantly reduced (p < 0.05) due to wall thickening, but was not restored to control levels. This provided a useful model for development and further analysis of chronic venous insufficiency and assessment of possible intervention strategies.