Introduction. Oncogenic human papillomaviruses (HPVs) are the central causal agent of cervical cancer. Vaccines were developed, and are currently being tested in phase II/III trials. The host immune response is type-specific, and likely not all oncogenic types will be targeted by vaccine formulations. A theoretical concern is that infection with less prevalent oncogenic HPV types may replace the disease prevented by a vaccine against the most common oncogenic HPVs. This underscores the importance of having a better understanding of the epidemiology of infections with multiple HPV types, in terms of their dynamics and risk factors. The objectives of this project were to: (1) describe the occurrence of HPV coinfections; (2) determine whether prior HPV infections are associated with acquisition and clearance of other HPV types, and; (3) identify predictors of HPV coinfection. / Methodology. In a prospective cohort of 2075 Brazilian women, cervical specimens were collected for cytology and HPV DNA detection. Information on potential risk factors was obtained by interview at baseline, and at return visits. Follow-up visits were scheduled every 4 months in the first year and every 6 months thereafter. / Results. The prevalence of HPV coinfection was 7.6% among all women, and 21% among HPV-positive women. HPV coinfection was less common among cytologically normal women, and peaked among women with lower grade cytological abnormalities. Both prevalence and incidence of HPV coinfection decreased markedly with increasing age. HPV infection at enrolment was associated with a higher risk of infection with any other HPV type. The adjusted hazard ratios and 95% confidence intervals (95% CI) of acquisition of any HPV type were: 2.2 (95% CI: 1.5--3.2) for oncogenic types excluding HPV 16 and 18 detected at enrolment, and 2.6 (95% CI: 1.7--4.2) for HPV 16 or 18 detected at enrolment. Predictors of HPV coinfection included younger age, higher number of sexual partners, a history of condyloma, and younger age at first sexual intercourse. / Conclusion. HPV coinfections were relatively common, especially among younger women. Prior HPV infections increased the risk of subsequent infections with other types. These results may have implications for vaccine development and public health decisions about vaccination programs.

Tissue eosinophilia is a prominent feature of allergic inflammatory diseases that may be in part mediated through the regulation of eosinophil survival at inflammatory site. T helper 2 type cytokines, such as interleukin (IL)-5, Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), are the important mediators of allergic inflammation leading to prolonged survival of eosinophils. Due to the proposed central role of eosinophils in asthma and other allergic airway diseases, there is considerable interest, to determine the mechanisms that regulate eosinophil functions and accumulation. IL-12 and IL-18 attract considerable attention for their immunomodulatory roles on T helper cell subsets, and their potential to affect on eosinophil function. / The general aim of this study was to determine the effect of IL-12 and IL-18 on eosinophil functions. Results from this thesis demonstrate that eosinophils express functional receptors for IL-12 and IL-18. Acting alone or in synergy, IL-12 and IL-18 induced eosinophil apoptosis, in vitro. The apoptotic effect of IL-12 was reversed by IL-5, suggesting that IL-5 and IL-12 have counter-regulatory effects on eosinophil survival. Our regulation studies demonstrated that Phorbol-Myristate-Acetate (PMA) induced optimal expression of IL-18 and IL-12 receptors by eosinophils. IL-18 receptor expression by eosinophils was markedly increased following stimulation with interferon (IFN)-gamma, Tumor Necrosis Factor (TNF), or IL-12. Up-regulation of IL-18 receptor upon IL-12 stimulation was particularly important, which may explain IL-12 and IL-18 synergy on eosinophil apoptosis. We also investigated IL-12 and IL-18 expression by eosinophils. Eosinophils did not express IL-18. However, there was constitutive IL-12 expression by eosinophils. Release of IL-12 was also confirmed in eosinophil supernatants, which suggested an autocrine mechanism of IL-12 action on eosinophils. / Extending our understanding of the role of IL-12 and IL-18 in allergic inflammation, we have defined a novel pathway by which IL-12 and/or IL-18 may actually regulate eosinophils functions, and exert an inhibitory effect on eosinophil survival. Our findings provide critical new insights into mechanisms regulating eosinophil survival. To gain an even more detailed understanding of regulatory signals mediating eosinophil survival, we need to define the mechanisms involved in IL-12 and IL-18 signalling. We have just begun to identify the molecules involved, and these include IL-12 and IL-18 receptors. Activation via IL-12 and/or IL-18 receptor-mediated mechanisms may provide a novel strategy for reducing the numbers or inhibiting the function of these cells in allergic diseases or other diseases characterized by increased numbers of, or mediators from, eosinophils.

<p> Breast cancer is a disease that continues to affect people of all ages, but especially older adults. The number of people impacted by this disease continues to grow and there is a rapid increase in women who are newly diagnosed with breast cancer. The primary risk factors for breast cancer are age and gender. The purpose of this project was to develop a breast cancer comprehensive resource packet for adults, age 65 years and older, who have been newly diagnosed with breast cancer. This packet incorporates patient education, possible coping strategies about living with breast cancer, useful tips for healthy living, and information on how to transition from living with breast cancer to becoming a cancer survivor. This packet provides a resource tool for breast cancer patients including beneficial tips directly from breast cancer survivors that may create an easier transition of living with breast cancer for those newly diagnosed.</p>

<p> In recent years, large-scale 'omic' studies have helped to understand disease pathogenesis; however, these studies were done largely at the 'transcriptome' level. Understanding the biological processes at the protein level is also equally important. In general, mass spectrometry (MS)-based quantitative proteomic strategies are used to study protein alterations in different biological states. Among these strategies, stable isotope labeling by amino acids in cell culture (SILAC) is most widely used for comparative proteomics. In SILAC, proteins in cell populations are metabolically encoded with 'heavy' isotopes of lysine and arginine and are used as internal standards for relative quantification of differentially altered proteins. In this dissertation, the use of SILAC was extended to study <i>in vivo</i> proteomic modulations in mice. A stable isotope (¹³C-lysine)-labeled 'SILAC mouse' was generated in order to quantify and compare protein alterations between normal and pathological conditions. </p><p> The primary objective of this dissertation was to identify novel and disease specific pathogenic mechanisms implicated in duchenne muscular dystrophy (DMD: a genetic muscle disease) and myositis (an autoimmune muscle disease) <i> in vivo</i> considering the entire complexity of the tissue. More importantly, the goal is to study global protein alterations in an unbiased manner in the affected skeletal muscle tissue. However, it is impractical to study in-depth disease pathology at tissue level using human muscle biopsy samples due to heterogeneity, complexity, and limited availability of muscle tissues at different stages in the disease process. To overcome these issues, mouse models that closely mimic the human disease phenotype i.e. a dystrophin deficient 'mdx' for DMD and a conditional major histocompatibility complex (MHC) class-I transgenic mice for myositis, were used. It was hypothesized that identification of precise proteomic alterations in the affected muscle, using mass spectrometry based untargeted-stable isotope labeled-proteomic strategy, would help to discover disease specific pathogenic mechanisms. </p><p> Firstly, the untargeted-labeled-proteomics approach using <i>in vivo</i> SILAC mouse proved to be a robust technique to uncover previously unidentified pathological pathways in mouse models of human skeletal muscle diseases. With respect to DMD, SILAC mouse proteomic profiling identified 73 significantly altered proteins in the early stage of the disease in mdx muscle compared to healthy muscle. Bioinformatics analyses of the altered proteins identified that integrin-linked kinase (ILK), actin cytoskeleton signaling, and mitochondrial energy metabolic pathways are significantly altered very early in the disease process in dystrophin deficient muscle. Disease specific protein modulations were further validated using an independent set of samples, SILAC spike-in strategy and specific antibody based biochemical assays. Moreover, the potential candidates of ILK pathway such as vimentin, desmin and ILK were confirmed to be significantly up-regulated in dystrophin-deficient human DMD samples suggesting the importance of these findings in relation to human disease pathology. </p><p> A novel association between the reduced mitochondrial activity and impaired sarcolemmal healing was identified in dystrophin deficient muscle. Live imaging of isolated single muscle fibers determined that reduced mitochondrial translocation to the site of injury negates the membrane repair processes even in the presence of compensatory up-regulation of repair proteins (dysferlin and annexin). Thus, current studies provided the first comprehensive understanding of the dystrophic muscle pathologies at the proteomic level. </p><p> In auto-immune myositis, SILAC mouse proteomic profiling identified significant alteration in the levels of 179 proteins. These proteins belong to the endoplasmic reticulum (ER) stress response, ubiquitin proteasome pathway (UPP), ER associated degradation (ERAD), oxidative phosphorylation, glycolysis, cytoskeleton, and muscle contractile apparatus categories. A significant increase in the ubiquitination of muscle proteins as well as a specific increase in ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) was observed in myositis, but not in normal or other dystrophic muscles. Furthermore, inhibition of UPP using a specific proteasome inhibitor, bortezomib, significantly improved muscle function and also significantly decreased TNF-&alpha; (pro-inflammatory cytokine) expression in the skeletal muscle of myositis mice. Treatment with bortezomib, decreased GRP-78 (ER stress sensor) levels and also enhanced muscle regeneration. Thus, it can be concluded that UPP and ERAD activation in myositis muscle contribute to muscle degeneration. UCHL-1 is a potential biomarker for myositis disease progression and inhibition of UPP offers a potential therapeutic strategy for myositis. </p><p> These studies not only provided information on the implicated pathways both in 'dystrophin-deficient' and 'myositis' muscle but also identified potential therapeutic targets. Nevertheless, future experiments will help to associate pathways identified using this proteomic strategy with gene expression profiling to comprehensively understand disease specific as well as general pathogenic pathways. These studies will pave the way to enhance development of improved therapies for these rare muscle diseases.</p>

<p> The purpose of this research was to better understand the effects of abdominopelvic irradiation in dogs. Three studies were performed to that end. The first was a clinical investigation, performed by retrospective data analysis, of safety and activity of intensity-modulated and image-guided radiation therapy (IM-IGRT) for treatment of genitourinary (GU) carcinomas in dogs. The second was a prospective study which developed dogs as a novel animal model for studying radiation-induced erectile dysfunction (RI-ED). The third study reviewed pathological changes associated with unexpected colorectal toxicities encountered in the development of the RI-ED model. </p><p> As mentioned, the objective of the first study was to assess local tumor control, overall survival and toxicosis following IM/IGRT for treatment of genitourinary carcinomas (CGUC) in dogs. Medical records of patients for which there was intent to treat with a course of definitive-intent IM/IGRT for CGUC were reviewed. Primary tumors were located in the prostate, urinary bladder or urethra of 21 dogs. The total radiation dose ranged from 54-58 Gy, delivered in 20 daily fractions. Grade 1 and 2 acute gastrointestinal toxicoses developed in 33% and 5% of dogs, respectively. Grade 1 and 2 acute genitourinary, and grade 1 acute integumentary toxicoses were documented in 5%, 5% and 20% of dogs, respectively. Four dogs experienced late grade 3 gastrointestinal or genitourinary toxicosis. The subjective response rate was 60%. The median event-free survival was 317 days; the overall median survival time was 654 days. Neither local tumor control nor overall survival were statistically dependent upon location of the primary tumor. In conclusion, IM/IGRT is generally well-tolerated and provides an effective option for locoregional control of CGUC. And, as compared with previous reports in the veterinary literature, inclusion of IM/IGRT in multimodal treatment protocols for CGUC can result in superior survival times. </p><p> The etiopathology of RI-ED is poorly understood, though this is a common complication of men treated for prostate cancer. Purported mechanisms include cavernosal, arteriogenic and neurogenic injuries. Radiation dose to the posterolateral prostatic neurovascular bundles (NVB) and penile bulb (PB) have been associated with RI-ED. Herein, a canine model is described that has been developed to study the pathogenesis of RI-ED. Stereotactic body radiotherapy (SBRT) was used to irradiate the prostate gland, NVB and/or PB of purpose-bred, intact male dogs. Manual evaluation was used to characterize erectile function and quality. Ultrasound of the internal pudendal arteries, prostate and penis, dynamic contrast-enhanced MRI of the NVB and prostate, and electrophysiology of sensory and motor nerves as well as muscle were performed before and after irradiation. Gross necropsy and histopathology was also performed. Erectile dysfunction was a repeatable finding in subjects for whom the prostate, neurovascular bundles and penile bulb were irradiated with 50 Gy, as documented via subjective and objective manual evaluations following SBRT. Irradiated dogs were found to have a decreased extravascular, extracellular volume in the glans penis, longer systolic rise times in the pudendal artery following papaverine injection, abnormal spontaneous EMG activity in the bulbocavernosus muscle, and slower pudendal nerve motor conduction velocities. Radiation dose-dependent changes in internal pudendal arterial function and dysfunction of the pudendal nerve due to axonal loss may contribute to RI-ED. Measurable endpoints have been developed for evaluation of RI-ED in dogs, that should be used in future studies to refine this novel animal model and perform additional studies aimed at further elucidating the etiopathologic processes underlying RI-ED. </p><p> The objective of the final study was to describe the dose-response relationship and time-dependency of late radiation-induced colorectal complications endured by dogs in the RI-ED study. The prostates of nineteen intact male mixed breed hounds were irradiated with one of four different dose/fractionation schemes. Subjects were monitored for signs of colorectal toxicosis for up to one year following irradiation. Gross necropsy and histopathology were performed upon euthanasia. All toxicoses were graded according to the RTOG criteria for gastrointestinal toxicity. The frequency and severity of colorectal ulceration were higher in dogs treated 5 fractions of 10 Gy delivered on consecutive days, as compared with those treated on an every other day schedule. The mechanism for this time-dependency is unclear, but likely related to completeness of epithelial regeneration. Vascular sclerosis and serosal thickening occurred in all treatment groups, in a dose-responsive fashion.</p>

Enhanced, abnormal adherence of sickle erythrocytes to the endothelium lining blood vessels is hypothesized to contribute to vasoocclusion in sickle cell patients, but the specific molecular mechanisms by which these erythrocyte-endothelial interactions occur have not yet been fully elucidated. Alteration of endothelial function, or frank endothelial damage, may also be involved in the pathogenesis of vascular occlusion. In this thesis, these two different aspects of vascular occlusion, namely adhesion and damage, were studied. A parallel plate flow chamber and computerized phase contrast microscopy coupled to digital image processing were used to visualize and analyze the adhesion of sickle red cells to 1, 4 and 24 hour IL-1$\beta$ stimulated endothelial cells. In addition, damage to activated endothelial cells due to short and long term perfusion of sickle erythrocytes was determined. Adhesion studies. Pretreatment of monolayers with 50pg/ml of IL-1$\beta$ for 1, 4 and 24 hours caused approximately 16-fold increases in adhesion of sickle cells to activated endothelium at all time points. RGD peptides, enzymes and monoclonal antibodies were used to determine the pathways involved in these sickle erythrocyte-endothelial interactions. Results indicate that there were different endothelial adhesion receptors involved at the different time points: an $\alpha$v$\beta$3 pathway after 1 hour of IL-1$\beta$ stimulation, another pathway, partially mediated by E-selectin, after 4 hours of IL-1$\beta$ stimulation, and a VCAM-1 pathway after prolonged exposure ($>$9 hours) of endothelial cells to IL-1$\beta$. The $\alpha$4$\beta$1 integrin and SLe$\sp{\rm x}$/SLe$\sp{\rm a}$ carbohydrates were established as the sickle cell determinants in these interactions. Damage studies. Perfusion of sickle cells over 4 and 24 hour IL-1$\beta$ stimulated endothelial monolayers for short periods of time resulted in damage to the endothelium. About 6-8% damage was observed on 4 and 24 hour IL-1$\beta$ stimulated endothelial cells due to the perfusion of sickle cells. In addition, there was a definite correlation between damage and adhesion on the 24 hour activated cells, but this correlation was much weaker on the 4 hour stimulated cells. Perfusion of sickle cells over 4 and 24 hour IL-1$\beta$ activated endothelial monolayers for long periods of time resulted in hemolysis and indicated that modifications to the apparatus were required in order to obtain meaningful results.

Pathologic arterial thrombosis is the leading cause of death in many countries. Current therapies provide only moderate protection from heart attack, stroke, and other clinical manifestations of thrombosis. To develop more potent therapeutics, molecular mechanisms that mediate platelet thrombosis are being researched. Thrombosis depends on complex interactions between the vascular wall, circulating components, and blood flow conditions. Under arterial shear conditions, platelet thrombosis requires von Willebrand factor (vWf) binding to platelet glycoprotein (GP) Ib-IX-V and GP IIb-IIIa complexes. In this work, dynamic experimental models of the vascular system are used in three studies investigating the role of GP Ib-IX-V and GP IIb-IIIa in arterial thrombosis. First, experimental results indicate two compounds that inhibit GP Ib-vWf binding have strong potential as anti-thrombotic agents. Findings with these compounds also suggest that soluble and immobilized vWf have different structural conformations. Second, a novel experimental model was developed to examine vWf-GP Ib-IX-V interactions that mediate platelet adhesion to exposed subendothelium. The system consists of vWf-coated glass slides, mammalian cells expressing full or partial GP Ib-IX-V complexes, and a parallel plate flow chamber with phase contrast video microscopy and digital image processing. Results with this system suggest the novel finding that optimal binding between immobilized vWf and GP Ib$\alpha$ requires the presence of GP V within the GP Ib-IX-V complex. The results also demonstrate that the role of immobilized vWf with respect to GP Ib-IX-V binding does not change after exposure to high shear. Third, platelet and leukocyte function were evaluated using whole blood samples from patients undergoing percutaneous transluminal coronary angioplasty (PTCA) and receiving one of three standard therapies: abciximab, a GP IIb-IIIa inhibitor, ticlopidine, an orally active anti-platelet agent, or both treatments. Combined abciximab/ticlopidine therapy produces the most prolonged inhibition of in vitro mural thrombosis and the most consistent reduction in shear-induced platelet aggregation. At 2 hours post-PTCA, abciximab therapy, with or without ticlopidine, promotes leukocyte rolling on the collagen/vWf-platelet surface. However, abciximab-enhanced leukocyte rolling is almost completely inhibited by monoclonal antibodies to P-selectin or P-selectin glycoprotein ligand-1. Basic mechanism studies like these increase understanding of thrombosis pathophysiology and accelerate anti-thrombotic agent development.

The prognosis of breast cancer is characterized by a high degree of variability, which is partially accounted for, by a set of clinical, pathological and biological parameters. The aim of my study was to test whether met, a proto-oncogene encoding a protein related to normal histological differentiation, undergoes structural changes in breast cancer. I have extracted tumor DNA from 83 breast cancer patients and performed Southern blot hybridization with the MetH probe. This study demonstrates that somatic alterations of met occur in 22% of informative women with breast cancer. No correlation was found between loss of heterozygosity (LOH) for met and conventional horizontal prognosis factors or status for c-erbB-2 proto-oncosene expression. Estrogen receptor (ER) showed correlation with progesterone receptor (PR), and S phase correlated with ploidy and size of the tumor. It remains to be confirmed whether overall survival (OS) and disease free survival (DFS) correlate with genetic alteration of met.

The E1A gene of human adenoviruses induces DNA synthesis but is only able to transform primary rodent cells abortively. Stable transformation is induced by E1A in combination with the E1B gene or another oncogene such as activated Ras. E1B encodes two major proteins, 55K and 19K that are each able to transform cells in cooperation with E1A but with greater efficiency when both are present. The E1B-19K protein is believed to function in a manner homologous to the Bcl-2 proto-oncogene by preventing apoptosis which is induced by the E1A proteins. The E1B-55K protein binds to the p53 tumour suppressor and is believed to function by abrogating p53 activity. Apoptosis induced by E1A is believed to be caused by the increase in the levels of p53 and subsequent induction of p53-dependent programmed cell death. / In the present studies, evidence is shown indicating that E1A-induced apoptosis during adenovirus type 5 infection can occur independently of p53 as well. The E1A gene encodes two major proteins, a 289-residue (289R) and 243-residue (243R) species which differ only by a unique 46 amino acid region in the larger polypeptide. This region corresponds to the CR3 domain of E1A which is a potent transactivation domain known to activate expression of many cellular and early viral genes. E1A-induced p53-independent apoptosis was found to be dependent on the expression of the 289R protein and the subsequent transactivation of one or more early viral genes. This p53-independent apoptosis can be blocked by the E1B-19K protein but not by 55K. As shown in previous studies, the 243R E1A protein was demonstrated to induce apoptosis that is dependent on p53. Direct evidence is provided here that E1B-55K can block p53-dependent apoptosis. / The E1B-55K protein is believed to function by binding p53 tethering a transcriptional repression domain to p53. Mutants of 55K which cannot bind p53 are also deficient for transformation. In these studies, three phosphorylation sites have been mapped to a highly conserved region in the carboxy-terminus of 55K at positions Ser-490, Ser-491 and Thr-495. A mutant (pm 490/1/5A) in which these phosphorylation sites were converted to alanines resulted in 55K becoming completely defective for cooperation with E1A to transform primary cells. Interestingly, the pm490/1/5A mutant was still able to bind to p53 as efficiently as the wild-type protein. Previous work has shown the 55K protein possesses an intrinsic transcriptional repression activity when localized to promoters, such as by fusion with the GAL4-DNA-binding domain, even in the absence of p53. Such repression activity was found to be totally absent in the carboxy-terminal mutants of 55K. In addition, the phosphorylation mutants of 55K were completely deficient for blocking p53-dependent apoptosis. Thus phosphorylation of the 55K protein appears to regulate transformation, transcriptional repression, and apoptosis by the p53 tumour suppressor.

Little is known about the pathway leading from a stressful stimulus such as hyperthermia or cadmium to a malformation, and the possibility of the heat shock response being linked to this phenomenon prompted the studies in this thesis. In order to determine the relationship between the ability of heat or cadmium to induce a heat shock response and the ability of these stressors to induce abnormal embryogenesis, rat embryos were cultured during organogenesis and the steady-state mRNAs of two heat shock proteins were used as indicators of the stress response in these embryos. The extent of mRNA accumulation for each heat shock protein varied depending on the tissue examined. When embryos were cultured in the presence of cadmium chloride, hsp27 and hsp70 mRNAs took much longer to accumulate compared to those observed following heat shock. Thus, the time course of heat shock protein mRNA accumulation was quite different in embryos exposed to cadmium from those exposed to hyperthermia. In addition, the concentrations of hsp27 and hsp70 mRNA appears to be differentially regulated in both embryo and yolk sac tissues for embryos treated with heat or cadmium. In conclusion, mammalian embryos are able to mount a heat shock response through the accumulation of hsp27 and hsp70 mRNAs in response to hyperthermia or cadmium treatments. The extent of this response appears to correlate with the teratogenicity effects of hyperthermia but not with those of cadmium.