Expression of Imprinted Genes Is Aberrant in Deceased Newborn Cloned Calves and Relatively Normal in Surviving Adult Clones

Cattle are the species used most frequently for the development of assisted reproductive technologies, such as nuclear transfer. Cattle cloning can be performed by a large number of laboratories around the world, and the efficiency of nuclear transfer in cattle is the highest among all species in which successful cloning has been achieved. However, an understanding of the expression of imprinted genes in this important species is lacking. In the present study, real time reverse transcription polymerase chain reaction (RT-PCR) was utilized to quantify the expression of the bovine Igf2, Igf2r, and H19 genes in eight major organs (brain, bladder, heart, kidney, liver, lung, spleen, and thymus) of somatic cell cloned calves that died shortly after birth, in three tissues (skin, muscle, and liver) of healthy clones that survived to adulthood, and in corresponding tissues of control animals from natural reproduction. We found that, deceased bovine cloned calves exhibited abnormal expression of all three genes studied in various organs. Large variations in the expression levels of imprinted genes were also seen among these clones, which were produced from the same genetic donor. In surviving adult clones, however, the expression of these imprinted genes was largely normal, except for the expression of the Igf2 gene in muscle, which was highly variable. Our data showed disruptions of expression of imprinted genes in bovine clones, which is possibly due to incomplete reprogramming of donor cell nuclei during nuclear transfer, and these abnormalities may be associated with the high neonatal mortality in cloned animals; clones that survived to adulthood, however, are not only physically healthy but also relatively normal at the molecular level of those three imprinted genes.

Tumor heterogeneity dictates sensitivity to gefitinib (Iressa(TM)/ZD1839) in solid tumor models

The epidermal growth factor receptor (EGFR) and its ligands are overexpressed in many human tumors, including bladder and pancreas, correlating with a more aggressive tumor phenotype and poor patient prognosis. We initiated the present study to characterize the heterogeneity of gefitinib responsiveness in a panel of human bladder and pancreatic cancer cell lines in order to identify the biological characteristics of EGFR-dependent proliferation that could be used to prospectively identify drug-sensitive tumors. A second objective was to elucidate how to best exploit these results by utilizing gefitinib in combination therapy. To these ends, we examined the effects of the EGFR antagonist gefitinib on proliferation and apoptosis in a panel of 18 human bladder cancer cell lines and 9 human pancreatic cancer cell lines. Our data confirmed the existence of marked heterogeneity in Iressa responsiveness with less than half of the cell lines displaying significant growth inhibition by clinically relevant concentrations of the drug. Gefitinib responsiveness was found to be p27 kip1 dependent as DNA synthesis was restored following exposure to p27siRNA. Unfortunately, Iressa responsiveness was not closely linked to surface EGFR or TGF-α expression in the bladder cancer cells, however, cellular TGF-α expression correlated directly with Iressa sensitivity in the pancreatic cancer cell lines. These findings provide the potential for prospectively identifying patients with drug-sensitive tumors. ^ Further studies aimed at exploiting gefitinib-mediated cell cycle effects led us to investigate if gefitinib-mediated TRAIL sensitization correlated with increased p27kip1 accumulation. We observed that increased TRAIL sensitivity following gefitinib exposure was not dependent on p27 kip1 expression. Additional studies initiated to examine the role(s) of Akt and Erk signaling demonstrated that exposure to PI3K or MEK inhibitors significantly enhanced TRAIL-induced apoptosis at concentrations that block target phosphorylation. Furthermore, combinations of TRAIL and the PI3K or MEK inhibitors increased procaspase-8 processing above levels observed with TRAIL alone, indicating that the effects were exerted at the level of caspase-8 activation, considered the earliest step in the TRAIL pathway. ^

Sex disparities among cases of hepatocellular carcinoma

Though Hepatocellular Carcinoma is the fifth most common type of cancer, it is the third most deadly and incidence is on the rise. The disease, however, does not affect men and women at similar incidence rates. This thesis was designed to compare the differences in male and female incidence of hepatocellular carcinoma and other common cancer types through age standardized sex ratios for select countries, world-wide. Men were demonstrated an increase in incidence of liver cancer about five to ten years before women in Gharbiah Governorate. Results showed that males had excess incident cases, in decreasing magnitudes, of esophageal, bladder, lung, mouth, liver, stomach, kidney, rectal, lymphoid leukemia, pancreas, non-Hodgkin lymphoma, myeloid leukemia, and colon cancers. There was a slight excess of female gallbladder cancer incidence compared to men, while many more women than men were diagnosed with thyroid cancer. Among different race/ethnicities in the United States, sex ratios resembled US ratios more than those in their countries of origin; this was seen most significantly amongst Hispanic Whites. Sex ratios were generally wider in more developed countries, though more research is needed to determine if this is consistent for all cancer types and countries. Many facets of the disproportionate sex ratios need further exploration, including areas of hormone levels and cultural or lifestyle variances that may lead to differences among men and women and developed and developing countries.^

Health significance of chlorination byproducts in drinking water: The Houston experience: A continuation study

Background. Houston, Texas, once obtained all its drinking water from underground sources. However, in 1853, the city began supplementing its water from the surface source Lake Houston. This created differences in the exposure to disinfection byproducts (DBPs) in different parts of Houston. Trihalomethanes (THMs) are the most common DBP and are useful indicators of DBPs in treated drinking water. This study examines the relationship between THMs in chlorinated drinking water and the incidence of bladder cancer in Houston. ^ Methods. Individual bladder cancer deaths, from 1975 to 2004, were assigned to four surface water exposure areas in Houston utilizing census tracts—area A used groundwater the longest, area B used treated lake water the longest, area C used treated lake water the second longest, and area D used a combination of groundwater and treated lake water. Within each surface water exposure area mortality rates were calculated in 5 year intervals by four race-gender categories. Linear regression models were fitted to the bladder cancer mortality rates over the entire period of available data (1990–2004). ^ Results. A decrease in bladder cancer mortality was observed amongst white males in area B (p = 0.030), white females in area A (p = 0.008), non-white males in area D (p = 0.003), and non-white females in areas A and B (p = 0.002 & 0.001). Bladder cancer mortality differed by race-gender and time (p ≤ 0.001 & p ≤ 0.001), but not by surface water exposure area (p = 0.876). ^ Conclusion. The relationship between bladder cancer mortality and the four surface water exposure areas (signifying THM exposure) was insignificant. This result could be attributable to Houston controlling for THMs starting in the early 1980’s by using chloramine as a secondary disinfectant in the drinking water purification process.^

Mutagenicity study of the urinary metabolites of 2-aminonaphthalene

The mutagenicity study of the urinary metabolites of 2-aminonaphthalene was conducted to determine whether differences in metabolism between different acetylator phenotypes could account for a proposed mechanism of bladder carcinogenesis. This required the use of fast and slow acetylator rabbits with phenotypic similarities to humans. In the absence of available slow acetylators, it was necessary to inhibit fast acetylators. The proposed mechanism was that slow acetylators were at greater potential risk of bladder carcinogenesis due to low rates of acetylation, a detoxification mechanism for certain aromatic amines. The alternate metabolic pathway will be hydroxylation. The fast acetylators were proposed to exhibit lower risk of bladder carcinogenicity as a result of higher acetylation rates and less mutagenic metabolites.^ This hypothesis was approached by determining from in vitro mutagenicity assays with Salmonella typhimurium strains TA98 and TA100 whether different metabolites were mutagenic. The acetylation rate of each rabbit and a suitable method of acetylation inhibition were determined through oral exposure to dapsone and the acetylation inhibitor, K-p-aminosalicylic acid. Residues of dapsone and its acetylated metabolite were extracted from blood samples and analyzed by ultra-violet spectrometry using standard curves for each metabolite. The urine samples were concentrated on XAD-2 resin and analyzed both as whole urine concentrates and as isolated metabolites from spots on high performance thin layer chromatography plates. The major isolated spots were identified and quantified through extraction and analysis by high performance liquid chromatography when possible.^ Acetylation rate determination and inhibition were successfully demonstrated in rabbits. Significant mutagenicity was noted for several critical metabolites. None of the mutagenic metabolites were detected in higher concentration in the inhibited acetylators and thus, no clear relationship of metabolite concentration to bladder carcinogenesis was evident for the compounds analyzed. There was some evidence that the inhibitor may have affected critical enzyme systems other than acetylation alone. This would account for the lower concentrations of mutagenic hydroxylated compounds observed. ^

Illustrated Catalogue of Standard Surgical Instruments and Allied Lines

Section "A": Dissecting and Post-Mortem Instruments Diagnostic Instruments and Apparatus Microscopes and Microscopic Accessories Laboratory Apparatus and Glass Ware Apparatus for Blood and Urine Analysis Apparatus for Phlebotomy, Cupping and Leeching Apparatus for Infusion and Transfusion Syringes for Aspiration and Injection Osteological Preparations Section "B": Anaesthetic, General Operating, Osteotomy, Trepanning, Bullet, Pocket Case, Cautery, Ligatures, Sutures, Dressings, Etc. Section "B" continued Section "C": Eye, Ear, Nasal, Dermal, Oral, Tonsil, Tracheal, Laryngeal,Esophageal, Stomach, Intestinal, Gall Bladder Section "C": continued Section "D": Rectal, Phimosis, Prostatic, Vesical, Urethral, Ureteral, Instruments Section "E": Gynecic, Hysterectomy, Obstetrical, Instrument Satchels, Medicine Cases Section "F": Electric Cautery Transformers, Electro-Cautery Burners and Accessories, Electric Current Controllers, Electro-Diagnostic Outfits, Electrolysis Instruments Electro-Therapeutic Lamps, Faradic Batteries, Galvanic Batteries Section "G": Office Furniture, Office Sterilizing Apparatus, Hospital Supplies, Surgical Rubber Goods, Sick Room Utensils, Invalid Rolling Chairs, Invalid Supplies Section "H": Artificial Limbs, Deformity Apparatus, Fracture Apparatus, Splints, Splint Material, Elastic Hosiery, Abdominal Supporters, Crutches, Trusses, Suspensories, Etc. Index

William Osler: Original Papers 1898-1906

Part 1: 1898-1899 On Chronic Symmetrical Enlargement of the Salivary and Lachrymal Glands, 1898 Leprosy in the United States, with the Report of a Case, 1898 An Acute Myxaedematous Condition, with Tachycardia, Glycosuria, Melaena, Mania and Death, 1898 On some of the Intestinal Features of Typhoid Fever, 1898 Cerebro-Spinal Fever, 1898 The Arthritis of Cerebro-Spinal Fever, 1898 In Memoriam, William Pepper, 1899 After Twenty-Five Years, 1899 The Diagnosis of Typhoid Fever, 1899 Interstitial Processes in the Central Nervous System, 1899 Part 2: 1900 The Home Treatment of Consumption, 1900 On Splenic Anaemia, 1900 The Chronic Intermittent Fever of Endocarditis, 1900 A Case of Multiple Gangrene in Malarial Fever, 1900 Latent Cancer of the Stomach, 1900 On the Study of Tuberculosis, 1900 Fatal Angina Pectoris without Lesions of the Coronary Arteries of a Young Man, 1900 On the Advantages of a Trace of Albumin and a Few Casts in the Urine of Certain Men above Fifty Years of Age, 1900 Part 3: 1901-1902 Congenital Absence of the Abdominal Muscles with Distended Hypertrophied Urinary Bladder, 1901 Intermittent Claudication, 1902 On the Diagnosis of Bilateral Cystic Kidney, 1902 On Amebic Abscess of the Liver, 1902 Note on the Occurrence of Ascites in Solid Abdominal Tumors, 1902 Amebic Dysentery, 1902 Notes on Aneurism, 1902 William Beaumont; a Pioneer American Physiologist, 1902 Part 4: 1903 On the Educational Value of the Medical Society, 1903 On obliteration of the Superior Vena Cava,1903 Chronic Cyanosis, with Polycythemia and Enlarged Spleen: A New Clinical Entity, 1903 The Home and its Relation to the Tuberculosis Problem, 1903 Unity, Peace, and Concord, 1903 Typhoid Fever and Tuberculosis, 1903 Part 5: 1904-1906 Ochronosis, 1904 The “Phthisiologia” of Richard Morton, M.D., 1904 On the Surgical Importance of the Visceral Crises In the Erythema Group of Skin Diseases, 1904 Aneurysm of the Abdominal Aorta, 1905 Back Notes

Improving Cervical Cancer Nodal Boost Radiation Therapy by Quantifying Uncertainties and Exploring Advanced Radiation Therapy Modalities

Radiation therapy for patients with intact cervical cancer is frequently delivered using primary external beam radiation therapy (EBRT) followed by two fractions of intracavitary brachytherapy (ICBT). Although the tumor is the primary radiation target, controlling microscopic disease in the lymph nodes is just as critical to patient treatment outcome. In patients where gross lymphadenopathy is discovered, an extra EBRT boost course is delivered between the two ICBT fractions. Since the nodal boost is an addendum to primary EBRT and ICBT, the prescription and delivery must be performed considering previously delivered dose. This project aims to address the major issues of this complex process for the purpose of improving treatment accuracy while increasing dose sparing to the surrounding normal tissues. Because external beam boosts to involved lymph nodes are given prior to the completion of ICBT, assumptions must be made about dose to positive lymph nodes from future implants. The first aim of this project was to quantify differences in nodal dose contribution between independent ICBT fractions. We retrospectively evaluated differences in the ICBT dose contribution to positive pelvic nodes for ten patients who had previously received external beam nodal boost. Our results indicate that the mean dose to the pelvic nodes differed by up to 1.9 Gy between independent ICBT fractions. The second aim is to develop and validate a volumetric method for summing dose of the normal tissues during prescription of nodal boost. The traditional method of dose summation uses the maximum point dose from each modality, which often only represents the worst case scenario. However, the worst case is often an exaggeration when highly conformal therapy methods such as intensity modulated radiation therapy (IMRT) are used. We used deformable image registration algorithms to volumetrically sum dose for the bladder and rectum and created a voxel-by-voxel validation method. The mean error in deformable image registration results of all voxels within the bladder and rectum were 5 and 6 mm, respectively. Finally, the third aim explored the potential use of proton therapy to reduce normal tissue dose. A major physical advantage of protons over photons is that protons stop after delivering dose in the tumor. Although theoretically superior to photons, proton beams are more sensitive to uncertainties caused by interfractional anatomical variations, and must be accounted for during treatment planning to ensure complete target coverage. We have demonstrated a systematic approach to determine population-based anatomical margin requirements for proton therapy. The observed optimal treatment angles for common iliac nodes were 90° (left lateral) and 180° (posterior-anterior [PA]) with additional 0.8 cm and 0.9 cm margins, respectively. For external iliac nodes, lateral and PA beams required additional 0.4 cm and 0.9 cm margins, respectively. Through this project, we have provided radiation oncologists with additional information about potential differences in nodal dose between independent ICBT insertions and volumetric total dose distribution in the bladder and rectum. We have also determined the margins needed for safe delivery of proton therapy when delivering nodal boosts to patients with cervical cancer.

Mechanisms Underlying the Heterogeneous Sensitivities of Cancer Cells to Proteasome Inhibitors

The mechanisms underlying cellular response to proteasome inhibitors have not been clearly elucidated in solid tumor models. Evidence suggests that the ability of a cell to manage the amount of proteotoxic stress following proteasome inhibition dictates survival. In this study using the FDA-approved proteasome inhibitor bortezomib (Velcade®) in solid tumor cells, we demonstrated that perhaps the most critical response to proteasome inhibition is repression of global protein synthesis by phosphorylation of the eukaryotic initiation factor 2-α subunit (eIF2α). In a panel of 10 distinct human pancreatic cancer cells, we showed marked heterogeneity in the ability of cancer cells to induce eIF2α phosphorylation upon stress (eIF2α-P); lack of inducible eIF2α-P led to excessive accumulation of aggregated proteins, reactive oxygen species, and ultimately cell death. In addition, we examined complementary cytoprotective mechanisms involving the activation of the heat shock response (HSR), and found that induction of heat shock protein 70 kDa (Hsp72) protected against proteasome inhibitor-induced cell death in human bladder cancer cells. Finally, investigation of a novel histone deacetylase 6 (HDAC6)-selective inhibitor suggested that the cytoprotective role of the cytoplasmic histone deacetylase 6 (HDAC6) in response to proteasome inhibition may have been previously overestimated.

Vertical distribution of relative numbers of the main groups of sound-scattering fishes in the Central Indian Ocean

Findings made in 31 catches with an Isaacs-Kidd midwater trawl in the light (09.00-16.00) and dark (21.00-04.00) periods of a day within a survey area of about 100 sq. miles with approximate center coordinates of 13°S and 78°E have been used to investigate vertical distribution of the main groups of sound-scattering fishes (35 species of the family Myctophidae and 16 species of other families). It has been shown that during daylight hours all fishes sink to depths deeper than 400 m. Data are presented concerning the fish population of night-time sound-scattering layers at depths of 70-150 m and about 400 m and of the daytime ones at depths of about 450 m.

Acoustic multi-frequency indicator of four major groups on a spatial grid in the Bay of Biscay

The first data set contains the mean and cofficient of variation (standard deviation divided by mean) of a multi-frequency indicator I derived from ER60 acoustic information collected at five frequencies (18, 38, 70, 120, and 200 kHz) in the Bay of Biscay in May of the years 2006, 2008, 2009 and 2010 (Pelgas surveys). The multi-frequency indicator was first calculated per voxel (20 m long × 5 m deep sampling unit) and then averaged on a spatial grid (approx. 20 nm × 20 nm) for five 5-m depth layers in the surface waters (10-15m, 15-20m, 20-25m, 25-30m below sea surface); there are missing values in particular in the shallowest layer. The second data set provides for each grid cell and depth layer the proportion of voxels for which the multi-frequency indicator I was indicative of a certain group of organisms. For this the following interpretation was used: I < 0.39 swim bladder fish or large gas bubbles, I = 0.39-0.58 small resonant bubbles present in gas bearing organisms such as larval fish and phytoplankton, I = 0.7-0.8 fluidlike zooplankton such as copepods and euphausiids, and I > 0.8 mackerel. These proportions can be interpreted as a relative abundance index for each of the four organism groups.

Mejoras en el procesamiento de imágenes de TC para el tratamiento con radioterapia adaptativa del cáncer de próstata

El cáncer de próstata es el tipo de cáncer con mayor prevalencia entre los hombres del mundo occidental y, pese a tener una alta tasa de supervivencia relativa, es la segunda mayor causa de muerte por cáncer en este sector de la población. El tratamiento de elección frente al cáncer de próstata es, en la mayoría de los casos, la radioterapia externa. Las técnicas más modernas de radioterapia externa, como la radioterapia modulada en intensidad, permiten incrementar la dosis en el tumor mientras se reduce la dosis en el tejido sano. Sin embargo, la localización del volumen objetivo varía con el día de tratamiento, y se requieren movimientos muy pequeños de los órganos para sacar partes del volumen objetivo fuera de la región terapéutica, o para introducir tejidos sanos críticos dentro. Para evitar esto se han desarrollado técnicas más avanzadas, como la radioterapia guiada por imagen, que se define por un manejo más preciso de los movimientos internos mediante una adaptación de la planificación del tratamiento basada en la información anatómica obtenida de imágenes de tomografía computarizada (TC) previas a la sesión terapéutica. Además, la radioterapia adaptativa añade la información dosimétrica de las fracciones previas a la información anatómica. Uno de los fundamentos de la radioterapia adaptativa es el registro deformable de imágenes, de gran utilidad a la hora de modelar los desplazamientos y deformaciones de los órganos internos. Sin embargo, su utilización conlleva nuevos retos científico-tecnológicos en el procesamiento de imágenes, principalmente asociados a la variabilidad de los órganos, tanto en localización como en apariencia. El objetivo de esta tesis doctoral es mejorar los procesos clínicos de delineación automática de contornos y de cálculo de dosis acumulada para la planificación y monitorización de tratamientos con radioterapia adaptativa, a partir de nuevos métodos de procesamiento de imágenes de TC (1) en presencia de contrastes variables, y (2) cambios de apariencia del recto. Además, se pretende (3) proveer de herramientas para la evaluación de la calidad de los contornos obtenidos en el caso del gross tumor volumen (GTV). Las principales contribuciones de esta tesis doctoral son las siguientes: _ 1. La adaptación, implementación y evaluación de un algoritmo de registro basado en el flujo óptico de la fase de la imagen como herramienta para el cálculo de transformaciones no-rígidas en presencia de cambios de intensidad, y su aplicabilidad a tratamientos de radioterapia adaptativa en cáncer de próstata con uso de agentes de contraste radiológico. Los resultados demuestran que el algoritmo seleccionado presenta mejores resultados cualitativos en presencia de contraste radiológico en la vejiga, y no distorsiona la imagen forzando deformaciones poco realistas. 2. La definición, desarrollo y validación de un nuevo método de enmascaramiento de los contenidos del recto (MER), y la evaluación de su influencia en el procedimiento de radioterapia adaptativa en cáncer de próstata. Las segmentaciones obtenidas mediante el MER para la creación de máscaras homogéneas en las imágenes de sesión permiten mejorar sensiblemente los resultados de los algoritmos de registro en la región rectal. Así, el uso de la metodología propuesta incrementa el índice de volumen solapado entre los contornos manuales y automáticos del recto hasta un valor del 89%, cercano a los resultados obtenidos usando máscaras manuales para el registro de las dos imágenes. De esta manera se pueden corregir tanto el cálculo de los nuevos contornos como el cálculo de la dosis acumulada. 3. La definición de una metodología de evaluación de la calidad de los contornos del GTV, que permite la representación de la distribución espacial del error, adaptándola a volúmenes no-convexos como el formado por la próstata y las vesículas seminales. Dicha metodología de evaluación, basada en un nuevo algoritmo de reconstrucción tridimensional y una nueva métrica de cuantificación, presenta resultados precisos con una gran resolución espacial en un tiempo despreciable frente al tiempo de registro. Esta nueva metodología puede ser una herramienta útil para la comparación de distintos algoritmos de registro deformable orientados a la radioterapia adaptativa en cáncer de próstata. En conclusión, el trabajo realizado en esta tesis doctoral corrobora las hipótesis de investigación postuladas, y pretende servir como cimiento de futuros avances en el procesamiento de imagen médica en los tratamientos de radioterapia adaptativa en cáncer de próstata. Asimismo, se siguen abriendo nuevas líneas de aplicación futura de métodos de procesamiento de imágenes médicas con el fin de mejorar los procesos de radioterapia adaptativa en presencia de cambios de apariencia de los órganos, e incrementar la seguridad del paciente. I.2 Inglés Prostate cancer is the most prevalent cancer amongst men in the Western world and, despite having a relatively high survival rate, is the second leading cause of cancer death in this sector of the population. The treatment of choice against prostate cancer is, in most cases, external beam radiation therapy. The most modern techniques of external radiotherapy, as intensity modulated radiotherapy, allow increasing the dose to the tumor whilst reducing the dose to healthy tissue. However, the location of the target volume varies with the day of treatment, and very small movements of the organs are required to pull out parts of the target volume outside the therapeutic region, or to introduce critical healthy tissues inside. Advanced techniques, such as the image-guided radiotherapy (IGRT), have been developed to avoid this. IGRT is defined by more precise handling of internal movements by adapting treatment planning based on the anatomical information obtained from computed tomography (CT) images prior to the therapy session. Moreover, the adaptive radiotherapy adds dosimetric information of previous fractions to the anatomical information. One of the fundamentals of adaptive radiotherapy is deformable image registration, very useful when modeling the displacements and deformations of the internal organs. However, its use brings new scientific and technological challenges in image processing, mainly associated to the variability of the organs, both in location and appearance. The aim of this thesis is to improve clinical processes of automatic contour delineation and cumulative dose calculation for planning and monitoring of adaptive radiotherapy treatments, based on new methods of CT image processing (1) in the presence of varying contrasts, and (2) rectum appearance changes. It also aims (3) to provide tools for assessing the quality of contours obtained in the case of gross tumor volume (GTV). The main contributions of this PhD thesis are as follows: 1. The adaptation, implementation and evaluation of a registration algorithm based on the optical flow of the image phase as a tool for the calculation of non-rigid transformations in the presence of intensity changes, and its applicability to adaptive radiotherapy treatment in prostate cancer with use of radiological contrast agents. The results demonstrate that the selected algorithm shows better qualitative results in the presence of radiological contrast agents in the urinary bladder, and does not distort the image forcing unrealistic deformations. 2. The definition, development and validation of a new method for masking the contents of the rectum (MER, Spanish acronym), and assessing their impact on the process of adaptive radiotherapy in prostate cancer. The segmentations obtained by the MER for the creation of homogenous masks in the session CT images can improve significantly the results of registration algorithms in the rectal region. Thus, the use of the proposed methodology increases the volume overlap index between manual and automatic contours of the rectum to a value of 89%, close to the results obtained using manual masks for both images. In this way, both the calculation of new contours and the calculation of the accumulated dose can be corrected. 3. The definition of a methodology for assessing the quality of the contours of the GTV, which allows the representation of the spatial distribution of the error, adapting it to non-convex volumes such as that formed by the prostate and seminal vesicles. Said evaluation methodology, based on a new three-dimensional reconstruction algorithm and a new quantification metric, presents accurate results with high spatial resolution in a time negligible compared to the registration time. This new approach may be a useful tool to compare different deformable registration algorithms oriented to adaptive radiotherapy in prostate cancer In conclusion, this PhD thesis corroborates the postulated research hypotheses, and is intended to serve as a foundation for future advances in medical image processing in adaptive radiotherapy treatment in prostate cancer. In addition, it opens new future applications for medical image processing methods aimed at improving the adaptive radiotherapy processes in the presence of organ’s appearance changes, and increase the patient safety.

Wild-type p53 triggers a rapid senescence program in human tumor cells lacking functional p53

The p53 tumor suppressor gene has been shown to play an important role in determining cell fate. Overexpression of wild-type p53 in tumor cells has been shown to lead to growth arrest or apoptosis. Previous studies in fibroblasts have provided indirect evidence for a link between p53 and senescence. Here we show, using an inducible p53 expression system, that wild-type p53 overexpression in EJ bladder carcinoma cells, which have lost functional p53, triggers the rapid onset of G1 and G2/M growth arrest associated with p21 up-regulation and repression of mitotic cyclins (cyclin A and B) and cdc2. Growth arrest in response to p53 induction became irreversible within 48-72 h, with cells exhibiting morphological features as well as specific biochemical and ultrastructural markers of the senescent phenotype. These findings provide direct evidence that p53 overexpression can activate the rapid onset of senescence in tumor cells.

STEAP: A prostate-specific cell-surface antigen highly expressed in human prostate tumors

In search of novel genes expressed in metastatic prostate cancer, we subtracted cDNA isolated from benign prostatic hypertrophic tissue from cDNA isolated from a prostate cancer xenograft model that mimics advanced disease. One novel gene that is highly expressed in advanced prostate cancer encodes a 339-amino acid protein with six potential membrane-spanning regions flanked by hydrophilic amino- and carboxyl-terminal domains. This structure suggests a potential function as a channel or transporter protein. This gene, named STEAP for six-transmembrane epithelial antigen of the prostate, is expressed predominantly in human prostate tissue and is up-regulated in multiple cancer cell lines, including prostate, bladder, colon, ovarian, and Ewing sarcoma. Immunohistochemical analysis of clinical specimens demonstrates significant STEAP expression at the cell–cell junctions of the secretory epithelium of prostate and prostate cancer cells. Little to no staining was detected at the plasma membranes of normal, nonprostate human tissues, except for bladder tissue, which expressed low levels of STEAP at the cell membrane. Protein analysis located STEAP at the cell surface of prostate-cancer cell lines. Our results support STEAP as a cell-surface tumor-antigen target for prostate cancer therapy and diagnostic imaging.

Differential gene expression in p53-mediated apoptosis-resistant vs. apoptosis-sensitive tumor cell lines

Induction of wild-type p53 in the ECV-304 bladder carcinoma cell line by infection with a p53 recombinant adenovirus (Ad5CMV-p53) resulted in extensive apoptosis and eventual death of nearly all of the cells. As a strategy to determine the molecular events important to p53-mediated apoptosis in these transformed cells, ECV-304 cells were selected for resistance to p53 by repeated infections with Ad5CMV-p53. We compared the expression of 5,730 genes in p53-resistant (DECV) and p53-sensitive ECV-304 cells by reverse transcription–PCR, Northern blotting, and DNA microarray analysis. The expression of 480 genes differed by 2-fold or more between the two p53-infected cell lines. A number of potential targets for p53 were identified that play roles in cell cycle regulation, DNA repair, redox control, cell adhesion, apoptosis, and differentiation. Proline oxidase, a mitochondrial enzyme involved in the proline/pyrroline-5-carboxylate redox cycle, was up-regulated by p53 in ECV but not in DECV cells. Pyrroline-5-carboxylate (P5C), a proline-derived metabolite generated by proline oxidase, inhibited the proliferation and survival of ECV-304 and DECV cells and induced apoptosis in both cell lines. A recombinant proline oxidase protein tagged with a green fluorescent protein at the amino terminus localized to mitochondria and induced apoptosis in p53-null H1299 non-small cell lung carcinoma cells. The results directly implicate proline oxidase and the proline/P5C pathway in p53-induced growth suppression and apoptosis.