Orientador: Luís Fernando Barbisan / Banca: Daniel Araki Ribeiro / Banca: Lígia Souza Lima Silveira da Mota / Resumo: O consumo de mate em altas temperaturas tem sido considerado um fator de risco para o desenvolvimento do carcinoma epidermóide de esôfago (CE) na América do Sul. Desta forma, os efeitos da ingestão de mate sobre danos de DNA e a carcinogênese de esôfago, induzidos pela dietilnitrosamina (DEN) e injúria térmica, foram avaliados em ratos Wistar machos. Na fase de iniciação, os animais foram iniciados com injeções i.p. da DEN (8 x 80 mg/Kg p.c.) e submetidos a injúria térmica (água a 65°C, lmIlrato, instilado no interior do esôfago) e receberam, concomitantemente, mate (2.0% p/v, grupo teste) ou chá-verde (2.0% p/v, grupo controle positivo) como única fonte de líquidos por oito semanas. Nenhum tratamento adicional foi introduzido durante a fase de pós-iniciação (nona a vigésima semana do experimento). Amostras de sangue periférico foram coletadas quatro horas após a última administração da DEN para o teste do cometa na oitava semana e amostras de esôfago e fígado foram coletadas na oitava e vigésima semanas do experimento. Na oitava semana, a ingestão de mate e chá-verde por si não foi genotóxica e reduziu de forma significativa os níveis de danos no DNA de leucócitos de sangue periférico nos animais tratados com a DEN. Além disso, uma redução significativa nos níveis de proliferação celular no epitélio do esôfago e no parênquima hepático e no número de lesões hepáticas pré-neoplásicas foram também observadas nos grupos iniciados e que receberam mate ou chá-verde. Na vigésima semana, uma menor incidência de neoplasias de esôfago e fígado foi observada nos grupos que receberam previamente mate e chá-verde quando comparado ao grupo iniciado pela DEN e submetido à injúria térmica. Os resultados do presente estudo indicam que a ingestão de mate se mostrou benéfica contra danos no DNA e a carcinogênese de esôfago e fígado induzidos pela DEN. / Abstract: Drinking hot mate has been associated with risk for esophageal squamous cell carcinoma m South America. Thus, the modifying effects of mate tea intake on DNA damage and esophageal carcinogenesis induced by diethylnitrosamine (DEN) plus thermal injury were evaluated in male Wistar rats. In the initiation phase, rats were treated with DEN injections (8 x 80 mg/Kg b.w.) plus thermal injury (water 65°C, lml/rat, instilled into the esophagus) and concomitantly received mate tea (2.0% w/v, test group) or green tea (2.0% w/v, positive control group) as the sole source of drinking fluid for 8 weeks. Any additional treatment was introduced at post-initiation until week 20. Peripheral blood was collected 4 hr after the last DEN application for comet assay at week 8 and samples from esophagus and liver were collected at weeks 8 and 20. At week 8, mate or green tea intake itselfwere non-genotoxic and significantly decreased DNA damage levels in peripheral blood leucocytes from DEN-treated animaIs. Also, a significant reduction of cell proliferation rates in both esophageal epithelium and liver parenchyma and on the number of putative preneoplastic liver lesions were observed in initiated and mate or green tea-treated animaIs at week 8. A significant lower incidence of esophageal and liver neoplasms and tumor multiplicity was observed in the groups previously treated with mate or green tea when compared to the DEN initiated/thermal injury group at week 20. These data indicate that mate tea presented protective effects against DNA damage and esophageal and liver carcinogenesis induced by DEN. / Mestre

An attempt has been made to demonstrate an association between the carcinogenic activity of a chemical compound and its capacity to induce DNA damage and chromosome aberrations which may result in mutations and/or neoplastic transformation. Twenty-five 4-nitroquinoline 1-oxide (4NQO) derivatives and five related compounds of 4-nitropyridine 1-oxide (4NPO) of varying carcinogenicity were examined. [Formulae omitted] The induction of DNA damage, chromosome aberrations and clone forming capacity were used as end points. Monolayer cultures of embryonal Syrian-hamster cells and an established line of baby hamster kidney cells (BHK-21) were employed in this study. DNA damage, as measured by the unscheduled incorporation of tritiated thymidine (³H-TdR), was assayed by the autoradiographic procedure. To distinguish DNA repair synthesis from DNA replication synthesis at S-phase, cultured embryonal hamster cells were arrested at G₁ by growing them in an arginine deficient medium (ADM) prior to the application of the various carcinogens. The unscheduled uptake of radioisotope was estimated by counting the number of grains per diploid nucleus of carcinogen treated cells. The highly oncogenic derivatives of 4NQO and 4NPO elicited an elevated level of unscheduled ³H-TdR incorporation in treated cells, while the weakly oncogenic compounds induced only a smaller amount of DNA repair synthesis. The non-oncogenic N-oxides failed to provoke any detectable ³H-TdR uptake. Chromosome aberrations were studied in ADM-arrested cells which were exposed to the various compounds and then triggered into division by transferring them into the regular growth medium. A direct proportionality was observed between the degree of carcinogenicity of a compound and the frequency of induced chromosome aberrations. The clone forming ability of treated cells was employed as a means to compare the cytotoxicity of the 4NQO and 4NPO derivatives. Potent carcinogens were highly cytotoxic; weakly carcinogenic compounds showed only a slight lethal effect and non-oncogenic derivatives did not affect cell survival. This study demonstrated the capacity of carcinogens to induce alterations at the chromosome and DNA level. The possible role of DNA repair and chromosome aberrations in neoplastic transformation was discussed. The use of DNA repair synthes as an economic and relevant tool for identifying mutagens and/or carcinogens has been suggested. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

The National Cancer Institution reported over 80,000 diagnoses of bladder cancer (BCa) in the United States in 2018. Despite these numbers, minimal research toward developing new diagnostic techniques and treatment options are underway. Evidence suggests a significant increase in non-specific a-naphthyl acetateesterase levels in BCa patient’s urine. There has been little research focused on identification of the esterase present. It is also suggested that elevated oxidative stress resulting in production of reactive oxygen species (ROS) is common in tumorigenic bladder cells as a result of increased metabolic activity. Oxidized protein hydrolase (OPH) is an 80kD serine protease, previously found to be elevated in many other types of cancer. OPH degrades proteins damaged by ROS and also exhibits a highly specific esterase activity toward (AcApNA) N-acetyl-alanyl-p-nitroanilide and ANAA (α-naphthyl N-acetylalaninate) containing substrate. Investigation of OPH expression in BCa could result in development of new diagnostic techniques and possible application toward prodrugs targeting cells with elevated ROS and/or OPH. Due to lack of commercial OPH, a positive control for this protein is needed for testing. To do this E. coli(BL-21 DE-3) were cultured and inserted with pET-21a (+) plasmids containing a human OPH gene insert prior to a His7 tag. After being selectively grown on ampicillin media, the bacteria were induced by IPTG and digested using lysozyme. The soluble rOPH suspended in the supernatant was separated from the pellet by centrifugation and further purified using Ni-NTA resin chromatography columns specific for the His7 tag sequence. The UM-UC-3 bladder cancer cell line, commonly used in published research to screen efficiency of chemotherapeutics, were cultured in accordance to ATCC. These cells were then compared against none tumorigenic bladder cancer cells and rOPH in a series of tests. Sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) were transferred for western blot analysis using antibodies specific for human OPH to investigate the expression levels present in cells. Native-PAGE electrophoresis showed OPH esterase activity across these cells using S-ANAA substrate as a specific esterase colorimetric stain. With these results, possible treatment options can be investigated with use of novel prodrug chemotherapy specifically targeting OPH in BCa cells, ultimately leading to apoptosis in effected cells. These events may also lead to possible biomarkers used for easier and earlier diagnosis of BCa across various spectrums.

Multiple myeloma is characterized by proliferation of plasma cells in the bone marrow, producing monoclonal immunoglobulin. It accounts for 17% of hematologic malignancies in the US. Diagnosis is often suspected in the setting of bone lytic lesions, anemia, hypercalcemia or renal failure. Rarely, multiple myeloma can present with soft tissue involvement which can be difficult to diagnose. Below we present one such presentation. Our patient is a 53-year-old who was initially diagnosed with multiple myeloma six years back when he presented to hospital with back and right leg pain. On admission he was found to have multiple lytic lesions involving the appendicular and axial skeleton. On further workup, bone marrow biopsy showed 30% plasma cells with IgG kappa monoclonal protein elevation. Patient was diagnosed with ISS stage II multiple myeloma. He was treated with standard regimen with Velcade, Revlimid and dexamethasone with excellent response. Patient was evaluated for stem cell transplant however did not qualify for it due to social challenges. Patient was continued on maintenance therapy with Velcade and Revlimid for 8 cycles prior to clinical relapse with lytic lesions in the C-spine. At this point patient was switched to different therapeutic regimen with pomalidomide, carfilzomib and dexamethasone and had excellent response for 35 cycles on this regimen. Patient had interruption in treatment for 3 months due to other medical comorbidities. A repeat bone marrow biopsy which was done in November of 2019 revealed extensive bone marrow involvement with 70% plasma cells concerning for relapse. Patient was started on single agent daratumumab in December 2019 however had a difficult course interrupted by right-sided abdominal pain, persistent nausea and decreased appetite requiring hospital admission. Further workup revealed a 2.7 cm lesion in the liver as well as a 4.9 x 7.3 cm T11 left paraspinal soft tissue mass. Biopsy of the liver lesion revealed sheets of kappa restricted abnormal plasma cells concerning for progression of disease. Given the involvement of the visceral organ and the extent of his disease, it was decided to switch patient's treatment from single agent daratumumab to a multi agent chemotherapy regimen with dexamethasone, cyclophosphamide, etoposide and cisplatin. Patient received his 1st cycle inpatient and had marked symptomatic improvement and was discharged home. His M-protein spike reduced from 3.9 to 1.8 g/dl post once cycle of treatment. Soft tissue involvement by multiple myeloma is rare event. Though malignant plasma cells may diffusely infiltrate the liver parenchyma, the nodular spread is unique. In review by Talamo et al, out of 2,584 patients with MM, only 11 had liver plasmacytomas. This phenomenon is driven by lack of expression of adhesion molecules, increased heparanase-1 expression and loss of chemokine receptors on myeloma cells. Such alterations in cell architecture lead to more aggressive disease behavior. At present time treatment for this unique patient population does not differ from other MM cases. It is important for clinicians to recognize the possibility of such event.

Skin cancer is a major health problem worldwide. It is the most common cancer in the United States and poses a significant healthcare burden. Excessive UVR exposure is the most common cause of skin cancer. Despite various precautionary measures to avoid direct UVR exposure, the incidence of skin cancer and mortality related to it remains high. Furthermore, the current treatment options are expensive and have side effects including toxicity to normal cells. Thus, a safe and effective approach is needed to prevent and treat skin cancer. Chemopreventive strategy using naturally occurring compounds, such as resveratrol, is a promising approach to reduce the incidence of UVR-induced skin cancer and delay its progression. This review highlights the current body of evidence related to chemopreventive role of resveratrol and its molecular mechanisms in UVR-induced skin carcinogenesis.

Urinary bladder paraganglioma (UBP) is a rare neuroendocrine neoplasm. It accounts for less than 1% of urinary bladder tumors and less than 6% among all types of paragangliomas. More commonly, UBP occurs in the female population aged 20-40 years old. UBP is classified into functional and non-functional types, and the majority is functional, leading to symptoms and signs of excess catecholamine, including hypertension, palpitation, syncope, and headache. Non-functional UBP comprises about 15% of UBPs and lacks the excess secretion of catecholamine, which often leads to misdiagnosis as urothelial cancer due to its rarity and nonspecific symptoms - increased urinary frequency/urgency and painless gross hematuria. Here, we present a rare case of a non-functional UBP. A 65-year-old male with BPH presented to ER with a 6-month history of urinary retention. He also was experiencing intermittent hematuria and dysuria during this time but otherwise remained asymptomatic without headache, dyspnea, wheezing, or diarrhea. Physical exam showed normal BP and no suprapubic tenderness on palpation. UA showed gross hematuria. Subsequent cystoscopy showed thickening of the bladder dome and an 8 mm lesion. Transurethral resection of bladder tumor (TURBT) was performed, and pathology showed 1 cm tumor confined to submucosa with questionable margins. Chromogranin, synaptophysin, CD56, GATA3, CD10 were stained positive; cytokeratin AE1/AE3, cytokeratin 34betaE12, SOX10, S100, and calretinin were negative. From morphology and immunochemistry, the diagnosis of UBP was made. Free metanephrine, plasma normetanephrine, 24-hour urine metanephrine and normetanephrine levels were not elevated. Post-TURBT MRI abdomen showed no other suspicious lesions. A wide re-resection of the bladder dome was performed due to the questionable margins from the initial surgery, and pathology showed benign bladder tissue with unremarkable immunostains, indicating no overt features of residual paraganglioma. Due to its paucity and uncertain biological behavior, the prognosis of UBP is not well established. While most UBP are benign, 10-15% of cases are malignant. High expression of VEGF and or abnormal vessel architecture in the tumor cells raise suspicion of malignancy. However, typically, definitive evidence of malignancy in paraganglioma is its invasion of adjacent organs or distant metastasis. The local recurrence rate ranges from 5-15%, thus necessitating long-term surveillance for 10-years. Systemic chemotherapy, including cyclophosphamide, vincristine, dacarbazine (CVD), or temozolomide, is necessary for distant metastatic or symptomatic disease. Iobenguane I-131 or Lutathera can be utilized with positive MIBG or 68Ga DOTATATE scan, respectively. Otherwise, surgical extirpation remains the choice of curative intent, and a multidisciplinary approach consisting of urologists, medical/radiation oncologists, and endocrinologists would be warranted for this rare entity of disease.

Precision medicine involves a personalized approach to healthcare acknowledging an individual-to-individual variability in genetics, environment, and lifestyle. Research in precision medicine can be pulled from basic, clinical or epidemiological sciences from which imaging, omics, or other types of data can be mined to contribute to the ‘information commons’ from which patient-specific patterns are drawn. Advancement in basic sciences that focus on imaging and other high content information processing for precision medicine is of particular importance in fields like oncology and nephrology where, diseases like focal segmented glomerular sclerosis have neither seen drug development in the past two decades, nor have an affirmative treatment plan. One reason is the lack of high-content high-throughput platforms for drug testing with a physiologically appropriate microenvironment. This work aims to build a high-content platform for podocyte-based drug discovery. Having demonstrated that cells in patterns demonstrate a phenotypic change representative of the in vivo condition, we first start by building a robust 96-well plate with our microfabricated platform as its base. We demonstrate a relevant increase in expression of podocyte specific proteins like synaptopodin in our patterned podocytes along with a decrease in cell-to-cell variability when compared with their unpatterned counterparts. Next, we demonstrate the use of our platform as a tool for drug discovery by showing that we achieve a reproducible actin-based dose response curve using human podocyte cell lines, something that has never been done to our knowledge. We see that our platform pushes immortalized podocytes towards cell cycle arrest at a much earlier timepoint during differentiation with improved functional performance metrics, such as lower motility and increased cytoskeletal segregation. We show that our platform may be able to extend its versatility by synergizing the effects of substrate shape and stiffness, and we show a potential application in studying the effect of this platform on the expression of Yes associated protein (YAP). We demonstrate the flexibility of this platform using another case study, this time with cancer cells. It is well known that the progression of neoplastic cells to metastasis is a major contributing factor to poor prognosis. The metastatic cascade involves invasion and migration coupled by angiogenesis and intravasation. The subsequent cells that survive circulation and attach to the endothelium extravasate and colonize in a distal location. Current techniques, such as Transwell and scratch assays that attempt to quantify metastatic potential are difficult to scale and consequently may be challenging to use in large-scale drug testing. We show that using our platform, we are able to rapidly quantify the metastatic potential of cancer cells in situ with high sensitivity, independent of cell seeding density. By changing the dimensions of our microfabricated patterns, we are able to vary the mechanical resistance that the cell experiences traversing and use this to mimic cell invasion across different microenvironments. Importantly, we show that we can quantify the effect of the metastatic potential in response to a pharmacological intervention and thus demonstrate that we can use this platform for drug testing. In conclusion, we present a novel multifaceted platform and demonstrate its versatility with two different applications. In the context of drug discovery, we show that the platform serves as a superior model for podocyte injury. The reproducible Puromycin aminonucleocide (PAN) actin-based dose response curves obtained using this platform, opens avenues to investigate the effect of various targeted therapies for podocyte-based kidney diseases. In the context of screening for metastatic potential of cancer cells, we show that our platform exhibits a superior sensitivity to existing screening techniques. Additionally, the potential of using a patient’s own cells in the future for either application presents exciting avenues for precision medicine.

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