Orientador: Daniel Galera Bernabé / Coorientador: Sandra Helena de Oliveira Penha / Coorientador: Roelf Justino Cruz Rizzolo / Banca: Flávia Lombardi Lopes / Banca: Glauco Issamu Miyahara / Resumo: A ocorrência de eventos estressores nas fases iniciais de vida (estresse precoce de vida - EPV) pode afetar negativamente funções fisiológicas e psicológicas na fase adulta. Apesar de investigações pré-clínicas terem mostrado que o estresse crônico pode afetar a progressão do câncer, não há estudos que investigaram os efeitos do EPV na progressão do câncer bucal. No presente estudo, utilizamos um modelo animal de carcinogênese bucal induzida pelo carcinógeno 4-Nitroquinolona-1-Óxido (4NQO) para avaliar o impacto do EPV induzido por separação materna (SM) sobre a incidência e progressão do carcinoma espinocelular (CEC) de boca. As ninhadas submetidas ao protocolo de SM foram separadas de suas mães durante 3 horas por dia, do dia pós-natal 1 ao 21. Após os animais atingirem a idade adulta (90 dias), os grupos SM e controle foram tratados com 4NQO durante 120 dias. Análise histopatológica foi realizada para avaliar a incidência de CEC de boca e grau de malignidade do tumor entre os animais estressados e não estressados. Também foram avaliados o volume e espessura tumoral e o peso do baço e das glândulas adrenais. Os níveis níveis plasmáticos de norepinefrina foram analisados por ELISA e a expressão de RNAm para os genes relacionados à progressão do CEC de boca (IL-6, TNF-alpha, VEGF, p53 e CDKN2A) foram analisados por PCR em tempo real. A SM no período pós-natal aumentou em aproximadamente 60% a ocorrência de CEC de boca na idade adulta. Os ratos submetidos à SM desenvolveram tu... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Early life stress (ELS) may negatively affect the behavior and physiological functions in adulthood. Despite pre-clinical investigation have shown that chronic stress may affect cancer progression, there are no studies which have investigated ELS effects on oral cancer progression. In the present study, we used an oral carcinogenesis animal model induced by carcinogen 4-Nitroquinoline-1-oxide (4NQO) to assess the impact of ELS induced by maternal separation (MS) on the oral squamous cell carcinoma (OSCC) occurrence and progression. The litters underwent MS protocol were separated from their dam for 3 hours daily, during postnatal day 1-21. After animals reach adulthood (90 days), MS and control groups were treated with 4NQO during 120 days. Histopathological analysis was performed to evaluate the OSCC incidence and malignant degree between stressed and non-stressed rats. The volume and tumor thickness and the spleen and adrenal glands weight were also evaluated. Plasma norepinephrine levels were analyzed by ELISA and mRNA expression for OSCC progression-related genes (IL-6, TNF-alpha, VEGF, p53 and CDKN2A) were analyzed by real-time PCR. MS in neonatal period increased in almost 60% chemically induced OSCC occurrence in adulthood. Rats exposed to MS developed thicker (p=0.02) and larger tumors (p=0.03) compared to non-stressed rats. OSCCs from ELS rats showed worst pattern of invasion (p=0.004) and more perineural invasion (p=0.04) ELS also significantly reduced spleen (p=0.003) and adrenal glands (p=0.03) weight in cancer rats. ELS induced overexpression of IL-6 (p=0.04) and attenuated p53 expression (p=0.04) in OSCCs. Our results provides the first evidences that ELS may increase oral cancer onset and progression, and suggest that this effect can be associated with altered expression of IL-6 and p53 / Mestre

Growth hormone (GH) is a vital pituitary hormone controlling somatic cell growth and development. GH has a multitude of effects on the body: deficiency can lead to dwarfism while excess can cause conditions such as gigantism. Human patients with mutations in the transcription factor Pit-1 show decreased GH and prolactin levels. However, Pit-1 is known to control multiple pituitary hormones, so what other factors lead to the specificity of transcriptional regulation of the GH gene via its promoter? In order to study this, our lab has been analyzing rat pituitary cell lines to understand the role of ZNF292 (formerly called Zn-16), a selective GH transcription regulator with 16 zinc fingers that bind to the GH promoter DNA. This work has used rat MtT/S cells that are unique in that they exclusively express GH. MtT/S cells were procured from Riken Cell Bank in Japan, cultured, and examined for GH hormone and RNA expression. Results confirmed that the MtT/S cells are terminally differentiated as somatotrophs. To understand the role of ZNF292 in transcription of the GH gene, recent rat genomic data was analyzed to determine the positions of 7 exons upstream from the large exon 8 that contains the important zinc finger-encoding portions of the protein. First, MtT/S RNA was reverse transcribed into DNA, then PCR amplification was performed using primer pairs encompassing various sections of the exon 1 – 7 region. Specific PCR products were found with distinct products ranging in size from 130 to 960, all of which agreed with the predicted sizes of these exons. It had previously been theorized that ZNF292 contained a single large exon; however, these results show that splicing of the primary transcript does occur in this upstream region. Characterizing this exonic region was performed because it has been shown that ZNF292 produces circular RNA (circRNA) of unknown function in human endothelial cells and in certain types of cancer. CircRNAs are thought to be created by the “back-splicing” of exons, so that rather than a linear transcript, the ends are circularized for a portion of the transcript. Having determined the sequence and organization of these upstream exons, we are now testing primer sets that will demonstrate productive amplification only from circRNAs. Further, we are removing linear RNAs using RNAse R treatment to selectively enhance circRNA presence in the reactions. Finally, data from RNA-Seq analysis of the MtT/S cells will be scrutinized to determine if additional exon/intron boundaries or alternative splice sites exist in this upstream 7 exon region. The study of circular RNAs could be very important in understanding its role in acting as a microRNA sponge or RNA-binding protein sponge during their regulation of downstream gene transcription. Also, analysis of this mechanism shows potential as a clinical tool in cancer treatment because ZNF292 functions as a tumor suppressor in colorectal and chronic lymphatic leukemia.

Additional Hydroxyl group on CT6 (3,4-dihydroxy-5,7-dimethoxyflavone), a flavone extracted from Chromolaena Tacotana potentially confers additional activity against pancreatic cancer as compared to CT7 (4-hydroxy-5,7-dimethoxyflavone) Parker Wade1, Miranda Green1, April Weaver1, Omri Coke1, Ruben D. Torrenegra2, and Victoria Palau1 1Department of Pharmaceutical Sciences, College of Pharmacy, East Tennessee State University, Johnson City, TN. 2Department of Chemistry, Universidad de Ciencias Aplicadas y Ambientales, Bogota, Colombia and Pancreatic cancer is one of the deadliest types of cancers, with a mortality rate of about 95%. This high mortality rate signifies there is a need for further research into finding treatment options for those affected by pancreatic cancer. Recent studies have found cytotoxic effects on cancerous cells elicited from compounds, such as flavones, in plants indigenous to Western South America, specifically Colombia. The flavones 3,4-dihydroxy-5,7-dimethoxyflavone (CT6) and 4-hydroxy-5,7-dimethoxyflavone (CT7) were isolated from Chromolaena Tacotana, member of the asteraceae family. The molecular structures of the flavones differ only by an additional hydroxyl group on CT6. Both of these compounds were tested on MIA PaCa2 and Panc28 pancreatic cancer cells at concentrations ranging from 5μM to 80μM. Cell viability after dosing of CT6 and CT7 was determined using MTT and spectrophotometry analysis. MIA PaCa2 is more poorly differentiated than Panc28. CT6 conferred greater activity on both cell lines compared to CT7. Percent cell viability of the Panc28 cell line reached a low of 35.55% (p=0.0001) with CT6, compared to 84.25% (p=0.0275) with CT7. Percent cell viability of the MIA PaCa2 cell line reached a low of 46.72% (p=0.000001)with CT6. However, CT7 showed no significant difference, with percent cell viability reaching 103.73% (p=0.5605) when compared to the control for this cell line. While CT6 exerted cytotoxic activity on both Panc28 and MIA PaCa2, CT6 had significantly more cytotoxic activity on Panc28, which could be related to the greater differentiation status of this cell line. More in depth studies will need to be conducted to determine the exact reasons for greater activity of CT6 on Panc28 cells. This could be due to the compound’s target, mitochondrial activity of the cell lines, and the minor structural differences between the two compounds.

Marginal zone (MZ) lymphomas (MZLs) represent a group of lymphomas originating from B lymphocytes of the “marginal zone” which is the external part of the secondary lymphoid follicles. The WHO classifies MZL into 3 entities; extranodal MZL, splenic MZL and nodal MZL. Extranodal marginal zone lymphoma (EMZL) can arise in different tissues, including the stomach, salivary gland, lung, small bowel, thyroid, ocular adnexa and skin. We present a 25 years old female with a history of angioedema and chronic cutaneous eczema who developed an unusual EMZL. Patient presented with a history of rapidly enlarging skin nodule on her left elbow that had been present for almost one year. Over a period of 2-3 weeks she felt the nodule rapidly changed in size and shape. Excisional biopsy of the mass revealed a lymphoid infiltrate based in the reticular dermis and focally extending into the subcutaneous adipose tissue with formation of disrupted lymphoid follicles positive for CD20, CD23 and BCL2 but negative for CD10, Cyclin D1 and SOX11. Diagnosis was consistent with extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma). Patient on presentation did not have any B symptoms other cutaneous lesions, lymphadenopathy or hepatosplenomegaly. PET scan revealed no evidence of abnormal uptake leading to a final Stage IE definition. Patient initiated definitive radiation therapy. EMZL accounts for 5 -10 % of non-Hodgkin lymphoma. It has been described often in organs that are normally devoid of germinal centers. It may arise in reactive lymphoid tissue induced by chronic inflammation in extranodal sites. Primary cutaneous marginal zone lymphoma (PCMZL) is associated with infectious etiologies such as Borrelia burgdorferi and less commonly with viral infections or in relation to autoimmune disorders. Autoimmune disorders, specifically Sjögren's syndrome is associated with a 30-fold increased risk of marginal zone lymphoma. Localized disease can be treated by local radiotherapy, intralesional injections or excision. Widespread skin disease is usually treated with a CD20 directed monoclonal antibody-Rituximab. Patients with PCMZL usually have an indolent clinical course. Extracutaneous dissemination of MALT Lymphoma is uncommon and happens in 6-8 % of patients. The 5 years overall survival is between 98-100%. Family physicians and dermatologists should have a high index of suspicion for this rare lymphoma subtype especially in patients with inflammatory chronic skin conditions and atopy.

Most cardiac tumors are metastatic tumors, which are 20–40 times more common than primary tumors of the heart. Most primary tumors of the heart are benign, with atrial myxomas being the most common. Primary cardiac tumors are extremely rare with an incidence of less than 0.1 percent. Virtually all types of sarcomas have been reported in the heart as isolated case reports. We present a rare case of biatrial high-grade osteosarcoma. A fifty-four-year-old Hispanic female presented with shortness of breath and was cyanotic on the exam while visiting Mexico. Due to abnormal chest x-ray, echocardiogram concerning for bilateral atrial myoma she was referred to a cardiothoracic surgeon. She underwent bi atrial intracardiac tumor resection in Mexico. Several months prior to her resection she noted numbness on the side of the face that was evaluated by her physicians in the United States with a Brain MRI and carotid Ultrasound/Doppler that was unrevealing. She also remembered an episode of uncontrolled hypertension two years prior to surgery requiring admission to a local hospital in East Tennessee with cardiology evaluation that did not include an echocardiogram. Surgical pathology showed extensive undifferentiated spindle cell proliferation with multifocal osteoid production and foci of osseocartilaginous differentiation. There was prominent necrosis and moderately high mitotic rate (10-19/HPF). Tumor cells were positive for SatB2 and negative for vascular, muscular, or neural markers. This is consistent with a primary cardiac high-grade osteosarcoma. These occur very rarely, usually in the atria, and behave aggressively. Post resection staging PET-CT showed hypermetabolic mixed lytic and sclerotic lesion of T10 concerning for metastasis disease. She received approximately 6 cycles of adriamycin and ifosfamide chemotherapy. Adriamycin was discontinued due to left ventricular dysfunction with an ejection fraction of 30-35%, multiple segmental abnormalities, diffuse left ventricular hypokinesis, and moderate to severe mitral valve regurgitation. Despite intracardiac tumor, resection, concern for metastatic disease, chemotherapy, and systolic dysfunction patient is asymptomatic and does have robust performance status. A follow-up PET-CT five months after cessation of treatment reveals no significant evidence of uptake other than abnormalities in the T10 vertebra. A repeat echocardiogram continues to reveal a depressed ejection fraction of 35%. The patient is completely asymptomatic, seemingly fit with an ECOG performance status of 0-1. Osteosarcomas are aggressive with a high incidence of recurrence and metastasis. Fewer than 50 cases of primary cardiac osteosarcomas have been reported in the literature. Currently, it is postulated that they arise from undifferentiated mesenchymal stem cells in the endocardium that transform into active osteoblasts. Even though complete resection can be achieved in some cases, long-term results are usually poor. No standard therapy has been established due to the tumor's low incidence rate and lack of clinical trial data. Our case highlights the importance of evaluating common symptoms thoroughly since it may be a harbinger of rare and serious disorders. This case reflects the heterogeneous nature of sarcoma histology, the consequent tumor biology and hence varied clinical course and prognosis.

Small cell variant of medullary thyroid carcinoma is an extremely rare histologic entity with a paucity of data. As such, there is a lack of clinical experience regarding this disease. In this case, a 52-year-old patient with small cell variant medullary thyroid carcinoma was experiencing intractable nausea, vomiting, and diarrhea. The initial workup was extensive yet unrevealing. He was refractory to all treatments. On further laboratory analysis, the calcitonin was substantially high and the thyroid stimulating hormone level was mildly elevated. This case is presented to highlight a possible treatment for this rare cancer through thyroxine suppression therapy. This case is presented due to the lack of literature available on small cell medullary thyroid carcinoma and also to discuss a possible direct relationship between thyroid stimulating hormone and calcitonin levels in this disease population.

Acute promyelocytic leukemia (APL) is a clinically distinct and rare type of acute myeloid leukemia and represents an oncologic emergency. Even rarer is the incidence of APL in pregnancy with less than 60 cases described in the literature. A 33-year-old pregnant female at 34 week gestation presented to hospital with reports of abdominal pain. On admission she was found to have acute onset pancytopenia with a WBC count of 1.2, Hemoglobin of 9.7g/dl, and platelet count of 26000. Initial history, exam, and investigations including a peripheral smear, coagulation panel, liver function, vitamin b12 and folate levels did not reveal possible etiology of pancytopenia. Given worsening pancytopenia, bone marrow biopsy was done which showed 58% promyelocytes and 11% blasts with numerous Auer rods present. Cytogenetics showed abnormal female karyotype with t(15:17) and FISH analysis revealed PML/RARA fusion in 76.5% of analyzed cells. The above findings were diagnostic of APL. After multidisciplinary discussion with high risk obstetrics physician, it was decided to immediately induce labor for immediate initiation of treatment of APL. She had a prolonged labor requiring aggressive blood product support and initiation of All trans retinoic acid (ATRA) before delivery given concerns of coagulopathy. Induction treatment with Arsenic trioxide (ATO) was started the day after her delivery. Repeat bone marrow biopsy on day 24 showed complete morphologic remission. Shortly thereafter, she started cycle 1 of consolidation with ATRA and arsenic trioxide. APL is characterized by a translocation between chromosome 15 and 17. Coagulopathy is a pathognomonic feature of this leukemia and often the reason for high mortality in early course of disease. APL when treated with ATRA and ATO, has excellent remission rate and 99% overall survival at 2 years. APL in pregnancy is associated with increased risk of preterm delivery, perinatal mortality, and miscarriage. Following pregnancy, there is an increased risk of bleeding, infection, or placental abruption. ATRA, one of the pillars around which treatment of APL revolves, is highly teratogenic during the first trimester and has low risk later in pregnancy. Treatment is directed by the trimester of pregnancy. Termination of pregnancy or treatment with single agent conventional chemotherapy is preferred in the first trimester whereas treatment with ATRA prior to delivery and use of chemotherapy after delivery is the preferred approach in the 2nd and 3rd trimester. This case is an example of individualized approach with a multidisciplinary team need in the setting of scarce data.

Merkel cell carcinoma (MCC) is a rare neuroendocrine malignancy of the skin that is highly aggressive and often metastasizes early. MCC is diagnosed based on histopathological findings and is most commonly treated with surgical resection, which may be accompanied by chemotherapy and/or radiation. This report describes a 55-year-old male with history of recurrent malignant melanoma of the right pinna and subsequent excision. Three years following the excision of melanoma, he presents with a lesion to the right forehead as well as a right-sided neck mass that were found to be metastatic Merkel cell carcinoma. Although there have been reports describing the development of second cancers following the treatment of MCC, the development of MCC after the treatment of other malignancies has not been well-described. Merkel cell carcinoma remains a highly aggressive and frequently metastatic malignancy that should not be overlooked, especially when developed after the diagnosis and treatment of other primary cutaneous malignancies such as melanoma.

Despite the recent advances in cancer therapy, the successful detections and treatments of cancer still remains a challenge. The existing strategies for early cancer detection are often limited due to their poor sensitivity and specificity. Also, the non-selective action of therapeutic interventions hinders treatment success. Our research was therefore directed towards the engineering of excellent bi-functionalized nanoparticles (NPs) based on carbon dots (CDs) that would improve early cancer detection and overcome the limitations of chemotherapy. With the actively targeting agent, these new NPs are expected to effectively deliver pharmacological agents directly to cancer cells. CDs are carbon-based NPs that are utilized as bioimaging agents and drug delivery systems (DDS) due to their excellent biocompatibility, non-toxicity, unique imaging, and facile surface modification. Using folic acid (FA) as targeting agent, the prepared novel CDs will carry doxorubicin (DOX) covalently and non-covalently to the cancer cells with overexpressed folate receptors. The CDs were first synthesized via the hydrothermal bottom-up approach using citric acid and ethylenediamine as precursors. The prepared CDs were then functionalized by FA via a non-cleavable peptide bond followed by complexation with DOX covalently or non-covalently to obtain the desired FA-CD-DOX NPs. All the NPs and intermediates were characterized using ultraviolet-visible spectroscopy (UV-vis), fluorescence spectroscopy (FL), and Fourier transform infrared spectroscopy (FTIR). Assessment of the drug loading capacity (DLC) and drug loading efficiency (DLE) with UV-vis indicated that the non-covalent NPs have low DLC but high DLE compared to the relatively low DLE and high DLC of covalent NPs. In vitro drug release studies were also carried out in phosphate buffered saline (PBS) systems with various pH. It was found that even though both non-covalent and covalent complexes released more DOX at pH 5.0 than at pH 7.0, the DOX release rate was faster in the non-covalent FA-CD-DOX NPs compared to the covalent FA-CD-DOX. Based on these results, we project increased accumulation of drugs in the more acidic (pH 4.5-5.0) microenvironment of cancer cells compared to that of normal healthy cells under physiological pH (7.4). This new FA-CD-DOX NPs could work as efficient theranostic systems to detect and treat cancer.

Indiana University-Purdue University Indianapolis (IUPUI) / Because colorectal cancer (CRC) is the fourth most common cancer and the second leading cause of cancer death in the US, identifying biomarkers that might inform disease prevention and early diagnosis is of great public health importance. Mitochondria are key cytoplasmic organelles containing an independent genome, i.e., mitochondrial DNA (mtDNA). It has been increasingly recognized that mtDNA copy number (mtDNAcn) is a biomarker for mitochondrial function and cellular oxidative stress. To date, the few studies that have assessed associations between mtDNAcn and CRC outcomes have yielded inconsistent findings. Further, no epidemiologic study has examined the relationship between insulinemic potential of lifestyle and mtDNAcn. Therefore, in this dissertation, three studies were conducted using data from the Nurses’ Health Study and the Health Professionals Follow-Up Study. First, the association between pre-diagnostic leukocyte mtDNAcn and CRC risk was studied in a nested casecontrol study (324 cases/658 controls). Lower mtDNAcn was significantly associated with increased risk of CRC and proximal colon cancer. That inverse association remained significant among individuals with ≥ 8 years’ follow-up since blood collection, suggesting that mtDNAcn might serve as a long-term predictor of CRC risk. Second, possible associations of pre-diagnostic mtDNAcn with overall and CRC-specific survival were examined among 587 CRC patients. MtDNAcn was not significantly associated with survival overall or in subgroups by cancer location, grade, or stage. Among current smokers, there was an inverse association between one standard deviation (SD) decrease in mtDNAcn and increased overall death risk. Among patients diagnosed at or before 70.5 years of age and those with anti-inflammatory diets, reduced mtDNAcn was associated with lower CRC-specific death risk. Lastly, the cross-sectional association between empirical lifestyle index for hyperinsulinemia (ELIH) and mtDNAcn was investigated among 2,835 subjects without major chronic diseases (cancers, diabetes, and cardiovascular diseases). A significant inverse association was found: least-squares means ± SD of mtDNAcn z-score decreased dramatically across ELIH quintiles. Overall, the findings from this dissertation will contribute to the evaluation of mtDNAcn as a potential biomarker for CRC risk and prognosis, and inform future interventions designed to reduce the insulinemic potential of lifestyle factors to preserve mitochondrial function. / 2022-04-06