Diffuse large B cell lymphoma (DLBCL) is the most common non-Hodgkin’s lymphoma. Patients who fail conventional therapy (~50%) have a poor prognosis and few treatment options. It is essential to understand the underlying biological processes, the progression of the disease, and utilize this information to develop new therapeutics. DLBCL patients with high C-MYC expression have a poor prognosis and new therapeutics for these patients are needed. This thesis describes work testing the hypothesis that JQ1, which can indirectly inhibit C-MYC in some tumors, can be used as an effective treatment for DLBCL. Some tumors have an unknown mechanism causing high C-MYC expression, leading me to investigate the underlying mechanisms. YY1 is a transcriptional regulator of c- Myc and has been implicated in DLBCL and as a potential regulator of the germinal center (GC) reaction. DLBCL arises from GC cells or post-GC cells. I tested the hypothesis that YY1 regulates the GC reaction. SMURF2 is an E3-ubiquitin ligase for YY1 and a tumor suppressor for DLBCL. I was interested in examining the mechanism underlying the suppression of DLBCL by SMURF2 leading to the hypothesis that SMURF2 regulates the GC. This thesis shows JQ1 leads to cell death and cellular senescence in human DLBCL cells. I conclude that BRD4 inhibition by JQ1 or derivatives could provide a new therapeutic avenue for DLBCL patients. I also show loss of YY1 perturbs the GC by decreasing the dark zone and increasing apoptosis. Finally I show modulation of SMURF2 does not affect the GC, suggesting SMURF2 utilizes a different mechanism to act as a tumor suppressor and may not modulate YY1 in the context of the GC.

Excess food consumption and/or lack of exercise have dramatically contributed to the prevalence of overweight (BMI≥25) and obesity (BMI≥30) in modern society. The obesity epidemic has been linked to the rise in type 2 diabetes. In recent years, evidence has pointed to a close association between mitochondrial dysfunction in white adipose tissue (WAT) and insulin resistance, a key feature of type 2 diabetes. In order to dissect the cause and effect relationship between WAT mitochondrial dysfunction and insulin resistance, we established an in vitro cell line system to investigate this issue. We artificially introduced mitochondrial dysfunction in 3T3-L1 adipocytes by depleting the mitochondrial transcription factor A (Tfam) during adipogenesis, without changing the overall adipocyte differentiation program. We found that these Tfam-depleted 3T3-L1 adipocytes showed symptoms of insulin resistance, evidenced by impaired insulin stimulated GLUT4 translocation and glucose uptake. This result suggested that mitochondrial dysfunction could be a primary contributor to insulin resistance in fat tissue. However, the exact mechanism underlying this finding remains unclear. As part of a comprehensive understanding of insulin signaling in fat cells, we also investigated the involvement of the endosomal protein WDFY2 in the regulation of Akt isoform-specific effect on glucose uptake. In 3T3-L1 adipocytes, both Akt1 and Akt2 isoforms are expressed, but only Akt2 plays an indispensible role in insulin-stimulated GLUT4 translocation and glucose uptake. Previous studies implied that endosomal proteins may take a part in determining Akt substrate specificity. Here we found that WDFY2 preferentially co-localized with Akt2 and that knockdown of WDFY2 inhibited insulin-stimulated glucose uptake in 3T3-L1 adipocytes, suggesting that endosomes are involved in this regulation. The effect of WDFY2 knockdown on insulin-stimulated glucose uptake worked through the down-regulation of Akt2, but not Akt1, protein level. We concluded that, endosomal protein WDFY2, by preferentially interacting with Akt2, regulates insulin signaling in glucose uptake in 3T3-L1 adipocytes. Our findings may help to develop specific therapeutic interventions for treatment of insulin resistance and type 2 diabetes.

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal malignancies in the United States, with an average five-year survival rate of just 6.7%. One unifying aspect of PDAC is mutational activation of the KRAS oncogene, which occurs in over 90% of PDAC. Therefore, inhibiting KRAS function is likely an effective therapeutic strategy for this disease, and current research in our lab and others is focused on identifying downstream effectors of KRAS signaling that may be therapeutic targets. miRNAs are powerful regulators of gene expression that can behave as oncogenes or tumor suppressors. Dysregulation of miRNA expression is commonly observed in human tumors, including PDAC. The mir-17~92 cluster of miRNAs is an established oncogene in a variety of tumor contexts, and members of the mir-17~92 cluster are upregulated in PDAC, but their role has not been explored in vivo. This dissertation encompasses two studies exploring the role of miRNAs in pancreatic tumorigenesis. In Chapter II, I demonstrate that deletion of the mir-17~92 cluster impairs PDAC precursor lesion formation and maintenance, and correlates with reduced ERK signaling in these lesions. mir-17~92 deficient tumors and cell lines are also less invasive, which I attribute to the loss of the miR-19 family of miRNAs. In Chapter III, I find that Dicer heterozygosity inhibits PDAC metastasis, and that this phenotype is attributable to an increased sensitivity to anoikis. Ongoing experiments will determine whether shifts in particular miRNA signatures between cell lines can be attributed to this phenotype. Together these findings illustrate the importance of miRNA biogenesis, and the mir-17~92 cluster in particular, in supporting PDAC development and progression.

Animals have evolved complicated and overlapping mechanisms to guard against the development of cancer and infection by pathogenic organisms. ARF, a potent tumor suppressor, positively regulates p53 by antagonizing p53’s negative regulator, MDM2, which in turn results in either apoptosis or cell cycle arrest. ARF also has p53-independent tumor suppressor activity. The CtBP transcriptional co-repressors promote cancer cell survival and migration/invasion. CtBP senses cellular metabolism via a regulatory dehydrogenase domain, and is a target for negative regulation by ARF. ARF targets CtBP to the proteasome for degradation, which results in the up regulation of proapoptotic BH3-only proteins, and p53-independent apoptosis. CtBP inhibition by ARF also up regulates PTEN, reducing cancer cell motility, making CtBP a potential therapeutic target in human cancer. The CtBP dehydrogenase substrate 4-methylthio-2-oxobutyric acid (MTOB) can act as a CtBP inhibitor at high concentrations, and is cytotoxic to cancer cells from a wide variety of tissues. MTOB induced apoptosis was independent of p53, and correlated with the de-repression of the pro-apoptotic CtBP repression target Bik. CtBP over-expression, or Bik silencing, rescued MTOB-induced cell death. MTOB did not induce apoptosis in mouse embryonic fibroblasts (MEFs), but was increasingly cytotoxic to immortalized and transformed MEFs, suggesting that CtBP inhibition may provide a suitable therapeutic index for cancer therapy. In human colon cancer cell peritoneal xenografts, MTOB treatment decreased tumor burden, and induced tumor cell apoptosis. To verify the potential utility of CtBP as a therapeutic target in human cancer the expression of CtBP and its negative regulator ARF was studied in a series of resected human colon adenocarcinomas. CtBP and ARF levels were inversely-correlated, with elevated CtBP levels (compared with adjacent normal tissue) observed in greater than 60% of specimens, with ARF absent in nearly all specimens exhibiting elevated CtBP levels. Targeting CtBP with a small molecule like MTOB may thus represent a useful and widely applicable therapeutic strategy in human malignancies. ARF has long been known to respond to virally encoded oncogenes. Recently, p14ARF was linked to the innate immune response to non-transforming viruses in mice. Therefore a wider role for the ARF pathway in viral infection was considered. Previous studies linking p53 to multiple points of the Human Immunodeficiency Virus-1 (HIV-1) life cycle suggested that ARF may also play a role in the HIV life cycle. In this study the interdependency of ARF and HIV infection was investigated. ARF expression was determined for a variety of cell types upon HIV infection. In every case, ARF levels exhibited dynamic changes upon HIV infection-in most cases ARF levels were reduced in infected cells. The impact of ARF over-expression or silencing by RNAi on HIV infection was also examined. Consistently, p24 levels were increased with ARF overexpression, and decreased when ARF was silenced. Thus ARF and HIV modulate each other, and ARF may paradoxically play a positive role in the HIV life cycle.

La vía PI3K/AKT/mTOR participa en múltiples procesos celulares fundamentales para la célula. Algunas mutaciones genéticas de los componentes de esta vía se han asociado a diversas enfermedades humanas: las más importantes son los carcinomas de mama, tiroides y endometrio, el glioblastoma multiforme, el cáncer de próstata y los linfomas. La vía canónica PI3K/AKT/mTOR se ha estudiado ampliamente en los últimos años. Sin embargo, el conocimiento de la complejidad de sus componentes principales y su interrelación con los elementos de otras vías va en aumento. Por ello, es importantes actualizar cada cierto tiempo la información disponible para la comprensión de este mecanismo. Así mismo, se están y se han desarrollado numerosos ensayos con medicinas selectivas en búsqueda de un tratamiento más inteligente para las enfermedades asociadas a alteraciones de esta vía. Por tanto, realizamos una revisión de esta vía de transducción con el objetivo de tener una visión cercana de su funcionamiento, sus alteraciones y enumerar algunas moléculas promisorias para ser utilizadas en futuros tratamientos. / The PI3K/AKT/mTOR pathway is involved in multiple cellular processes which are essential for the cells. Some genetic mutations of the components of this pathway have been associated with various human diseases, the most important of which are breast, thyroid and endometrium carcinomas; glioblastoma multiforme; prostate cancer and lymphomas. The PI3K/AKT/mTOR canonical pathway has been extensively studied in recent years. However, as the complexity of its main components and their correlation with the components of other pathways are increasing, it is important to update from time to time the available information to understand this mechanism. Furthermore, many trials have been conducted with selective medicines aimed to look for a more intelligent treatment for diseases associated with alterations in this pathway. Therefore, we review this transduction pathway to take a close look at its functioning and alterations, and to list some promising molecules for future treatments. / Revisión por pares

No description available.

No / Androgens stimulate many hair follicles to alter hair colour and size via the hair growth cycle; in androgenetic alopecia tiny, pale hairs gradually replace large, pigmented ones. Since stem cell factor (SCF) is important in embryonic melanocyte migration and maintaining adult rodent pigmentation, we investigated SCF/c-Kit signalling in human hair follicles to determine whether this was altered in androgenetic alopecia. Quantitative immunohistochemistry detected three melanocyte-lineage markers and c-Kit in four focus areas: the epidermis, infundibulum, hair bulb (where pigment is formed) and mid-follicle outer root sheath (ORS). Colocalisation confirmed melanocyte c-Kit expression; cultured follicular melanocytes also exhibited c-Kit. Few ORS cells expressed differentiated melanocyte markers or c-Kit, but NKI/beteb antibody, which also recognises early melanocyte-lineage antigens, identified fourfold more cells, confirmed by colocalisation. Occasional similar bulbar cells were seen. Melanocyte distribution, concentration and c-Kit expression were unaltered in balding follicles. Androgenetic alopecia cultured dermal papilla cells secreted less SCF, measured by ELISA, than normal cells. This identifies three types of melanocyte-lineage cells in human follicles. The c-Kit expression by dendritic, pigmenting, bulbar melanocytes and rounded, differentiated, non-pigmenting ORS melanocytes implicate SCF in maintaining pigmentation and migration into regenerating hair bulbs. Less differentiated, c-Kit-independent cells in the mid-follicle ORS stem cell niche and occasionally in the bulb, presumably a local reserve for long scalp hair growth, implicate other factors in activating stem cells. Androgens appear to reduce alopecia hair colour by inhibiting dermal papilla SCF production, impeding bulbar melanocyte pigmentation. These results may facilitate new treatments for hair colour changes in hirsutism, alopecia or greying.

No / Zinc oxide (ZnO) nanoparticles are the mostly used engineered metal oxide nanoparticles in consumer products. This has increased the likelihood of human exposure to this engineered nanoparticle (ENPs) through different routes. At present, the majority of the studies concerning ZnO ENPs toxicity have been conducted using in vitro and in vivo systems. In this study, for the first time we assessed the effect of ZnO ENPs on the major cellular pathways in the lymphocytes of healthy individuals as well as in susceptible patients suffering from lung cancer, chronic obstructive pulmonary disease (COPD) and asthma. Using the differential expression analysis, we observed a significant (P < 0.05) dose-dependent (10, 20 and 40 microg/ml for 6h) increase in the expression of tumour suppressor protein p53 (40, 60 and 110%); Ras p21 (30, 52 and 80%); c-Jun N-terminal kinases; JNKs) (28, 47 and 78%) in lung cancer patient samples treated with ZnO ENPs compared to healthy controls. A similar trend was also seen in COPD patient samples where a significant (P < 0.05) dose-dependent increase in the expression of tumour suppressor protein p53 (26, 45 and 84%), Ras p21 (21, 40 and 77%), JNKs (17, 32 and 69%) was observed after 6h of ZnO ENPs treatment at the aforesaid concentrations. However, the increase in the expression profile of tested protein was not significant in the asthma patients as compared to controls. Our results reiterate the concern about the safety of ZnO ENPs in consumer products and suggest the need for a complete risk assessment of any new ENPs before its use.

No / Vitiligo is characterized by a patchy loss of inherited skin color affecting approximately 0.5% of individuals of all races. Despite the absence of the protecting pigment and the overwhelming evidence for hydrogen peroxide (H(2)O(2))-induced oxidative stress in the entire epidermis of these patients, there is neither increased photodamage/skin aging nor a higher incidence for sun-induced nonmelanoma skin cancer. Here we demonstrate for the first time increased DNA damage via 8-oxoguanine in the skin and plasma in association with epidermal up-regulated phosphorylated/acetylated p53 and high levels of the p53 antagonist p76(MDM2). Short-patch base-excision repair via hOgg1, APE1, and polymerasebeta DNA repair is up-regulated. Overexpression of Bcl-2 and low caspase 3 and cytochrome c levels argue against increased apoptosis in this disease. Moreover, we show the presence of high epidermal peroxynitrite (ONOO(-)) levels via nitrotyrosine together with high nitrated p53 levels. We demonstrate by EMSA that nitration of p53 by ONOO(-) (300 x 10(-6) M) abrogates DNA binding, while H(2)O(2)-oxidized p53 (10(-3) M) enhances DNA binding capacity and prevents ONOO(-)-induced abrogation of DNA binding. Taken together, we add a novel reactive oxygen species to the list of oxidative stress inducers in vitiligo. Moreover, we propose up-regulated wild-type p53 together with p76(MDM2) as major players in the control of DNA damage/repair and prevention of photodamage and nonmelanoma skin cancer in vitiligo.

A síndrome de Li-Fraumeni (LFS) e sua variante like (LFL) são associadas a mutações germinativas no gene TP53 e predispõe ao alto risco para múltiplos tumores em idade jovem. Analisamos 91 famílias LFS/LFL do sul/sudeste do Brasil para mutações germinativas e haplótipos de TP53 (PIN2, PIN3 e PEX4) e MDM2 (309T-G). A mutação R337H ocorreu em 44,4% das famílias avaliadas. Em 750 controles da região a freqüência populacional da mutação foi 0,3%. A genotipagem de oito indivíduos não relacionados R337H-positivos para 29 TAG SNPs intragênicos demonstrou o mesmo haplótipo raro estabelecendo efeito fundador para R337H. O alelo duplicado no PIN3 apresenta impacto modificador e retardo de 17,1 anos na ocorrência de tumores em famílias com mutação no TP53, enquanto o SNP309 MDM2 modula a idade dos sarcomas de partes moles. / Li-Fraumeni syndrome (LFS) and its variant like (LFL) are associated with germline mutations in the TP53 gene and predispose to a variety of cancers at an earlier age. We analyzed 91 LFS/LFL families from southern Brazil for germline mutations in TP53 and polymorphisms in TP53 (PIN2, PIN3, PEX4) and MDM2 (309T-G). The germline TP53 mutation R337H was found in 44.4% of all families included. In 750 controls from the same region, mutation prevalence was 0.3%. Genotyping of eight unrelated R337H-positive individuals for 29 intragenic TAG SNPs showed that they all shared the same rare haplotype confirming the founder effect for the mutation. Duplication of PIN3 had a modifier effect on the age of tumor onset (delay of 17.1 years) in TP53 mutation carriers whereas MDM2 SNP309 modulated age of onset for soft-tissue sarcomas.