*1-Dimensional Advective-Diffusion Model in Porous Media Infusion of therapeutic agents into tissue is makes use of two mass transport modes: advective transport, and molecular diffusion. Bulk infusion into a 0.6% wt agarose phantom was modeled as an infinite, homogenous, and isotropic porous medium saturated with the same solvent used in the infused dye tracer. The source is assumed to be spherical and isotropic with constant flow rate and concentration. The Peclet numberdecreases with power function Pe = 15762t0.337 due to the decrease in mean dye-front pore velocity as V goes to Vfinal. Diffusive mass transport does not become significant during any relevent time period. *Arborizing Fiberoptic Microneedle Catheter We have developed an arborizing catheter that allows multiple slender fused-silica CED cannulae to be deployed within a target volume of the brain via a single needle tract, and tested it in a widely accepted tissue phantom. The arborizing catheter was constructed by bonding and encapsulating seven slender PEEK tubes in a radially symmetric bundle with a progressive helical angle along the length, then grinding a conicle tip where the helical angle is greatest. The catheter was tested by casting 0.6% wt agarose around the device with all needles deployed to a tip-to-tip distance of 4 mm. Phantom temperature was maintained at 26 ± 2°C. 5% wt Indigo Carmine dye was infused at a rate of 0.3 uL/min/needle for 4 hours. N=4 infusions showed a Vd/Vi of 139.774, with a standard deviation of 45.01. This is an order of magnitude greater than single-needle infusions under similar conditions [45]. The arborizer showed the additional benefit of arresting reflux propagating up the lengths of individual needles, which has historically been a weakness of single-needle CED catheter designs. *In Vivo Co-Delivery of Single Walled Carbon Nano-horns and Laser Light to Treat Human Transitional Cell Carcinoma of the Urinary Bladder in a Rodent Model Using a rodent model we explored a treatment method for Transitional Cell Carcinoma (TCC) in the urinary bladder in which Single Walled Carbon Nanohorn (SWNH) solution and 1064 nm laser light are delivered into tumorous tissue via a co-delivery Fiberoptic Microneedle Device (FMD). Preliminary treatment parameters were determined by injecting SWNH solutions with concentrations of 0 mg/mL, 0.17 mg/mL, or 0.255 mg/mL into ex vivo porcine skin and irradiating each for three minutes at laser powers of 500 mW, or 1000 mW. The combination with the greatest temperature increase without burning the tissue, 0.17 mg/mL at 1000 mW, was selected for the in vivo treatment. TCC tumors were induced in a rodent model by injecting a solution of 106 AY27 urothelial carcinoma cells into the lateral aspect of the left hind leg of young, female F344 rats. When tumors reached 5-10 mm3, rats were anesthitized and treated. SWNH solution was injected directly into the tumor and irradiated until the target temperature of 60degC was achieved. The rats were then recovered from anestesia and monitored for 7-14 days, at which point they were humanely sacrificed, and the tumors prepared for histological examination. Histological assessment of areas of FMD treatment correlated well with gross morphological appearance. Foci of tumor necrosis showed sharp (1-2 mm) delineation from areas of viable tumor (not treated) and normal tissue. We believe we have demonstrated the feasibility of using the FMD for treatment of urothelial carcinoma using an animal model of this disease, and are encouraged to continue development of this treatment and testing in larger animal models. / Master of Science

Glioma, the tumor of glial cells, is one of the common types of primary central nervous system (CNS) neoplasms. Astrocytoma is the most common of all gliomas and originates from astrocytic glial cells. Astrocytoma tumors belong to two main categories: benign tumors, comprising of grade I Pilocytic astrocytoma and malignant tumors which diffusely infiltrate throughout the brain parenchyma. Diffusely infiltrating astrocytomas are graded into diffuse astrocytoma (DA; grade II), anaplastic astrocytoma (AA; grade III) and glioblastoma (GBM; grade IV) in the order of increasing malignancy. Patients with grade II astrocytoma have a median survival time of 6 to 8 years after surgical intervention. While the more aggressive grade III (AA) and grade IV (GBM) are together called malignant astrocytomas, the treatment protocols and length of survival are distinctly different between these grades. The median survival time for grade III patients is 2 to 3 years whereas patients with grade IV have a median survival of 12-15 months. GBMs have been further divided into primary GBM and secondary GBM on the basis of clinical and histopathological criteria. Primary GBM presents in an acute de novo manner with no evidence of an antecedent lower grade tumor and it accounts for >90% of all GBMs. In contrast, secondary GBM results from the progressive malignant transformation of a grade II or grade III astrocytoma. The current WHO grading system of astrocytomas is based on the histopathological characteristics of the underlying tumor tissue. Diagnoses by pathologists are dependent on specific histologic features: increased mitosis, nuclear atypia, microvascular proliferation and/or necrosis, which associate with biologically aggressive behaviour (WHO 2007). Though grading based on histology is largely reproducible and well accepted, subjectivity involved and substantial disagreement between pathologists has remained a major concern. Because of inherent sampling problems (mainly due to tumor location in the brain) and inadequate sample size available for histological evaluation, there exists a very high possibility of error in grading. Recent studies have attempted to characterize the molecular basis for the histological and prognostic differences between grade III and grade IV astrocytoma. While reports have shown the grade specific profile of gene expression, there is no molecular signature that can accurately classify grade III and grade IV astrocytoma samples. In the current work, we have identified molecular signatures for the accurate classification of grade III and grade IV astrocytoma patients by using transcriptome and miRNome data. The receptor tyrosine kinase pathway is known to be overexpressed in 88% of glioblastoma patients. The expression and activation of the receptors is reported to be deregulated by events like amplification and activating mutations. The aberrant expression of RTKs could also be due to the deregulation of miRNAs, which, in the untransformed astrocytes regulate and fine-tune the levels of the RTKs. In the current study, we have identified that tumor suppressor miRNA miR-219-5p regulates RTK pathway by targeting EGFR and PDGFRα. Part I. Transcriptome approach: Identification of a 16-gene signature for classification of malignant astrocytomas In order to obtain a more robust molecular classifier to accurately classify grade III and grade IV astrocytoma samples, we used transcriptome data from microarray study previously performed in our laboratory. The differential regulation of 175 genes identified from microarray was validated in a cohort of grade III and grade IV patients by real-time qRT-PCR. In order to identify the classification signature that can classify grade III and grade IV astrocytoma samples, we used the expression data of 175 genes for performing Prediction Analysis of Microarrays (PAM) in the training set of grade III and grade IV astrocytoma samples. PAM analysis identified the most discriminatory 16-gene expression signature for the classification of grade III and grade IV astrocytoma. The Principal Component Analysis (PCA) of 16-genes astrocytoma patient samples revealed that the expression of 16-genes could classify grade III and grade IV astrocytoma samples into two separate clusters. In the training set, the 16-gene signature was able to classify grade III and grade IV patients with an accuracy rate of 87.9% as tested by additional analysis of Cross-Validated probability by PAM. The 16-gene signature obtained in the training set was validated in the test set with diagnostic accuracy of 89%. We further validated the 16-gene signature in three independent cohorts of patient samples from publicly available databases: GSE1993, GSE4422 and TCGA datasets and the classification signature got validated with accuracy rates of 88%, 92% and 99% respectively. To address the discordance in grading between 16-gene signature and histopathology, we looked at the clinical features (age and survival) and molecular markers (CDKN2A loss, EGFR amplification and p53 mutation) that differ substantially between grade III and grade IV in discordant grade III and grade IV samples. The grading done by 16-gene signature correlated with known clinical and molecular markers that distinguish grade III and grade IV proving the utility of the 16-gene signature in the molecular classification of grade III and grade IV. In order to identify the pathways that 16 genes of the classification signature could regulate, we performed protein-protein interaction network and subsequently pathway analysis. The pathways with highest significance were ECM (extracellular matrix) and focal adhesion pathways, which are known to be involved in the epithelial to mesenchymal transition (EMT), correlating well with the aggressive infiltration of grade IV tumors. In addition to accurately classifying the grade III and grade IV samples, the 16-gene signature also demonstrated that genes involved in epithelial-mesenchymal transition play key role in distinguishing grade III and grade IV astrocytoma samples. Part II. miRNome approach microRNAs (miRNAs) have emerged as one of the important regulators of the interaction network that controls various cellular processes. miRNAs are short non-coding RNAs (mature RNA being 21-22nt long) that regulate the target mRNA by binding mostly in the 3’ UTR bringing about either translational repression or degradation of the target. miRNAs are shown to play key roles in cell survival, proliferation, apoptosis, migration, invasion and various other characteristic features that get altered in human cancers. miRNAs are characterized to have oncogenic or tumor suppressor role and the aberrant expression of miRNAs is reported in multiple human cancer types. Part A. Genome-wide expression profiling identifies deregulated miRNAs in malignant astrocytoma With an aim to identify the role of miRNAs in the development of in malignant astrocytoma, we performed a large-scale, genome-wide microRNA (miRNA) (n=756) expression profiling of 26 grade IV astrocytoma, 13 grade III astrocytoma and 7 normal brain samples. Using Significance Analysis of Microarrays (SAM), we identified several differentially regulated miRNAs between control normal brain and malignant astrocytoma, grade III and grade IV astrocytoma, grade III astrocytoma and grade IV secondary GBM, progressive pathway and de novo pathway of GBM development and also between primary and secondary GBM. Importantly, we identified a most discriminatory 23-miRNA expression signature, by using PAM, which precisely distinguished grade III from grade IV astrocytoma samples with an accuracy of 90%. We re-evaluated the grading of discordant samples by histopathology and identified that one of the discordant grade III samples had areas of necrosis and it was reclassified as grade IV GBM. Similarly, out of two discordant grade IV samples, one sample had oligo component and it was reclassified as grade III mixed oligoastrocytoma. Thus, after the revised grading, the prediction accuracy increased from 90% to 95%. The differential expression pattern of nine miRNAs was further validated by real-time RT-PCR in an independent set of malignant astrocytomas (n=72) and normal samples (n=7). Inhibition of two glioblastoma-upregulatedmiRNAs (miR-21 and miR-23a) and exogenous overexpression of two glioblastoma-downregulatedmiRNAs (miR-218 and miR-219-5p) resulted in reduced soft agar colony formation but showed varying effects on cell proliferation and chemosensitivity. Thus, we have identified the grade specific expression of miRNAs in malignant astrocytoma and identified a miRNA expression signature to classify grade III astrocytoma from grade IV glioblastoma. In addition, we have demonstrated the functional relevance of miRNA modulation and thus showed the miRNA involvement and their importance in astrocytoma development. Part B. miR-219-5p inhibits the receptor tyrosine kinase pathway by targeting mitogenic receptor kinases in glioblastoma The receptor tyrosine kinase (RTK) pathway, being one of the important growth promoting pathways, is known to be deregulated in 88% of the patients with glioblastoma. In order to understand the role of miRNAs in regulating the RTK pathway, we undertook a screening procedure to identify the potential miRNAs that could target different members of the RTK pathway. From the screening study involving bioinformatical prediction of miRNAs and subsequent experimental validation by modulation of miRNA levels in glioma cell lines, we identified miR-219-5p as a candidate miRNA. The overexpression of miR-219-5p reduced the protein levels of both EGFR and PDGFRα. We confirmed the binding of miR-219-5p to the 3’ UTRs by using reporter plasmids. We also confirmed the specificity of miR-219-5p binding sites in the 3’ UTR of EGFR by site directed mutagenesis of binding sites which abrogated the miRNA-UTR interaction. The expression of miR-219-5p was significantly downregulated in grade III as well as in grade IV astrocytoma samples in the miRNA microarray experiment and we further validated the downregulation in an independent cohort of grade III and grade IV astrocytoma patients by real-time qRT-PCR. The ectopic overexpression of miR-219-5p in glioma cell lines inhibited cell proliferation, colony formation, anchorage independent growth and the migration of glioma cells. In addition, overexpression of miR-219-5p decreased MAPK and PI3K pathways, in concordance with its ability to target EGFR and PDGFRα. Additionally, for the further characterization of miR-219-5p – EGFR interaction and its effect on MAPK and PI3K pathways, we used U87 glioma cells that stably overexpress wild-type EGFR and constitutively active ΔEGFR (both lacking 3’-UTR and thus being insensitive to miR-219-5p overexpression) along with U87 parental cells. In these cell lines with the overexpression of EGFR lacking 3’-UTR, miR-219-5p was unable to inhibit - MAPK and PI3K pathways and also glioma cell migration suggesting that these effects were indeed because of its ability to target EGFR. Further, in the glioblastoma patient cohort (TCGA dataset), we found significant negative correlation between EGFR protein levels, both total EGFR and phospho EGFR and miR-219-5p levels in the glioblastoma tissue samples suggesting a role of miR-219-5p in increasing the protein levels of EGFR in glioblastoma. In summary, we have identified and characterized miR-219-5p as the RTK regulating tumor suppressor miRNA in glioblastoma.

Insulin-like growth factor binding proteins (IGFBPs) modulate the bioavailability of IGFs in circulation. IGFBPs 1-6 bind IGFs with high affinity and can either potentiate or inhibit IGF signaling in a context dependent manner. IGFBP2 is a 36 kDa protein and the second most abundant IGFBP in serum. Numerous studies in the recent past have implied a pro-tumorigenic role of IGFBP2. Elevated expression of IGFBP2 has been observed in multiple malignancies, including glioblastoma multiforme (GBM), ovarian, pancreatic, gastric, prostate, colon, breast, thyroid cancer and leukemia. In addition, increased expression of IGFBP2 in both tissues and serum of patients has been correlated with poor prognosis in prostate, glioblastoma and colon cancers. Pro-tumorigenic actions of IGFBP2 have been supported by in vitro studies, where IGFBP2 increases the tumorigenic potential of adrenocortical tumor cells, epidermoid carcinoma cells, glioma cells and ovarian cancer cells. Further, using xenograft animal models, the role of IGFBP2 in the progression of glioma has been established. In breast cancer, IGFBP2 was found to be over expressed in ductal carcinoma in situ and invasive breast cancer samples. IGFBP2 over expression has been shown to confer drug resistance and an increased expression has been reported to correlate with lymph node metastasis in T1 breast carcinomas. These reports implicate IGFBP2 in breast cancer biology. However, its role in breast cancer progression is not well defined. With this background, the following objectives were set for the current study: Functional characterization of IGFBP2 with respect to its possible role in breast cancer progression. Elucidation of the molecular mechanisms of IGFBP2 actions. Towards this, immunohistochemistry was performed on 132 invasive ductal carcinoma (IDC) grade III tumors using IGFBP2 specific antibody. It was observed that IGFBP2 expression was significantly higher in tumors in comparison to normal tissues that showed no detectable staining for IGFBP2. It was also observed that expression of IGFBP2 significantly correlated with the expression of ER. To understand the functional significance of IGFBP2 over expression in breast cancer, IGFBP2 was characterized with respect to proliferation, survival and tumor forming ability (in vitro and in vivo) in BT474 breast cancer cells. The knockdown of IGFBP2 expression resulted in suppression of colony formation (nearly 70%) in these breast cancer cells, which could be partially reversed upon exogenous addition of IGFBP2 protein. Proliferation assays using stable clones with knockdown of IGFBP2 in BT474 cells showed a significant decrease in proliferation as compared to vector transfected cells in the presence of serum. Culturing of IGFBP2 knockdown breast cancer cells in serum free medium resulted in their growth arrest in G0/G1 phase of cell cycle as compared to control cells, which progressed through the cell cycle. Prolonged culturing of IGFBP2 knockdown cells in serum free condition (up to 72 h) resulted in the increase of cells in sub G1 phase of the cell cycle. Prolonged depletion of growth factors (serum free conditions) could result in apoptosis of these G1 arrested IGFBP2 knockdown cells. When serum starved IGFBP2 knockdown cells were treated with IGFBP2 protein, the cells arrested in G0/G1 phase were able to progress through the cell cycle and concomitant decrease in sub G1 fraction was observed. Knockdown of IGFBP2 resulted in significantly decreased number and visibly smaller colonies in anchorage independent conditions in vitro. Consistent with this observation, in vivo tumor xenograft formation with IGFBP2 knockdown cells also showed significant reduction in tumor weight as compared to vector generated tumors. These results imply that IGFBP2 has potent growth promoting effects on breast cancer and acts as a mitogen/survival factor for breast cancer cells. To elucidate the molecular mechanisms underlying the pro-tumorigenic effects of IGFBP2, the transcriptome profile following IGFBP2 perturbation in breast cancer cells was determined. IGFBP2 knockdown resulted in significant changes in the expression of genes associated with cellular proliferation and tumorigenicity. The down regulated genes were found to be associated with several events, notably cell cycle, p53 and Wnt signaling, as revealed by Gene Set Enrichment Analysis (GSEA). To further validate these results in breast cancer tissues, whole genome expression analysis was performed in 19 breast tumor samples which were categorized as IGFBP2 positive or negative based on immunohistochemical staining pattern. In comparison to IGFBP2 negative tumors, IGFBP2 positive tumors showed increased expression of genes belonging to MAPK, focal adhesion and Wnt signaling pathway. In order to identify the genes commonly regulated by IGFBP2 in cell lines and tumors, the gene expression profiles of IGFBP2 positive versus IGFBP2 negative tumors and IGFBP2 knockdown breast cancer cells were compared. 347 genes were found to be common among IGFBP2 regulated genes in tumors and cell line. The most significant networks representing the web of interactions among these genes were found to be associated with cellular growth and proliferation, cellular movement and nucleic acid metabolism, indicating an association of IGFBP2 expression phenotype to the distinct changes in expression of genes associated with the regulation of cellular growth and migration. Silencing of IGFBP2 in BT474 cells resulted in a reduced IGF signaling as evidenced by the reduced phosphorylation of IGF1R and concomitantly that of ERK. This effect could be reversed upon addition of the IGFBP2 protein, implying that IGFBP2 potentiates IGF signaling in breast cancer cells. Besides IGF ligand and their receptors, regulation of proliferation associated genes like CENPF, TOP2A, CCND1 and FOXM1 by IGFBP2 was observed, thus providing a molecular basis for the pro-proliferative effects of IGFBP2 on breast cancer cells. Addition of IGFBP2 to immortal breast cells resulted in reduced IGF1R signaling and reduced pERK and pAKT signaling. Additionally, the genes involved in cellular proliferation were down regulated upon IGFBP2 treatment in immortal cells. IGFBP2 knockdown clones had reduced expression of FOXM1, a key regulator of cell cycle for G1/S and G2/M transition, and M phase progression. The regulation of CENPF and CCND1 genes was established following over expression of FOXM1 in IGFBP2 knockdown cells. One of the important and novel finding of this study is the regulation of Wnt signaling pathway genes such as CCND1, MMP7, FGF18, MYCBP, FN1 and survivin by IGFBP2. In support of this, β-catenin protein was found to be regulated by IGFBP2 in breast cancer and GBM cells, as evidenced by knockdown and over expression studies. Furthermore, regulation of β-catenin by IGFBP2 was found to involve integrin-FAK and IGF1R signaling. Another important finding of this study is the correlation of IGFBP2 over expression with elevated β-catenin levels in breast tumors. When expression of both IGFBP2 and β-catenin was correlated with the lymph node status of breast cancers, a significant association of IGFBP2 and β-catenin staining with increased lymph node metastasis was observed in comparison with tumors that did not show staining for either protein. Altogether, in this study employing genomic, cellular and molecular approaches, a pro- tumorigenic role for IGFBP2 in breast cancer has been established. Furthermore, this study provides novel insights into the molecular mechanisms employed by IGFBP2 involving IGF1R, FAK and Wnt signaling pathways during breast cancer progression.

Bakgrund: Svårläkta sår är ett växande problem då vi lever allt längre och de är vanligare hos äldre personer. Ett svårläkt sår är ett sår som inte läkt inom en sex veckors period. Det finns flera bakomliggande orsaker till uppkomsten av denna typ av sår, malignitet och venös insufficiens är två exempel. Sjuksköterskan bör ha evidensbaserad kunskap för att kunna ge en god omvårdnad till de patienter som lider av ett svårläkt sår. Syfte: Syftet med litteraturöversikten är att beskriva patienters upplevelser av att leva med maligna tumörsår eller venösa bensår. Metod: En litteraturöversikt har genomförts med hjälp av Fribergs metod. Samtliga tio vetenskapliga artiklar som använts i litteraturöversikten har använt sig av kvalitativ metod. Databaserna som använts vid datainsamlingen är Academic Search Complete, Cinahl Complete och PubMed. Resultat: Resultatet sammanfattas i fyra huvudteman: Upplevelsen av symtom, En förändrad identitet, Social isolering och Att leva med ett svårläkt sår. Det var vanligt bland deltagarna att såren hade en negativ inverkan på deras liv och orsakade dem en förändrad kroppsuppfattning. Symtomen upplevdes besvärande och orsakade dem oro, ångest och skam över den egna kroppen, som ledde till att de isolerade sig från omgivningen. Deltagarna använde sig av olika coping-strategier för att hantera sin situation och klara av det dagliga livet. Diskussion: Resultatet diskuterades utifrån Jocalyn Lawlers somologiska teori som beskriver hur en patient kan uppleva en omvårdnadssituation och hur sjuksköterskan kan främja ett vårdande möte genom att se hela patienten. / Background: Chronic wounds are an increasing problem in society because we live longer, and the problem is more common when we get older. The criteria of a chronic wound are that it has not healed within six weeks. There are several underlying reasons for this type of wounds; malignancy and venous insufficiency are two examples. A nurse should have evidence based knowledge to be able to provide good care for patients suffering from this type of wounds. Aim: The aim of the study was to describe the patients’ experience of living with malignant wounds or venous leg ulcers. Method: A literature study has been carried out using Friberg’s method. Ten scientific articles used in the literature study were qualitative. The databases that were used in data collection were Academic Search Complete, Cinahl Complete and PubMed. Results: The results are summarized into four main themes: Perception of symptoms, A changed identity, Social isolation and Live with a chronic wound. The study showed that the wounds had a negative impact on the patients’ lives and caused them an altered body image. The symptoms were experienced as bothersome and caused anxiety, fear and shame. That led to social isolation. Participants used a variety of coping strategies to manage their situation to manage with their daily life. Discussion: The results were analyzed on the basis of Jocalyn Lawler’s somology theory that describes how a patient may experience a nursing situation and how the nurse can promote a caring encounter when seeing the whole patient.

INTRODUÇÃO: Os gliomas são tumores neuroepiteliais e correspondem a aproximadamente 24,6% de todos os tumores primários cerebrais. Na última década, grande esforço tem sido feito em meio acadêmico para caracterização molecular dos gliomas na tentativa de descrever comportamento clínico, definir prognóstico e predizer resposta terapêutica. Ferramentas de bioinformática estimam que os microRNAs possam regular cerca de 60% dos genes humanos, incluindo um número significativo de oncogenes, genes supressores tumorais e genes relacionados a quimio e radioressistência. Uma problemática atual na prática clínica é a ausência de ferramentas moleculares para predizer a evolução dos gliomas classificados como baixo grau, uma vez que, durante o seguimento clínico-radiológico pós-cirúrgico, esses pacientes podem ter recidiva tumoral e confirmação histopatológica de glioma de alto grau. A transformação tumoral em glioma de alto grau implica em pior prognóstico e redução das possibilidades terapêuticas. OBJETIVOS: O objetivo deste trabalho é analisar a expressão dos microRNAs miR-124a, miR-138, miR-155, miR-1275 em amostras de tumor primário humano e sangue periférico de pacientes com diagnóstico de gliomas de baixo e alto graus (astrocitoma grau I,astrocitoma grau II, astrocitoma grau III, glioblastoma, oligodendroglioma grau II e oligodendroglioma grau III). PACIENTES E MÉTODOS: As análises da expressão dos microRNAs foram realizadas utilizando-se a técnica de PCR em tempo real. Foram analisados 65 pacientes adultos (entre 18 e 65 anos de idade) com diagnóstico histopatológico confirmado de glioma com material biológico tumoral criopreservado armazenado junto ao Banco de Tumores do Sistema Nervoso Central do HCFMRP. Foram coletadas informações contidas no prontuário médico referentes às características clínicas, epidemiológicas, de evolução clínica e radiológica e tempo para recidiva e óbito. Considerando-se um nível de significância de 5%, a associação das variáveis qualitativas categóricas e expressão de microRNAs foi realizada pelo teste de Mann-Whitney. A análise de sobrevida foi realizada pelo método não-paramétrico de Kaplan-Meier. RESULTADOS: Os microRNAs em estudo não apresentaram hiperexpressão em tecido tumoral de pacientes diagnosticados com Glioblastoma. Apenas o miR-1275 apresentou expressão aumentada em pacientes com diagnóstico de astrocitoma pilocítico e oligodendroglioma grau II. Além disso, tumores de linhagem oligodendroglial, de baixo e alto graus, demonstraram hiperexpressão do miR-1275. Em sangue periférico, observou-se significativa hipoexpressão dos miR-1275, miR-124 e miR-138 em amostra de glioblastoma. Observou-se, ainda, acentuada hipoexpressão do miR-138 em amostras de oligodendroglioma grau III. CONCLUSÃO: Os microRNAs miR-124a, miR-138, miR-155 e miR-1275 não apresentaram hiperexpressão em tecido tumoral de pacientes diagnosticados com GBM. O miR-1275 apresentou expressão aumentada em pacientes com diagnóstico de astrocitoma pilocítico e o miR-155 foi hiperexpresso apenas em amostras de oligodendroglioma grau II. Além disso, tumores de linhagem oligodendroglial, de baixo e alto graus, demonstraram hiperexpressão do miR1275 e miR-124a. Em sangue periférico, observou-se significativa hipoexpressão dos miR-1275, miR-124 e miR-138 em amostra de glioblastoma assim como acentuada hipoexpressão do miR-138 em amostras de oligodendroglioma grau III / INTRODUCTION: Gliomas are neuroepithelial tumors and correspond to approximately 24.6% of all primary brain tumors. In the last decade, great effort has been made in academic circles to characterize gliomas in an attempt to describe clinical behavior, define prognosis and predict therapeutic response. Bioinformatics tools estimate that microRNAs can regulate about 60% of human genes, including a significant number of oncogenes, tumor suppressor genes, and chemo and radioresistance genes. A current problem in clinical practice is the absence of molecular tools to predict the evolution of gliomas classified as low grade, since during postoperative radiological follow-up these patients may have tumor recurrence with histopathological confirmation of high glioma degree. Tumor transformation in high-grade glioma has a worsening of prognosis and reduction of therapeutic possibilities OBJECTIVES: The objective of this project is to analyze the expression of miR124a, miR-138, miR-155, miR-1275 in human primary and peripheral blood samples from patients diagnosed with low and high grade gliomas (astrocytoma grade I, astrocytoma grade II, astrocytoma grade III, glioblastoma, oligodendroglioma grade II and oligodendroglioma grade III). PATIENTS AND METHODS: MicroRNA expression analyzes were performed using the real-time PCR technique. Sixty-five adult patients (18-65 years of age) with confirmed histopathological diagnosis of glioma with cryopreserved tumor biological material stored at the Bank of Tumors of the Central Nervous System of HCFMRP were analyzed. Information collected in the medical records regarding clinical, epidemiological, clinical and radiological characteristics and time for recurrence and death were collected. Considering a level of significance of 5%, the association of categorical qualitative variables and microRNA expression were performed by the Mann-Whitney test. Survival analysis was performed using the Kaplan-Meier non-parametric method. RESULTS: The microRNAs under study did not present hyperexpression in tumor tissue of patients diagnosed with glioblastoma. Only miR-1275 showed increased expression in patients diagnosed with pilocytic astrocytoma and grade II oligodendroglioma. In addition, tumors of low and high grade oligodendroglial lineage demonstrated overexpression of miR-1275. In peripheral blood, significant hypoexpression of miR-1275, miR-124 and miR-138 were observed in a glioblastoma sample. There was also a marked hypoexpression of miR-138 in samples of grade III oligodendroglioma. CONCLUSION: The microRNAs miR-124a, miR-138, miR155 and miR-1275 did not show hyperexpression in tumor tissue of patients diagnosed with GBM. MiR-1275 showed increased expression in patients diagnosed with pilocytic astrocytoma and miR-155 was hyperexpressed only in samples of grade II oligodendroglioma. In addition, tumors of oligodendroglial lineage, of low and high grades, demonstrated hyperexpression of miR-1275 and miR-124a. In peripheral blood, significant hypoexpression of miR-1275, miR-124 and miR-138 were observed in the GBM sample as well as marked hypoexpression of miR-138 in samples of grade III oligodendroglioma

Nos últimos anos, o desenvolvimento da computação tem auxiliado profissionais da saúde no tratamento, prevenção e diagnóstico de doenças. Um dos grandes desafios do campo tem sido o desenvolvimento de métodos para a discriminação do câncer de pele do tipo melanoma maligno em relação à outras lesões benignas. Para tal, pesquisadores usam técnicas de processamento e análise de imagens, explorando métricas baseadas na regra ABCD, para o desenvolvimento de métodos de diagnóstico de melanoma através de imagens. Enquanto diversos estudos abordam a coloração e textura do melanoma, um tratamento sistemático da irregularidade dos melanomas ainda não foi relatado. O presente trabalho traz um estudo dos fatores que influenciam a discriminação de lesões melanocíticas malignas e benignas, tomando como referência a assimetria das lesões e as irregularidades contidas em sua borda. Foram coletadas 143 imagens de casos clínicos de melanoma maligno, nevos regulares e nevos atípicos. Após tratamentos iniciais das imagens e posterior segmentação das lesões, extraiu-se 52 métricas referentes ao propósito do trabalho. A visualização da projeção LDA das três categorias revelou boa discriminação entre as categorias melanoma com relação as demais, reforçando a premissa original da acentuada irregularidade dos melanomas. Este resultado foi confirmado pela validação cruzada da projeção, com acertos da ordem de 75% para o grupo melanoma e 54% e 40% para os respectivos grupos nevo regular e nevo atípico. Deste resultado prevê-se uma das aplicações do sistema, na discriminação geral entre melanoma dos demais nevos. Para tal, uniu-se os grupos benignos em uma única categoria para a validação cruzada, gerando cálculos de sensibilidade e especificidade da ordem de 90% e 73% respectivamente. Outro importante resultado foi a comparação desses valores com as informações sobre o diâmetro das lesões. Conclui-se que ambas medidas (borda e tamanho) são igualmente relevantes no diagnóstico do melanoma, reflexo da própria patologia do melanoma, na qual acentuam-se ambas características em relação as demais lesões. Porém, a junção de ambas informações num único processamento não melhora a qualidade do diagnóstico, que nos permite prever que possa ser mais vantajoso proceder duas validações distintas com pesos iguais para o diagnóstico final. Por fim, um teste realizado com clínicos gerais e especialistas em melanoma revelou uma importante contribuição do método no auxílio de triagens ambulatoriais de casos suspeitos, principalmente para médicos com baixa ou nenhuma experiência em diagnóstico de melanoma. / Recent development in computer science have helped health professionals in the heath area in the treatment, prevention and diagnostic of illnesses. One of the leading challenges in this field has been the development of methods for the skin cancer discrimination between the types of malignant melanoma in relation to other benign lesions. For this, researchers have been using techniques of image processing and analysis, exploring metrics based on the ABCD rule, to the development of methods of diagnostic of melanoma through images. While plenty of studies are about the color and texture of the melanoma, a systematic treatment of the irregularity of the melanoma has not been reported yet. The present work presents a study of the factors which influence the discrimination among the malignant melanocytic and benign lesions, having as a reference the asymmetry of the lesions and the irregularities in their edges. It was collected 143 images of clinic cases of malignant melanoma, regular nevus and atypical nevus. After initial treatment of the images, followed by a segmentation of the lesions, it was extracted 52 metrics referent to the purpose of this study. The visualization of the projection LDA in three categories revealed a good discrimination among the categories of melanoma in relation to the others, reinforcing the original premise of the melanomas´ sharp irregularity. This result was confirmed by the cross-validation of the projection, with successes of 75 % to the melanoma group and 54 %-40 % to the regular nevus group and atypical nevus group, respectively. This result predicts one of the applications of the system in the general discrimination between the melanoma and the other nevus. In this regard, it was joined together the benign groups in one category to the cross-validation, generating calculations of sensibility and specificity of 90 % and 73 % respectively. Another important result was the comparison among these metrics with the information about the diameter of the lesions. It was concluded that both measurements (edges and size) are equally relevant in the diagnostic of the melanoma, reflection of the melanoma´s own pathology, in which both characteristics are enhanced in relation to the other lesions. However, the combination of both measurement in only one processing does not improve the quality of the diagnostic, which allows the prediction that it can be more advantageous to produce two distinct validations with the same weighs to the final diagnostic. To sum up, an accomplished test with doctors who are general practioner and specialists in melanoma revealed a significant contribution in the method to help in the triage of medical consultations in suspicious cases, mainly to doctors with little or no experience in the diagnostic of melanoma.

O exame de tomografia computadorizada é um grande aliado na elaboração do diagnóstico, planejamento do tratamento e proservação de lesões malignas do complexo maxilomandibular. Em muitos casos é considerado o exame de eleição para estes fins. Um dos problemas que ocorrem com a TC, é o aparecimento de artefatos devido a restaurações metálicas, implantes osseointegrados ou dispositivos metálicos de fixação óssea. Estes artefatos atrapalham a interpretação das imagens, dificultando a visualização de lesões. O propósito deste estudo foi avaliar imagens de TC com seções de cortes axiais e coronais e determinar se o tipo de seção pode minimizar o problema dos artefatos metálicos dentários. Para isto, dois avaliadores calibrados analisaram 72 imagens (36 em cortes axiais e 36 em cortes coronais) e graduaram quais imagens eram mais bem interpretadas. Os resultados não mostraram diferenças significantes entre os cortes axiais e coronais para se chegar ao diagnóstico, na presença de artefatos metálicos na imagem, assim como demonstrou boa concordância entre os observadores. Conclui-se que a presença dos artefatos metálicos dentários não foi considerada suficiente para impedir a localização de neoplasias de tecido mole. Não foi detectada uma interferência maior de artefatos dentários metálicos tanto nas imagens axiais como nas coronais com relação à interpretação das lesões. A imagem coronal apresentou uma maior interferência em comparação com a imagem axial. Palavras-chave: tomografia computadorizada, artefatos metálicos, neoplasias malignas dos maxilares. / Computed tomography is an important tool to determinate the diagnosis, treatment and follow up malignant lesions of the maxillomandibular complex. In many cases, it is the election exam to all of these purposes. One problem with CT is the artifacts that appear in the presence of metallic restorations, implants and metallic fixation screws or plates. These artifacts lead to misinterpretations of the images, making difficult to visualize lesions in these areas. The aim of this research was to evaluate CT images with different sections (axial and coronal) e to determine if the type of section can minimize the problem of dental metallic artifacts. Two calibrate examiners (oral and maxillofacial radiologists) analyzed 72 images (36 in axial slices and 36 in coronal slices) and graduate which images were best viewed. The results attested that there were no significant differences between axial and coronal images to reach the diagnosis in the presence of dental metallic artifacts, and there were a good concordance with both examiners. We conclude that the presence of dental metallic artifacts wasn’t sufficient to disturb the precise location neoplasm of the soft tissue. It wasn’t detected any dental metallic interference possible to misdiagnosis neither on axial images nor on coronal images. Coronal images presented a higher interference when comparing with axial images.

A Candida é um microrganismo dimórfico encontrado comumente no trato gastrointestinal, na pele e nas membranas mucosas dos seres humanos. Na forma de levedura, o fungo permanece em equilíbrio e não é patogênico, podendo estar presente na cavidade oral de indivíduos saudáveis. A candidíase oral geralmente é classificada em quatro grandes grupos, e a candidíase crônica hiperplásica (CCH) é representada por uma infecção crônica, caracterizada pela hiperplasia epitelial do hospedeiro. Especificamente, essas lesões são difíceis de serem diferenciadas de leucoplasias e têm sido associadas com o aumento da chance de desenvolvimento de displasias e lesões malignas. O objetivo deste trabalho foi realizar um levantamento dos casos diagnosticados no Serviço de Patologia Cirúrgica da FOUSP, com vistas a demonstrar a ocorrência dessas infecções em boca e avaliar seus aspectos histológicos característicos. Os casos foram selecionados utilizando o sistema Laudo e Imagem do Serviço de Patologia Cirúrgica da FOUSP e para caracterização das amostras, os dados clínicos do paciente e os aspectos da lesão foram coletados. A análise histopatológica das lâminas foi realizada posteriormente através da observação em microscópio de luz, e as colorações de hematoxilina-eosina (HE) e do ácido periódico de Schiff (PAS) foram utilizadas para avaliação das características microscópicas e a presença de Candida, respectivamente. Os profissionais que realizaram as biópsias foram contatados para obter informações a respeito da evolução da lesão e do paciente. De um total de 58675 biópsias realizadas entre janeiro de 2002 e dezembro de 2017, 36 casos foram diagnosticados como CCH. As mulheres foram mais afetadas, havendo predileção pela raça branca e pela quinta e sexta décadas de vida. De um modo geral, clinicamente, as lesões se apresentavam como um nódulo ou placa branca assintomática localizada em língua ou mucosa jugal. Histologicamente, significância estatística foi notada na presença de projeções epiteliais, exocitose, infiltrado inflamatório discreto e mononuclear. Hifas de Candida foram vistas em todas as amostras, distribuídas perpendicularmente à superfície epitelial. Dos 36 diagnósticos realizados, apenas 11 \"follow-ups\" foram obtidos e desses, apenas cinco pacientes tiveram melhoras quando tratados com antifúngico tópico ou sistêmico. Conclui-se que as lesões de CCH correspondem a uma mínima fração dos diagnósticos realizados no Serviço de Patologia da FOUSP e apresentam geralmente aspectos histológicos característicos. Entretanto, a infecção pelo microrganismo como causa da lesão ou sua presença como organismo oportunista em algumas lesões pode ser esclarecida com mais estudos, principalmente prospectivos. / Candida is a dimorphic microorganism commonly found in the gastrointestinal tract, skin and mucous membranes of humans. In the yeast phase, the fungus is not pathogenic and may be present in the oral cavity of healthy individuals. Oral candidiasis is generally classified into four groups, and the hyperplastic variant is represented by a chronic infection, characterized by an epithelial hyperplasia of the host. Specifically, these lesions are difficult to differentiate from leukoplakias and they also have been associated with an increased chance of developing dysplasias and malignant lesions. The aim of this work was to analyze the incidence of CHC diagnosed at the Oral Pathology Service of FOUSP, intending to demonstrate the occurrence of these infections in the oral cavity as well as to evaluate the histology of the specimens to define the anatomopathological aspects that characterize them. The cases were accessed through the \"Laudo e Imagem\" system and for the characterization of the samples, the clinical data of the patients and the aspects of the lesions were collected. Subsequently, the histopathological analysis of the sections was performed under light microscopy, and the hematoxylin-eosin and periodic acid Schiffer staining were used to evaluate the microscopic characteristics and the presence of Candida, respectively. The professionals who performed the biopsies were contacted to obtain information about the evolution of the lesion and the patient. Out of 58675 diagnoses done between January 2002 and December 2017, 36 cases were of CHC. Women were the most affected and there was a predilection for the white race as well as for the fifth and sixth decades of life. In general, clinically, the lesions appeared as an asymptomatic nodule or white plaque on the tongue or buccal mucosa. Histologically, statistical significance was noted in the presence of epithelial hyperplasia, exocytosis and mononuclear inflammatory infiltrate. Candida hyphae, arranged perpendicular to the epithelial surface, were seen in all samples. Of the 36 diagnoses, only 11 \"follow-ups\" were obtained and of these, only 5 patients had improvement when treated with topical or systemic antifungal drugs. It was concluded that the CHC lesions correspond to a minimal fraction of the diagnoses performed at the Oral Pathology Service of FOUSP, and although their histological characteristics is suggestive of infection by Candida, further studies, mainly prospective, are needed to better understand the lesion.

Les adénomes hépatocellulaires (AHC) sont des tumeurs bénignes rares dérivant de la prolifération d'hépatocytes et se développent dans un contexte hépatique par ailleurs normal, principalement chez des femmes suite à la prise de contraceptifs oraux. D'autres études ont identifié des facteurs de risque supplémentaires comme le syndrome métabolique et des mutations germinales d’HNF1α. Les travaux réalisés dans le laboratoire ont permis le classement des AHC en cinq sous-groupes : 1) les AHC-H avec des mutations inactivatrices du facteur de transcription HNF1α, 2) AHC-I avec une activation de la voie inflammatoire, 3) B-AHC portant des mutations activatrices du gène CTNNB1, codant pour la β-caténine 4) Sh-AHC avec une activation de la voie sonic hedgehog et 5) U-AHC sans altération moléculaire spécifique trouvée à ce jour. Les AHC montrent un faible risque de transformation maligne en carcinome hépatocellulaire (CHC). Le CHC est la deuxième cause de décès liés au cancer dans le monde entier, ce qui en fait un problème majeur de santé publique. Le CHC se développe dans 90% des cas sur un foie cirrhotique et est lié à une infection virale (VHB et HCV), à une consommation d'alcool et au syndrome métabolique. L'une des modifications génétiques les plus fréquentes dans les CHC est la réactivation de la télomérase (TERT), une enzyme clé nécessaire à la maintenance des télomères. Tout d'abord, dans nos travaux, nous avons cherché à établir une corrélation génotype-phénotype des AHC activés pour la β-caténine pour mieux comprendre l'hétérogénéité des tumeurs et le risque de transformation maligne. Notre étude a permis d'identifier trois sous-groupes d’AHC mutés CTNNB1 selon le type de mutation : mutations fortement, modérément et faiblement activatrices. De façon intéressante, chaque type de mutation présentait immuno marquage spécifique de la GS et un risque de transformation maligne diffèrent. En plus, nous avons identifié la mutation du promoteur de TERT comme un événement tardif au cours de la tumorigenèse nécessaire à la transformation des AHC-B en CHC. Deuxièmement, en raison de l’importance de la réactivation de TERT dans les tumeurs du foie, nous avons testé un nouveau traitement potentiel basé sur des oligonucléotides antisens conduisant à l’inhibition de l’expression transcriptionelle de TERT. En accord avec l'érosion progressive des séquences télomériques qui se produisent à chaque division cellulaire, nous avons pu observer un effet sur la prolifération cellulaire après traitement à long terme. Nous avons montré que les effets observés étaient liés au raccourcissement des télomères. De plus, nous avons identifié AAV2 comme un nouveau virus lié au développement de rares cas de CHC. Nos résultats sont soutenus par l'expansion clonale des cellules tumorales avec insertion virale, la surexpression des gènes insérés et l'insertion observée uniquement dans des gènes déjà connus pour être impliqués dans la tumorigenèse comme TERT, TNFSF10, MLL4, CCNA2 et CCNE1. En conclusion, dans nos études, nous avons affiné la classification des AHC, avec des possibles applications dans la prise en charge clinique des patients pour identifier le meilleur traitement selon le type de mutation et le risque de transformation maligne. De plus, nous avons obtenu des résultats prometteurs concernant l'utilisation de TERT comme cible thérapeutique. Cependant, des études supplémentaires sont nécessaires pour mieux évaluer les effets du traitement par ASO contre le TERT sur la physiologie cellulaire et les avantages possibles avec des combinaisons thérapeutiques. Enfin, nos résultats remettent en question la sécurité des thérapies génique basées sur l'AAV actuellement en cours, mais des études supplémentaires sont actuellement en cours au laboratoire pour comprendre les conséquences et les mécanismes impliqués dans de l'insertion virale. / Hepatocellular adenomas (HCA) are rare benign tumors deriving from proliferation of hepatocytes occurring in normal liver background mainly of female patients with history of oral contraceptive assumption. Further studies identified additional risk factors as metabolic syndromes and germline mutations. Works performed in the laboratory allowed the classification of HCA in five subgroups: 1) H-HCA with inactivating mutation of transcription factor HNF1α, 2) I-HCA with activation of the inflammatory pathway, 3) B-HCA carrying activating mutations of CTNNB1 gene, coding for β-catenin 4) Sh-HCA with activation of the sonic hedgehog pathway and 5) U-HCA with no specific alteration found to date. HCA show an average low risk of malignant transformation in hepatocellular carcinoma (HCC). HCC is the second cause of cancer-related deaths worldwide making it a major problem of public health. HCC development occurs in 90% of the cases on cirrhotic background and is linked to virus infection (HBV and HCV), alcohol intake and metabolic syndromes. One of the most frequent genetic alterations in HCC is the reactivation of TERT, a key enzyme necessary for telomeres maintenance. First, in our works we aimed to perform a correlation between genotype and phenotype of β-catenin activated HCA to better understand tumor heterogeneity and risk of malignant transformation. Our study allowed the identification of three subgroups of CTNNB1 mutated HCA according to the mutation type: highly, moderately and weakly activating mutations. Interestingly each type of mutation showed specific IHC staining for GS and risk of malignant transformation. Additionally we identified TERT promoter mutation as a late event in tumorigenesis necessary for HCA transformation in HCC with a significant correlation with CTNNB1 alterations. Second, due to the relevance of TERT reactivation in tumors, we tested a new potential treatment based on antisense oligonucleotides leading to TERT transcript ablation and lack of protein production. According to the slow erosion of the telomere sequences that occurs at each cell division, we were able to observe some effect on cell proliferation with long-term treatment. We showed that the observed effects were linked to telomere shortening. Further, we identified AAV2 as a new virus linked to tumor formation. Our results are supported by the clonal expansion of cells with viral insertion, overexpression of the inserted genes and insertion observed only in genes already known to be involved in tumorigenesis like TERT, TNFSF10, MLL4, CCNA2 and CCNE1. Taken together, in our studies we refined HCA classification, with possible direct applications in the clinical care of the patients in identifying the best treatment according to mutation type and risk of malignant transformation. Additionally we obtained promising results regarding using TERT as a therapeutic target. Still, additional studies are needed to better assess the effects of ASO treatment against TERT on cell physiology and possible advantages in using combination therapies. Finally, our findings challenge the safety of AAV based targeted therapies currently ongoing but additional studies to understand the effective consequences and mechanisms of viral insertion are object of current work in the laboratory.

Die Talkumpleurodese stellt ein effektives Verfahren zur Behandlung von rezidivierenden malignen Pleuraergüssen dar. Sie ist ein rein palliatives Verfahren. Das Auftreten des malignen Pleuraergusses stellt den Beginn des letzten Lebensabschnittes eines Tumorpatienten dar. Es ist verbunden mit dem Kardinalsymptom Dyspnoe. Um diese zu lindern, wird im blinden Aktionismus die operative Pleurodese angeboten. Dieses führt zu einer hohen perioperativen Mortalität. In 36 Monaten (1997-1999) wurden in unserer Klinik 115 operative Talkum-Pleurodesen durchgeführt. Die perioperative Letalität lag bei 7,8 %. Die häufigsten Primärtumoren waren Mammakarzinom (MÜL 12,4 Monate), Bronchialkarzinm (MÜL 6,2 Monate), CUP (MÜL 10,7 Monate) Ovarialkarzinom (MÜL 2,2 Monate) und Mesotheliom (MÜL 10,9 Monate). Die Patienten mit Mammakarzinom, Bronchialkarzinom und Ovarialkarzinom litten postoperativ deutlich gehäufter unter erneuter Luftnot. Es ist daher nötig, Standards festzulegen, um ein geeignetes Patientengut zu behandeln. In der Literatur finden sich eindeutige Zusammenhänge zum Karnofsky-Index des Patienten sowie zu den Laborparametern pH (kritisch / Talc pleurodesis is the most effective treatment für malignant pleural effusions. It is a palliative procedure. The appearance of a malignant pleural effusions marks the beginning of the last part of life of a patient with cancer. The cardinal symptom is dyspnoea. To allay, we offer surgical pleurodesis, offen in blind action. This is causing high mortality. We performed 115 thoracoscopic talc pudrages between 1997 and 1999 (36 month). The inpatient mortality was 7,8%. The most offen origins of metastatic carcinoma were Breast cancer (MS 12,4 month), Lung cancer (MS 6,2 month), Carcinoma of unknown primary (MS 10,7 month), Malignant mesothelioma (MS 10,9 month) and Ovarian cancer (MS 2,2 month). The patients with Breast-, Lung- and Ovarian cancer suffered the must from dyspnoea after pleurodesis. That''s why it is necessary, to find standards to choose suitable patients, which profit. In literature there are definite connection between Karnofsky Performance Scale of a patient, pleural fluid pH (critically