MicroRNA-Based Therapeutics in Hepatocellular Carcinoma: In Vitro and in Vivo Studies

Hepatocellular carcinoma (HCC) is a serious public health problem, without an effective cure. It has been demonstrated that the deregulation of microRNAs expression contribute to tumorigenesis. In HCC, miR-221 was shown to be up-regulated in more than 70% of the cases and was associated with higher tumor stage, metastasis and a shorter time to recurrence after surgery. A tumor promoting function of miR-221 was proved in a transgenic mouse model, which was predisposed to the development of liver cancers. These findings suggested that miR-221 could represent a potential target for anti-tumor approaches. Conversely, miR-199a is of interest because its level was shown to be reduced in almost 100% of HCC, it was significantly correlated with poor prognosis and was shown to target c-Met, an oncogene involved in invasion and metastasis. In the present thesis, novel Adenoviruses and Adeno-Associated viral vector (AAVVs) were developed. They were genetically modified to drive the expression of multiple binding sites for miR-221, the “miR-221 sponge”, as well as miRNA precursor for miR-199a. Analysis of miR-221 sponge in HCC cells revealed the capability to reduce miR-221 endogenous levels, which was accompanied by an increase in CDKN1B/p27 protein, a known target of miR-221. An increase in apoptosis was detected in Hep3B cells after infection with any of adeno or AAVs in comparison with control viruses. Moreover, restoring the level of miR-199a could also reduce viability and increase apoptosis of HepG2 cells. Therapeutic efficacy of miR-221 antisense oligonucleotides and a tumor suppressive role of miR-199a alone or in combination with sorafenib were also evaluated. We showed that combining an antimiR-221/mimic miR-199a with sorafenib improves the response of HCC cells to molecular targeted treatment through enhancing the inhibition of cell proliferation and induction of apoptosis and arresting the cell cycle in G1. To validate the therapeutic potential of miR-199a, we demonstrated that in vivo delivery of miR-199a oligonucleotide leads to a reduction of the number and size of tumor nodules as sorafenib, without any apparent toxicity. This study showed that methods for modulating microRNA activities could elicit measurable anti-tumor effects, that deserve further study to improve existing therapies

[¹⁸F]fluorination of biorelevant arylboronic acid pinacol ester scaffolds synthesized by convergence techniques

Aim: The development of small molecules through convergent multicomponent reactions (MCR) has been boosted during the last decade due to the ability to synthesize, virtually without any side-products, numerous small drug-like molecules with several degrees of structural diversity.(1) The association of positron emission tomography (PET) labeling techniques in line with the “one-pot” development of biologically active compounds has the potential to become relevant not only for the evaluation and characterization of those MCR products through molecular imaging, but also to increase the library of radiotracers available. Therefore, since the [18F]fluorination of arylboronic acid pinacol ester derivatives tolerates electron-poor and electro-rich arenes and various functional groups,(2) the main goal of this research work was to achieve the 18F-radiolabeling of several different molecules synthesized through MCR. Materials and Methods: [18F]Fluorination of boronic acid pinacol esters was first extensively optimized using a benzaldehyde derivative in relation to the ideal amount of Cu(II) catalyst and precursor to be used, as well as the reaction solvent. Radiochemical conversion (RCC) yields were assessed by TLC-SG. The optimized radiolabeling conditions were subsequently applied to several structurally different MCR scaffolds comprising biologically relevant pharmacophores (e.g. β-lactam, morpholine, tetrazole, oxazole) that were synthesized to specifically contain a boronic acid pinacol ester group. Results: Radiolabeling with fluorine-18 was achieved with volumes (800 μl) and activities (≤ 2 GBq) compatible with most radiochemistry techniques and modules. In summary, an increase in the quantities of precursor or Cu(II) catalyst lead to higher conversion yields. An optimal amount of precursor (0.06 mmol) and Cu(OTf)2(py)4 (0.04 mmol) was defined for further reactions, with DMA being a preferential solvent over DMF. RCC yields from 15% to 76%, depending on the scaffold, were reproducibly achieved. Interestingly, it was noticed that the structure of the scaffolds, beyond the arylboronic acid, exerts some influence in the final RCC, with electron-withdrawing groups in the para position apparently enhancing the radiolabeling yield. Conclusion: The developed method with high RCC and reproducibility has the potential to be applied in line with MCR and also has a possibility to be incorporated in a later stage of this convergent “one-pot” synthesis strategy. Further studies are currently ongoing to apply this radiolabeling concept to fluorine-containing approved drugs whose boronic acid pinacol ester precursors can be synthesized through MCR (e.g. atorvastatin)

Influencia multifactorial en el desarrollo de contractura capsular, rotura protésica y complicaciones menores de las prótesis-expansoras en la reconstrucción de mama

Estudiamos de forma retrospectiva la evolución de 303 implantes mamarios tipo Becker en 231 pacientes reconstruídas en el Servicio de Cirugía Plástica del C.H.U.S. Se estudiaron posibles variables que pudieran influir en la presencia de complicaciones (contractura capsular, rotura protésica y complicaciones menores) y también en la supervivencia de estos implantes a largo plazo. De todas las variables de estudio, la radioterapia actuó como variable independiente aumentando el índice de complicaciones y disminuyendo significativamente la supervivencia de estos implantes. En base a los resultados obtenidos podemos concluir que, ante una paciente que haya recibido tratamiento radioterápico después de una mastectomía y que solicite una reconstucción mamaria, debe recomendarse reconstrucción únicamente con tejido autólogo. El uso de implantes debe ser evitado debido al alto índice de complicaciones que presenta

A deep learning based approach for classification of CerbB2 tumor cells in breast cancer

Bu çalışmada, meme kanserinde CerbB2 tümör hücre skorlarının derin öğrenme modellerine dayalı olarak sınıflandırılmasına yönelik, özgün bir yaklaşım önerilmektedir. Bildirinin bir diğer katkısı da, özgün meme kanseri dokusundan veri kümesinin oluşturulmasıdır. Derin öğrenme modellerinin eğitilmesi, doğrulanması ve sınanması amacıyla, doku örnek görüntüleri üzerinden hücre parçaları oluşturulmuştur. Oluşturulan hücre parçalarına ilişkin CerbB2 tümör skorları, evrişimsel sinir ağları (ESA) yardımıyla yüksek başarımda sınıflandırılmıştır.This study proposes a unique approach to classify CerbB2 tumor cell scores in breast cancer based on deep learning models. Another contribution of the study is the creation of a dataset from original breast cancer tissues. On the purpose of training, validating and testing with deep learning models cell fragments were generated from sample tissue images. CerbB2 tumor scores were generated for the cell fragments were classified with high performance by the aid of convolutional neural networks (CNN)