Excisão de Pterígio Primário com Autotransplante Conjuntival e Cola Biológica

Introdução: O Pterígio é uma patologia frequente e o seu tratamento cirúrgico é consensualmente aceite. Diferentes opções cirúrgicas e variantes estão descritas: enquanto alguns métodos caíram em desuso pela sua elevada taxa de recidiva, outros foram abandonadas pelos seus custos e complicações associados. A realização de autotransplante conjuntival tornou-se a técnica de eleição e o recurso a cola biológica tornou o procedimento mais fácil e rápido. Apresentamos a nossa experiência com esta técnica e reportamos as vantagens da utilização de cola biológica num estudo prospectivo a 5 anos. Material e métodos: 101 casos de 92 doentes com pterígio primário foram submetidos a autotransplante conjuntival (e limbar se área limbar envolvida superior a 6mm) com recurso a cola biológica de fibrina (Tissucol® / Tisseellyo®) - 94 casos, ou polímero de polietileno glicol (PEG; Ocuseal®) - 7 casos. Objectivo: primário- determinar a taxa de recidiva por tempo de tratamento. Secundários- identificar perda de retalho, retracção conjuntival, complicações cirúrgicas e reacções adversas ao uso de cola biológica. Resultados: A taxa de recidiva foi de 6% (4% no primeiro ano pós-cirurgia). A taxa de perda de retalho foi de 2%, de retracção conjuntival de 17%. Não se observaram complicações cirúrgicas graves nem reacções adversas ao uso de cola biológica. Num caso verificou-se hemorragia conjuntival. Conclusões: A cirurgia de pterígio com autotransplante conjuntival e uso de cola biológica é um procedimento seguro, eficaz e com baixa taxa de recidiva

Branching into RNAi: Synthesis, Characterization and Biology of Branch and Hyperbranch siRNAs

The cancer epidemic continues to afflict millions of humans world-wide each year and despite a renewed hope with the development of new and improved forms of therapy, a cure for cancer remains an elusive goal. This is partly related to the rise of resilient forms of tumors that have evolved with resistance towards conventional chemotherapy and radiation treatments. Moreover, these non-specific therapeutic regimens are highly toxic, leading to severe immunosuppressive effects which poisons the body and compromises the road towards remission. In an effort to mitigate these limitations, cancer-targeting approaches are currently experiencing a renaissance in the translation of new medicines from pre-clinical to bedside use. Notably, gene therapy has recently gained widespread traction in cancer research in the advent of the first RNA interference (RNAi) application in humans. RNAi solicits the use of a double-stranded RNA substrate, aptly named short-interfering RNA (siRNA), which binds to and triggers the degradation of a targeted complementary mRNA strand within the catalytic site of the RNA-Induced Silencing Complex (RISC). In this manner, malignant mRNA expression is silenced, thereby inhibiting the translation of proteins that can lead to the production of pathological disorders such as cancer. In spite of their utility, several challenges still remain towards the development of a fruitful cancer-targeting gene therapy approach. Here, a new class of siRNA motifs is presented to increase substrate efficacy in the RNAi application. Our biological target is a member of the heat shock family of chaperone proteins, the Glucose Regulated Protein of 78 kilodaltons (GRP78) which signals tumor initiation, proliferation and resistance towards chemotherapy. Moreover, GRP78 is overexpressed and cell surface localized on a wide range of resilient tumor types but not on healthy cells, making it a viable bio-marker for the development of the proposed cancer-targeting gene therapy approach. Significantly, an efficient solid-phase synthesis method is described for the production of linear, V-shape, Y-branch and hyperbranch GRP78-silencing siRNAs. The novel V-shape, Y-branch and hyperbranch motifs were then studied by CD spectroscopy and thermal denaturation experiments. CD spectroscopy was used to characterize the requisite A-type double-stranded RNA helix for RNAi application; whereas thermal denaturation experiments were used to validate siRNA hybrid stabilities. With stable siRNA hybrids in hand, their biological activity was assessed in HepG2 hepatoblastoma cells, which constitutes a morbid form of pediatric liver cancer and a valid tumor model for studying our GRP78-targeting strategy. The GRP78 silencing activity of the putative branch and hyperbranch siRNAs is discussed and related to its underlying mechanisms for inducing apoptosis in cancer. Biological studies confirmed potent suppression of GRP78 expression (50-60%) while compromising cancer cell viability by ~20%. The development of an effective cancer-targeting gene therapy approach is highlighted by preliminary results that showcase the utility of a cancer-targeting peptide (CTP) to condense and deliver siRNA within cancer cells for therapeutic treatment. The latter forms the basis of our cancer-targeting gene therapy approach. Thus, branched and hyperbranched siRNAs may serve as potent siRNA candidates in cancer gene therapy applications

Continuum diffusion on networks: Trees with hyperbranched trunks and fractal branches

The probability that a random walker returns to its origin for large times scales as t(-d/2), where d is the spectral dimension. We calculate d for a class of tree structures using a renormalization technique on an infinite continued fraction. We consider a wide range of homogeneous networks based on replacing the branches of a self-similar tree with arbitrary fractals and composite fractals. We also consider a new class of inhomogeneous hyperbranched trees

Ectodysplasin target gene Fgf20 regulates mammary bud growth and ductal invasion and branching during puberty

Mammary gland development begins with the appearance of epithelial placodes that invaginate, sprout, and branch to form small arborized trees by birth. The second phase of ductal growth and branching is driven by the highly invasive structures called terminal end buds (TEBs) that form at ductal tips at the onset of puberty. Ectodysplasin (Eda), a tumor necrosis factor-like ligand, is essential for the development of skin appendages including the breast. In mice, Eda regulates mammary placode formation and branching morphogenesis, but the underlying molecular mechanisms are poorly understood. Fibroblast growth factor (Fgf) receptors have a recognized role in mammary ductal development and stem cell maintenance, but the ligands involved are ill-defined. Here we report that Fgf20 is expressed in embryonic mammary glands and is regulated by the Eda pathway. Fgf20 deficiency does not impede mammary gland induction, but compromises mammary bud growth, as well as TEB formation, ductal outgrowth and branching during puberty. We further show that loss of Fgf20 delays formation of Eda-induced supernumerary mammary buds and normalizes the embryonic and postnatal hyperbranching phenotype of Eda overexpressing mice. These findings identify a hitherto unknown function for Fgf20 in mammary budding and branching morphogenesis.Peer reviewe

Poly(Arginine) Derived Cancer-Targeting Peptides for the Development of a Cancer-Targeted Gene Therapy Approach in HepG2 Liver Cancer Cells

Cancer is a disease that has eluded medicinal approaches for many years and as a result new and improved therapeutic approaches are in constant demand. Although chemotherapy and radiation treatments have assisted in suppressing the growth of tumors, their poor selectivity and efficacy are major limitations for effective therapy en route towards the development of a cure for the cancer epidemic. With the mission of conquering cancer at heart, researchers have pursued a new form of cancer therapy, aptly named, a cancer targeting approach. This method revolves around the selection of a suitable biomarker, typically a cell surface receptor overexpressed or selectively localized on the surface of cancer cells but not on normal tissues. This biomarker forms the basis of a cancer targeting approach, in which high affinity and specific binding ligands (e.g. peptides) have been selected by phage display bio-panning methods or combinatorial chemistry to target cancer cells in-vivo. Building on this approach, the identification of a cyclic peptide, Pep42, H2N-CTVALPGGYVRVC-CONH2, has been selected as a specific and high-affinity binding ligand of the cell surface receptor, Glucose Regulated Protein 78 (GRP78). GRP78 is a member of the heat shock family of chaperone proteins, assisting in protein folding events under physiological stress induced conditions that are mitigated by the unfolded protein response (UPR) mechanism. In cancer, GRP78 is overexpressed and cell surface localized where it functions as a hub for cell signaling pathways that lead to cancer cell initiation, proliferation and resistance towards chemotherapy. Thus, GRP78 has been classified as a valid biomarker for the development of targeted anti-cancer approaches. Towards this goal, an Fmoc-based solid phase peptide synthesis (Fmoc-SPPS) method has been optimized on a polyethylene glycol (PEG) resin for the production of poly(arginine) derived Pep42 sequences in good yields (14-46%) and purities (\u3e95%) following RP-LCMS. The effect of the length (0-12) and stereochemistry (L/D) of the poly(arginine) sequences on Pep42 structure and stability were next evaluated by CD spectroscopy. Interestingly, peptides displayed varying folded conformations, transitioning between helical and turn structures, that were found to be contingent on the poly(arginine) sequence, solvent and disulfide bond formed within the Pep42 motif. Moreover, the peptide folds were found to be resilient towards thermal denaturation due to the covalent disulfide bond. Structure-activity relationships were next assessed in HepG2 hepatoblastoma cells, in which the cancer-targeting peptides were found to bind to the GRP78 receptor and internalize within the HepG2 cells. This discovery has led to their applications in cancer-targeted gene therapy, from which a GRP78-silencing short-interfering RNA (siRNA) was effectively transfected within the HepG2 cells for potent oncogene knockdown effects (50-60%, 40 pmol siRNA) while triggering modest cell death effects (5-10%). This thesis will highlight my efforts and contribution in collaboration with Drs. Blake and Bitsaktsis research groups towards the development of an effective cancer-targeted gene therapy approach

Dendritic nanocarriers based on hyperbranched polymers

Hyperbranched polymers are obtained through one-step polymerization reactions and exhibit properties that are very similar to those of perfect dendrimer analogues. Therefore, hyperbranched polymers are a suitable alternative for perfect dendrimers as building blocks for dendritic nanocarrier systems. With regard to using soluble hyperbranched polymers as carrier systems, their flexible chains are a major benefit as they can adopt and compartment guest molecules. Upon encapsulation, the properties of the host decides the fate of the guest, e.g., solubility, but the host can also shield a guest from the environment and protect it, e.g., from degradation and deactivation. With regard to the advantages of using hyperbranched polymers as nanocarrier systems and their scalable synthesis, we will discuss different types of hyperbranched polymers and their application as nanocarrier systems for drugs, dyes, and other guest molecules

Poly(Arginine) Derived Cancer-Targeting Peptides for the Development of a Cancer-Targeted Gene Therapy Approach in HepG2 Liver Cancer Cells

Cancer is a disease that has eluded medicinal approaches for many years and as a result new and improved therapeutic approaches are in constant demand. Although chemotherapy and radiation treatments have assisted in suppressing the growth of tumors, their poor selectivity and efficacy are major limitations for effective therapy en route towards the development of a cure for the cancer epidemic. With the mission of conquering cancer at heart, researchers have pursued a new form of cancer therapy, aptly named, a cancer targeting approach. This method revolves around the selection of a suitable biomarker, typically a cell surface receptor overexpressed or selectively localized on the surface of cancer cells but not on normal tissues. This biomarker forms the basis of a cancer targeting approach, in which high affinity and specific binding ligands (e.g. peptides) have been selected by phage display bio-panning methods or combinatorial chemistry to target cancer cells in-vivo. Building on this approach, the identification of a cyclic peptide, Pep42, H2N-CTVALPGGYVRVC-CONH2, has been selected as a specific and high-affinity binding ligand of the cell surface receptor, Glucose Regulated Protein 78 (GRP78). GRP78 is a member of the heat shock family of chaperone proteins, assisting in protein folding events under physiological stress induced conditions that are mitigated by the unfolded protein response (UPR) mechanism. In cancer, GRP78 is overexpressed and cell surface localized where it functions as a hub for cell signaling pathways that lead to cancer cell initiation, proliferation and resistance towards chemotherapy. Thus, GRP78 has been classified as a valid biomarker for the development of targeted anti-cancer approaches. Towards this goal, an Fmoc-based solid phase peptide synthesis (Fmoc-SPPS) method has been optimized on a polyethylene glycol (PEG) resin for the production of poly(arginine) derived Pep42 sequences in good yields (14-46%) and purities (\u3e95%) following RP-LCMS. The effect of the length (0-12) and stereochemistry (L/D) of the poly(arginine) sequences on Pep42 structure and stability were next evaluated by CD spectroscopy. Interestingly, peptides displayed varying folded conformations, transitioning between helical and turn structures, that were found to be contingent on the poly(arginine) sequence, solvent and disulfide bond formed within the Pep42 motif. Moreover, the peptide folds were found to be resilient towards thermal denaturation due to the covalent disulfide bond. Structure-activity relationships were next assessed in HepG2 hepatoblastoma cells, in which the cancer-targeting peptides were found to bind to the GRP78 receptor and internalize within the HepG2 cells. This discovery has led to their applications in cancer-targeted gene therapy, from which a GRP78-silencing short-interfering RNA (siRNA) was effectively transfected within the HepG2 cells for potent oncogene knockdown effects (50-60%, 40 pmol siRNA) while triggering modest cell death effects (5-10%). This thesis will highlight my efforts and contribution in collaboration with Drs. Blake and Bitsaktsis research groups towards the development of an effective cancer-targeted gene therapy approach