This study evaluated the potential toxicity, genotoxicity, and cellular uptake of various sizes and concentrations of chitosan (CS) nanoparticles cultured with normal human dental pulp cells. Normal human dental pulp cells (hDPCs) were derived from human dental pulp tissues and cultured with (50–67) nm and (318–350) nm CS-nanoparticles in concentrations of 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL, 2 mg/mL, and 4 mg/mL as study groups and 0 mg/mL as a control group for time intervals of 16 hours, 24 hours, 3 days, 7 days and 14 days. Attachment efficiency and proliferation rate were assessed by measuring the optical density of crystal violet-stained cells. Cell viability was determined by the activity of mitochondrial dehydrogenase enzymes. Genotoxicity was assessed using the cytokinesis-block micronucleus method and by measuring the fluorescent intensity of phosphorylated H2AX nuclear foci. Cellular uptake was determined by tagging chitosan nanoparticles with FITC stain and then measuring the fluorescence intensity of FITC-tagged chitosan nanoparticles using a spectrophotometer. Statistical analysis was performed using chi-square, one-way ANOVA, and post-hoc Tukey tests. All concentrations of the (50–67) nm group significantly reduced attachment efficiency in comparison with control (P< 0.01) and with (318–350) nm group (p<0.01). Proliferation rate and cell viability were significantly reduced in cells exposed to various concentrations of (50-67) nm chitosan when compared to (318-350) nm group (P<0.05) and control group (P<0.05). For both size groups, higher concentrations significantly showed lower proliferation rate and cell viability when compared to lower concentration (P< 0.01). CS-nanoparticles were able to internalize hDPCs and significantly induced micronuclei, nuclear buds, and pH2AX at concentrations of 0.5 mg/mL and 2 mg/mL as compared to 0.1 mg/mL (P<0.01) and control groups (P< 0.01). At both the 0.5 mg/mL and 2 mg/mL concentrations, (50–67) nm chitosan significantly induced higher proportions of micronuclei (P= 0.001), nuclear buds (P= 0.009), and pH2AX nuclear foci (P= 0.00004) compared to (318–350) nm chitosan. In conclusion, CS-nanoparticles at sizes (50–67) nm and (318–350) nm at a concentration of (0.5–4) mg/mL internalized hDPCs and exhibited cytotoxic and genotoxic effects in dose-dependent and size-associated manners.

Making up just over 3% of all new cancer cases in the United States, pancreatic cancer is not inherently a common malignant disease. Yet, it continuously is shown to be one of the most lethal and common causes of cancer death. Early detection is critical among all cancer types. However, oncologists and researchers have struggled to find effective strategies or tests to detect cancer of the pancreas early on in development. Thus, the cancer is often found late stage and requires significant chemotherapy intervention. These multi-drug treatment cocktails have shown benefit, but only in added months and not years to a patient’s life. Significant adverse effects often limit the full effective doses of treatment. In order to limit these adverse effects, as well as increase the effectiveness of treatment, we have designed, optimized, and tested unique drug carriers known as polymersomes. Using characteristics of the localized environment surrounding pancreatic tumors and the cells found therein, we created targeted therapies that are responsive and relatively selective toward cancerous cells. Herein, are found two distinct polymersomes. The first, is a low oxygen reactive drug carrier with an additional small peptide molecule that is able to penetrate dense tumor tissue and has shown decreased tumor growth of as much as 260% as compared to control samples in an animal model of pancreatic cancer. The chemical make-up of this polymersome allows for extended circulation time and a high accumulation at the tumor site. A second design, uses an intracellular enzyme to destabilize the polymersomes’ structure, which in turn, releases a selected chemotherapy drug near its intended site of action. This strategy, has shown a 10 fold increase in potency of the chemotherapy drug, as compared to when the drug is given alone and showed decreased toxicity to non-cancerous cells. It is certain that thoughtful drug delivery strategies and not just drug molecule design will be instrumental in the paradigm shift of pancreatic cancer from likely death to survival. / NIH grant 1 R01GM 114080; Grand Challenge Initiative; Office of the Dean, College of Health Professions

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In this Ph.D. project, we aim to understand degradation of nanomachines by studying the mechanisms that lead to the in vitro degradation of molecular shuttles, which are nanoscale active systems composed of kinesin motor proteins and cytoskeletal filaments called microtubules. In addition, we aimed to improve learning outcomes by designing a hybrid college-level engineering course combining case-based and lecture-based teaching. The creation of complex active nanosystems integrating cytoskeletal filaments propelled by surface-adhered motor proteins often relies on microtubules’ ability to glide for up to meter-long distances. Even though theoretical considerations support this ability, we show that microtubule detachment (either spontaneous or triggered by a microtubule crossing event) is a non-negligible phenomenon that has been overlooked until now. Furthermore, we show that under our conditions (100, 500, 1000 motors per µm2 and 0.01 or 1 mM ATP), the average gliding distance before spontaneous detachment ranges from 0.3 mm to 8 mm and depends on the gliding velocity of the microtubules, the density of the kinesin motors on the glass surface, and time. Wear, defined as the gradual removal of small amounts of material from moving parts of a machine, as well as breakage, defined as the rupture of a material, are two major causes of machine failure at the macroscale. Since these mechanisms have molecular origins, we expect them to occur at the nanoscale as well. Here, we show that microtubules propelled by surface-adhered kinesin motors are subject to wear and breakage just like macroscale machines. Furthermore, the combined effect of wear, breakage and microtubule detachment from the surface of the flow cell permit to predict how molecular shuttles degrade in vitro. Taking a step back and looking at science in a broader sense, we can say that science does not only consist of acquiring knowledge, but also relies on one’s ability to transmit his/her knowledge. In this regard, one of the biggest challenges in education is to be efficient, that is to say to design a teaching method that would both maximize the student’s retention of information and prepare them to apply their knowledge to real-life situations. We considered this challenge as an integral part of this Ph.D. project, and we tackled it by designing a novel type of engineering course in which the students’ involvement in the learning process plays a central role. To do so, we combined, in a single engineering course, both of the approaches to learning that are used in Engineering education and in Business schools. The final chapter of this manuscript summarizes the findings of the two projects presented here and discusses the future research that can be conducted on the basis of this thesis.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Recently nanoparticles (NPs) have been introduced and used in combination with therapeutic approaches to develop nanotechnology-enabled medicine. These nanostructures allow for the exploitation of the physiochemical properties which may be beneficial in cancer treatment. The use of NPs in nanomedicine has proven successful in modern chemotherapeutics and has demonstrated promising potential in in vivo and in vitro radiosensitization studies. This is a baseline study aimed to determine the cytotoxic effects of AuNPs for potential radiosensitization analysis. The study analysed the effects of different AuNP sizes (30, 50 and 80nm), concentrations (5, 10 and 15 μg/ml) over various time periods in CHOK1 and A549 cells.

Orientador: Taís Maria Bauab / Banca: Marlus Chorilli / Banca: Margarete Teresa Gottardo de Almeida / Resumo: O uso indiscriminado e generalizado de drogas antifúngicas levou a um rápido desenvolvimento de cepas resistentes aos medicamentos empregados na terapêutica clínica, caracterizando-se como a principal causa de inúmeros casos de inadequação do pacienta à terapia medicamentosa disponível. Os produtos naturais vêm ganhando destaque nos últimos anos por fornecer uma nova abordagem medicamentosa para o tratamento de processos patológicos, dentre esses, os infecciosos. No decorrer da evolução genética e fisiológica dos micro-organismos, as ciências microbiológicas encontram-se em expansão, preocupando-se em introduzir novas triagens terapêuticas frente às afecções fúngicas, com especial atenção àquelas adquiridas por espécies do gênero Candida. Isto se deve das Candidas serem as leveduras mais frequentemente envolvidas nas infecções micóticas capazes de causar dano ao organismo humano, como a candidíase vulvovaginal. O uso de produtos naturais na descoberta de novos antifúngicos mostra-se de extrema importância, a exemplo, destaca-se a espécie Syngonanthus nitens que vem se mostrando ativa na descoberta do potencial antifúngico contra Candida spp. A nanotecnologia farmacêutica apresenta-se como uma importante ferramenta para aprimorar a biodisponibilidade de produtos de origem natural, ao oferecer sistemas de liberação de fármacos modernos como os sistemas precursores de cristais líquidos mucoadesivos (SPCLM) empregados para veiculação de extratos vegetais. As plantas pertencentes a família Eriocaulaceae mostram-se promissoras na atividade antifúngica, o que instiga o aprofundamento destas investigações, assim como a descoberta de novos métodos de aplicabilidade. Frente ao atual cenário, este trabalho teve o objetivo de avaliar o potencial antifúngico do extrato metanólico de escapos de Syngonanthus nitens incorporado e não incorporado em SPCLM voltado ao tratamento da candidíase vulvovaginal. O ... / Abstract: The indiscriminate and widespread use of antifungal drugs with potential led to a rapid development of resistant strains to drugs used in clinical, therapeutic and this is characterized as the main cause of numerous cases of inappropriate patient to drug therapy available. Natural products have been gaining prominence in recent years for providing a new drug therapy for the treatment of pathological processes, among these, infectious. During the genetic and physiological evolution of micro-organisms, microbiological sciences are expanding, worrying to introduce new therapeutic trials ahead to fungal diseases, with special attention to those acquired by Candida species, this being one of yeasts more often involved in mycotic infections can cause harm to human body, such as vulvovaginal candidiasis. The use of natural products in the discovery of new antifungal is extremely important, the example, is the Syngonanthus nitens species (gold grass) that has proved active in the discovery of antifungal potential against Candida spp. The pharmaceutical nanotechnology presents itself as an important tool to improve the bioavailability of natural product origin, offering modern drug release systems as precursors of mucoadhesive liquid crystal systems (PMLCS) employees for placement of plant extracts. The plants belonging to family Eriocaulaceae show promise in antifungal activity, which encourages the deepening of these investigations, as well as the discovery of new methods of applicability. Front of the current scenario, this work aimed to evaluate the antifungal potential of the methanol extract of Syngonanthus nitens scapes incorporated and not incorporated in PMLCS focusing on the treatment of vulvovaginal candidiasis. The study was conducted by applying in vitro and in vivo antifungal methods intended to elucidate the profile, having obtained satisfactory results as regards the applicability thereof in the treatment and prevention of VVC. The ... / Mestre

Carbon nanotubes (CNT) have unparalleled mechanical properties, spanning several orders of magnitude over both length and time scales. Computational and experimental results vary greatly, partly due to the multitude of variables. Coupling physics-based molecular dynamics (MD) with informatics methodologies is proposed to navigate the large problem space. The adaptive intermolecular reactive empirical bond order (AIREBO) is used to model short range, long range and torsional interactions. A powerful approach that has not been used to study CNT mechanical properties is the derivation of descriptors and quantitative structure property relationships (QSPRs). For the study of defected single-walled CNTs (SWCNT), two descriptors were identified as critical: the density of non-sp2 hybridized carbons and the density of methyl groups functionalizing the surface. It is believed that both of these descriptors can be experimentally measured, paving the way for closed-loop computational-experimental development. Informatics can facilitate discovery of hidden knowledge. Further evaluation of the critical descriptors selected for Poisson’s ratio lead to the discovery that Poisson’s ratio has strain-varying nonlinear elastic behavior. CNT effectiveness in composites is based both on intrinsic mechanical properties and interfacial load transfer. In double-walled CNTs, inter-wall bonds are surface defects that decrease the intrinsic properties but also improve load transfer. QSPRs can be used to model these inverse effects and pinpoint the optimal amount of inter-wall bonds.

Single molecule biosensing techniques offer unique advantages and opportunities for basic biological studies and medical diagnostic applications. However, their signal levels are intrinsically very weak and can be easily masked by the noise from the sensor itself or the measurement electronics. Thus, the biosensing systems and devices must be carefully characterized and optimized to reduce noise. This thesis first presents optimizations that enable high bandwidth, single channel recordings of the calcium-induced calcium release channel ryanodine receptor 1. By directly integrating a suspended bilayer with a complementary metal oxide semiconductor transimpedance amplifier, the total input capacitance and, therefore, high frequency noise are lowered, enabling gating events to be recorded at an order of magnitude higher bandwidth than the previous state of the art. Next, low frequency noise optimizations for carbon nanotube transistors are explored using hexagonal boron nitride substrates. These devices have improved 1/f noise performance compared to equivalent devices on silicon oxide and demonstrate evidence of contact limited noise. Finally, a basic characterization of 1/f noise in carbon nanotubes is developed using correlated transport and noise measurements in crossed carbon nanotube homojunction devices. These methods of optimizing and characterizing noise can aid in the development of single molecule biosensors with improved temporal resolution and error rates.

Induction of apoptosis in cancer cells has increasingly been the focus of many therapeutic approaches in oncology field. Since its identification as a TNF family member, TRAIL (TNF-related apoptosis-inducing ligand) paved a new path in apoptosis inducing cancer therapies. Its selective ability to activate extrinsic and intrinsic cell death pathways in cancer cells only, independently from p53 mutations responsible for conventional therapeutics resistance, spotted TRAIL as a potent cancer apoptotic agent. Many recombinant preparations of TRAIL and death receptor targeting monoclonal antibodies have been developed and being tested pre-clinically and clinically both as a single agent and in combinations. Of note, the monoclonal antibodies were not the only type of antibodies developed to target TRAIL receptors. Recent technology has brought forth several single chain variable domains (scFv) designs fused recombinantly to TRAIL as well. Also, it is becoming progressively more understandable that field of nanotechnology has revolutionized cancer diagnosis and therapy. The recent breakthroughs in materials science and protein engineering have helped considerably in strategically loading drugs into nanoparticles or conjugating drugs to their surface. In this review we aim to comprehensively highlight the molecular knowledge of TRAIL in the context of its pathway, receptors and resistance factors. We also aim to review the clinical trials that have been done using TRAIL based therapies and to review various scFv designs, the arsenal of nano-carriers and molecules available to selectively target tumor cells with TRAIL.

Since its identification as a member of the tumour necrosis factor (TNF) family, TRAIL (TNF-related apoptosis-inducing ligand) has emerged as a new avenue in apoptosis-inducing cancer therapies. Its ability to circumvent the chemoresistance of conventional therapeutics and to interact with cancer stem cells (CSCs) self-renewal pathways, amplified its potential as a cancer apoptotic agent. Many recombinant preparations of this death ligand and monoclonal antibodies targeting its death receptors have been tested in monotherapy and combinational clinical trials. Gene therapy is a new approach for cancer treatment which implies viral or non-viral functional transgene induction of apoptosis in cancer cells or repair of the underlying genetic abnormality on a molecular level. The role of this approach in overcoming the traditional barriers of radiation and chemotherapeutics systemic toxicity, risk of recurrence, and metastasis made it a promising platform for cancer treatment. The recent first Food Drug Administration (FDA) approved oncolytic herpes virus for melanoma treatment brings forth the potency of the cancer gene therapy approach in the future. Many gene delivery systems have been studied for intratumoural TRAIL gene delivery alone or in combination with chemotherapeutic agents to produce synergistic cancer cytotoxicity. However, there still remain many obstacles to be conquered for this different gene delivery systems. Nanomedicine on the other hand offers a new frontier for clinical trials and biomedical research. The FDA approved nanodrugs motivates horizon exploration for other nanoscale designed particles' implications in gene delivery. In this review we aim to highlight the molecular role of TRAIL in apoptosis and interaction with cancer stem cells (CSCs) self-renewal pathways. Finally, we also aim to discuss the different roles of gene delivery systems, mesenchymal cells, and nanotechnology designs in TRAIL gene delivery.