Nanomaterials for Double-Stranded RNA Delivery

RNA interference has enormous potential as a potent, specific, and environmentally friendly alternative to small molecule pesticides for crop protection. The use of exogenous double-stranded RNA offers flexibility in targeting and use in crops in which transgenic manipulation is not an option. The combination of RNAi with nanotechnology offers further advantages that are not available with dsRNA alone. In this work, I have evaluated several different combinations of nanomaterials and polymers for use in RNAi-based pest control systems. First, I have characterized the use of chitosan/dsRNA polyplex nanoparticles for gene knockdown using the model nematode Caenorhabditis elegans. Though chitosan/dsRNA polyplexes are equally as effective as naked dsRNA for gene knockdown on a concentration basis, these materials are assimilated into cells in a manner independent of dsRNA specific transport proteins. The mechanism of uptake is likely clathrin-mediated endocytosis. In addition, I identify a significant and yet unreported side-effect associated with chitosan exposure, the dysregulation of a major myosin isoform. Next, I have determined the efficacy of chitosan/dsRNA polyplex nanoparticles under different environmental conditions. The presence of inorganic ions (phosphate and nitrate) at realistic environmental concentrations does not alter the efficacy of the nanoparticles for gene knockdown, nor do they inhibit knockdown by naked dsRNA. These conditions did not cause any significant changes to the hydrodynamic diameter or zeta potential of the particles themselves between treatments. By contrast, a pH higher than six and the presence of natural organic matter significantly reduce the efficacy of the nanomaterials at gene knockdown but leave knockdown by naked dsRNA unaffected. Though some changes in polyplex size are observed in the pH treatments, these changes are comparatively small, and particles remain well within the size that can be ingested by C. elegans. At pH 8, the charge of the particles is effectively neutral. Similarly, concentrations of natural organic matter \u3e2.5 mg/L cause a charge reversal of the particles, from strongly cationic to strongly anionic. Large aggregates are also visible in each of these treatments. Lastly, I characterize the efficacy of a suite of different polymer and solid core nanomaterials for dsRNA delivery, similar to the above. Poly-L-lysine, poly-L-arginine, Ge-doped imogolite, and poly-L-arginine-citrate coated Au nanoparticles all fail to cause any appreciable knockdown in the same C. elegans reporter system. Uptake of the polymers was exceedingly poor, and though the Au particles appear to have been ingested, there is no evidence of significant gene knockdown. Furthermore, poly-L-arginine caused significant injury to the mouthparts of C. elegans exposed to these materials. Layered double-hydroxide nanoparticles were successful at gene knockdown, and appear to function slightly better than naked dsRNA alone, and were translocated in C. elegans in a similar fashion to naked dsRNA. Taken together, these findings aid in the development of safe and effective RNAi biological control agents

Lysine-functionalized nanodiamonds: synthesis, characterization and potential as gene delivery agents

Detonation nanodiamonds (NDs), due to their 4-5 nm primary particle size, stable inert core, reactive surface, ability to form hydrogel, are emerging as intracellular delivery vehicle for small and large molecules. Despite several favorable characteristics, the use of NDs in biological systems is impeded by their high aggregation propensity in polar liquid medium. To develop NDs as potential gene delivery vectors, pristine carboxylated NDs (pNDs) were functionalized with lysine through covalent conjugation. Raman and FTIR spectroscopic determinations confirmed the functionalization of NDs with lysine molecules, while thermogravimetric analysis estimated a surface loading of 1.7 mmol/g. Through lysine-functionalization, the dispersion stability of NDs in water increased considerably, showing a zeta potential of +49 mV. The average particle size of pNDs as measured by dynamic light scattering was substantially reduced from 1281 to 21 nm after lysine functionalization. Atomic force microscopy further substantiated the disaggregation of pNDs achieved through lysine functionalization. The lysine-functionalized NDs (fNDs) were able to electrostatically bind and block the migration of the nucleic acids at a weight ratio of 5:1 and 20:1 of fNDs:pDNA and fNDs:siRNA, respectively, with a shift in zeta potential from negative to positive value. The particle size of the complexes stabilized around 110 nm for fNDs-pDNA and less than 280 nm for fNDs-siRNA at the weight ratios of 50:1 fNDs:nucleic acid. While the Raman-fluorescence maps were equivocal with regards to the cellular association of NDs, backscattering maps clearly indicated the interaction of the fNDs with the cells. Cellular internalization of a few fNDs was suggested by laser confocal scanning microscopy. MTT assay demonstrated no significant in vitro cytotoxicity of pNDs and fNDs in the concentration range from 4 to 250 µg/mL. Flow cytometeric assessment of the gene expression (GFP intensity measurements) suggested that a strong binding of siRNA with fNDs might have prevented the release of nucleic acid into the cytoplasm of the cells. Overall, in this study, stable aqueous dispersion of NDs was generated using a mechanochemical approach feasible at a small laboratory scale, and early evidence was presented that the fNDs can be optimized for safe delivery of nucleic acids into mammalian cells

Overcoming the stability, toxicity, and biodegradation challenges of tumor stimuli-responsive inorganic nanoparticles for delivery of cancer therapeutics

Introduction: Stimuli-responsive nanomaterials for cancer therapy have attracted much interest recently due to their potential for improving the current standard of care. Different types of inorganic nanoparticles are widely employed for the development of these strategies, but in some cases safety concerns hinder their clinical translation. This review aims to provide an overview of the challenges that inorganic nanoparticles face regarding their stability, toxicity and biodegradability, as well as the strategies that have been proposed to overcome them. Areas covered: The available information about the in vitro and in vivo biocompatibility, as well as the biodegradability of the following nanoparticles is presented and discussed: superparamagnetic iron oxide nanoparticles, gold nanoparticles, graphene and mesoporous nanoparticles made of silicon or silicon oxide. The toxicology of inorganic nanoparticles is greatly affected by many physicochemical parameters, and their surface modification emerges as the main intervention to improve their biocompatibility and tailor their performance for specific biomedical applications. Expert opinion: Even though many different studies have been performed regarding the biological behavior of inorganic nanoparticles, long-term in vivo data is still scarce, limiting our capacity to evaluate the proposed nanomaterials for clinical use. The role of biodegradability in different therapeutic contexts is also discussed

Optimized GeLC-MS/MS for Bottom-Up Proteomics

Despite tremendous advances in mass spectrometry instrumentation and mass spectrometry-based methodologies, global protein profiling of organellar, cellular, tissue and body fluid proteomes in different organisms remains a challenging task due to the complexity of the samples and the wide dynamic range of protein concentrations. In addition, large amounts of produced data make result exploitation difficult. To overcome these issues, further advances in sample preparation, mass spectrometry instrumentation as well as data processing and data analysis are required. The presented study focuses as first on the improvement of the proteolytic digestion of proteins in in-gel based proteomic approach (Gel-LCMS). To this end commonly used bovine trypsin (BT) was modified with oligosaccharides in order to overcome its main disadvantages, such as weak thermostability and fast autolysis at basic pH. Glycosylated trypsin derivates maintained their cleavage specifity and showed better thermostability, autolysis resistance and less autolytic background than unmodified BT. In line with the “accelerated digestion protocol” (ADP) previously established in our laboratory modified enzymes were tested in in-gel digestion of proteins. Kinetics of in-gel digestion was studied by MALDI TOF mass spectrometry using 18O-labeled peptides as internal standards as well as by label-free quantification approach, which utilizes intensities of peptide ions detected by nanoLC-MS/MS. In the performed kinetic study the effect of temperature, enzyme concentration and digestion time on the yield of digestion products was characterized. The obtained results showed that in-gel digestion of proteins by glycosylated trypsin conjugates was less efficient compared to the conventional digestion (CD) and achieved maximal 50 to 70% of CD yield, suggesting that the attached sugar molecules limit free diffusion of the modified trypsins into the polyacrylamide gel pores. Nevertheless, these thermostable and autolysis resistant enzymes can be regarded as promising candidates for gel-free shotgun approach. To address the reliability issue of proteomic data I further focused on protein identifications with borderline statistical confidence produced by database searching. These hits are typically produced by matching a few marginal quality MS/MS spectra to database peptide sequences and represent a significant bottleneck in proteomics. A method was developed for rapid validation of borderline hits, which takes advantage of the independent interpretation of the acquired tandem mass spectra by de novo sequencing software PepNovo followed by mass-spectrometry driven BLAST (MS BLAST) sequence similarity searching that utilize all partially accurate, degenerate and redundant proposed peptide sequences. It was demonstrated that a combination of MASCOT software, de novo sequencing software PepNovo and MS BLAST, bundled by a simple scripted interface, enabled rapid and efficient validation of a large number of borderline hits, produced by matching of one or two MS/MS spectra with marginal statistical significance

Engineering mechanobiology: the bacterial exclusively-mechanosensitive ion channel MscL as a future tool for neuronal stimulation technology

The development of novel approaches to stimulate neuronal circuits is crucial to understand the physiology of neuronal networks, and to provide new strategies to treat neurological disorders. Nowadays, chemical, electrical or optical approaches are the main exploited strategies to interrogate and dissect neuronal circuit functions. However, although all these methods have contributed to achieve important insights into neuroscience research field, they all present relevant limitations for their use in in-vivo studies or clinical applications. For example, while chemical stimulation does not require invasive surgical procedures, it is difficult to control the pharmacokinetics and the spatial selectivity of the stimulus; electrical stimulation provides high temporal bandwidth, but it has low spatial resolution and it requires implantation of electrodes; optical stimulation provides subcellular resolution but the low depth penetration in dense tissue still requires the invasive insertion of stimulating probes. Due to all these drawbacks, there is still a strong need to develop new stimulation strategies to remotely activate neuronal circuits as deep as possible. The development of remote stimulation techniques would allow the combination of functional and behavioral studies, and the design of novel and minimally invasive prosthetic approaches. Alternative approaches to circumvent surgical implantation of probes include transcranial electrical, thermal, magnetic, and ultrasound stimulation. Among v these methods, the use of magnetic and ultrasound (US) fields represents the most promising vector to remotely convey information to the brain tissue. Both magnetic and low-intensity US fields provide an efficient mean for delicate and reversible alteration of cells and tissues through the generation of local mechanical perturbations. In this regard, advances in the mechanobiology research field have led to the discovery, design and engineering of cellular transduction pathways to perform stimulation of cellular activity. Furthermore, the use of US pressure fields is attracting considerable interest due to its potential for the development of miniaturized, portable and implantation-free US stimulation devices. The purpose of my PhD research activity was the establishment of a novel neuronal stimulation paradigm adding a cellular selectivity to the US stimulation technology through the selective mechano-sensitization of neuronal cells, in analogy to the well-established optogenetic approach. In order to achieve the above mentioned goal, we propose the cellular overexpression of mechanosensitive (MS) ion channels, which could then be gated upon the application of an US generated pressure field. Therefore, we selected the bacterial large conductance mechanosensitive ion channel (MscL), an exclusively-MS ion channel, as ideal tool to develop a mechanogenetic approach. Indeed, the MscL with its extensive characterization represents a malleable nano-valve that could be further engineered with respect to channel sensitivity, conductance and gating mechanism, in order to obtain the desired biophysical properties to achieve reliable and efficient remote mechanical stimulation of neuronal activity. In the first part of the work, we report the development of an engineered MscL construct, called eMscL, to induce the heterologous expression of the bacterial protein in rodent primary neuronal cultures. Furthermore, we report the structural and functional characterization of neuronal cells expressing the eMscL channel, at both single-cell and network levels, in order to show that the functional expression of the engineered MscL channel induces an effective vi neuronal sensitization to mechanical stimulation, which does not affect the physiological development of the neuronal itself. In the second part of the work, we report the design and development of a water tank-free ultrasound delivery system integrated to a custom inverted fluorescence microscope, which allows the simultaneous US stimulation and monitoring of neuronal network activity at single resolution. Overall, this work represents the first development of a genetically mechanosensitized neuronal in-vitro model. Moreover, the developed US delivery system provides the platform to perform high-throughput and reliable investigation, testing and calibration of the stimulation protocols. In this respect, we propose, and envisage in the near future, the exploitation of the engineered MscL ion channel as a mature tool for novel neuro-technological applications

Identification of genes regulated during neuronal apoptosis using oligonucleotide microarrays.

Neuronal apoptosis occurs extensively during the normal development of the mammalian nervous system and ensures that appropriate connections are made between neurons and their target cells. Developing sympathetic neurons and the PC6-3 cell line, a PC 12 sub-clone, are useful in vitro systems for studying neuronal apoptosis. Both sympathetic neurons and neuronally differentiated PC6-3 cells depend on nerve growth factor (NGF) for survival and die by apoptosis after NGF withdrawal in a transcription and translation-dependent manner. The c-Jun protein is known to be required for neuronal cell death following survival factor withdrawal, however the direct targets of this basic/leucine zipper transcription factor are unknown. The aim of this thesis was to identify and study c-Jun target genes involved in neuronal apoptosis. In experiments using Affymetrix GeneChip oligonucleotide microarrays, 78 genes were identified that had an altered pattern of expression after NGF withdrawal from neuronal PC6-3 cells. One of the induced genes, ATF-3, a basic/leucine zipper transcription factor that can dimerise with c-Jun, was confirmed as an up-regulated transcript during PC6-3 cell apoptosis and this occurred after the induction of c-Jun. In sympathetic neurons, atf-3 RNA and protein levels increase between 8 and 16 hours after NGF withdrawal and remain elevated at 24 hours. This protein induction occurs after that of c-Jun and is inhibited by the c-Jun N-terminal kinase inhibitor SP600125 and the mixed-lineage kinase (MLK) inhibitor CEP-11004. This suggests that induction of ATF-3 may require JNK activity in neurons and that the atf-3 gene might be a target of c-Jun. The function of ATF-3 in sympathetic neurons was investigated in microinjection experiments. Injection of expression vectors for c-Jun and ATF-3 decreased the survival of sympathetic neurons in the presence of NGF, as measured at 72 hours after injection. In contrast, overexpression of ATF-3 or the ATF-3 basic/leucine zipper domain in the absence of NGF increased the survival of sympathetic neurons. In addition, when ATF-3 expression was knocked down, using a pSUPER ATF-3 RNAi expression vector, the amount of cell death observed after 48 hours of NGF deprivation in neurons was increased

Transcriptomic and proteomic analysis of Ascaris suum larvae during their hepato-tracheal migration

The gastro-intestinal nematodes Ascaris lumbricoides and Ascaris suum are amongst the most prevalent parasites of humans and pigs, respectively. Ascaris infections cause serious public health problems and significant economic losses in the pig industry. Traditionally ascariasis is controlled by mass treatment with anthelmintics. However, due to the short activity of the anthelmintics and an environment often highly contaminated with Ascaris eggs, reinfections can occur rapidly. In addition, the development of anthelmintic resistance which has been observed in other nematodes, suggests that current repeated doses of massive chemotherapy treatment will probably also lead to drug resistance in Ascaris spp. eventually. Therefore, investigation of the alternative means of ascariasis control such as vaccination is worthwhile for pursuing. To promote the rational development of an effective vaccine, a better understanding of the molecular biology and host-parasite relationships of Ascaris is required. In chapter two, a transcriptome dataset was generated and analyzed in order to identify both the highest transcribed and stage-specific transcripts for each larval stage, the metabolic changes and chemosensation pathways active in the larvae and, finally, the expression of potential molecular mimicry candidates. Illumina RNA sequencing of the A. suum infective-stage, liver-stage, lung-stage and intestinal-stage larvae resulted in 95,463,423 sequences yielding 18,543 contigs. Within the top 250 most highly transcribed contigs per stage, accounting for approximately 60% of the total transcription in each stage, cuticle collagens were by far the most abundant gene family. The analysis also identified a set of interesting stage-specific genes, such as venom allergen, chitinase, thioredoxin and cecropin, with a potentially important role in the host-parasite interaction processes. Analysis of the metabolic pathways showed that the degradation of complex carbohydrates likely forms an essential part of the energy metabolism of this parasite, as almost 10% of the total transcriptome of the L4 stage encodes for enzymes involved in this pathway. In comparison to Caenorhabditis elegans, a reduced number of olfactory molecules were identified in A. suum, in particular the aerotaxis molecules seem to absent, suggesting that they are less important for this parasite. Finally, 12 transcript sequences were identified as potential molecular mimicry candidates, including a suppressor of cytokine signaling family. Overall, the outcome of this transcriptomic analysis provides novel and valuable information regarding the biology of A. suum larvae, which can be used as a foundation for further research. In chapter three, we identified the excretory-secretory proteins of the migratory stages of A. suum utilizing LC-MS/MS. In total 106 proteins were identified, some of which are known as important players in the parasite-host interface. Interestingly, an abundance of glycosyl hydrolases was observed in the ES material of the intestinal L4 stage larvae. By combining the proteomic analysis with in-depth genomic, transcriptomic and enzymatic analyses we could show that the glycosyl hydrolase protein family has undergone a massive expansion in A. suum and that most of the glycolytic activity is present in the intestinal tissue of the adult parasites. Again, as already indicated by the transcriptomic analysis, this could suggest that the degradation of complex carbohydrates forms an essential part of the energy metabolism of this parasite once it establishes in the small intestine. In chapter four, we employed two different approaches, i.e. biotin labeling and enzymatic shaving, combined with LC-MS/MS to study the cuticle surface associated proteins of the infective stage larvae of A. suum. In total, 17 proteins were identified as surface associated proteins by the labeling approach. Many of these molecules had been previously reported as surface exposed proteins in other helminth species. On the other hand, the MS/MS spectra for the shaving approach only resulted in the identification of 3 genuine surface attached proteins, i.e. a cuticle collagen, a tubulin and a protein with unknown function. These results extended the knowledge on the biology of Ascaris larvae and their cuticle proteins, which play important roles in host-parasite interactions and hopefully it will benefit the development of novel intervention strategies. In conclusion, the transcriptomic and proteomic investigations performed in this thesis have led to a significant progress in our understanding of Ascaris biology. The most important observations were the potentially important role of carbohydrate metabolism in the larvae during their hepato-tracheal migration and the identification of the molecules essential to parasite survival and development. The study provides a basis for further molecular investigations aimed at exploring the biological role of the proteins identified and their potential as vaccine and/or therapeutic targets

The RNA 5’ phosphatase PIR-1 cooperates with dicer to produce endogenous small RNAs and suppress viral replication in C. elegans

Tese de doutoramento, Ciências Biomédicas (Ciências Biopatalógicas), Universidade de Lisboa, Faculdade de Medicina, 2015Most organisms utilize small RNAs (sRNA) to control diverse aspects of development, reproduction and physiology by regulating gene expression at the transcriptional and post-transcriptional levels. The essential ribonuclease Dicer is a key enzyme in the production of several types of sRNAs. Prior work on the nematode Caenorhabditis elegans (C. elegans) has uncovered PIR-1 – a small protein conserved in all metazoans – as an interacting partner of Dicer in vivo. The human ortholog of PIR-1 has RNA 5' tri- and diphosphatase activities in vitro, but its biological role remains unclear. With the intent of finding its function, we characterized various aspects of C. elegans PIR-1. We found this enzyme to be essential for general growth and development, germline proliferation, and sperm maturation. We confirmed that PIR-1 associates with Dicer in vivo and expanded its repertoire of known interactions. Profiling of sRNAs from pir-1 loss-of-function animals by high-throughput sequencing revealed that PIR-1 is required for the production of 26G-RNAs during spermatogenesis, a class of Dicer-dependent sRNAs. 26G-RNAs are essential to promote appropriate sperm development, in agreement with the pir-1 mutant sperm defect. Additionally, we discovered a second, 26G-RNA-independent role for PIR-1, in which it cooperates with Dicer and other canonical RNA interference (RNAi) pathway components to suppress the replication of the C. elegans Orsay RNA virus. By demonstrating that PIR-1 functions as its human counterpart in vitro, and that a pir-1 transgene with a mutated phosphatase active site cannot rescue any of the mutant defects, we concluded that PIR-1 acts as an RNA phosphatase in vivo. This is the first study in which concrete biological functions are assigned to this enzyme. Given its high degree of conservation, these results provide a solid basis for studies on the multiple functions of PIR-1 in more complex animals.A maioria dos organismos utiliza pequenos RNAs (sRNA) para controlar diversos aspectos do seu desenvolvimento, reprodução e fisiologia, através da regulação da expressão génica ao nível transcricional e pós-transcricional. A ribonuclease essencial Dicer desempenha um papel central na produção de vários tipos de sRNAs. Um estudo anterior realizado no nemátode Caenorhabditis elegans (C. elegans) revelou que PIR-1 – uma pequena proteína conservada em todos os metazoários – se associa à Dicer in vivo. A proteína ortóloga humana PIR1 funciona in vitro como uma tri- e di-fosfatase 5' de RNA. A sua função biológica, porém, não é clara. Com o objectivo de encontrar a sua função, procedemos à caracterização da PIR-1 de C. elegans. Esta enzima é essencial para o desenvolvimento geral e crescimento, proliferação da linha germinal e para a maturação de espermatozóides. Neste estudo comprovámos a interacção entre PIR-1 e Dicer in vivo, e expandimos o repertório de proteínas com as quais a PIR-1 se associa. Sequenciação “high-throughput” dos sRNAs de um mutante de pir-1, revelou que esta proteína é necessária, juntamente com a Dicer, para a produção de 26G-RNAs, que promovem a espermatogénese. Adicionalmente, descobrimos que em cooperação com a Dicer e outros componentes da via da interferência por RNA (RNAi), a PIR-1 suprime a replicação do vírus Orsay, que infecta especificamente C. elegans. Ao demonstrar que a PIR-1 possui a mesma actividade que a proteína humana in vitro, e que um transgene de pir-1 com o centro catalítico inactivo não permite a supressão de nenhum dos defeitos do mutante, concluímos que a PIR-1 funciona como uma fosfatase de RNA in vivo. Este é o primeiro estudo a atribuir funções biológicas a esta enzima. Dada a sua conservação, estes resultados formam uma base sólida para estudos futuros sobre as múltiplas funções da fosfatase PIR-1 em animais de maior complexidade