Horizontal transfer of RNAi between honeybees and Varroa destructor

Dissertação de mestrado em Biotecnologia Farmacêutica, apresentada à Faculdade de Farmácia da Universidade de CoimbraEste trabalho abrange uma revisão do estado de arte do processo de liofilização e da sua aplicação na área da Nanotecnologia. O foco principal desta revisão é a caracterização dos produtos liofilizados inovadores existentes no mercado e em fase de desenvolvimento clínico, com aplicação em Oncologia. Na área farmacêutica, a exploração das propriedades únicas das nanopartículas tornou-se diferenciadora pela demonstração de efeitos na biodisponibilidade de moléculas ativas e também na proteção contra a degradação enzimática. Mais recentemente, as nanopartículas têm sido usadas para proporcionar um aumento da especificidade e efetividade no direcionamento dos fármacos aos locais alvo, permitindo a diminuição da dose eficaz e da toxicidade geral. Com tudo isto, o desenvolvimento de nanoestruturas para transporte de fármacos tem sido objeto de um interesse crescente, tendo em vista colmatar alguns pontos negativos existentes nos sistemas de dosagem tradicionais. No entanto, a estabilidade das nanopartículas pode constituir um problema, pelo que a sua concepção não é propriamente linear. No caso específico das nanodispersões coloidais, fatores físicos e químicos contribuem para uma baixa estabilidade a longo termo, provocando a destabilização do sistema que limita a sua aplicação clínica. A liofilização surge neste cenário como um processo tecnológico que pode minimizar os problemas de estabilidade das suspensões aquosas de nanopartículas. Trata-se de uma técnica de desidratação muito eficiente quando comparada com as demais, que tem vindo a apresentar melhorias na estabilidade destas suspensões coloidais, e por esta razão merece destaque. Cada produto possui um ciclo de liofilização único em função das suas particularidades, o que faz com que este processo seja uma operação unitária. Para ser percetível a forma como o processo de liofilização é executado cumprindo as boas práticas laboratoriais (BPL/GLP) e de fabrico (BPF/GMP) são explícitos, neste estudo, os requisitos normativos a ter em conta. A liofilização, além de ser útil ao nível da estabilidade, é ainda utilizada para outras finalidades que estão descritas no estudo. De forma a elucidar toda a operação envolvida neste processo é feita uma breve abordagem das etapas envolvidas, dos métodos utilizados, das ix aplicações farmacêuticas e dos principais pros e contras, com o intuito de entender a importância da formulação de nanopartículas liofilizadas. Será ainda dedicado um capítulo ao estudo dos componentes e das condições do processo para conseguir obter resistência às tensões a que as suspensões de nanopartículas são submetidas durante a fase de congelamento na liofilização. Nesta fase, o efeito crioprotetor ou lioprotetor é determinante, dado que na reidratação do produto é mandatório que as propriedades originais das nanopartículas sejam mantidas. A respeito dos avanços recentes com aplicação deste processo, são ainda apresentadas as formulações liofilizadas que estão inseridas no mercado e os ensaios clínicos e pré-clínicos existentes para o tratamento de doenças do foro oncológico, com referência aos produtos protegidos por patente. A relevância deste trabalho traduz-se no facto de o resultado da pesquisa ser orientado em ambiente empresarial e académico, com vista à inserção no processo de valorização da propriedade intelectual de uma spin-off da Universidade de CoimbraThis work aims at conducting a review of the state of art of the lyophilization process and its application in the field of Nanotechnology. The main focus is the characterization of existing innovative freeze-dried products on the market and in clinical evaluation, with applications in Oncology. In the pharmaceutical area, the unique properties of nanoparticles began to demonstrate positive effects on the bioavailability of active molecules and also in protecting against enzymatic degradation. More recently, nanoparticles have been used to provide increased specificity and effectiveness in the targeting of drugs to specific locations, thereby reducing the effective dose and toxicity. With all this, the development of nanostructures for drug delivery has provided the solution for existing weaknesses in traditional dosing systems. However, the stability of the nanoparticles is not exactly a linear topic. In the case of colloidal nanodispersions, physical and chemical factors contribute to a low long term stability, leading to destabilization of the system and thus creating an obstacle to its clinical application. Lyophilization in this scenario appears as a technological process that can minimize stability problems of aqueous suspensions of nanoparticles. It is a very efficient dewatering technique, which has been presenting improvements in the stability of these colloidal suspensions, and therefore noteworthy. Each product has a single lyophilization cycle on the basis of their features, which makes this process a unitary operation. To be noticeable how the freeze-drying process is performed in compliance with the good laboratory practice (BPL/GLP) and manufacturing practice (BPF/GMP), the regulatory requirements to be considered are explicit in this study. This technique, besides being useful for stability issues, it is still used for other purposes which are described in the study. In order to elucidate the entire operation involved in this process, it is made a brief approach of the steps involved, the methods used, the pharmaceutical applications and the main pros and cons of the formulation of lyophilized nanoparticles. The study also comprises a chapter on the components and process conditions to achieve resistance to the stresses to which the nanoparticulate suspensions are subjected during the xi freezing stage of the lyophilization. At this stage, the cryoprotectant or lioprotetor effect is decisive for the rehydration, in order to preserve that the original properties of the nanoparticles. Regarding the recent advances in the application of this process, this study presents the most recent lyophilized formulations on the market and in clinical and preclinical trials for the treatment of oncological diseases, with reference to the proprietary products. The main conclusions of this work will be included in the valuing process of the intellectual property of a spin-off from the University of Coimbra, combining an academic and business perspective on a pharmaceutical process of industrial application

Gene Expression Patterns of Oxidative Phosphorylation Complex I Subunits Are Organized in Clusters

After the radiation of eukaryotes, the NUO operon, controlling the transcription of the NADH dehydrogenase complex of the oxidative phosphorylation system (OXPHOS complex I), was broken down and genes encoding this protein complex were dispersed across the nuclear genome. Seven genes, however, were retained in the genome of the mitochondrion, the ancient symbiote of eukaryotes. This division, in combination with the three-fold increase in subunit number from bacteria (N = ∼14) to man (N = 45), renders the transcription regulation of OXPHOS complex I a challenge. Recently bioinformatics analysis of the promoter regions of all OXPHOS genes in mammals supported patterns of co-regulation, suggesting that natural selection favored a mechanism facilitating the transcriptional regulatory control of genes encoding subunits of these large protein complexes. Here, using real time PCR of mitochondrial (mtDNA)- and nuclear DNA (nDNA)-encoded transcripts in a panel of 13 different human tissues, we show that the expression pattern of OXPHOS complex I genes is regulated in several clusters. Firstly, all mtDNA-encoded complex I subunits (N = 7) share a similar expression pattern, distinct from all tested nDNA-encoded subunits (N = 10). Secondly, two sub-clusters of nDNA-encoded transcripts with significantly different expression patterns were observed. Thirdly, the expression patterns of two nDNA-encoded genes, NDUFA4 and NDUFA5, notably diverged from the rest of the nDNA-encoded subunits, suggesting a certain degree of tissue specificity. Finally, the expression pattern of the mtDNA-encoded ND4L gene diverged from the rest of the tested mtDNA-encoded transcripts that are regulated by the same promoter, consistent with post-transcriptional regulation. These findings suggest, for the first time, that the regulation of complex I subunits expression in humans is complex rather than reflecting global co-regulation

Real Time PCR Primers employed in this study.

<p>Real Time PCR Primers employed in this study.</p

The tested complex I subunits, their genome affiliation (mtDNA or nuclear DNA), and their location in complex I.

<p>The tested complex I subunits, their genome affiliation (mtDNA or nuclear DNA), and their location in complex I.</p

Cluster analysis of the tissue expression pattern in complex I genes.

<p>Numbers above branches represent p-values reflecting the significance of the clustering (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009985#s4" target="_blank">methods</a> section). Tissues most contributing to the branching order are mentioned below each branch. Relative expression patterns of mtDNA and nDNA-encoded complex I subunits were normalized to a reference gene (GAPDH) and hence do not represent absolute quantification of transcripts levels in the tested tissues but rather a relative expression pattern within each tissue.</p

Bidirectional Transfer of RNAi between Honey Bee and <em>Varroa destructor</em>: <em>Varroa</em> Gene Silencing Reduces <em>Varroa</em> Population

<div><p>The mite <em>Varroa destructor</em> is an obligatory ectoparasite of the honey bee (<em>Apis mellifera</em>) and is one of the major threats to apiculture worldwide. We previously reported that honey bees fed on double-stranded RNA (dsRNA) with a sequence homologous to that of the Israeli acute paralysis virus are protected from the viral disease. Here we show that dsRNA ingested by bees is transferred to the <em>Varroa</em> mite and from mite on to a parasitized bee. This cross-species, reciprocal exchange of dsRNA between bee and <em>Varroa</em> engendered targeted gene silencing in the latter, and resulted in an over 60% decrease in the mite population. Thus, transfer of gene-silencing-triggering molecules between this invertebrate host and its ectoparasite could lead to a conceptually novel approach to <em>Varroa</em> control.</p> </div

List of <i>Varroa</i> dsRNA sequences used in <i>Varroa</i> gene silencing.

<p>This table lists the <i>Varroa</i> dsRNA sequences numbers, genes function, and the mixtures they are contained in. The full sequences are located in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003035#ppat.1003035.s003" target="_blank">Table S1</a>.</p

Mean (+ SE) total number of bees (capped brood and adults) in four treatments.

<p>Treatments did not differ significantly.</p

Demonstration of dsRNA transfer from bee to <i>Varroa</i> and from <i>Varroa</i> to bee.

<p>(A) Indirect dsRNA transmission from bee to <i>Varroa</i> parasitizing bee brood. RT-PCR of <i>Varroa</i>-extracted RNA. Numbers represent days from the beginning of dsRNA feeding. M = size markers. C = positive control (GFP-carrying plasmid). + indicates samples collected from dsRNA-GFP-treated hives. – indicates samples collected from untreated, control hives. (B) DsRNA transmission from <i>Varroa</i> to bee. RT-PCR was performed on RNA extracted from a dsRNA-GFP-carrying <i>Varroa</i> (V+) and from <i>Varroa</i> devoid of dsRNA-GFP (V−). B+ represents amplification of RNA from bees infested with dsRNA-GFP-carrying <i>Varroa</i> and B– represents amplification from bees infested with dsRNA-GFP-devoid <i>Varroa</i>. M and C: as in legend for A.</p