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

Implementing the sterile insect technique with RNA interference – a review

RNA interference (RNAi) of insect pests is reviewed and its potential for implementing Sterile Insect Technique (SIT)-related control is considered. The molecular mechanisms that support RNAi in pest species are reviewed in detail, drawing on literature from a range of species including Drosophila melanogaster and Homo sapiens. The underlying genes that enable RNAi are generally conserved across taxa, although variance exists in both their form and function. RNAi represents a plausible, non-GM system for targeting populations of insects for control purposes, if RNA interference (RNAi) effector molecules can be delivered environmentally (eRNAi). We consider studies of eRNAi from across several insect orders and review to what extent taxonomy, genetics and differing methods of double stranded (ds)RNA synthesis and delivery can influence the efficiency of gene knockdown. Several factors, including the secondary structure of the target mRNA and the specific nucleotide sequence of dsRNA effector molecules, can affect the potency of eRNAi. However, taxonomic relationships between insects cannot be used to reliably forecast the efficiency of an eRNAi response. The mechanisms by which insects acquire dsRNA from their environment require further research, but the evidence to date suggests that endocytosis and transport channels both play key roles. Delivery of RNA molecules packaged in intermediary carriers such as bacteria or nanoparticles may facilitate their entry into and through the gut, and enable the evasion of host defense systems, such as toxic pH, that would otherwise attenuate the potential for RNAi

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

Transcriptome analysis of complex I-deficient patients reveals distinct expression programs for subunits and assembly factors of the oxidative phosphorylation system

Contains fulltext : 154334.pdf (publisher's version ) (Open Access)BACKGROUND: Transcriptional control of mitochondrial metabolism is essential for cellular function. A better understanding of this process will aid the elucidation of mitochondrial disorders, in particular of the many genetically unsolved cases of oxidative phosphorylation (OXPHOS) deficiency. Yet, to date only few studies have investigated nuclear gene regulation in the context of OXPHOS deficiency. In this study we performed RNA sequencing of two control and two complex I-deficient patient cell lines cultured in the presence of compounds that perturb mitochondrial metabolism: chloramphenicol, AICAR, or resveratrol. We combined this with a comprehensive analysis of mitochondrial and nuclear gene expression patterns, co-expression calculations and transcription factor binding sites. RESULTS: Our analyses show that subsets of mitochondrial OXPHOS genes respond opposingly to chloramphenicol and AICAR, whereas the response of nuclear OXPHOS genes is less consistent between cell lines and treatments. Across all samples nuclear OXPHOS genes have a significantly higher co-expression with each other than with other genes, including those encoding mitochondrial proteins. We found no evidence for complex-specific mRNA expression regulation: subunits of different OXPHOS complexes are similarly (co-)expressed and regulated by a common set of transcription factors. However, we did observe significant differences between the expression of nuclear genes for OXPHOS subunits versus assembly factors, suggesting divergent transcription programs. Furthermore, complex I co-expression calculations identified 684 genes with a likely role in OXPHOS biogenesis and function. Analysis of evolutionarily conserved transcription factor binding sites in the promoters of these genes revealed almost all known OXPHOS regulators (including GABP, NRF1/2, SP1, YY1, E-box factors) and a set of novel candidates (ELK1, KLF7, SP4, EHF, ZNF143, and TEL2). CONCLUSIONS: OXPHOS genes share an expression program distinct from other genes encoding mitochondrial proteins, indicative of targeted nuclear regulation of a mitochondrial sub-process. Within the subset of OXPHOS genes we established a difference in expression between mitochondrial and nuclear genes, and between nuclear genes encoding subunits and assembly factors. Most transcription regulators of genes that co-express with complex I are well-established factors for OXPHOS biogenesis. For the remaining six factors we here suggest for the first time a link with transcription regulation in OXPHOS deficiency

ANALISIS SENTIMEN PADA JASA OJEK ONLINE MENGGUNAKAN METODE NAÏVE BAYES

Ojek online adalah sebuah terobosan baru di bidang transportasi yang semakin diminati masyarakat. Analisis sentimen pada jasa ojek online merupakan proses mengekstraksi pendapat, sentimen, evaluasi, dan emosi orang tentang pelayanan ojek online yang tertulis. Di media sosial masyarakat mengeluarkan beragam opini tentang pelayanan dari transportasi ini dengan jumlah yang banyak, sehingga terdapat kesulitan untuk menentukan opini yang bersifat positif, negatif ataupun netral. Penelitian terdahulu mengenai analisis sentimen pada Twitter menggunakan metode Naïve Bayes dengan data sebanyak 3000 data yang terdiri dari 1000 kalimat netral, 1000 kalimat positif dan 1000 kalimat negatif dengan hasil akurasi hingga 88%. Sedangkan penelitian yang akan dilakukan yaitu membuat sistem yang mampu mengklasifikasikan sentimen ke dalam sentimen positif, netral atau negatif serta menyalurkan opini tersebut ke setiap jasa yang bersangkutan dengan opini yang muncul. Hasil yang didapatkan dari akurasi naïve bayes memperoleh ketepatan 80%. Kata kunci: Naïve Bayes, Ojek Online, Opini, Twitte