Abstrakt Bakgrund: Barn som drabbas med tumörer behöver genomgå strålbehandling. För att uppnå optimala behandlingsresultat krävs sederingar eller generella anestesi. Tidigare studier har indikerat att strålningsspecifika effekter påverkade kognitiva funktioner hos barn. Syfte: Karakterisera biomarkörer för skador orsakade av strålning och undersöka de neurotoxiska effekterna som uppstår vid hög exponering för gammastrålning. Metod: Del1: Design av 8 primrar för musgenerna Hes1, Hes5, Timp1, Aldh11l1 . RNA-pool används från fem olika grupper. QPCR-metoden används för att bestämma effektiviteten hos de 8 primrarna. Del2: Experimenten utfördes på NMRI-möss på postnatal dag 10. Genuttryck undersöktes för 5 kontrollgrupper och 5 exponerade grupper för hög dos gammastrålning 2Gy. DNA-extraktion utfördes på hippocampus och prefrontalcortex från mössens hjärna. qPCR-metoden används för att studera uppreglering och nedreglering av gener. Resultat: Effektiviteten för primrarna Hes 5 = 80%, Hes1:2= 93% vilket uppfyllde kriterierna 80–110%. Resultat för genererad nedreglering av BDNF i hippocampus och uppreglering av Hes 5 och Jagged-1 i prefrontal cortex, vilket visade en signifikant skillnad mellan gruppernas medelvärden. Däremot observerade ingen signifikant skillnad i uttrycket av generna Nrf2, Grin2, Jagged-1 i hippocampus. Slutsats: Strålningsdoser som används för att behandla hjärntumörer hos barn kan påverka hur vissa allmänna uttryck och leda till dynamiska förändringar i uttrycket som styr normal hjärnutveckling.

由於生物科技的蓬勃發展，加上人類基因體的序列草圖在電腦快速的運算能力配合之下已逐步完成，許多生物科技企業或生物學家，已早早開始投入生物資訊的領域中，而生物科技的領域是廣闊無垠的，只有在電腦的配合下，才能快速且準確的得到所想要的結果。而PCR技術的出現，對整個生物界可說是一埸不小的衝擊與革命。大大減少了以往所需的時間和資源。 有鑒於以上的發展，本研究乃著手收集PCR相關文獻，整理分析相關知識及相關生技公司職員討論後作為研究基礎，本研究從資訊的角度切入，以Microsoft .Net為實作平台，C# 和ASP.Net 程式語言，提供生物學家一個方便使用的跨平台網路介面，並能讓生物學家從任何地方、任何時候來使用。並導入OOA/OOP的系統架構概念，將可以提高系統功能的再使用性與整體架構的擴充性。 在本研究中以PCR(Polymerase Chain Reaction)中的引子設計為主要實作目標。且設計一個簡化版本的Multiplex PCR提供生物學家使用。而主要會影響Primer Design的優劣因素有以下這些：Melting Temperature, Annealing Score, GC content, Primer length,因此本研究使用這些因素來做為本研究選擇Primer的因素，並設計了Primer Design Algorithm來篩選出合適的引子。接著設計Primer Scoring Model用來評量引子的分數，並提供生物學家一個選擇的標準，也加入了權重的觀念，好讓使用者調整適合實驗的參數之重要性。 / The invention of PCR (Polymerase Chain Reaction) has revolutionized bioinformatics and molecular technology. PCR can amplify specific region of DNA sequence in few minutes. One of these critical parameters is the selection of short oligonucleotides, which called primer. To design the suitable primers is still not part of any standardized algorithm, and several different sets of rules or criteria have been proposed to control the selection of primer sequence. Our research results include the followings: we have created a general-purpose application that design for primer design with relative PCR domain knowledge. We have also implemented a prototype system with Microsoft new OOP language, C#. We also provide a simplify version of Multiple PCR, it could handle two DNA templates to produce qualified multiple primer sets. This program can scan entire gene sequences for all possible primer pairs obeying these primer design rules and displays information such as the length and melting temperature of each primer, which may be of additional help in choosing primers. This program allows the stringency of certain criteria to be varied for better suit the requirement of particular experiment. We try to provide a convenient computerized tool which has a friendly user interface for biologists or scientists to use easily. The design of our system is based on OOA (Object-Oriented Analysis) and OOP (Object-Oriented Programming). Therefore programmers can easily change some algorithm or updated biology data to improve the efficiency of our primer design system.

Recombinant DNA technology has been commonly used in a number of fields to synthesize new products or generate products with a new pathway. Conventional cloning methods are expensive and require significant time and labor; λ-PCR, a new cloning method developed in the Senger lab, has a number of advantages compared to other cloning processes due to its employment of relatively inexpensive and widely available materials and time-efficiency. While the amount of lab work required for the cloning process is minimal, the importance of accurate primer design cannot be overstated. The target of this study was to create an effective procedure for λ-PCR primer design that ensures accurate cloning reactions. Additionally, synthetic ribosome binding sites (RBS) were included in the primer designs to test heterologous protein expression of the cyan fluorescent reporter with different RBS strengths. These RBS sequences were designed with an online tool, the RBS Calculator. A chimeric primer design procedure for λ-PCR was developed and shown to effectively create primers used for accurate cloning with λ-PCR; this method was used to design primers for CFP cloning in addition to two enzymes cloned in the Senger lab. A total of five strains of BL21(DE3) with pET28a + CFP were constructed, each with the same cyan fluorescent protein (CFP) reporter but different RBS sequences located directly upstream of the start codon of the CFP gene. Expression of the protein was measured using both whole-cell and cell-free systems to determine which system yields higher protein concentrations. A number of other factors were tested to optimize conditions for high protein expression, including: induction time, IPTG concentration, temperature, and media (for the cell-free experiments only). Additionally, expression for each synthetic RBS sequence was investigated to determine an accurate method for predicting protein translation. NUPACK and the Salis Lab RBS Calculator were both used to evaluate the effects of these different synthetic RBS sequences. The results of the plate reader experiments with the 5 CFP strains revealed a number of factors to be statistically significant when predicting protein expression, including: IPTG concentration, induction time, and in the cell-free experiments, type of media. The whole-cell system consistently produced higher amounts of protein than the cell-free system. Lastly, contrasts between the CFP strains showed each strain's performance did not match the predictions from the RBS Calculator. Consequently, a new method for improving protein expression with synthetic RBS sequences was developed using relationships between Gibbs free energy of the RBS-rRNA complex and expression levels obtained through experimentation. Additionally, secondary structure present at the RBS in the mRNA transcript was modeled with strain expression since these structures cause deviations in the relationship between Gibbs free energy of the mRNA-rRNA complex and CFP expression. / Master of Science / Recombinant DNA technology has been used to genetically enhance organisms to produce greater amounts of a product already made by the organism or to make an organism synthesize a new product. Genes are commonly modified in organisms using cloning practices which typically involves inserting a target gene into a plasmid and transforming the plasmid into the organism of interest. A new cloning process developed in the Senger lab, λ-PCR, improves the cloning process compared to other methods due to its use of relatively inexpensive materials and high efficiency. A primary goal of this study was to develop a procedure for λ-PCR primer design that allows for accurate use of the cloning method. Additionally, this study investigated the use of synthetic ribosome binding sites to control and improve expression of proteins cloned into an organism. Ribosome binding sites are sequences located upstream of the gene that increase the molecule’s affinity for the rRNA sequence on the ribosome, bind to the ribosome just upstream of the beginning of the gene, and initiate expression of the gene. Tools have been developed that create synthetic ribosome binding sites designed to produce specific amounts of protein. For example, the tools can increase or decrease expression of a gene depending on the application. These tools, the Salis Lab RBS Calculator and NUPACK, were used to design and evaluate the effects of the synthetic ribosome binding sites. Additionally, a new method was created to design synthetic ribosome binding sites since the methods used during the design process yielded inaccuracies. Each strain of E. coli contained the same gene, a cyan fluorescent protein (CFP), but had different RBS sequences located upstream of the gene. Expression of CFP was controlled via induction, meaning the addition of a particular molecule, IPTG in this system, triggered expression of CFP. Each of the CFP strains were tested with a variety of v conditions in order to find the conditions most suitable for protein expression; the variables tested include: induction time, IPTG (inducer) concentration, and temperature. Media was also tested for the cell-free systems, meaning the strains were grown overnight for 18 hours and lysed, a process where the cell membrane is broken in order to utilize the cell’s components for protein expression; the cell lysate was resuspended in new media for the experiments. ANOVA and multiple linear regression revealed IPTG concentration, induction time, and media to be significant factors impacting protein expression. This analysis also showed each CFP strain did not perform as the RBS Calculator predicted. Modeling each strain’s CFP expression using the RBS-rRNA binding strengths and secondary structures present in the RBS allowed for the creation of a new model for predicting and designing RBS sequences.

FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / A tese à composta por dois capÃtulos. O primeiro capÃtulo inclui o trabalho cujo objetivo foi investigar o potencial uso de pares de transcritos correlativos baseados em microarranjos como marcadores de fertilidade masculina usando a displasia da bainha fibrosa (DFS) como modelo afetado. Atualmente à bastante reconhecido que a tecnologia de microarranjos pode ser limitada pelos custos e que a qualidade dos transcritos permanece relativamente desconhecida. Para responder essas questÃes, nÃs analizamos pares de transcritos estÃveis por qPCR com um processo sistemÃtico de desenho de primers sistemÃtico. Nesse estudo experimental, nÃs utilizamos amostras de homens com fertilidade comprovada e de homens com diagnÃtico de DFS. Nossa abordagem foi baseada nas sequÃncias de primers dos seis genes de interesse, os quais foram desenhados utilizando os programas Oligo7 e Primer3Plus. A especificidade do primer foi inicialmente analisada in silico atravÃs de pesquisas nos bancos de dados ENSEMBL, University of California Santa Cruz (UCSC), e National Center for Biotechnology Information (NCBI) para uso de sequÃncias especÃficas aos genes alvos. A habilidade dos pares de transcritos em classificar as amostras de homens de fertilidade comprovada das amostras de DFS foi avaliada. Nossos resultados mostraram que em conjunÃÃo com a identificaÃÃo de quatro novos pares estÃveis, a comparaÃÃo dos coeficientes de correlaÃÃo dos valores de C(t) dos DSF revelou a interrupÃÃo de quatro pares estÃveis identificados nas amostras de homens normais. Esta seleÃÃo de pares estÃveis resolve a questÃo sobre a DSF. Em conclusÃo, os resultados mostram efetivamente que o desenho de primers e qPCR podem fornecer um ensaio molecular de baixo custo para avaliar a fertilidade masculina. O segundo capÃtulo divide-se em dois estudos e avalia em carneiros os efeitos de uma dieta suplementada com farelo de castanha de caju. No estudo 1, nosso objetivo foi detectar a presenÃa de transcritos para Heat Shock Protein (HSP70), clusterina (CLU), proteÃna semelhante à subunidade alfa do complexo T (TCP1) e proteÃna do complexo T subunidade 8 (CCT8) no espermatozÃide de ovinos, seguindo a mesma metodologia para qPCR utilizada no capitulo 1. As sequÃncias de primers foram desenhadas utilizando os programas Primer3Plus e Oligo Analyzer. Gene para protamina 2 (PRM2) foi usado como controle interno de reaÃÃo. O sÃmen foi coletado de machos pÃberes Morada Nova utlizando eletroejaculador. As amostras selecionadas para extraÃÃo de RNA espermÃtico seguiram as recomendaÃÃes do ColÃgio Brasileiro de ReproduÃÃo Animal quanto aos parÃmetros de motilidade, vigor e concentraÃÃo. Nossos resultados mostraram a presenÃa de mRNA para a HSP70 nos espermatozÃides de ovinos. Maiores estudos sÃo necessÃrios a fim de confirmar ou refutar a presenÃa das chaperonas TCP1 e CCT8 no espermatozÃide ovino. A presenÃa do transcrito da HSP70 no espermatozÃide de ovinos abre perspectivas para estudos futuros sobre os efeitos do mRNA HSP70 no desenvolvimento embrionÃrio, de modo a avaliar se essa expressÃo ocorre de modo espontÃneo, programado e seqÃencial, e se esses mecanismos se refletem na fertilidade e no desenvolvimento embrionÃrio. O segundo estudo tem como principal objetivo avaliar os efeitos de uma dieta contendo farelo de castanha de caju (FCC) na expressÃo de genes relacionados ao metabolismo dos lipÃdios no mÃsculo Longissimus dorsi de carneiros Morada Nova. Vinte carneiros maduros sexualmente foram divididos em dois grupos baseando-se no peso vivo. Os animais foram mantidos em baias individuais. Durante trÃs meses, o grupo castanha (GCA) foi alimentado com raÃÃo contendo FCC, enquanto o grupo controle (GCO) recebeu raÃÃo à base de milho e soja. As duas dietas eram isocalÃricas e isoprotÃicas, adicionadas de suplemento mineral. Os carneiros tambÃm receberam feno de Tifton e Ãgua à vontade. A quantidade de alimento ofertado (raÃÃo e feno) foi ajustada diariamente para sobra de 10%. Sete genes codificantes de proteÃnas envolvidas direta ou indiretamente foram selecionados como alvos, incluindo: GH, ACACA, CAST, CAPN3, LPL, SCD e FASN. Para normalizaÃÃo, foram selecionados cinco genes candidatos: ACTB, GAPDH, RPL4, RPS18 e TBP. Dentre os sete genes alvos selecionados anteriormente, os alvos GH, ACACA e CAST foram removidos. Os dois primeiros foram removidos devido amplificaÃÃo de alinhamento mÃltiplo (baixa especificidade do primers), enquanto CAST apresentou baixa eficiÃncia de amplificaÃÃo. Da lista de gene alvo final, a expressÃo de somente dois genes foi afetada pela dieta, SCD (p<0.01) e FASN (p<0.05), enquanto LPL (p=0,1022) e CAPN3 (p=0,0939) nÃo apresentaram diferenÃa significativa (p<0.05). Os genes SCD e FASN foram reprimidos no GCA comparada ao GCO. Este à o primeiro relato de que uma raÃÃo contendo FCC afetou a expressÃo gÃnica de proteÃnas envolvidas na deposiÃÃo de lipÃdios no mÃsculo em ovinos. Considerando que uma dieta contendo FCC altera a expressÃo de genes lipogÃnicos sem afetar o ganho de peso nem a eficiÃncia reprodutiva de ovinos, faz da castanha de caju uma importante alternativa para o sistema de produÃÃo de ovinos criados em regiÃes tropicais. / This thesis presents two chapters. In the first chapter, its objective was to investigate the potential use of correlative microarray-based transcript pairs as candidate markers for male fertility using dysplasia of the fibrous sheath (DFS) as an affected model. It is widely recognized that microarray technology may be limited by cost and that the quality of the transcript remains relatively unknown. To address these issues, we analyzed the stable transcript pairs by qPCR with a systematic primer design process. On this experimental study, we used men with proven fertility and men with a diagnosis of DFS. Our approach was based on primer sequences for six genes of interest were designed using Oligo7 and Primer3Plus. Primer specificity was initially assessed in silico by searching the ENSEMBL, University of California Santa Cruz, and National Center for Biotechnology Information databases for nontarget complementary sequences throughout the genome. The ability of transcript pairs to classify samples from males of proven fertility away from DFS was assessed. Our results showed that in conjunction with identifying four new stable transcript pairs, comparison of the DFS qPCR C(t) correlation coefficients revealed the disruption of four stable fertile sample transcript pairs. This suite of transcript pairs resolves DFS. In conclusion, the results show that with effectively designed primers, qPCR may provide an affordable molecular assay to assess male fertility status. Second chapter includes two studies regarding evaluations of ram feeded with supplemented diet with cashew nut. On the frist study, our goal was detect transcripts for Heat Shock Protein (HSP70), Clusterin (CLU), Ovis aries T-complex protein 1 alfa subunit-like protein (TCP1) e Ovis aries chaperonin containing TCP1, subunit 8 (theta) (CCT8) on ram sperm by. For primer designing we used published ESTs from NCBI and manually annotated by us using Primer3Plus and OligoAnalizer. PRM2 was used as internal qPCR control. Semen samples from mature Morada Nova ram were collected by eletroejaculator, washed in PBS and prepared for further RNA extraction. Selected samples followed quality recommendatios from ColÃgio Brasileiro de ReproduÃÃo Animal regarding motility, vigor and concentration. Our results showed presence of mRNA HSP70 on ram sperm and they can possible be envolved in early embryo development, oocyte activation and post fertilization events. Further analyses will be necessary to confirm presence of TCP1 and CCT8 on ram sperm. Our findings indicate new perpectives about the effects of these chaperones during embryo development mesuring if its expression reflects male fertility on the early embryo development. On the second study the the main goal is to evaluate the effects of a lipid-enriched diet containing cashew nut brain on the expression of genes related to lipid metabolism in the longissimus dorsi muscle of Morada Nova rams. Twenty sexually mature and reproductively sound rams were divided in two groups based on ram live weight, and each ram was kept on individual pens. During three months, group 1 (G1) rams were fed with a lipid-rich diet, containing cashew nut bran (CNB), while group 2 (G2) was fed with a meal based on corn and soy. Both diets were isocaloric and isoproteic, and had a mineral mix added-in. The rams also were offered Tifton grass hay and had free access to water. The amount of diet offered (ration plus hay) was adjusted everyday to a maximum waste of 10%. Seven genes coding for proteins directly or indirectly involved in lipid metabolism were initially selected as targets, incluiding GH, ACACA, CAST, CAPN3, LPL, SCD, and FASN. Also, five genes were selected as reference genes, ACTB, GAPDH, RPL4, RPS18 and TBP. From the seven genes originally selected as targets, GH, ACACA and CAST were removed, leaving the final list with four targets. The first two genes were removed due to alternative pairing of the primers (low specificity), while CAST showed low amplification efficiency during PCR reaction. From the final target list, the expression of only two genes was affected by diet, SCD (p<0.01) and FASN (p<0.05), while LPL (p=0,1022) and CAPN3 (p=0,0939) were not different at the p<0.05 level. Both SCD and FASN genes were down-regulated in G1 (lipid-rich diet containing CNB) compared to G2. These genes are involved in lipogenic pathways, related to tissue lipid deposition; therefore, these results were expected. This is the first time that a fat-rich diet based on CNB was shown to affect gene expression of proteins involved in fat deposition in carcass muscles of rams. Longissimus dorsi is one of the finest meat cuts. Considering that human diets rich in poli-unsaturated fatty acids (PUFA) can decrease the risk of heart and other chronic diseases, a change in the fatty acid profile of this muscle could contribute to a healthier diet, aggregating value to the end-product of the lamb meat market. The effects of CNB-based diet on the gene expression of SCD and FASN support the notion that such diet, as previously shown for other sources of lipid in ruminants, can potentially change the fatty acid composition of L. dorsi, but this hypothesis needs to be experimentally verified by profiling fatty acids in animals fed CNB versus carbohydrate-based diets. CNB use as an ingredient in animal feeding is environmentally-friendly, since it contributes to by-product recycling from the agroindustrial plants in Northeast Brazil. Also, considering that CNB-based diet changes lipogenic gene expression without affecting weight gain or reproductive status of the rams, as shown in another work from our team, makes CNB a very important alternative food in ram production systems in tropical regions.