Clavicipitaceous fungal endophytes provide several ecological benefits to their hosts. Besides improving host’s growth characteristics, Neotyphodium coenophialum, the endophyte of tall fescue (Lolium arundinaceum), produces ergot alkaloids that have been proposed to be involved in fescue toxicosis. One approach to address the toxicosis problem is to genetically manipulate and modify N. coenophialum by knocking out a pair of homologous genes, (dmaW1 and dmaW2), encoding dimethylallyltryptophan synthase, the enzyme for the first and determinant step in ergot-alkaloid biosynthesis. In this study, disruption of dmaW2 was attempted using several disruption methods. Out of 1522 transformants screened, three putative knockouts were identified. Southern blot analysis of digested genomic DNA indicated that homologous gene replacement at dmaW2 locus took place while dmaW1 was still present. Chromosome separation followed by Southern-blot hybridization showed that the dmaW genes in N. coenophialum are located on different chromosomes. The aim of this study was to obtain a nontoxic endophyte free of marker genes that could be used to inoculate popular tall fescue cultivars. Therefore the Cre/loxP system developed in this study allows reusing the marker gene for sequential transformations. Protoplasts from Neotyphodium coenophialum, Neotyphodium uncinatum, or Epichloë festucae isolates, containing a floxed hygromycin phosphotransferase (hph) gene (loxP::hph::loxP), were transfected with a Crerecombinase expression plasmid and then cultured without selection. The marker was excised in 0.5-2% of the colonies, leaving a single loxP sequence. This strategy will help to reduce the concerns related to field release or commercialization of economically important grasses associated with manipulated fungal strains. It is expected that the technology will likely be adapted and applied in other fungal species. Manipulation of the ergot alkaloid (EA) gene cluster from C. purpurea and C. fusiformis by introducing and expressing its genes in different fungal-grass symbionts was also investigated. Heterologous expression of the ergot alkaloid cluster could result either in the synthesis of compounds similar to the ones produced by the host or in synthesis of novel compounds with new modes of action. Even though the results indicated that several EA genes were expressed in the new symbiota, none of the ergot alkaloids intermediates were detected.

Human Herpes Simplex viruses and Simian Herpes Simplex viruses share a high degree of genome homology, but despite this, important differences arise when the viruses are compared at the level of gene expression and virulence in non-host primates. In Human Herpes viruses (HSV-1 and HSV-2); 5 genes (RL02, US01, RS01, UL54 and US12) are expressed with an immediate early kinetics, i.e. their transcriptional activation does not require de novo synthesis of host or viral factors. The five immediate early (IE) genes regulate the cascade of expression of the other early and late HSV genes. Literature indicates that in HSV-1 infections, ICP4, ICP27 and to a lesser extent, ICP0, are mandatory for the full expression of the early and late gene classes. In contrast, our data on the Simian simplexviruses SA8, HVP-2 and B virus indicate that ICP0 (RL2) is the only gene with true IE kinetics. It is possible that in Simian Herpes viruses, ICP0 is necessary for the expression of all other viral genes, and to test this hypothesis I have cloned and expressed in Vero cells the ICP0 protein for the simian simplexvirus SA8 and studied its effect on the SA8 genes that are homologous to the immediate early genes in HSV. Results demonstrate that ICP0 does not appear to be sufficient to activate the transcription of the other IE genes but it is likely that ICP0 functionality is a necessary component in the activation process.

Human Herpes Simplex viruses and Simian Herpes Simplex viruses share a high degree of genome homology, but despite this, important differences arise when the viruses are compared at the level of gene expression and virulence in non-host primates. In Human Herpes viruses (HSV-1 and HSV-2); 5 genes (RL02, US01, RS01, UL54 and US12) are expressed with an immediate early kinetics, i.e. their transcriptional activation does not require de novo synthesis of host or viral factors. The five immediate early (IE) genes regulate the cascade of expression of the other early and late HSV genes. Literature indicates that in HSV-1 infections, ICP4, ICP27 and to a lesser extent, ICP0, are mandatory for the full expression of the early and late gene classes. In contrast, our data on the Simian simplexviruses SA8, HVP-2 and B virus indicate that ICP0 (RL2) is the only gene with true IE kinetics. It is possible that in Simian Herpes viruses, ICP0 is necessary for the expression of all other viral genes, and to test this hypothesis I have cloned and expressed in Vero cells the ICP0 protein for the simian simplexvirus SA8 and studied its effect on the SA8 genes that are homologous to the immediate early genes in HSV. Results demonstrate that ICP0 does not appear to be sufficient to activate the transcription of the other IE genes but it is likely that ICP0 functionality is a necessary component in the activation process.

The skin is the largest organ in the body. Its function is to protect the body from potential harm and to maintain homeostasis. The epidermis is the outermost layer of the skin. Stratum corneum is the outermost layer of the epidermis, composed of corneocytes and surrounding lipids. The lipids are produced by different enzymes that all play a role in the formation and function of the skin permeability barrier. Mutations in genes coding for these enzymes can lead to barrier dysfunction and could cause autosomal recessive congenital ichthyosis (ARCI). Nine genes have been identified as ARCI-causative and two of them are CYP4F22 and CERS3.   The purpose of this project was to study co-localization of CYP4F22 with CERS3 and also mutated CYP4F22 enzymes, by transfecting plasmids into HeLa and HaCaT cells and performing PLA on HEKn cells. Co-localization could indicate potential interactions and by studying these more in the future, novel treatment strategies can be developed for ARCI patients.   Transfection attempts showed a low transfection grade of wild type genes in both HeLa and HaCaT cells. Tendencies towards co-localization was seen in both cell types and some HeLa cells showed strong correlation after image analysis. Transfection of mutated genes failed, unfortunately. PLA showed co-localization in normal keratinocytes. The obtained results indicated a co-localization, but results need to be confirmed by immunoprecipitation and immunoblotting in the future.

Master’s thesis deals with the transient transfection of the serum free animal cell culture using polyethyleneimines. In the theoretical part formation of recombinant DNA molecules, used expresion vectores, used DNA transfer and detection of recombinant proteins are discussed. The experimental part deals with efficiency of the polyethylenimine mediated transient transfection under various experimental conditions. 293HEK/EBNA cell line was chosen as an experimental model. First the most effective plasmide - pCEP4/SEAP was selected. Then three transfection methodes were tested: Muller (2005), Durocher et al. (2007) and Backliwal et al. (2008). The highest recombinant protein expresion was reached using the method of Backliwal et al. (2008).

Efficient production of the recombinant proteins (r-proteins) must be based on previous testing of an expression of a small amount of the r-proteins. This work focuses on optimizing the expression of the r-proteins in 12-well plates. It includes testing of an appropriate speed of shaking, production and transfection volume. It compares all the current testing vessels (it compares a 50-ml centrifugation tube to new tested plates that can substitute the unsuitable tubes). It also compares these new tested plates to production square bottles in order to compare the r-protein expression in the plates to the r-protein expression in the bottles. It monitors effects of carbon dioxide on a number of vital cells, their viability, a relative frequency of positive cells on GFP in various cultivation vessels (plates, tubes, bottles), and pH of HEK 293 cellular cultivation during the 4-day cultivation process as well. On the basis of the results and statistical processing of the results, we have set the optimal agitation speed of 230 rpm for the 12-well plates. We have also set the appropriate production and transfection volume of 2 and 0.5 ml for the 12-well plates. In order to evaluate variables and compare cultivations in all the vessels, the tubes could be substituted by the plates. There is a statistically significant impact of carbon dioxide on the number of cells, their variability, relative frequency of cells (positive on GFP) and pH of the cellular HEK 293 cultivation in the cultivation vessels. There is the strongest r-protein expression in carbon dioxide conditions. The results of this work allow to employ the 12-well plates when we aim to test the expression of the r-proteins in a small amount and in carbon dioxide conditions. On the basis of the findings, the expression of the r-proteins in the 12-well plates and carbon dioxide conditions can substitute the expression of the r-proteins in the production bottle and in carbon dioxide free conditions.

A prolactina humana (hPRL) é um hormônio sintetizado pela hipófise com inúmeras funções tais como: lactação, reprodução e regulação osmótica. Este hormônio é frequentemente dosado em casos de problemas na lactação, infertilidade, além de estudos que elucidam sua ligação em alguns tipos de câncer (mama, próstata e útero). A hPRL é encontrada na forma não glicosilada (NG-hPRL) (23 kDa) e glicosilada (G-hPRL) (25 kDa), sendo a isoforma glicosilada um modelo ideal de análise de perfil de N-glicanos, já que possui um único sítio de glicosilação localizado na Asparagina 31. A glicosilação está relacionada diretamente à solubilidade, à estabilidade, ao enovelamento, à meia-vida e atividade biológica in vivo. As células de ovário de hamster chinês (CHO) e as células embrionárias de rim humano (HEK293) são os hospedeiros mais utilizados para expressão de proteínas recombinantes, já que podem ser cultivadas em altas densidades e por possuírem similaridade nas modificações pós-traducionais. O objetivo foi expressar, purificar e realizar uma caracterização físico-química e biológica da hPRL glicosilada de células HEK293, incluindo análise da estrutura de carboidratos. Para tanto, foi realizada uma transfecção em células HEK293T (aderidas) com o vetor pcDNA 3.4-TOPO. Foi obtida uma expressão de 21,26 ± 8,3 μg/mL de hPRL no meio condicionado sem soro. A hPRL foi purificada por cromatografia de afinidade a metais imobilizados (IMAC), eluindo 92% da hPRL em uma única fração que, analisada por HPSEC, apresentou pureza de 97%. O perfil de N-glicanos da amostra apresentou seis espécies, todas com terminação em ácido-siálico, do tipo complexo, sendo bi, tri e tetra-antenárias, com relativa predominância da espécie N2G2S1 (29,4%). A bioatividade in vitro da G-hPRL HEK293 demonstrou ser ≅ 16 vezes menor que a G-hPRL produzida em células CHO. / Human prolactin (hPRL) is a hormone synthesized by the pituitary gland with innumerable functions such as lactation, reproduction and osmotic regulation. This hormone is often determined in cases of lactation problems, infertility, and studies that elucidate its connection in some types of cancer (breast, prostate and uterus). The hPRL is found in the non-glycosylated (NG-hPRL) (23 kDa) and glycosylated (G-hPRL) (25 kDa) form, being the glycosylated isoform an ideal model for N-glycan profile analysis, since it has a single glycosylation site located in Asparagine 31. Glycosylation is directly related to solubility, stability, folding, half-life and biological activity in vivo. Chinese hamster ovary (CHO) cells and human embryonic kidney (HEK293) cells are the most widely used hosts for expression of recombinant proteins, since they can be grown at high densities and have similarity in post-translational modifications. The objective of this work was to express, purify and perform a physicochemical and biological characterization of the glycosylated hPRL from HEK293 cells, including analysis of the carbohydrate structure. For this purpose, a transfection was performed on HEK293T (adhered) cells with the 3.4-TOPO pcDNA vector. Expression of 21.26 ± 8.3 μg/mL hPRL in the serum free conditioned medium was obtained. The hPRL was purified by immobilized metal affinity chromatography (IMAC), eluting 92% of the hPRL in a single fraction which analyzed by HPSEC, showed 97% purity. The N-glycans profile of the sample showed six species, all with sialic acid termination, complex type, being bi, tri and tetra antennary, with a relative predominance of N2G2S1 (29.4%). In vitro bioactivity of G-hPRL HEK293 demonstrated to be ≅ 16-fold lower than G-hPRL produced in CHO cells.

A prolactina humana (hPRL) é um hormônio sintetizado pela hipófise com inúmeras funções tais como: lactação, reprodução e regulação osmótica. Este hormônio é frequentemente dosado em casos de problemas na lactação, infertilidade, além de estudos que elucidam sua ligação em alguns tipos de câncer (mama, próstata e útero). A hPRL é encontrada na forma não glicosilada (NG-hPRL) (23 kDa) e glicosilada (G-hPRL) (25 kDa), sendo a isoforma glicosilada um modelo ideal de análise de perfil de N-glicanos, já que possui um único sítio de glicosilação localizado na Asparagina 31. A glicosilação está relacionada diretamente à solubilidade, à estabilidade, ao enovelamento, à meia-vida e atividade biológica in vivo. As células de ovário de hamster chinês (CHO) e as células embrionárias de rim humano (HEK293) são os hospedeiros mais utilizados para expressão de proteínas recombinantes, já que podem ser cultivadas em altas densidades e por possuírem similaridade nas modificações pós-traducionais. O objetivo foi expressar, purificar e realizar uma caracterização físico-química e biológica da hPRL glicosilada de células HEK293, incluindo análise da estrutura de carboidratos. Para tanto, foi realizada uma transfecção em células HEK293T (aderidas) com o vetor pcDNA 3.4-TOPO. Foi obtida uma expressão de 21,26 ± 8,3 μg/mL de hPRL no meio condicionado sem soro. A hPRL foi purificada por cromatografia de afinidade a metais imobilizados (IMAC), eluindo 92% da hPRL em uma única fração que, analisada por HPSEC, apresentou pureza de 97%. O perfil de N-glicanos da amostra apresentou seis espécies, todas com terminação em ácido-siálico, do tipo complexo, sendo bi, tri e tetra-antenárias, com relativa predominância da espécie N2G2S1 (29,4%). A bioatividade in vitro da G-hPRL HEK293 demonstrou ser ≅ 16 vezes menor que a G-hPRL produzida em células CHO. / Human prolactin (hPRL) is a hormone synthesized by the pituitary gland with innumerable functions such as lactation, reproduction and osmotic regulation. This hormone is often determined in cases of lactation problems, infertility, and studies that elucidate its connection in some types of cancer (breast, prostate and uterus). The hPRL is found in the non-glycosylated (NG-hPRL) (23 kDa) and glycosylated (G-hPRL) (25 kDa) form, being the glycosylated isoform an ideal model for N-glycan profile analysis, since it has a single glycosylation site located in Asparagine 31. Glycosylation is directly related to solubility, stability, folding, half-life and biological activity in vivo. Chinese hamster ovary (CHO) cells and human embryonic kidney (HEK293) cells are the most widely used hosts for expression of recombinant proteins, since they can be grown at high densities and have similarity in post-translational modifications. The objective of this work was to express, purify and perform a physicochemical and biological characterization of the glycosylated hPRL from HEK293 cells, including analysis of the carbohydrate structure. For this purpose, a transfection was performed on HEK293T (adhered) cells with the 3.4-TOPO pcDNA vector. Expression of 21.26 ± 8.3 μg/mL hPRL in the serum free conditioned medium was obtained. The hPRL was purified by immobilized metal affinity chromatography (IMAC), eluting 92% of the hPRL in a single fraction which analyzed by HPSEC, showed 97% purity. The N-glycans profile of the sample showed six species, all with sialic acid termination, complex type, being bi, tri and tetra antennary, with a relative predominance of N2G2S1 (29.4%). In vitro bioactivity of G-hPRL HEK293 demonstrated to be ≅ 16-fold lower than G-hPRL produced in CHO cells.

V této diplomové práci je popsána přechodná transfekce buněk 293 HEK adaptovaných na růst při suspenzní kultivaci bez přítomnosti séra za použití polyethyleniminů (PEI). Buňky byly transfekovány plasmidem pcDNA5/SEAP, který exprimuje sekretovanou formu lidské placentální alkalické fosfatázy. K porovnání účinnosti jednotlivých transfekcí byla měřena koncentrace exprimované fosfatázy v buněčném supernatantu. Cílem této práce bylo optimalizovat různé faktory ovlivňující účinnost transfekcí s důrazem na nalezení optimálního poměru DNA:PEI.

This thesis work describes the role of Rab proteins in amyloid processing and clearance in different cell pathways. It also describes an experimental approach used to analyze the expression effects of Rab10 in amyloid beta production. Since the main theory behind neurodegeneration in Alzheimer's disease claims that high levels of amyloid beta 42 (Aβ42) molecules trigger widespread neuronal death, control of Aβ42 has been a main target in Alzheimer's disease research. In addition, several studies show increased levels of particular Rab proteins in Alzheimer's pathogenesis. However, no review consolidates current findings in neurodegeneration of Alzheimer's with Rab protein dysfunction. The first chapter of this thesis aims to address this need by providing a current review of Rab proteins associated with APP and neurodegeneration. The second chapter constitutes an experimental approach used to characterize the effects of Rab10 and Sar1A GTPases in APP and amyloid processing. We found that Rab10 expression does not affect APP production but significantly changes Aβ generation, particularly the toxic Aβ42 and Aβ42:40 ratio. On the other hand, we found no significant effect of Sar1A expression on either APP or amyloid beta generation. These findings partially confirm the work done by Kauwe et al (2015) and provide preliminary evidence for two potential targets for protective effects in neurodegeneration.