Cytoplasmic dynein is a multisubunit complex involved in retrograde transport of cellular components along microtubules. The heavy chains (HC) are very large catalytic subunits which possess microtubule binding ability. The intermediate chains (IC) are responsible for targeting dynein to its appropriate cargo by interacting with the dynactin complex. The light intermediate chains (LIC) are previously unexplored subunits that have been proposed to modulate dynein activity by regulating the motor or the IC-dynactin interaction. The light chains (LC) are a newly identified class of subunit which are also thought to have regulatory functions. In the first part of this work, I analyzed the relationship between the four SDS-PAGE gel bands that comprise the light intermediate chains. 1- and 2-D electrophoresis before and after alkaline phosphatase treatment revealed that the four bands are derived from two different polypeptides, each of which is phosphorylated. Peptide microsequencing of these subunits yielded sequences that indicated similarity between them. cDNA cloning of the rat LICs revealed the presence of a conserved P-loop sequence and a very high degree of homology between the two different rat LICs and among LICs from different species. The second series of experiments was designed to analyze the association of pericentrin with cytoplasmic dynein. First, various dynein and dynactin subunits were co-associate with pericentrin in these experiments. Co-precipitation from 35S labeled cell extracts revealed a direct interaction between LIC and pericentrin. Comparison of pericentrin binding by LICl and LIC2 showed that only LICl was able to bind. Further investigation of the relationship between LICl and LIC2 demonstrated that each LIC will self-associate, but they will not form heterooligomers. Additionally, using co-overexpression and immunoprecipitation of LICl, LIC2, and HC, I have shown that binding of the two LICs to HC is mutually exclusive. Finally, I investigated the relationships between dynein HC, IC, and LIC by examining the interactions among the subunits. IC and LIC were both found to bind to the HC, but not to each other. Despite the lack of interaction between IC and LIC, they are, in fact, present in the same dynein complexes and they have partially overlapping binding sites within the N-terminal sequence of the HC. The HC dimerization site was determined to extend through a large portion of the N-terminus, and it includes both the IC and LIC binding sites, although these subunits are not required for dimerization. Together these studies implicate the light intermediate chains in dynein targeting. Targeting of dynein to its cargo has been thought to be performed by the dynactin complex, and for one particular cargo, the kinetochore, there is considerable evidence to support this model. The results presented here suggest that the light intermediate chains appear to function in a separate, non-dynactin-based targeting mechanism.

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Os venenos de serpentes contém uma mistura complexa de toxinas (proteínas e enzimas) que destroem os processos bioquímicos ou fisiológicos da presa. A crotamina é um dos principais componentes do veneno da cascavel Sul Americana Crotalus durissus terrificus. Essa proteína pertence à família dos polipeptídeos miotóxicos de baixo peso molecular (SBMPs). Uma importante atividade da crotamina é a habilidade de penetrar rapidamente em células proliferamente ativas, podendo ser usada como nanocarreadora, levando DNA plasmidial e drogas para dentro de células tumorais. Outra atividade interessante desta toxina é a indução de morte celular dose dependente em células cancerígenas proliferamente ativas sem prejudicar células normais, demonstrando promissora atividade anticâncer. A crotamina apresenta forte atividade antifúngica e moderada atividade contra procariotos, age bloqueando canais de potássio dependente de voltagem, sendo seletiva para os canais Kv1.1, Kv1.2 e Kv1.3, e induz paralisia dos membros posteriores de camundongo. Dada a importância das atividades da crotamina, a toxina foi usada para produzir na forma recombinante. De forma geral, as toxinas animais são peptídeos secretados para o lúmen da glândula de veneno e são ricos em pontes dissulfeto, comumente expressas em sistemas procarióticos na forma de corpúsculos de inclusão ou em sistemas de expressão periplasmática. Foram construídos três genes sintéticos, um para a expressão em citoplasma e dois para a expressão em periplasma bacteriano utilizando o vetor pRSET-A (Invitrogen ®). Para a expressão de crotamina em citoplasma bacteriano, a região do DNA que codifica a região do peptídeo maduro foi clonada em fusão com uma cauda simples de 6 x His separada por uma seqüência codificante do sítio de clivagem de enterokinase em substituição à proteína de fusão do vetor comercial pRSETA. Para a expressão em periplasma a região codificante da crotamina madura foi clonada sob o controle da seqüência sinal da OmpA de E. coli. Todos os códons raros da crotamina madura foram otimizados de acordo com os códons preferenciais de E. coli. Em uma das construções para a expressão em periplasma, além da crotamina madura, o peptídeo sinal de OmpA também teve seus códons raros otimizados. Os plasmídeos desenhados para a expressão da crotamina madura em citoplasma e periplasma foram transformados em bactéria competente BL21(DE3) e BL21-AI e a indução da proteína foi feita por IPTG e arabinose, respectivamente. A expressão foi analisada por SDS-PAGE e Western Blotting, mostrando que foi possível expressar as três construções em BL21-AI. / Snake venoms contain a complex cocktail of toxins (proteins and enzymes) which disrupt physiological or biochemical processes of the pray. Crotamine is one of the major components present in the venom of the South American rattlesnake Crotalus durissus terrificus. It belongs to the small basic myotoxins peptide SBMPs. An important activity of crotamine is the ability to rapidly penetrate proliferative cells, acting as a nanocarrier, carrying plasmid DNA and drugs into tumor cells. Another interesting activity of this toxin is the ability to promote cell death of actively proliferating cancer cells in a dose dependent manner. Crotamine shows strong antifungical activity and modest activities against prokaryotes, it acts by blocking voltage-dependent potassium channels being selective for channels Kv1.1, Kv1.2 e Kv1.3 and inducing paralysis of the hind limbs of mice. Given the importance of the activities of crotamine, the toxin was chosen for production in the recombinant form. Generally, animal toxins are peptides secreted into the lumen of the venom gland and are rich in disulfide bridges. When expressed in prokaryotic system, animal toxins are commonly found in the form of inclusion bodies. They can also be expressed in periplasmic sytem. Three synthetic genes were constructed, one for expression in the cytoplasm and two for expression in the bacterial periplasm using pRSET-A (Invitrogen ®) system. For cytoplasm expression, the DNA region encoding the mature peptide was cloned in fusion with a 6 x His tag separated by a site. For expression in the periplasm, the mature crotamine coding region was cloned in fusion with E. coli OmpA signal sequence. All crotamine and OmpA signal peptide rare codons were optimized according to E. coli preferred codon usage. The plasmids designed for crotamine expression in periplasm and cytoplasm have been transformed in strains BL21(DE3) and BL21-AI were used as hosts, and the induction of recombinant protein was done by IPTG and arabinose, respectively. The induced culture products were analyzed by SDSPAGE and Western Blot, showing that it was possible to express the three constructs in BL21-AI.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22065号 / 工博第4646号 / 新制||工||1724(附属図書館) / 京都大学大学院工学研究科マイクロエンジニアリング専攻 / (主査)教授 井上 康博, 教授 中部 主敬, 教授 横川 隆司 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

<p> Two studies are presented in this thesis. First part is about the origin of tRNA genes in Trypanosoma and Leishmania. These organisms have special mitochondrial DNA, termed kinetoplast DNA (kDNA), which is unique in its structure and function. kDNA is a massive network which is composed of thousands of connected DNA circles. Unlike most other mitochondrial genomes, there is no gene encoding tRNAs in their kDNAs. So all the tRNAs used in mitochondria must be encoded on nuclear genes and transported from the cytoplasm into the mitochondria. So our question of interest is where the tRNA genes in their nucleus come from. We carry out phylogenetic analysis of these genes and the corresponding ones in bacteria, mitochondria and eukaryotic nuclei. There is no evidence indicating gene transfer from mitochondria to nucleus on the basis of this analysis. These results are consistent with the simplest hypothesis, i.e. that all tRNA genes of Trypanosoma and Leishmania have the same origin as nuclear genes of other eukaryotes.</p> <p> The second part is about the comparison of eukaryote phylogenies obtained from mitochondrial rRNA and protein sequences. We carried out phylogenetic analysis for the species which have complete mitochondrial genomes by using both concatenated mitochondrial rRNA and protein sequences. We got phylogenies for three groups, fungi/metazoan, plant/algae and stramenopile/alveolate group. The analysis is useful for the further study of position of the genetic code changes and the mechanisms involved.</p> / Thesis / Master of Science (MSc)

Electroporation affects the dielectric properties of cells. Dielectric measurement techniques can provide a label-free and non-invasive modality to study this phenomenon. In this thesis we introduce a dielectrophoresis (DEP) based technique to study changes in the cytoplasm conductivity of single Chinese hamster ovary (CHO) cells immediately after electroporation. Using a microfluidic chip, we study changes in the DEP response of single CHO cells a few seconds after electroporation. First, in order to quantify our DEP measurement results and relate them to the cells internal conductivity, we introduce a dielectric model for CHO cells. This is achieved by measuring the DEP response of many individual cells in the β-dispersion frequency region and curve fitting to the measured data. Second, we present quantitative results for changes in the cytoplasm conductivity of single cells subjected to pulsed electric fields with various intensities. We observe that when electroporation is performed in media with lower ionic concentration than cells cytoplasm, their internal conductivity decreases after electroporation depending on the intensity of applied pulses. We also observe that with reversible electroporation there is a limit on the decrease in the cells’ internal conductivity. We hypothesize the reason is the presence of large and relatively immobile negative ions inside the cell which attract mobile positive ions (mainly sodium and potassium) to maintain cell electrical neutrality. We monitor the temporal response of cells after electroporation to measure the time constant of changes due to ion transport and observe this ranges from seconds to tens of seconds depending on the applied pulse intensity. This result can be used to infer information about the density and resealing time of very small pores (not measurable with conventional marker molecules). Lastly, we measure the electroporation of cells in media with different conductivities. Our results show that electroporation in very low conductivity media requires stronger pulses to achieve a similar poration extent as in high conductivity media. The outcome of this thesis can be used to improve our understanding of the dynamics of electroporation as well as its modelling in order to make more accurate predictions or optimize the process for specific applications. / February 2017

Durante o desenvolvimento de holometálobos alguns tecidos são eliminados/remodelados durante a metamorfose. A autofagia age nesse processo degradando componentes citoplasmáticos, inicialmente isolando-os em dupla membrana, estrutura chamada autofagossomo e esses conteúdos são degradados por hidrolases lisossomais. Porém, aspectos apoptóticos podem estar presentes nesse processo, como o envolvimento de caspases e a fragmentação nuclear. Alterações morfológicas na glândula salivar e no corpo gorduroso, que são bons exemplos de órgãos que sofrem morte celular programada (MCP) no desenvolvimento de R. americana, foram analisados por microscopia de luz e eletrônica. Durante a remoção desses órgãos, núcleos apresentam morfologia condensada e com fragmentação confirmada por TUNEL. Ambos tecidos mostraram formação de autofagossomos, mas a glândula salivar completa o processo de MCP durante a metamorfose. Genes antiapoptóticos e autofágicos que têm importante papel na MCP foram caracterizados. MCP em R. americana apresenta cooperação de aspetos da autofagia e da apoptose. / In the development of holometabolous insects, some tissues are eliminated/remodelated during metamorphosis. Autophagy acts in this process by degrading cytoplasm contents, initially by surrounding them within a double membrane, a structure called autophagosome and its contents are degraded by lysosomal hydrolases. However, some features of apoptotic cell death may be present in this process, such as the involvement of caspases and nuclear fragmentation. Morphological changes of salivary gland and fat body, good examples of organs that suffer programmed cell death (PCD) during R. americana development, were analyzed by light and electron microscopy. During the removal of these organs, nuclei present fragmented and condensed morphology, confirmed by TUNEL assay. Both tissues show the formation of autophagosomes, but the salivary gland completes the process of PCD during metamorphosis. Antiapoptotic and autophagic genes that play important function in the PCD, were characterized. R. americana PCD occurs with the cooperation of autophagy and apoptosis features.

The development of T cells in the thymus is a tightly regulated process. Any defect in thymic differentiation could result in autoimmune disorders, inability to ward off infections or neoplasm. Early thymocyte development requires signals mediated through the preTCR complex by the associated CD3 chains (gamma, delta, epsilon, and zeta). Research conducted towards this project has revealed that signaling modules within the intracytoplasmic domain of CD3epsilon is absolutely required for this process. Interestingly, our results emphasized the importance of the proline-rich sequence motif in preTCR mediated signaling events, such as the proliferation of double negative thymocytes and the regulation of TCR surface expression on double positive thymocytes in a stage-specific manner. The outcomes of this project may provide a better understanding of the mechanism of preTCR-mediated thymocyte differentiation and the role of CD3 chains in these processes.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010.