Programmed cell death (PCD) is a highly evolved mechanism of cellular suicide that is aberrantly activated following neural injury. Two fundamental PCD signaling pathways termed the extrinsic (caspase-8-mediated) and intrinsic (caspase-9-mediated) pathways, have been described. While each pathway is initiated by distinct cellular stimuli, both pathways culminate in the activation of downstream executioner caspases. Previous efforts to isolate the in vivo contribution of each pathway have been hindered by the embryonic lethality of casp8 and casp9 null mice. In the present study, I overcame this obstacle to directly assess the contribution of each pathway following two well-characterized forms of acute neural injury; excitotoxic destruction of CA1 pyramidal neurons, and the loss of motor neurons following facial nerve transection. To determine the role of caspase-8, I constructed several lines of mice in which caspase-8 was conditionally ablated within the relevant neuronal populations. The results obtained from these animals definitively demonstrate that caspase-8 is not required by either motor neurons or CA1 pyramidal neurons to undergo PCD following injury. Therefore, these findings have provided the first direct experimental evidence to counter the widely held dogma of caspase-8 as the central effector of death receptor-mediated signaling within neurons. With respect to the intrinsic pathway, several lines of evidence suggest that the apoptosome predominantly regulates the death of motor neurons. I tested this hypothesis by performing facial axotomies in mice containing a point mutation introduced (“knocked in”) into the genomic locus of cytochrome c which abolishes its ability to activate the intrinsic pathway. Homozygous cytochrome c knock-in mice displayed a significant enhancement in motor neuron survival in comparison to control littermates following injury. However, the level of motor neuron protection differed from that previously reported in mice either overexpressing anti-apoptotic or lacking pro-apoptotic members of the Bcl-2 family. Therefore, the results of this study directly demonstrate the influence of the apoptosome on injury-induced neuronal PCD isolated from upstream Bcl-2 family-mediated effects. In addition, my results have provided the first evidence that activation of the apoptosome is required for the release of apoptosis inducing factor (AIF) from the mitochondria of injured motor neurons in vivo.

Programmed cell death (PCD) is a highly evolved mechanism of cellular suicide that is aberrantly activated following neural injury. Two fundamental PCD signaling pathways termed the extrinsic (caspase-8-mediated) and intrinsic (caspase-9-mediated) pathways, have been described. While each pathway is initiated by distinct cellular stimuli, both pathways culminate in the activation of downstream executioner caspases. Previous efforts to isolate the in vivo contribution of each pathway have been hindered by the embryonic lethality of casp8 and casp9 null mice. In the present study, I overcame this obstacle to directly assess the contribution of each pathway following two well-characterized forms of acute neural injury; excitotoxic destruction of CA1 pyramidal neurons, and the loss of motor neurons following facial nerve transection. To determine the role of caspase-8, I constructed several lines of mice in which caspase-8 was conditionally ablated within the relevant neuronal populations. The results obtained from these animals definitively demonstrate that caspase-8 is not required by either motor neurons or CA1 pyramidal neurons to undergo PCD following injury. Therefore, these findings have provided the first direct experimental evidence to counter the widely held dogma of caspase-8 as the central effector of death receptor-mediated signaling within neurons. With respect to the intrinsic pathway, several lines of evidence suggest that the apoptosome predominantly regulates the death of motor neurons. I tested this hypothesis by performing facial axotomies in mice containing a point mutation introduced (“knocked in”) into the genomic locus of cytochrome c which abolishes its ability to activate the intrinsic pathway. Homozygous cytochrome c knock-in mice displayed a significant enhancement in motor neuron survival in comparison to control littermates following injury. However, the level of motor neuron protection differed from that previously reported in mice either overexpressing anti-apoptotic or lacking pro-apoptotic members of the Bcl-2 family. Therefore, the results of this study directly demonstrate the influence of the apoptosome on injury-induced neuronal PCD isolated from upstream Bcl-2 family-mediated effects. In addition, my results have provided the first evidence that activation of the apoptosome is required for the release of apoptosis inducing factor (AIF) from the mitochondria of injured motor neurons in vivo.

Dentre os mecanismos relacionados à interação bactéria-planta, a biossíntese bacteriana de ácido indol acético (AIA) exerce um papel fundamental na promoção do crescimento vegetal, uma vez que é capaz de influenciar inúmeros processos fisiológicos nas plantas. Diferentes vias metabólicas são utilizadas pelas bactérias para a biossíntese do AIA, sendo a via do ácido indol-3-pirúvico (IPyA) a mais comumente descrita. Nesta via encontra-se o gene indol-3-piruvato descarboxilase (ipdC) com vital função na produção de AIA utilizando como precursor o aminoácido L-triptofano. Nesse contexto, estudos moleculares acerca das vias metabólicas e dos genes envolvidos nesse processo são preponderantes para o entendimento da inter-relação das vias regulatórias com a síntese do fitormônio. A rizobactéria Bacillus sp. (RZ2MS9) vem apresentando satisfatória atividade na promoção de crescimento vegetal. O sequenciamento do seu genoma apontou a presença de uma vasta gama de genes relacionados à promoção do crescimento, com destaque para genes codificadores de auxinas. Assim, o estudo teve por objetivo comprovar a função do gene ipdC na biossíntese do AIA pela via dpendente do L-triptofano através do nocaute sítio dirigido do gene ipdC na Rizobactéria Promotora do Crescimento em Plantas (RPCP) Bacillus sp. (RZ2MS9). Para tanto, foi realizado o nocaute sítio dirigido por meio da técnica de CRISPR-Cas9. O nocaute do gene ipdC foi eficiente, gerando mutantes disruptivos para o referido gene. A biossíntese do AIA pela linhagem ΔipdC apresentou reduções nas concentrações do fitormônio, de acordo com o tempo de crescimento, sendo 87,96% em 24 horas, 88,25% em 48 horas e 58,27% em 72 horas do crescimento em comparação à linhagem selvagem (WT). Além disso, a biossíntese do AIA na ausência do aminoácido L-triptofano também foi avaliada, não sendo constatada síntese do fitormônio em nenhum dos tempos crescimento, tanto na linhagem selvagem, quanto na linhagem ΔipdC. O presente estudo foi pioneiro no nocaute do gene ipdC em uma linhagem de Bacillus utilizando a técnica de CRISPR-Cas9. Os resultados obtidos contribuem para um melhor entendimento da influência do gene ipdC e da via IPyA na biossíntese do AIA pela linhagem RZ2MS9 e futuramente sera comprovado seu papel na promoção de crescimento vegetal. / Among the mechanisms related to the bacterium-plant interaction, the bacterial biosynthesis of indole acetic acid (AIA) plays a fundamental role in the promotion of plant growth, since it is capable of influencing innumerable physiological processes in plants. Different metabolic pathways are used by bacteria for the biosynthesis of IAA, with the indole-3-pyruvic acid (IPyA) pathway being the most commonly described. In this pathway, the indole-3-pyruvate decarboxylase (ipdC) gene has a vital role in the production of IAA using the amino acid L-tryptophan as a precursor. In this context, molecular studies about the metabolic pathways and the genes involved in this process are preponderant for the understanding of the interrelationship of the regulatory pathways with the phytormonium synthesis. The rhizobacterium Bacillus sp. (RZ2MS9) has been showing satisfactory activity in promoting plant growth. The sequencing of its genome pointed to the presence of a wide range of genes related to growth promotion, especially genes encoding auxins. Thus, the objective of the present study was to verify the function of the ipdC gene in the IAA biosynthesis L-tryptophan dependent through the knockout of the ipdC in the plant growth-promoting rhizobateria (PGPR) Bacillus sp. (RZ2MS9). Therefore, the knockout was realized using the CRISPR-Cas9. The knockout of the ipdC gene was efficient, generating disruptive mutants for the said gene. IAA biosynthesis by the ΔipdC strain showed reductions in phytormonium concentrations, according to the growth time, being 87.96% in 24 hours, 88.25% in 48 hours and 58.27% in 72 hours of growth compared to the Wild Type (WT). In addition, the biosynthesis of IAA in the absence of the amino acid L-tryptophan was also evaluated, with no phytormonium synthesis being observed at any growth time, both in the wild type and ΔipdC strain. The present study pioneered the knockout of the ipdC gene in a Bacillus strain using the CRISPR-Cas9. The results obtained contribute to a better understanding of the influence of the ipdC gene and the IPyA pathway in the IAA biosynthesis through the RZ2MS9 strain, and its role in plant growth promoting will be demonstrated in the future.

Dentre os mecanismos relacionados à interação bactéria-planta, a biossíntese bacteriana de ácido indol acético (AIA) exerce um papel fundamental na promoção do crescimento vegetal, uma vez que é capaz de influenciar inúmeros processos fisiológicos nas plantas. Diferentes vias metabólicas são utilizadas pelas bactérias para a biossíntese do AIA, sendo a via do ácido indol-3-pirúvico (IPyA) a mais comumente descrita. Nesta via encontra-se o gene indol-3-piruvato descarboxilase (ipdC) com vital função na produção de AIA utilizando como precursor o aminoácido L-triptofano. Nesse contexto, estudos moleculares acerca das vias metabólicas e dos genes envolvidos nesse processo são preponderantes para o entendimento da inter-relação das vias regulatórias com a síntese do fitormônio. A rizobactéria Bacillus sp. (RZ2MS9) vem apresentando satisfatória atividade na promoção de crescimento vegetal. O sequenciamento do seu genoma apontou a presença de uma vasta gama de genes relacionados à promoção do crescimento, com destaque para genes codificadores de auxinas. Assim, o estudo teve por objetivo comprovar a função do gene ipdC na biossíntese do AIA pela via dpendente do L-triptofano através do nocaute sítio dirigido do gene ipdC na Rizobactéria Promotora do Crescimento em Plantas (RPCP) Bacillus sp. (RZ2MS9). Para tanto, foi realizado o nocaute sítio dirigido por meio da técnica de CRISPR-Cas9. O nocaute do gene ipdC foi eficiente, gerando mutantes disruptivos para o referido gene. A biossíntese do AIA pela linhagem ΔipdC apresentou reduções nas concentrações do fitormônio, de acordo com o tempo de crescimento, sendo 87,96% em 24 horas, 88,25% em 48 horas e 58,27% em 72 horas do crescimento em comparação à linhagem selvagem (WT). Além disso, a biossíntese do AIA na ausência do aminoácido L-triptofano também foi avaliada, não sendo constatada síntese do fitormônio em nenhum dos tempos crescimento, tanto na linhagem selvagem, quanto na linhagem ΔipdC. O presente estudo foi pioneiro no nocaute do gene ipdC em uma linhagem de Bacillus utilizando a técnica de CRISPR-Cas9. Os resultados obtidos contribuem para um melhor entendimento da influência do gene ipdC e da via IPyA na biossíntese do AIA pela linhagem RZ2MS9 e futuramente sera comprovado seu papel na promoção de crescimento vegetal. / Among the mechanisms related to the bacterium-plant interaction, the bacterial biosynthesis of indole acetic acid (AIA) plays a fundamental role in the promotion of plant growth, since it is capable of influencing innumerable physiological processes in plants. Different metabolic pathways are used by bacteria for the biosynthesis of IAA, with the indole-3-pyruvic acid (IPyA) pathway being the most commonly described. In this pathway, the indole-3-pyruvate decarboxylase (ipdC) gene has a vital role in the production of IAA using the amino acid L-tryptophan as a precursor. In this context, molecular studies about the metabolic pathways and the genes involved in this process are preponderant for the understanding of the interrelationship of the regulatory pathways with the phytormonium synthesis. The rhizobacterium Bacillus sp. (RZ2MS9) has been showing satisfactory activity in promoting plant growth. The sequencing of its genome pointed to the presence of a wide range of genes related to growth promotion, especially genes encoding auxins. Thus, the objective of the present study was to verify the function of the ipdC gene in the IAA biosynthesis L-tryptophan dependent through the knockout of the ipdC in the plant growth-promoting rhizobateria (PGPR) Bacillus sp. (RZ2MS9). Therefore, the knockout was realized using the CRISPR-Cas9. The knockout of the ipdC gene was efficient, generating disruptive mutants for the said gene. IAA biosynthesis by the ΔipdC strain showed reductions in phytormonium concentrations, according to the growth time, being 87.96% in 24 hours, 88.25% in 48 hours and 58.27% in 72 hours of growth compared to the Wild Type (WT). In addition, the biosynthesis of IAA in the absence of the amino acid L-tryptophan was also evaluated, with no phytormonium synthesis being observed at any growth time, both in the wild type and ΔipdC strain. The present study pioneered the knockout of the ipdC gene in a Bacillus strain using the CRISPR-Cas9. The results obtained contribute to a better understanding of the influence of the ipdC gene and the IPyA pathway in the IAA biosynthesis through the RZ2MS9 strain, and its role in plant growth promoting will be demonstrated in the future.

In the developing mouse oocyte, as well as in the preimplantation embryo, betaine (N,N,N-trimethylglycine) plays an important role first as a mechanism for cell volume regulation and second as a major methyl donor. Thus, the presence of betaine has implications both during development, and throughout the lifespan. It has previously been observed that betaine accumulates in the mouse oocyte as it matures, however its origin in the egg is unknown. Here I explore the enzyme choline dehydrogenase (CHDH; EC 1.199.1) as a method by which the mouse oocyte synthesizes the betaine that we observe prior to initiation transport activity in the preimplantation embryo. I carefully monitored betaine transport throughout meiotic maturation to confirm that no other previously unobserved membrane transport existed in the maturing oocyte. However, no betaine transport into oocytes was detected during meiotic maturation suggesting de novo synthesis. Previous data suggests that the enzyme is expressed (at the transcript level) in the developing oocyte, and becomes active during meiotic maturation. I demonstrated the presence of CHDH protein in the oocyte and preimplantation embryo. I then examined whether the mouse oocyte synthesizes betaine autonomously and addressed whether CHDH is a requirement for this process. Chdh knockout oocytes did not accumulate betaine in vivo, while normal betaine levels were observed in Chdh wildtype oocytes. CHDH-mediated synthesis of betaine was directly confirmed by detection of increased betaine in oocytes matured in vitro in the presence of choline. Chdh-/- oocytes failed to produce betaine when similarly cultured in choline. This establishes the production of betaine as an autonomous process in maturing oocytes. Overall, I have built upon previous data to demonstrate that betaine accumulation is a feature of meiotic maturation that occurs by de novo synthesis of the molecule, a process that requires transient activation of the enzyme choline dehydrogenase.

The World Health Organization (WHO) states that 30,000 children under the age of five die each day worldwide. Around a quarter of these die from diarrheal disease caused by microbial infection. In addition to this high mortality rate, there are data emerging on the morbidity effects of diarrheal disease, for example a few episodes of diarrhea in the first two years of life can remove 10 IQ points and lead to growth deficiency. Vibrio cholerae, the causative agent of the diarrheal disease cholera, is a serious problem in third world countries, where sanitary and hygiene infrastructure is very poor, and claims several thousand lives every year. In order to better understand the pathogenicity regulation in V. cholerae, structural and functional investigations of a hypothetical protein family present in pathogenicity islands and a transcriptional regulator protein for DNA-binding were investigated. Two adjacent genes, vc1804 and vc1805, encode hypothetical proteins within the Vibrio pathogenicity island-2 (VPI-2) of Vibrio cholerae, and are part of a cluster of genes only present in pathogenic strains of the bacterium. Paralogous adjacent genes, vc0508 and vc0509, are also present within a second pathogenicity island, the Vibrio seventh pandemic island-2 (VSP-2), of V. cholerae O1 El Tor and O139 serogroup isolates. Sequence similarity suggests that the VC0508, VC0509, VC1804 and VC1805 proteins will share a similar fold. The crystal structures of VC0508, VC0509 and VC1805 have been determined to a resolution of 1.9, 2.4 and 2.1 Å, respectively. Several recombinant constructs of vc1804 were made, but no soluble proteins were expressed. This hypothetical protein family reveals structural homology to human mitochondrial protein p32. Human p32 is a promiscuous protein known to bind to a variety of partners including the globular head component of C1q. We have shown that VC1805 binds to C1q. One possibility is that VC1805 is involved in adherence of the bacterium to membrane-bound C1q in the gut. To explore the roles of VC0508, VC0509, VC1804 and VC1805 in vivo, gene knockout and animal model studies of those proteins are underway. The ferric uptake regulator (Fur), a metal-dependent DNA-binding protein, acts as both a repressor and activator of numerous genes involved in maintaining iron homeostasis in bacteria. It has also been demonstrated in Vibrio cholerae that Fur plays an additional role in pathogenesis, and this opens up the potential of Fur as a drug target for cholera. The first crystal structure of a Fur protein, from Pseudomonas aeruginosa, revealed a dimeric molecule with each monomer containing a dimerization domain, a helical DNA-binding domain and two metal binding sites: Zn1 is proposed to be a regulatory Fe-binding site, and Zn2 is proposed to be a structural Zn-binding site. Here we present the crystal structure of V. cholerae Fur (VcFur) that reveals a very different orientation of the DNA-binding domains. Accompanying these structural changes are alterations in the amino acids coordinating the zinc at the Zn2 site, and this lends support to this being the site regulated by iron. There is no evidence of metal binding to the cysteines that are conserved in many Fur homologues, including the much-studied E. coli Fur. An analysis of the metal binding properties shows that like other Fur proteins, VcFur can be activated by a range of divalent metals. EPR spectroscopy measurements of the movements of the DNA-binding domain, in the presence of DNA and different metals, are underway.

Chloroplasts are organelles that convert light energy to chemical energy through photosynthesis. The movement of chloroplasts within the cell for the optimization of light absorption is crucial for plant survival. Cellular motor proteins and cytoskeletal tracks can facilitate transport of organelles. As an ancient superfamily of microtubule-dependent motors, kinesins participate in various cellular activities including cytokinesis, vesicle and organelle movements. Based on phylogenetic relationships and functional analysis, the kinesin superfamily has been subdivided into more than 14 families, most of which can be found in plants. With the ever increasing amount of genomic information, it is important and beneficial to systematically characterize and document kinesins within an organism. As a result of my collaborative work with other members of the Vidali lab, a detailed phylogenetic characterization of the 76 kinesins of the kinesin superfamily in the moss Physcomitrella patens is reported here. We found a remarkable conservation of families and subfamily classes with Arabidopsis, which is important for future comparative analyses of functions. Some of the families are composed of fewer members, while other families are greatly expanded in moss. To improve the comparison between species, and to simplify communication between research groups, we proposed a classification of subfamilies based on our phylogenetic analysis. As part of my efforts in studying chloroplasts motility, I investigated the function of two members of Physcomitrella kinesin family 14 class V proteins, Ppkin14-Va and -Vb. These two proteins are orthologs of the Arabidopsis KAC proteins which mediate actin-based chloroplast movement in Arabidopsis thaliana. In contrast, in the Physcomitrella both actin filaments (AFs) and microtubules (MTs) participate in chloroplast movement. Our results show that Ppkin14-Vs are important for maintaining chloroplast dispersion. They also function during chloroplast light avoidance responses via an AF-dependent, rather than MT-dependent mechanism. Although two Ppkin14- Vs do not act as MT-based motors, our phylogenetic study on moss kinesins provides an important source of information to track other potential kinesins that are predicted to move chloroplasts on MTs.

Aspergillus fumigatus has long been a focus of research, as it is the cause of the majority of Aspergillus infections. A. fumigatus is widely distributed in the environment and mainly distributed in air as conidia and is the main source of lung infection. A. fumigatus airborne counts were determined monthly during two years from the outside air environment at the University of Manchester campus and compared to total fungal airborne counts. Total fungal airborne counts were strongly seasonally associated with peak counts occurring during the summer months reaching 1,100-1400 CFU m-3and were correlated positively with mean temperature (R2=0.697). In contrast, Aspergillus fumigatus counts were not seasonally associated and gave persistent low levels of between 3-20 CFU m-3and were not correlated with mean temperature. A random selection of Manchester environmental isolates collected over one year along with clinical patient isolates and environmental isolates from the air from Dublin were analysed for genetic diversity using two combined RAPD primers. RAPD analysis revealed that the Manchester environmental isolates represented a genetically diverse population while the clinical isolates were less diverse and formed three major clusters. The Dublin isolates were the least diverse, probably due to their isolation at a single time point. When the pathogenicity of clinical and Dublin isolates were compared with a random selection of Manchester isolates in a wax moth model, as a group, clinical isolates were significantly more pathogenic than environmental isolates. Moreover, when relative pathogenicity of individual isolates was compared, clinical isolates were the most pathogenic, Dublin isolates the least pathogenic and Manchester isolates showed a range of pathogenicities suggesting that selection for the most pathogenic isolates from the environment occurs during patient infection. When the expression of secreted phospholipases in vitro during wax moth larvae of a range of isolates displaying varying degrees of pathogenicity was compared, two phospholipase C genes, AfplcA and AfplcC were strongly correlated with pathogenicity. AfplcC was by far the most highly expressed, however a ΔAfplcC knockout strain did not show attenuated virulence compared to the wild type in wax moth larvae.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第20059号 / 薬科博第66号 / 新制||薬科||8(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 竹島 浩, 教授 中山 和久, 教授 根岸 学 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Multiple myeloma, or myeloma, is a bone marrow cancer which characterizes by uncontrolled proliferation of mutant plasma cells. It is a disease that claims many lives every year, mostly due to the absence of curative treatment. Finding a suitable treatment is therefor of great importance. One way to study different diseases is to use a gene editing method for knockdown or knockout of specific genes. The main aim of this project was to design guide RNAs, to be able to use CRISPR/Cas9 for knockout of the two genes BMPR1A and BMPR2 in different myeloma cell lines (KJON, INA-6 and IH-1). This, to be able to study the expression and function of these genes. Further aim of the project was to investigate potential SMAD activation by treatment with different bone morphogenetic proteins (BMPs). However, due to limited time this could not be carried through. Six guide RNAs were designed and ligated into pLentiCRISPRv2. Plasmid amplification was done by transformation of Escherichia coli. To check the quality of the plasmids, PCR, gel electrophoresis and Sanger sequencing was performed. The results from the gel electrophoresis showed that nine of the twelve samples for BMPR1A and seven of the thirteen samples for BMPR2, that were tested, were positive. The results from the Sanger sequencing confirmed that all guides that were tested (BMPR1A 3.2.3, BMPR1A 4.2.2, BMPR2 1.1.4 and BMPR2 2.1.2), were properly ligated into the plasmids. The main aim of the project was successfully accomplished, but additional work is needed for any further conclusions.