Xanthomonas citri (X. citri), causador do cancro cítrico, afeta muitas áreas de cultivo de citros com impacto comercial. Transportadores ABC foram relatados como essenciais para a patogênese de X. citri. O gene nrtT codifica a proteína periplasmática responsável pelo suposto transporte de nitrato/nitrito/taurina. Neste trabalho, mostramos que a deleção de nrtT não afeta o crescimento de X. citri, porém reduz e atrasa os sintomas do cancro durante a infecção em folhas de citros. Observamos redução na produção de goma xantana e capacidade de aderência na linhagem mutante. A proteína NrtT foi expressa monomérica e monodispersa. Foram observadas alterações na estrutura secundária e aumento da estabilidade térmica de NrtT na presença de MOPS, indicando ser este um possível ligante de NrtT. A proteína PotF descrita como ligadora de putrescina não sofre alteração significativa na estabilidade térmica na presença de putrescina, entretanto, dados de SAXS mostram alterações na estrutura possivelmente decorrentes da ligação com putrescina. / Xanthomonas citri (X. citri), which causes citrus canker, affects many citrus growing areas with commercial impact. ABC transporters have been reported as essential for the pathogenesis of X. citri. The nrtT gene encodes the periplasmic protein responsible for the alleged transport of nitrate/nitrite/taurine. In this work, we show that nrtT deletion does not affect the growth of X. citri, but delays and reduces the symptoms of cancer during infection of citrus leaves. We observed a reduction in the production of xanthan gum and adhesion capacity in the mutant strain. The NrtT protein was expressed monomeric and monodisperse. There were changes in the secondary structure and increased thermal stability NrtT in the presence of MOPS, indicating that this was a possible binder NrtT. The PotF protein described as putrescine-binding is not significantly altered thermal stability in the presence of putrescine, however, SAXS data shows changes in the structure possibly resulting from connection with putrescine.

The serotonin transporter (SLC6A4) has been a prominent choice in studying multiple neuropsychiatric illnesses. However, underlying molecular mechanisms of the regulatory control of this gene leading to these illnesses remain incomplete. Since this locus is the target of virtually every antidepressant, understanding the molecular mechanisms would benefit the development of effective therapeutic agents. In an attempt to better understand the regulatory control of the human serotonin transporter, a series of investigations were conducted on the 5HTTLPR genotype, DNA methylation and SLC6A4 mRNA expression with respect to childhood sexual abuse and depression. Moreover, since vast majority of studies have only concentrated on the Long (L) and Short (S) polymorphisms, the characteristics of the Extra-Long (XL) allele with respect to transcriptional efficiency was investigated. Finally, due to the increase in gene expression studies, the normalization of gene expression with respect to multiple housekeeping genes was explored. Through this study, we demonstrate significant gene-environmental interaction effects at this locus. The extra-long variant was associated with increased gene transcriptional efficiency. Also, although the best gene expression normalization was achieved with one housekeeping gene, we present a strong explanation on the importance of utilizing more than one housekeeping gene. These results could certainly aid in understanding and treating disorders related to the human serotonin transporter.

The objective of this study was to investigate the role of organic cation transporters (OCTs) in the uptake of hydrophilic drugs into the olfactory bulb and subsequently to the brain. Two OCT2 substrates, amantadine and cimetidine were used as model drugs for this purpose. Bovine nasal explants (olfactory and respiratory tissue) were used as an in vitro model for preliminary screening to identify the role of transporters involved in the uptake of drug across these tissues. It was observed from both PCR and immunohistochemistry that OCTs, OCT2, OCTN1 and OCTN2 were present in the bovine respiratory and olfactory mucosa. Transport studies of amantadine in the presence and absence of OCT2 and OCTN2 inhibitors indicated that both these transporters play a role in the transport of amantadine across the bovine respiratory mucosa, whereas transport across the olfactory mucosa was predominantly via OCT2. This was followed by in vivo studies in rats where the blood, striatum and olfactory bulb concentrations of amantadine were determined following intranasal and intra-arterial administration. Shortly after nasal administration, the olfactory bulb concentrations exceeded the concentrations in the striatum suggesting the olfactory pathway to be the major route of uptake. Co-administration of the drug with an OCT2 inhibitor intranasally showed statistically significant reductions in the brain uptake of amantadine. A synergistic inhibitory effect on amantadine uptake was observed with the combined inhibition OCT2 and OCTN2. Additionally, the CNS exposure of these drugs following intranasal administration in the presence and absence of the OCT inhibitors was evaluated using the ratio of the free drug concentrations in the brain compared to plasma. While the plasma concentration profiles were similar both in the presence and absence of inhibition, the free drug ratios were highest when no inhibitor was included. Additionally similiar in vivo studies were also carried out for a second model drug, cimetidine, where cimetidine uptake into the rat brain was found to be significantly reduced in the presence of the OCT2 inhibitor, pentamidine. This demonstrates that there was a greater CNS exposure to each drug when OCT transporters were active, confirming their role in their direct CNS distribution from the nasal cavity to the brain. The results of this study suggest that OCT substrates might be good candidates for the delivery to the brain via the olfactory route.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13248号 / 論農博第2873号 / 新制||農||1070(附属図書館) / 学位論文||H31||N5172(農学部図書室) / (主査)教授 小川 順, 教授 阪井 康能, 教授 栗原 達夫 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23088号 / 医博第4715号 / 新制||医||1050(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 柳田 素子, 教授 岩田 想, 教授 浅野 雅秀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

L’agent pathogène Agrobacterium tumefaciens induit, chez les plantes, le développement de tumeurs dans lesquelles il prolifère, en intégrant un fragment de son plasmide Ti de virulence dans le génome de son hôte. Les tissus transformés synthétisent des composés originaux, appelés opines, qui sont utilisés comme nutriments spécifiques par la bactérie. Une vingtaine d’opines sont connues à ce jour, et chacune d’elle peut être métabolisée par des souches d’Agrobacterium tumefaciens possédant les gènes de transport et de catabolisme qui lui sont associés, ce qui apparait comme un avantage compétitif dans la colonisation de la tumeur. La présence de ces gènes dépend du type de plasmide Ti que la souche pathogène possède.Agrobacterium tumefaciens B6 possède un pTi de type octopine, qui porte les gènes de métabolisme des mannityl-opines, qui sont la mannopine, l’acide mannopinique, l’agropine et l’acide agropinique. La mannopine et l’acide mannopinique sont synthétisés par la même enzyme, et ont pour précurseurs respectivement la désoxy-fructosyl-glutamine (DFG) et le désoxy-fructosyl-glutamate (DFGA), tous deux opines de la famille de la chrysopine. La DFG est aussi un composé d’Amadori répandu et assimilable par de nombreux organismes. La mannopine sert de précurseur pour la synthèse de l’agropine. Enfin, la mannopine, l’acide mannopinique et l’agropine peuvent toutes trois se lactamiser spontanément en acide agropinique.Malgré la similarité chimique de ces quatre opines, chacune est transportée par une protéine périplasmique de fixation (PBP) associée à un transporteur ATP-binding Cassette (ABC) différent. La PBP sélectionne et fixe une opine pour l’apporter au transporteur ABC, qui permet le passage de l’opine dans le cytoplasme grâce à l’hydrolyse de deux molécules d’ATP. La spécificité du transporteur entier est déterminée par la PBP.Des études génétiques chez des souches possédant un pTi de type octopine ont montré que le système PBP-transporteur ABC AgaABCD est spécifique de l’acide agropinique, AgtABCD spécifique de l’agropine, MoaABCD spécifique de l’acide mannopinique et que MotABCD transporte la mannopine et également l’acide mannopinique. Chez la souche C58, qui ne possède pas un pTi de type octopine, le système de transport SocAB, codé par des gènes situés sur le plasmide cryptique At, transporte la DFG comme nutriment, et semble aussi capable d’importer la mannopine.Mon travail de thèse a permis, dans un premier temps, de caractériser les fortes affinités et la spécificité des PBP AgaA et AgtB pour l’acide agropinique, de la PBP MoaA pour l’acide mannopinique et de la PBP SocA pour la DFG, mais aussi la non spécificité de MotA pour la mannopine, l’acide mannopinique et la DFG, ce qui remet en question les affinités précédemment décrites pour AgtB et SocA. Dans un deuxième temps, ce travail a apporté les bases moléculaires et structurales des complexes PBP-mannityl-opines, complexes jamais caractérisés auparavant. Enfin, dans un troisième temps, la structure de la PBP AttC chez la souche C58, annotée comme mannopine-like, a été déterminée, et les expériences d’interaction ont montré qu’elle n’interagit avec aucune mannityl-opine, ce qui conduit à une révision de son annotation.Mes travaux apportent un éclairage nouveau sur l’import des mannityl-opines chez Agrobacterium tumefaciens. Le fait qu’aucun des transporteurs étudié ne permette l’import de l’agropine laisse penser qu’il existe une autre PBP ou un autre système de transport encore inconnu assurant cette fonction, ouvrant la voie vers de nouvelles études sur les pTi de type octopine et agropine. / Agrobacterium tumefaciens pathogenic agent confers the development of tumors in plants, in which it proliferates, integrating a fragment of its virulence Ti plasmid into its host genome. Transformed tissues synthesize original compounds, called opines, used as specific nutrients by the bacterium. More than twenty opines are known so far, and each one of them can be metabolized by A. tumefaciens strains possessing its associated transport and catabolism genes, which appears as a competitive advantage in the tumor colonization. The presence of these genes relies on the Ti plasmid type a pathogenic strain possesses. A. tumefaciens B6 possesses an octopine-type pTi, which harbors the metabolism genes of the mannityl-opines, which are mannopine, mannopinic acid, agropine and agropinic acid. Mannopine and mannopinic acid are synthesized by the same enzyme, and their precursors are deoxy-fructosyl-glutamine (DFG) and deoxy-fructosyl-glutamate (DFGA) respectively, both opines of the chrysopine family. DFG is also a wide-spread Amadori compound which can be uptaken by numerous organisms. Mannopine is a precursor for agropine synthesis. Finally, mannopine, mannopinic acid and agropine can spontaneously lactamize into agropinic acid.Despite the chemical similarity of these four opines, each one is transported by a different periplasmic binding protein (PBP) associated with an ATP-binding cassette (ABC) transporter. The PBP selects and binds one opine to bring it to the ABC transporter, which allows the passage of the opine to the cytoplasm due to two ATP molecules hydrolysis. The whole transporter specificity is determined by the PBP.Genetic studies in strains possessing an octopine-type pTi showed that AgaABCD PBP-ABC transporter system is specific to agropinic acid, AgtABCD to agropine, MoaABCD to mannopinic acid and that MotABCD transports mannopine and also mannopinic acid. In C58 strain, which do not possess an octopine-type pTi, SocAB transport system, coded by genes located on the cryptic pAt plasmid, allows the transport of DFG as a nutrient, and seems able to import mannopine too.My thesis work allowed, first, to characterize the strong affinities and the specificity of PBPs AgaA and AgtB to agropinic acid, PBP MoaA to mannopinic acid and PBP SocA to DFG, and also MotA unspecificity toward mannopine, mannopinique acid and DFG, which leads to a revision of the previously described affinities of AgtB and SocA. Secondly, this work brought molecular and structural basis of PBP-mannityl-opine complexes, never described before. Finally, the structure of PBP AttC, annotated as a mannopine binding-like protein in C58, was determined, and interactions experiments showed that it binds no mannityl-opines, leading to a revision of its annotation.My work sheds light on the mannityl-opines importation in Agrobacterium tumefaciens. The fact that none of the studied transport system allows agropine import lets think that there is another unknown PBP or another unknown whole transport system assuming this role, opening new ways to new studies about octopine- and agropine-type pTis.

A biofilm, which is associated with 80% of chronic infections in humans, is formed when bacteria aggregate, attach to a substrate and secrete a matrix protecting the bacteria from host cell defenses and antibiotics. Bartonella henselae (B. henselae) is the causative agent of cat scratch disease, persistent bacteremia, and one of the most frequently reported causes of blood-culture negative endocarditis (BCNE) in patients. The ability of B. henselae to adhere to the heart valve, form a biofilm and vegetation to cause endocarditis increases the morbidity and mortality rate in infected patients. The presence of a trimeric autotransporter adhesin (TAA) called Bartonella adhesin A (BadA) has been linked to biofilm formation in B. henselae. BadA is a protein of 3036 amino acids and a member of the TAAs found in Bartonella and other Gram-negative bacteria. The function of BadA has been studied in vitro and is critical for agglutination, host cell adhesion and activation of a pro-angiogenic host response. However, very little is known about badA gene regulation or the molecular basis of biofilm formation. This work aims to determine whether BadA is necessary for the establishment of biofilms and how the bacteria regulate badA expression. Using genetic mutations, real-time cell adhesion assay, RT-qPCR, and microscopy, it was shown that BadA is required for biofilm formation. Using an in-frame complete deletion strain of badA, a reduced ability to form a biofilm was observed which was restored in the deletion strain complemented with a partial badA. Analysis of the B. henselae transcriptome shows nine highly transcribed, homologous RNAs, termed Bartonella regulatory transcript (Brt1-9). The Brts are short-sized (<200 >nucleotides), highly expressed, and located in an intergenic region indicative of small RNAs (sRNA). The Brts are predicted to form a stable stem and loop structure with a potential terminator/riboswitch region on the 3′ end. Located ~20 nucleotides downstream of each Brt is a poorly transcribed helix-turn-helix DNA binding protein gene termed transcriptional regulatory protein (trps 1-9). High brt transcription stops just before the start of the trp implicating the 3’ loop of the Brt as a terminating loop. Replacement of the trp with a gfp reporter gene shows that in the absence of the 3′ end of Brt1, gfp is transcribed. Also consistent with our findings, an increase in both the transcription of trp1 and badA and the formation of a biofilm in mutants of the brt1 gene was observed. Furthermore, to determine the role of the Trp in regulating badA, an electrophoretic mobility shift assay was carried out. The data confirms that Trp1 binds the promoter region of badA gene to regulate gene expression. In summary, the brt1/trp1 regulon affects badA transcription and biofilm formation in B. henselae. Understanding the mechanism and condition(s) by which the brt/trp regulatory system regulates badA is a plausible approach to the development of treatments that target the formation of biofilm-related diseases and persistent bacteremia in humans.

Bakgrund: För att Sverige ska kunna nå sina uppsatta klimatmål innan år 2030, behövs stora förändringar ske i hur vi skickar och transporterar vårt gods. Idag står Sveriges vägtrafik för 90% av de koldioxidutsläpp som sker av alla inrikes transporter. För att lösa problemet med de långa avstånden ut mot glesbygden har man undersökt möjligheten till att försöka samordna varutransporterna som ska till glesbygdsområden. Tidigare varusamordningsprojekt har visat sig tillföra starka fördelar för transportföretag som bland annat bättre fyllnadsgrad och reducerade transportkostnader, samtidigt som att man minskar utsläppen kopplade till transporter. Syfte: Studiens syfte är att lägga en vetenskaplig grund för varusamordning samt presentera en beslutsmodell. Beslutsmodellen riktar sig mot kommunala beslutsfattare samt beslutsfattare inom företag som jobbar mot glesbygder. Metod: Studien har utförts enligt ramverket för en kvalitativ intervjustudie med en explorativ forskningsdesign. Detta för att författarna ska ha frihet att arbeta passande efter situationen då det är ett relativt outforskat ämne. Analys och resultat: I studiens resultat presenteras dels varusamordningens olika fördelar, dels dess nackdelar. Dessa för- och nackdelar är tänkta att synliggöra varusamordningens möjligheter samt tillkortakommanden. Vidare presenteras en beslutsmodell med förslag på beslutskriterier som tagits fram från de teoretiska och empiriska information som insamlades till studien. / Background: To reach the global goals of 2030, changes need to be made regarding how decision makers decide to freight their goods. In Sweden, road traffic is held accountable for 90% of the carbon dioxide emissions. To help solve the problem with vast distances in Sweden's rural areas, scientists have researched the possibility of coordinating the transports. The research has shown that transports receive a more appropriate fill rate, lower transportation costs, and because of those two factors, lowered their carbon dioxide emissions. Purpose: The purpose of this study is to lay a scientific foundation and present an example of a decision model. The decision model mainly aims towards decision makers in rural municipalities but also companies that work toward these rural areas. Methodology: This research undertook a qualitative research approach in an exploratory method. In order to ensure that the criteria’s and the pros and cons with coordinating freight transports the researchers interviewed key persons for the rural municipalities in a semi structured or structured nature. Findings: The study has found the most relevant pros and cons with a coordination of freight transports and the criteria’s needed to build an example of a decision model. Conclusions: The theoretical and empirical data has been similar in many, but one stands out, and that is the increase of lead times as a negative factor the rural areas, because of the coordination. The citizens and companies these rural areas are more interested in consistency and reliability. The study concluded the necessary criteria’s needed to build the decision model.

Man’s impact on the environment is something that increasingly becomes topical in today’s society. Because of this, several companies have started to take interest in the effects they’re responsible for, not least considering their image. Emissions of carbon dioxide generated from goods traffic are accountable for a large part of the total emissions of greenhouse gases in the world. This master thesis, assigned by and developed in cooperation with Coop, has therefore studied these emissions closer. One purpose with this report is to describe the logistical situation at Coop today, how the emissions from transports are calculated, and in what ways this could be improved. A mapping of working and calculating methods has thus been done. This led to a number of suggestions for improvement, amongst others a reworked calculation method for the emissions, and theoretical ideas for how emissions could be reduced in the future. Another purpose is to compare the emission quantities before and after the newly restructured logistical solution within the company, when some of the truck transports were replaced by transports by train. However, the complexity with several different systems for documentation and outspread responsibilities within the company, resulted in a lack of necessary data for the calculations to be complete. A limited result was calculated though. / I dagens samhälle blir människans påverkan på miljön alltmer aktuell. Därför är den påverkan som företag bidrar med något som alltfler av dessa börjar ta hänsyn till, inte minst med tanke på den egna imagen. Koldioxidutsläpp från godstransporter står för en stor del av de totala utsläppen av växthusgaser i världen. Detta examensarbete, på uppdrag och utvecklat i samarbete med Coop, har därför undersökt dessa utsläpp närmare. Ett syfte med arbetet är att ge en bild över hur Coops logistik ser ut i dagsläget, hur man beräknar sina transportutsläpp och hur detta skulle kunna förbättras. En kartläggning har därför gjorts av arbetssätt och beräkningsmetoder. Detta ledde sedan till en rad förbättringsförslag, med bl.a. en omarbetad beräkningsmetod för utsläppen och teoretiska idéer om hur utsläppen skulle kunna reduceras i framtiden. Vidare syftar arbetet till att göra en jämförelse av utsläppen före och efter den nyligen genomförda omstruktureringen av logistiken inom företaget, då en del av transporterna lades om från att köras med lastbilar till tåg. Komplexitet med många olika dokumenteringssystem och en bred ansvarsfördelning inom företaget gjorde dock att nödvändigt dataunderlag för kompletta beräkningar inte kunnat erhållas. Ett begränsat resultat erhölls dock.

Rationale Many studies demonstrate down-regulation of the norepinephrine transporter (NET) by desipramine (DMI) in vitro and in stress-naive rats. Little is known regarding regulation of the NET in stressed animals. Objective The present study was designed to investigate effects of DMI on the expression of NET and protein kinases in the stress rat. Methods Adult Fischer 344 rats were subjected to chronic social defeat (CSD) for 4 weeks. DMI (10 mg/kg, intraperitoneal (i.p.)) was administered concurrently with CSD or 1 or 2 weeks after cessation of CSD. Sucrose consumption, NET expression, and protein kinases were measured. Results CSD significantly increased messenger RNA (mRNA) and protein levels of NET in the locus coeruleus, as well as NET protein levels in the hippocampus, frontal cortex, and amygdala. These effects were nearly abolished when DMI was administered concurrently with CSD. CSD-induced upregulation of NET expression in the locus coeruleus, hippocampus, and amygdala lasted at least 2 weeks after cessation of CSD, an effect that was significantly attenuated by 1 or 2 weeks of DMI treatment starting from cessation of the CSD. Concurrent administration of DMI with CSD did not markedly interfere with CSD-induced decreases in protein levels of protein kinases A and C in these brain regions, but it did reverse the CSD-induced reduction in phosphorylated cAMP response element-binding (pCREB) protein levels in most brain regions. Conclusion These findings suggest that NET regulation by DMI occurs in both stressed and behaviorally naive rats, and DMI-induced changes in pCREB may be involved.