abstract: Emergence of multidrug resistant (MDR) bacteria is a major concern to global health. One of the major MDR mechanisms bacteria employ is efflux pumps for the expulsion of drugs from the cell. In Escherichia coli, AcrAB-TolC proteins constitute the major chromosomally-encoded drug efflux system. AcrB, a trimeric membrane protein is well-known for its substrate promiscuity. It has the ability to efflux a broad spectrum of substrates alongside compounds such as dyes, detergent, bile salts and metabolites. Newly identified AcrB residues were shown to be functionally relevant in the drug binding and translocation pathway using a positive genetic selection strategy. These residues—Y49, V127, D153, G288, F453, and L486—were identified as the sites of suppressors of an alteration, F610A, that confers a drug hypersensitivity phenotype. Using site-directed mutagenesis (SDM) along with the real-time efflux and the classical minimum inhibitory concentration (MIC) assays, I was able to characterize the mechanism of suppression. Three approaches were used for the characterization of these suppressors. The first approach focused on side chain specificity. The results showed that certain suppressor sites prefer a particular side chain property, such as size, to overcome the F610A defect. The second approach focused on the effects of efflux pump inhibitors. The results showed that though the suppressor residues were able to overcome the intrinsic defect of F610A, they were unable to overcome the extrinsic defect caused by the efflux pump inhibitors. This showed that the mechanism by which F610A imposes its effect on AcrB function is different than that of the efflux pump inhibitors. The final approach was to determine whether suppressors mapping in the periplasmic and trans-membrane domains act by the same or different mechanisms. The results showed both overlapping and distinct mechanisms of suppression. To conclude, these approaches have provided a deeper understanding of the mechanisms by which novel suppressor residues of AcrB overcome the functional defect of the drug binding domain alteration, F610A. / Dissertation/Thesis / Masters Thesis Biology 2016

abstract: Lignocellulosic biomass represents a renewable domestic feedstock that can support large-scale biochemical production processes for fuels and specialty chemicals. However, cost-effective conversion of lignocellulosic sugars into valuable chemicals by microorganisms still remains a challenge. Biomass recalcitrance to saccharification, microbial substrate utilization, bioproduct titer toxicity, and toxic chemicals associated with chemical pretreatments are at the center of the bottlenecks limiting further commercialization of lignocellulose conversion. Genetic and metabolic engineering has allowed researchers to manipulate microorganisms to overcome some of these challenges, but new innovative approaches are needed to make the process more commercially viable. Transport proteins represent an underexplored target in genetic engineering that can potentially help to control the input of lignocellulosic substrate and output of products/toxins in microbial biocatalysts. In this work, I characterize and explore the use of transport systems to increase substrate utilization, conserve energy, increase tolerance, and enhance biocatalyst performance. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2018

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O monitoramento da sensibilidade antifÃngica em espÃcies de Candida de origem veterinÃria à uma prÃtica recente e os mecanismos envolvidos ainda nÃo foram completamente elucidados. Considerando que o arsenal de drogas antifÃngicas à limitado e que o fenÃmeno de resistÃncia tem se tornado mais freqÃente, a compreensÃo deste fenÃmeno e a busca por alternativas terapÃuticas se fazem necessÃrias. Dessa forma, o presente trabalho teve como objetivo monitorar a sensibilidade in vitro de Candida spp. oriundas de animais, com Ãnfase na resistÃncia aos azÃlicos mediada por bombas de efluxo e na avaliaÃÃo da atividade do imidazÃlico levamisol sobre o crescimento destas leveduras. Para tanto, em um primeiro momento, foram avaliados 126 isolados de Candida, sendo 19 C. albicans, 17 C. famata, 5 C. guilliermondii, 8 C. krusei, 29 C. parapsilosis, 48 C. tropicalis, dos quais 22 foram isolados de rapinantes, 32 de periquitos do sertÃo, 56 de papagaios, 7 de araras canindà e 3 de um ouriÃo-cacheiro. Todas as cepas foram submetidas ao teste de microdiluiÃÃo em caldo, ante a anfotericina B, itraconazol, fluconazol, segundo metodologia padronizada pelo Clinical Laboratory Standards Institute (documento M27-A3). As concentraÃÃes inibitÃrias mÃnimas (CIMs) variaram de 0,03125 a 2 Âg/mL, 0,125 a 250 Âg/mL e de 0,03125 a 125 Âg/mL para anfotericina B, fluconazol e itraconazol, respectivamente. Dos 126 isolados avaliados por microdiluiÃÃo, 33 (26,2%) foram resistentes aos azÃlicos, sendo 7 (5,6%) resistentes somente a fluconazol, 1 (0.8%) resistente somente ao itraconazol e 24 (19%) resistentes a ambas as drogas. Em um segundo momento, todas estas cepas resistentes aos derivados azÃlicos, mais 20 C. albicans e 3 C. tropicalis resgatadas da nossa coleÃÃo de leveduras resistentes de origem veterinÃria, foram submetidas ao teste de inibiÃÃo de bomba de efluxo com prometazina, perfazendo um total de 56 cepas resistentes a azÃlicos. Dessa forma, as CIMs de fluconazol e itraconazol sofreram reduÃÃes significativas de 2 a 250 e de 16 a 4000 vezes, respectivamente. Investigou-se, ainda, a atividade antifÃngica do levamisol contra 12 C. albicans, 12 C. krusei, 12 C. parapsilosis e 12 C. tropicalis, sendo obtidas CIMs e concentraÃÃes fungicidas mÃnimas que variaram de 0,58 a 2,34 mg/mL e de 2,34 a 9,37 mg/mL, respectivamente. Paralelamente, foi demonstrado que o levamisol inibe a formaÃÃo do biofilme de C. albicans e C. tropicalis, bem como interfere na manutenÃÃo do biofilme maduro. Os dados apontam que a resistÃncia aos derivados azÃlicos Ã, pelo menos em parte, mediada por bombas de efluxo, bem como demonstram o potencial antifÃngico do imidazÃlico levamisol e sua capacidade de inibir o biofilme de cepas de Candida spp. oriundas de animais. / Monitoring of the antifungal susceptibility of Candida species from veterinary sources is a recent practice and the mechanisms involved in antifungal resistance have not been completely elucidated. Considering that the antifungal arsenal is limited and that antifungal resistance has become more frequent, the comprehension of this phenomenon and the pursuit for therapeutic alternatives are necessary. Thus, the present work aimed at monitoring the in vitro susceptibility of Candida spp. isolated from animals, with emphasis on efflux pump-mediated azole resistance and on the evaluation of the effect of the imidazole levamisole on the growth of these yeasts. For such, in a first approach, 126 Candida isolates (19 C. albicans, 17 C. famata, 5 C. guilliermondii, 8 C. krusei, 29 C. parapsilosis, 48 C. tropicalis), out of which 22 were recovered from raptors, 32 from cactus parakeets, 56 from Amazon parrots, 7 from blue-and-gold macaws and 3 from a Brazilian porcupine. All isolates were submitted to broth microdilution test against amphotericin B, itraconazole and fluconazole, according to the methodology recommended by the Clinical Laboratory Standards Institute (document M27-A3). The MICs ranged from 0.03125 to 2 Âg/mL, 0.125 to 250 Âg/mL and 0.03125 to 125 Âg/mL for amphotericin B, fluconazole and itraconazole, respectively. Out of 126 evaluated isolates, 33 (26.2%) were resistant to azoles, with 7 (5.6%) isolates resistant to fluconazole, 1 (0.8%) isolate resistant to itraconazole and 24 (19%) resistant to both drugs. In a second approach, all these azole resistant isolates, plus 20 C. albicans and 3 C. tropicalis that were recovered from our collection of resistant yeasts from veterinary sources, were submitted to the efflux pump inhibition assay with promethazine, with a total of 56 azole resistant isolates. Thus, MICs for fluconazole and itraconazole significantly reduced from 2 to 250 times for fluconazole and from 16 to 4000 times for itraconazole. The antifungal activity of levamisole against 12 C. albicans, 12 C. krusei, 12 C. parapsilosis and 12 C. tropicalis, was also evaluated, and MICs and minimum fungicidal concentrations varying from 0.58 to 2.34 mg/mL and from 2.34 to 9.37 mg/mL were obtained, respectively. Parallelly, it was demonstrated that levamisole significantly inhibits biofilm formation and interferes with the maintenance of mature biofilms. These data show that azole resistance is partially mediated by efflux-pumps and demonstrate the antifungal potential of the imidazole levamisole and its capacity of inhibiting the biofilm of strains of Candida spp. from animals.

Made available in DSpace on 2014-08-20T13:32:48Z (GMT). No. of bitstreams: 1 dissertacao_carolina_felix.pdf: 7826200 bytes, checksum: 4432d1e031f9b3b4bdbc3946dbb901e0 (MD5) Previous issue date: 2013-08-28 / Transport proteins related to drug efflux play an important role not only in the acquisition of drug-resistant phenotypes, but also in virulence of M. tuberculosis. The main objective of this study was to analyze the regulation of genes encoding the protein Rv1258c Tap considering the expression of genes Rv1255c and Rv1257c. RNA was extracted from cultures of M. bovis BCG overexpressing Rv1255c and Rv1257c, as well as a control strain containing the vector pVV16, and qRT-PCR of the Rv1258c gene was performed. RT-PCR was performed using RNA isolated previously from M. tuberculosis CDC1551, in order to verify that Rv1255c, Rv1256c, Rv1257c and Rv1258c genes are all connected to a transcript. The strain M. bovis BCG, expressing a red fluorescent protein under control of the promoter Rv1258c (pVVTapPro) was grown in the presence of streptomycin and glycine to verify the induction of the promoter. The qRT-PCR showed a downregulation of the gene in the strain overexpressing Rv1258c and Rv1257c, but no difference was observed in the strain overexpressing Rv1255c compared with the control. An increase in fluorescence was observed in the strain containing the promoter of the tap in the presence of 1 mg/L of streptomycin in comparison with the control. A two-fold induction of gene Tap was also observed in the presence of 1.6 mM glycine. The results suggest a role for Rv1257c in the regulation Tap expression. Moreover, the induction of Tap by streptomycin supports the importance of this protein in multidrugresistant M. tuberculosis. The effect of glycine on Tap promoter suggest a physiological role in cellular detoxification for this efflux pump. In conclusion, this study reinforces the need to obtain a deeper knowledge about the Tap protein operon and its regulation, in order to assist in the development of inhibitors of this efflux pump and possibly other mechanisms of induced resistance. / Proteínas transportadoras relacionada ao efluxo de drogas desempenham um papel importante não só na aquisição de fenótipos resistentes aos fármacos, mas também na virulência de M. tuberculosis. O principal objetivo deste estudo foi analisar a regulação do gene Rv1258c codificador da proteína Tap considerando a expressão dos genes Rv1255c e Rv1257c. RNA foi extraído a partir de culturas de M. bovis BCG superexpressando Rv1255c e Rv1257c, bem como de uma cepa controle contendo o vector pVV16 realizando o qRT-PCR do gene Rv1258c. RT-PCR foi realizada com RNA previamente isolado de M. tuberculosis CDC1551, a fim de verificar se o Rv1255c, Rv1256c, Rv1257c e Rv1258c genes estão todos ligados em um transcrito. A cepa M. bovis BCG, expressando uma proteína fluorescente vermelha sob o controle do promotor do Rv1258c (pVVTapPro) foi cultivado na presença de estreptomicina e glicina para verificar a indução deste promotor. O qRT-PCR demonstrou uma regulação negativa do gene Rv1258c na cepa sobre expressando Rv1257c, mas não foi observada diferença na cepa sobre expressando Rv1255c em comparação com o controle. Um aumento de fluorescência foi observada na cepa contendo o promotor da Tap na presença de 1 mg/L de estreptomicina, em comparação com o controle. Uma indução de duas vezes do gene Tap foi também observada na presença de glicina 1,6 mM. Os resultados sugerem um papel para o gene Rv1257c na regulação da expressão Tap. Além disso, a indução da Tap por estreptomicina apoia a importância desta proteína na multidroga-resistência de M. tuberculosis. O efeito da glicina no promotor da Tap sugere um papel fisiológico de detoxificação celular para essa bomba de efluxo. Em conclusão este estudo reforça a necessidade de se obter um conhecimento mais aprofundado a respeito do operon da proteína Tap e sua regulação, de maneira a auxiliar no desenvolvimento de inibidores dessa bomba de efluxo e possivelmente de outros mecanismos de resistência induzida.

Escherichia coli transporter protein AcrB and its homologues are the inner membrane components of the Resistance-Nodulation-Division (RND) family efflux pumps in Gram-negative bacteria. It is well accepted that soluble proteins are only marginally stable, but such insight is missing for membrane proteins. The lack of stability data, including thermodynamic stability and oligomer association affinity is a result of intrinsic difficulties in working with membrane proteins. In addition, the degradation of soluble proteins in E. coli has been extensively studied whereas the degradation process of membrane proteins remains unclear. A focus of my thesis is the validation and development of methods used to measure the thermo- and oligomeric- stability of membrane proteins. I investigated the mechanism of a popular thermal-stability assay developed specifically for the study of membrane proteins uses a thiol-specific probe, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). I found that, contrary to current understanding, the presence of a sulfhydryl group was not a prerequisite for the CPM thermal stability assay. The observed fluorescence increase is likely caused by binding of the fluorophore to hydrophobic patches exposed upon protein unfolding. I then applied these methods in the study of three projects. In the first project, I investigated how suppressor mutations restore the function of AcrBP223G, in which the Pro223 to Gly mutation compromised the function of AcrB via disrupting AcrB trimerization. The results suggested that the function loss resulted from compromised AcrB trimerization could be restored through various mechanisms involving the compensation of trimer stability and substrate binding. In the second project, I created two AcrB fusion proteins, with C-terminal yellow fluorescence protein (YFP) and cyan fluorescence protein (CFP), respectively. YFP and CFP form a fluorescence resonance energy transfer (FRET) pair. Using this pair of fusion proteins, I studied AcrB assembly both in detergent micelles and in lipid bilayers. A positive cooperativity was observed in kinetic studies of association of AcrB trimer. Reconstitution experiment revealed that the association showed a higher FRET efficiency and faster association rate in liposome than in DDM. In the last project, I developed a fluorescence method to study the degradation of AcrB-ssrA by the ClpXP system. Comparing to the degradation of GFP-ssrA, degradation of AcrB-CFP-ssrA showed a lower maximum velocity and tighter binding to the enzymes with a positive cooperativity.

Antibiotic resistance has been a developing problem for mankind in recent decades and multi-drug resistant bacteria are now encountered that are resistant to all treatment options available. In 2014, the World Health Organization announced that this problem is driving us towards a “post-antibiotic era” that will change the face of modern medicine as we know it. If lack of novel antibiotic development and FDA approval continues, by the year 2050, 10 million people will die each year to an antimicrobial resistant bacterial infection. With lack of pharmaceutical industry involvement in developing novel antibiotics, the responsibility now lies within the academic institutions to identify potential novel therapeutics to fuel the antibiotic drug discovery pipeline. Combinatorial chemistry is one technique used to expedite the discovery process by assessing a large chemical space in a relatively short time when compared to traditional screening approaches. Combinatorial libraries can be screened using multiple approaches and has shown successful application towards many disease states. We initially discovered broad spectrum antibacterial bis-cyclic guanidines using combinatorial libraries and expanded on the knowledge of the physiochemical attributes necessary to inhibit Gram negative bacterial pathogens. Following this success, we continued to assess the combinatorial libraries for adjunctive therapeutics that potentiate the activity of obsolete clinical antibiotics. The polyamine efflux pump inhibitors discovered in this subsequent study prove the benefits of using the large chemical space provided in the combinatorial libraries to identify a variety of therapeutics. Our studies always begin with identifying an active compound and active compounds undergo hit-to-lead optimization. This optimization studies are of utmost importance in developing a novel antibacterial agent for therapeutic applications. Our medicinal chemistry work described here is proof of the success of careful structure activity analyses to optimize a hit scaffold to create a more effective antibacterial agent. Overall, our work described here reveals the potential role of academic institutions in fending off the impending “post-antibiotic era”.

In spite of recent therapeutic advances, multiple myeloma (MM) remains a malignancy with very low curability. This has been partly attributed to the existence of a drug-resistant subpopulation known as cancer stem cells (CSCs). MM-CSCs are equipped with the necessary tools that render them highly resistant to virtually all conventional therapies. In this study, the growth inhibitory effects of withanolide D (WND), a steroidal lactone isolated from Withania somnifera, on drug-sensitive tumoral plasma cells and drug-resistant MM cells have been investigated. In MTT/XTT assays, WND exhibited similar cytostatic effects between drug-resistant and drug-sensitive cell lines in the nM range. WND also induced cell death and apoptosis in MM-CSCs and RPMI 8226 cells, as examined by the calcein/ethidium homodimer and annexin V/propidium iodide stainings, respectively. To determine whether P-glycoprotein (P-gp) efflux affected the cytostatic activity of WND, P-gp was inhibited with verapamil and results indicated that the WND cytostatic effect in MM-CSCs was independent of P-gp efflux. Furthermore, WND did not increase the accumulation of the fluorescent P-gp substrate rhodamine 123 in MM-CSCs, suggesting that WND may not inhibit P-gp at the tested relevant doses. Therefore, the WND-induced cytostatic effect may be independent of P-gp efflux. These findings warrant further investigation of WND in MM-CSC animal models.

Our previous work in yeast has demonstrated that overexpression of FPR1, and others FKBP immunophilins, conferred tolerance to weak organic acids such as acetic and sorbic acid. FK506 binding proteins (FKBP) where originally identified as the cellular targets of the immunosuppressant drugs rapamycin an FK506. FKBPs are peptidyl-prolyl cis-trans isomerases (PPase EC 5.1.2.8) that catalize the isomerization of peptidyl prolyl bonds between cis and trans configuration. FKBP are ubiquitous proteins that can be found either as a single catalytic proteins or being part of more complex proteins. To assess the implication of FKBP proteins in weak acid tolerance in plants we have generated lines of Arabidopsis thaliana overexpressing two different proteins: yeast FPR1 an Arabidopsis FKBP65 (ROF2). We isolated knock-out mutant rof2 and rof1 from T-DNA mutant seeds collection of Salk institute. Finally we crossed the single mutants to get the double mutant rof1 x rof2. In presence of acetic acid transgenic lines overexpressing any of these genes grew better than wild type plant. On the other hand an AtFKBP65 loss-of-function mutant line showed weak acid sensitivity. We Observed a similar behavior in presence of toxic cations (Norspermidine, Hygromycim B) suggesting a role in K+ transport and we have got the confirmation with the growth al low levels of K+. We excluded the direct participation of plasma membrane ATPase because its activity in rof2 knock out mutant is higher. Furthermore ROF2 overexpression lines show a lower activity than wild-type. With 35S:: ROF2-GFP construction it was possible to see a cytosolic and nuclear cellular distribution that in presence of weak acid condition change: the ROF"-GFP leave the nuclear region. In absence of stress we have observed a gain of apical dominance in 35S::AtFKBP65 mutants and its loss in FKBP65 knock-out line. The roots of AtFKBP65 knock-out mutants have reduced number of lateral roots and exogenous application of IAA was abl / Bissoli, G. (2013). Prolyl isomerases are important determinants of intracellular pH homeostasis in Arabidopsis thaliana [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/27596 / Palancia

Antibiotic resistance is a worldwide health threat, as bacteria continue to evade antibiotic treatment. In order to survive, bacteria utilize a number of resistance mechanisms, including efflux pumps, which efficiently export antibiotics outside of the cell to reduce intracellular damage. While such mechanisms are well known, there remains a significant gap in knowledge regarding how different environmental dynamics, such as the rate of antibiotic introduction or the diversity within a microbial community, play a role in resistance. In this work, we used the AcrAB-TolC efflux pump as a case study to explore how such complex dynamics promote antibiotic resistance and its evolution. First, through a combined effort using experiments and mathematical modeling, we discovered that the rate of antibiotic introduction impacts the fraction of resistant bacteria in a population. We then explored the impact of mixed populations on survival following antibiotic treatment. In mixed microcolonies, we found that resistant cells can harm their susceptible neighbors by exporting antibiotics to increase the local concentrations of these drugs. Next, we aimed to understand how these environmental effects may impact longer-term survival of an antibiotic treatment, focusing on the evolution of resistance over ~72 hours. Through a series of adaptive evolution experiments, we identified that near-MIC treatments were the most likely to promote antibiotic resistance, regardless of whether the strains contained the AcrAB-TolC pump at wild type or overexpressed levels, or whether the strains lacked the pump altogether. In studying antibiotic introduction rates on evolution, we found that slower introduction rates facilitated the evolution of high levels of resistance with a minimal fitness cost. Meanwhile, mixed populations demonstrated limited evolvability after rapid antibiotic introductions. This work provides important insights into the impacts of environmental factors, such as the rate of antibiotic introduction and the homogeneity of populations, on the promotion and evolution of antibiotic resistance. These lessons may help inform future policies on antibiotic use and mitigate the continued pattern of resistance evolution.

Oshima N, Yamada Y, Nagayama S, Kawada K, Hasegawa S, et al. (2014) Induction of Cancer Stem Cell Properties in Colon Cancer Cells by Defined Factors. PLoS ONE 9(7): e101735. doi:10.1371/journal.pone.0101735 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18547号 / 医博第3940号 / 新制||医||1006(附属図書館) / 31447 / 京都大学大学院医学研究科医学専攻 / (主査)教授 千葉 勉, 教授 野田 亮, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM