The Hedgehog protein (Hh) is a morphogen that is necessary for cell survival, growth and patterning in flies and mammals. In germline cells, alterations in the Hh signaling pathway can result in developmental disorders; in somatic cells, misregulation of the Hh signaling pathway can result in cancer. Most components of the signaling pathway were identified by genetic screens in Drosophila that were later found to be conserved in mammals. In the presence of the Hh signal, multiple Hh signaling components interact to mediate the induction of Hh target genes. In flies, Cubitus Interruptus (Ci) is the singular transcription factor of the pathway that is regulated by multiple upstream components of the pathway including Costal2 (Cos2). Cos2 is a scaffold protein that can both positively and negatively regulate Hh signaling by binding to Ci and various kinases such as Fused (Fu). We disrupted the binding of Cos2 to Fu using a physiological expressed genomic Costal2 transgene (gCosΔFu) and found that Fu must bind to Cos2 to promote efficient processing and activation of full-length Ci (Ci-155). Fu was thought to activate Ci-155 by phosphorylating Cos2 at sites S931 and S572, but we found that gCosS931A and gCosS572A did not reduce Ci activity in the fly wing disc. Instead, we hypothesize that Fu could directly phosphorylate Ci-155 or another unknown protein. To investigate if another protein was involved we developed a Hh sensitized genetic screen. We obtained multiple “hits” from the genetic screen but we did not find an obvious candidate that could be a substrate for Fu. Instead, we identified Mago Nashi and Srp54 which we found to be involved with the post-transcriptional regulation of ci RNA. We confirmed the existence of ci isoforms A and B and found that knockdown of Mago Nashi, resulted in an altered splicing pattern while knockdown of Srp54 reduced ci RNA levels. Mago Nashi inhibition and intronless Ci reduced Ci-155 protein levels, which suggests efficient splicing is necessary for normal Ci-155 levels. Furthermore, we found that reduced Ci-155 levels only affected Ci activity in sub-optimal Hh signaling conditions. In order to further dissect the mechanism Ci processing, activation and stabilization, we used physiologically expressed genomic Ci (gCi) and CRISPR Ci variants (crCi). First we examined Ci-S849A, which prevents Ci processing and we found that in the absence of processing, Ci-155 levels are uniformly high throughout the wing disc. Cos2 and PKA are necessary for Ci processing but we wanted to know if they had an additional role in Ci silencing We found that Cos2 but not PKA can silence and stabilize Ci-155 in the absence of processing. Activated Fu in the Ci-S849A wing disc highly activated and destabilized Ci-155, which was similar to Hh signaling at the AP Border. To test if Ci is the direct target of Fu, we are testing physiologically expressed Ci with point mutations and deletions that are near the Suppressor of Fused (Su(fu)) binding site to examine whether they are unresponsive to activated Fu. Su(fu) binds to Ci-155 to stabilize Ci- 155 levels and inhibit Ci activity, but the mechanism is not well understood. We developed Ci transgenes that have altered Su(fu) binding to determine if Su(fu) inhibits Ci by cytoplasmic anchoring, co-repressor recruitment, or by blocking a co-activator.

Gram-negative bacteria are able to cause many infections including blood stream infections (BSI).These bacteria may become resistant to antibiotics, often by acquiring genes in the presence of antibiotic selection pressure. Multi drug resistant Gram-negative bacteria have become an increasing problem worldwide. A study of antibiotic resistance in Gram-negative bacteria isolated from blood cultures from patients in the New Royal Infirmary of Edinburgh (NRIE) was performed. In addition, a study was performed on isolates from patients in an intensive care unit in Egypt. All isolates were investigated for susceptibility to an extensive range of antibiotics. Gram-negative bacteria from Edinburgh found to be resistant to either cefotaxime or ceftazidime were investigated further. Among the cefotaxime/ceftazidime resistant isolates, Polymerase Chain Reaction (PCR) analysis revealed the presence of CTX-M- β-lactamases. Seven E.coli isolates were found to have CTX-M-15 β-lactamases while the CTX-M-14 β-lactamase was detected in six Enterobacter cloacae. The insertion sequence ISEcp1 was detected upstream of the blaCTX-M-15 gene in some isolates while IS26 was found truncating the ISEcp1 in other isolates. Conjugation experiments found the blaCTX-M-15 gene was transferable to E. coli J62-2. All the isolates had detectable plasmids, a plasmid ~260kb carried the blaCTX-M-15 gene. Analysis of the -containing isolates by PFGE shows that those carrying the CTX-M-14 β-lactamase were identical indicating cross infection within the hospital. The CTX-M- 15 β-lactamase-containing isolates showed four isolates had ≥85% similarity but the others were diverse. Class 1 integrons were found in eight of the CTX-M β-lactamase containing isolates with the associated gene cassette and sul1 gene. The isolates from Egypt were found to be resistant to carbapenem, which is the final mainstream antibiotic option in the treatment of multidrug resistant Gram-negative bacteria. Further analysis revealed all carried the CTX-M-14 β-lactamase and two additionally carried the VIM-4 metallo β-lactamase, which accounted for the resistance to the carbapenems. Furthermore, the insertion sequence ISEcp1 was found upstream of the blaCTX-M-14 gene in two of the isolates. The blaVIM-4 gene was found to be part of the gene cassette in the class 1 integron associate with complex ISCR1. Two of the Egyptian isolates had a detectable plasmid, ~300kb in size, which carried both blaCTX-M-14 and blaVIM-4 genes. All the blood culture isolates were examined to ascertain the persistence of sulphonamide resistance despite the long-term prescribing reduction on this antibacterial. PCR was performed to detect sul1, sul2 and sul3 genes in all the isolates. Of the sulphonamide resistant isolates 25 carried the sul1, 27 carried the sul2 and none carried the sul3 genes. Eight isolates had both the sul1 and sul2 genes. Most of the isolates carried sul1 had Int1 as part of the same class 1 integron. Interestingly three isolates were PCR negative for sul1 but positive for sul2 and int1. Int2 and 3 were found in 3 and 2 isolates respectively. The class 1 integron contained different insert gene cassettes; dfrA (dfrA17, dfrA16, dfrA15), aadA (aadA5, aadA2, aadA1) and blaOXA-1 families in addition to the resident sul gene. In conclusion this thesis shows the diversity of the genetic environment and carriers of the CTX-M β-lactamases within the same hospital. Sulphonamide resistance in Gram-negatives persists despite the prescribing reduction of this antibacterial in a Scottish hospital and the recommended constraint on the use of sulphonamide.

Para infectar seus hospedeiros artrópodes, Metarhizium anisopliae produz uma série de enzimas hidrolíticas e diversas alterações morfológicas como a formação de estruturas denominadas de apressórios e blastosporos. Estas estruturas de infecção representam estágios cruciais de penetração e disseminação no inseto hospedeiro, respectivamente. Além disso, o apressório é uma estrutura conservada entre fungos entomopatogênicos e fitopatogênicos. O gene emp1 (codifica uma proteína de matriz extracelular de Magnaporthe grisea) parece ter um papel importante na diferenciação do apressório, bem como na patogenicidade e virulência. Um dos nossos objetivos foi avaliar a possível função de um ortólogo do gene emp1 em M. anisopliae pela análise do perfil transcricional e construção de mutantes funcionais por mutagênese insercional utilizando agrotransformação. Foram geradas linhagens transformantes contendo o cassete de inativação do gene emp1 (pPZP::bar::emp1), sendo este construído pela subclonagem das regiões flanqueadoras 5’ (1.515pb) e 3’ (1.513pb), fusionadas a um cassete de expressão do gene bar (3.500pb) que confere resistência a glifosinato de amônia. A freqüência de transformação observada nesta etapa de transformação foi superior a 60%, porém em apenas 1,5% dos transformantes há indícios de recombinação homóloga. Também foi analisado o perfil transcricional de emp1, sob três condições anteriormente padronizadas: células diferenciadas em apressório, hifas (crescimento vegetativo), e blastosporos, sendo observada a presença de transcritos deste gene em todas as condições testadas. Em uma segunda etapa deste trabalho, foi avaliado o perfil transcricional de quitinases putativas de M. anisopliae nos estágios de diferenciação celular de apressório, hifas e blastosporos. Esta classe de proteínas está diretamente envolvida no remodelamento da parede celular fúngica durante a diferenciação celular, como também na degradação da quitina da cutícula do hospedeiro durante a etapa de penetração. Originalmente foram caracterizados três genes para quitinases (chit1, chi2 e chi3), e a análise in silico do genoma revelou outras 20 quitinases putativas de Metarhizium. Essa diversidade pode ser responsável pelas diferentes funções que o conjunto de enzimas quitinolíticas tem na degradação de quitina durante o ciclo de vida e de infecção do fungo. É, portanto importante: (i) validar transcricionalmente as quitinases putativas e (ii) tentar atribuir função a cada uma delas. Para isso, procedemos a análise dos transcritos de cada uma das 23 quitinases putativas sendo sintetizados cDNAs a partir das três condições de diferenciação celular (apressório, hifas e blastosporos). Na condição de diferenciação a apressório dez espécies de transcritos foram detectadas sendo seis do subgrupo A, três do subgrupo B, uma do subgrupo D. Nenhum transcrito de quitinases do subgrupo C foi detectado nas condições testadas. Tanto em crescimento vegetativo quanto em blastosporos foram detectadas sete espécies de transcritos de quitinases do subgrupo A, e uma do subgrupo D. As quitinases do subgrupo B diferiram quanto a sua distribuição nas condições testadas: três espécies de transcritos foram detectadas durante o crescimento vegetativo e seis em blastosporos. O estudo envolvendo os diferentes genes putativos (emp1 e de quitinases) de M. anisopliae, sob as diversas abordagens, representa um importante precursor para nos fornecer indicativos sobre as funções destes genes no ciclo de vida e de infecção de Metarhizium. / To infect their arthropod hosts, Metarhizium anisopliae produces a series of hydrolytic enzymes and several morphological changes, including the formation of structures called appressoria and blastospores. These infective structures represent crucial stages of penetration and dissemination in the insect host, repectively. In addition, the appressorium is a structure conserved among phytopathogenic and entomopathogenic fungi. The emp1 gene (encodes an extracellular matrix protein) from Magnaporthe grisea showed an important role in appressorium differentiation, as well as pathogenicity and virulence. Our goal was evaluate the possible role of an ortholog gene emp1 in M. anisopliae by transcriptional analysis and construction of functional mutants by insertional mutagenesis mediated by agrotransformation. We generated transformants strains containing the gene inactivation cassette of emp1 (pPZP::bar::emp1), which was constructed by subcloning of flanking portions 5’ (1.515bp) and 3’ (1.513bp) fused in a expression cassette of bar gene (resistance to glufosinate ammonium). The frequency of transformation observed in this round was higher than 60%, but only 1,5% of the transformants there is evidence of homologous recombination. We also analysed the transcriptional profile of emp1 under three conditions previously standardized, such as differentiated cells in appressorium, hyphae (vegetative growth) and blastospores. Accordingly, we observed the presence of transcripts of this gene in all growth condition tested. In a second step of this work, we evaluated the transcriptional profile of putative chitinases of the M. anisopliae in those different stages of cellular diferentiation. This class of proteins is directly involved in fungal cell wall remodeling during cellular diferentiation, as well as degradation of chitin in the host cuticle during the penetration stage. Originally we characterized three genes of chitinases (chit1, chi2 and chi3), but apart from these, the in silico analysis of the genome revealed 20 putative chitinases. This diversity may be reponsible for differents functions that the set of chitinases enzymes have in the chitin degradation during the life and infection cycle of the fungus. It is therefore important: (i) validate transcriptionally the putative chitinases and (ii) attemping to assign function to each one of them. Chitinases transcript survey was performed using cDNAs from three cell types: appressorium, vegetative growth and blastospores. In the appressorium condition ten species of transcripts were detected being six from the subgroup A chitinases, three from group B and one from group D. No transcripts of subgroup C chitinases were detected. Both in vegetative growth and blastospores seven species of transcripts of the subgroup A, and one from group D were detected. Chitinases from subgroup B differed - three species of transcripts were detected during vegetative growth and six were detected in blastospores. This study of several putative genes (as emp1 and chitinases) of M. anisopliae, under different approaches, may represent an important preliminary outcome to shed a light in the functions of these genes during life cycle and infection of Metarhizium.

Luo, Xiao. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 118-134). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.iv / Abbreviations --- p.v / Contents --- p.vi / List of Figures --- p.ix / List of Tables --- p.xii / Chapter Chapter 1 : --- Literature Review --- p.1 / Chapter 1.1 --- Lentinula edodes --- p.1 / Chapter 1.1.1 --- Introduction and taxonomy --- p.1 / Chapter 1.1.2 --- Nutritional values and medical values --- p.2 / Chapter 1.2 --- Life cycle and morphology --- p.5 / Chapter 1.3 --- Lignocellulolytic system in wood-rotting fungi --- p.9 / Chapter 1.3.1 --- Structures of lignin --- p.9 / Chapter 1.3.2 --- Wood-rotting fungi --- p.11 / Chapter 1.3.3 --- Lignin degradation by white rot fungi --- p.12 / Chapter 1.3.4 --- Ligninolytic enzymes --- p.16 / Chapter 1.3.4.1 --- Lignin peroxidase --- p.16 / Chapter 1.3.4.2 --- Maganese peroxide --- p.16 / Chapter 1.3.4.3 --- Laccases --- p.19 / Chapter 1.3.5 --- Potential Industrial application of liglinolytic enzymes --- p.22 / Chapter 1.3.6 --- Ligninolytic enzymes in L. edodes --- p.23 / Chapter 1.4 --- Expression systems for fungal ligninolytic enzymes --- p.24 / Chapter 1.5 --- Aim of this project --- p.27 / Chapter 1.6 --- Long-term significance --- p.28 / Chapter Chapter 2: --- Sequence analysis of ligninolytic enzymes from Lentinula edodes --- p.29 / Chapter 2.1 --- Introduction --- p.29 / Chapter 2.2 --- Materials and methods --- p.32 / Chapter 2.2.1 --- Phylogenetic study and signal peptide prediction of the decuced ligninolytic enzymes --- p.32 / Chapter 2.2.2 --- Comparison ligninolytic enzymes of L. edodes and other basidiomycetes fungi --- p.32 / Chapter 2.3 --- Results --- p.34 / Chapter 2.3.1 --- Protein sequence analysis and signature sequences identification of L. edodes laccases --- p.34 / Chapter 2.3.2 --- Protein sequence analysis of L. edodes manganese peroxidases --- p.34 / Chapter 2.3.3 --- Phylogenetic study of ligninolytic genes from L.edodes --- p.35 / Chapter 2.4 --- Disscussion --- p.52 / Chapter Chapter 3: --- Transcription profiling of ligninolytic enzymes from Lentinula edodes --- p.56 / Chapter 3.1 --- Introduction --- p.56 / Chapter 3.2 --- Materials and Methods --- p.61 / Chapter 3.2.1 --- Strain cultivation --- p.61 / Chapter 3.2.2 --- "RNA extraction, mRNA isolation and cDNA synthesis" --- p.63 / Chapter 3.2.3 --- RNA Quality Estimation --- p.64 / Chapter 3.2.4 --- cDNA synthesis --- p.65 / Chapter 3.2.5 --- Primer verification --- p.66 / Chapter 3.2.6 --- Quantitative RT-PCR --- p.66 / Chapter 3.3 --- Results --- p.70 / Chapter 3.3.1 --- RNA quality estimation --- p.70 / Chapter 3.3.2 --- Quantification real time PCR --- p.70 / Chapter 3.3.3 --- Transcriptional profiling of laccases during the development of L edodes --- p.70 / Chapter 3.3.4 --- Transcriptional profiling of MnPs during the development of L edodes --- p.71 / Chapter 3.3.5 --- Transcript level analysis of laccases from in mycelia grown on lignocelluloses medium and non -lignocelluloses medium --- p.71 / Chapter 3.3.6 --- Transcript level analysis of MnPs in mycelia grown on lignocelluloses medium and non -lignocelluloses medium --- p.72 / Chapter 3.3.7 --- Differential expression of laccases from L. edodes grownin lignocelluloses medium during mycelia stage --- p.72 / Chapter 3.3.8 --- Differential expression of laccases from L. edodes grownin lignocelluloses medium during mycelia stage --- p.72 / Chapter 3.4 --- Discussion --- p.87 / Chapter 3.4.1 --- Transcriptional profiling of laccases and MnPs during four developmental stages --- p.87 / Chapter 3.4.2 --- Transcriptional profiling of laccases and MnPs in mycelium grown in lignocelluloses and non-lignocelluloses medium --- p.88 / Chapter 3.4.3 --- Temporal differential expression of laccases and manganese peroxidases --- p.90 / Chapter 3.5 --- Conclusion --- p.92 / Chapter Chapter 4: --- "Cloning and heterologous expression of Lentinula edodes laccase, lac1B, in yeast Pichia pastoris" --- p.93 / Chapter 4.1 --- Introduction --- p.93 / Chapter 4.2 --- Materials and Methods --- p.95 / Chapter 4.2.1 --- Strain cultivation --- p.95 / Chapter 4.2.2 --- First strand cDNA synthesis --- p.95 / Chapter 4.2.3 --- Construction of cDNA library --- p.95 / Chapter 4.2.4 --- Signal peptide prediction of Iac1 B --- p.96 / Chapter 4.2.5 --- Cloning of native laccase into Pichia pastoris expression vector --- p.96 / Chapter 4.2.6 --- Screening for positive colonies --- p.97 / Chapter 4.2.7 --- Construction of pool of recombinant vector --- p.97 / Chapter 4.2.8 --- Transformation of P. pastoris --- p.98 / Chapter 4.2.9 --- Screening for expression cassette into Pichia pastoris --- p.98 / Chapter 4.2.10 --- Enzyme Activity assay --- p.99 / Chapter 4.2.11 --- SDS-PAGE --- p.100 / Chapter 4.3 --- Results --- p.103 / Chapter 4.3.1 --- Screening for positive colonies with recombinant vector in TOP10 --- p.103 / Chapter 4.3.2 --- Screening for expression cassette from transform ants of P pastoris --- p.103 / Chapter 4.3.3 --- Enzyme activity assay --- p.103 / Chapter 4.3.4 --- SDS-PAGE --- p.104 / Chapter 4.4 --- Disscussion --- p.109 / Chapter Chapter 5: --- Concluding Remarks --- p.111 / Reference --- p.118

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How asexual reproduction affects genome evolution, and how organisms that are ancestrally sexual alter their reproductive machinery upon becoming asexual are both central unanswered questions in evolutionary biology. While these questions have been addressed to some extent in organisms such as asexual clams, rotifers, ostracods, arthropods, and fungi, the most powerful and direct tests of how sex and its absence influence evolution requires direct comparisons between closely related and otherwise similar sexual and asexual taxa. Here, I quantify the rates and patterns of molecular evolution in the meiosis-specific genes Msh4, Msh5, and Spo11 in multiple sexual and asexual lineages of Potamopyrgus antipodarum, a New Zealand freshwater snail. Because asexual P. antipodarum reproduce apomictically (without recombination), genes used only for meiosis should be under relaxed selection relative to meiosis-specific genes in sexual P. antipodarum, allowing me to directly study how asexuality affects the evolution of meiosis-specific genes. Contrary to expectations under relaxed selection, I found no evidence that these meiosis-specific genes are degrading in asexual P. antipodarum; instead they display molecular patterns consistent with purifying selection. The presence of intact meiosis-specific genes in asexual P. antipodarum hints that the asexuals may maintain the ability to perform meiosis despite reproducing apomictically. Asexual meiotic capability suggests that some meiotic components may persist or acquire a new role in these asexuals.

BRCA1 is a caretaker of genome integrity with various molecular functions, which are required for development and tumor suppression. These include the homology-directed repair (HDR) of DNA double strand breaks, stalled replication fork protection (SFP), transcription, chromatin remodeling and cell cycle checkpoint control. Recent studies reported that BRCA1 is required for reprogramming to pluripotency, but its specific role remains unknown. In this work, we use separation of function mutants for the roles of BRCA1 in HDR and SFP to show that BRCA1 is required to repair replication-associated DNA double strand breaks by homologous recombination during reprogramming. Deficiency in SFP proved inconsequential to induced pluripotent stem (iPS) cell generation and cells with this phenotype did not experience reduced reprogramming. Thus, the primary limiting factor for the transition to pluripotency is a specific class of DNA damage: double strand breaks, likely occurring in late replicating regions which require repair by homologous recombination. These findings identify an important role of DNA damage, linked to the progression of DNA replication, in limiting cell type transitions during reprogramming. Most studies on iPS cell generation have focused on gene expression as a limiting step, in part due to the wide availability of tools to analyze transcription. Since the progression of DNA replication and DNA damage during S-phase are cell type specific, we have started the development of a sequencing platform to map various aspects of replication progression, such as origin usage, polymerase direction,pausing and stalling. In this work, we demonstrate that nucleotide analogs, incorporated during DNA synthesis in mammalian cells, can be detected by Nanopore sequencing.

Memoria para optar al título de Ingeniero Civil Matemático / El presente trabajo muestra la implementación de técnicas de clasificación basadas en árboles de decisión para resolver y entender el problema de identificación de genes anotados en el ADN de la bacteria Escherichia Coli. Junto a lo anterior, se pretenden entender algunos principios biológicos subyacentes tras el mecanismo celular de identificación genética. Los métodos de clasificación que se implementan en este trabajo intentan simular la manera en que los complejos procesos celulares de transcripción y traducción genética identifican o encuentran las posiciones de inicio de los genes responsables de la posterior síntesis proteica. Se respeta la forma en que esta información es adquirida sin caer en el error de alejarse del marco biológico en cuestión. Para resolver el problema se crearon tres estrategias de clasificación basadas en la combinación de modelos de árboles de decisión y de un algoritmo de optimización sobre el área ocupada en el ADN por zonas génicas. La primera estrategia consiste en utilizar el algoritmo de optimización sobre candidatos a genes, obtenidos de una lectura secuencial en la doble hebra, para reducir la cantidad de potenciales genes. La solución obtenida es clasificada por los árboles de decisión. La segunda estrategia consiste en realizar el mismo proceso pero usando candidatos obtenidos desde una lectura en ambos sentidos de la doble hebra de ADN. La tercera estrategia consiste en iterar sucesivamente la optimización junto a los árboles utilizando la información incorrectamente clasificada por estos. Los resultados obtenidos se resumen como un conjunto de candidatos clasificados positivamente por los árboles de decisión y que cumplen con las restricciones impuestas por el algoritmo de optimización. / CMM - Conicyt PIA AFB170001