No description available.

No description available.

Blastocystis sp. é um protozoário intestinal comumente encontrado em amostras fecais de muitas espécies animais, incluindo humanos, sendo pouco estudado em pacientes imunocomprometidos, especialmente nos candidatos a transplante. O objetivo do presente estudo foi avaliar a ocorrência e a identificação molecular de subtipos (ST) de Blastocystis sp. em amostras fecais de pacientes candidatos a transplante. A Reação em Cadeia da Polimerase foi realizada utilizando primers específicos para o DNA ribossomal de Blastocystis. As sequências de DNA obtidas foram alinhadas e comparadas com outras sequências do banco de dados GenBank e MLST (Multilocus Sequence Typing). As amostras analisadas mostraram uma positividade de 16% (24/150) para Blastocystis sp. A maior ocorrência foi observada em candidatos a transplante renal (31,4%), seguida de candidatos a transplante hepático (10,4%) e candidatos a transplante de medula óssea (5,9%). O ST3 (45,8%) foi o mais prevalente entre os isolados, seguido pelo ST1 (37,5%), ST2 (12,5%) e ST7 (4,2%). Foi observado dentro dos subtipos: os alelos 4 e 78/81 (ST1); alelos 11 e 12 (ST2); alelos 34, 36, 37 e 54 (ST3) e alelo 96 (ST7). Este é o primeiro estudo de identificação molecular de Blastocystis sp. em candidatos a transplante. Os presentes resultados confirmam a maior ocorrência do subtipo 3 em candidatos a transplante, além de reforçar a importância de novas investigações de Blastocystis sp. nesses pacientes. / Blastocystis sp. is an intestinal protozoan commonly found in faecal samples of many animal species, including humans, but poorly studied in immunocompromised patients, especially in transplant candidates. The purpose this study was to evaluated the occurrence and molecular identification of Blastocystis sp. in faecal samples from transplant candidate patients. The Polymerase Chain Reaction was performed using specific primers for Blastocystis DNA ribosomal. The DNA sequences obtained were aligned and compared with other sequences from the GenBank and MLST database. The analyzed samples showed a positivity of 16% (24/150) for Blastocystis sp. The highest occurrence was observed in renal transplant candidates (31.4%), followed by hepatic transplant candidates (10.4%) and candidates for bone marrow transplantation (5.9%). The ST3 (45.8%) was the most prevalent among the isolates followed by ST1 (37.5%), ST2 (12.5%) and ST7 (4.2%). It was observed within the subtypes: alleles 4 and 78/81 (ST1); alleles 11 and 12 (ST2); alleles 34, 36, 37 and 54 (ST3); and allele 96 (ST7).This is the first study of Blastocystis sp. molecular identification in transplant candidates. These results confirmed the highest occurrence of the subtype 3 in transplant candidates, as well as reinforcing the importance of the new investigation of Blastocystis sp. in these patients.

Blastocystis sp. é um protozoário comumente encontrado em amostras fecais de humanos e animais, envolto por aspectos patogênicos e zoonóticos ainda controversos. Estudos recentes têm demonstrado a distribuição dos subtipos (STs) de Blastocystis sp., porém são escassos os relatos sobre a sua caracterização molecular na América Latina, sobretudo no Brasil. O objetivo do presente estudo foi investigar os STs presentes em isolados fecais de indivíduos acompanhados no Hospital das Clínicas de São Paulo da Faculdade de Medicina da Universidade de São Paulo (HC/FMUSP). Para tanto, amostras fecais positivas para Blastocystis sp. diagnosticadas na Seção de Parasitologia do Laboratório Central (HC/FMUSP) foram utilizadas para o isolamento de DNA. A reação em cadeia da polimerase (PCR) foi realizada utilizando iniciadores específicos para a subunidade 18S do DNA ribossomal de Blastocystis sp. A reação de sequenciamento dos produtos de PCR foi realizada, as sequências de DNA obtidas foram alinhadas e comparadas com outras sequências da base de dados GenBank e MLST. Foram identificados os STs (ST1, ST2, ST3 e ST6), sendo o ST3 o mais prevalente entre os isolados humanos seguido pelo ST1. Os alelos de número 34 e 36 foram os mais frequentes. Em conclusão, estes resultados contribuem para a caracterização molecular e a distribuição dos STs de Blastocystis sp. em amostras de fezes humanas no Brasil. / Blastocystis sp. is an organism described as enteroparasite protozoan, commonly found in stool samples from humans. Several subtypes have been described in humans, but pathogenic potential and aspects epidemiological are still controversial. The aim of the present study was to investigate Blastocystis subtypes (STs) from patients of Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC/FMUSP), Brazil. Blastocystis sp. positive stool samples diagnosed in Section of Parasitology of Central Laboratory (HC/FMUSP) were used for DNA isolation. Polymerase chain reaction (PCR) was performed using specific primers targeting the small subunit of rRNA gene. Direct DNA sequencing of PCR products was performed, and the DNA sequences were aligned and compared to other sequences present in GenBank and MLST database. Four STs were identified (ST1, ST2, ST3 and ST6), where the ST3 was the most prevalent ST among human isolates followed by ST1. Allele nos. 34 and 36 were the most frequent. Another important finding is the presence of ST6, rarely detected in human isolates. In conclusion, our results contribute to the molecular characterization and distribution of Blastocystis sp. STs in human stool specimens in Brazil.

Repair of damage to the DNA is essential for the maintenance of genomic stability, both during embryonic development and normal growth. The cell has therefore evolved a complex array of interconnected pathways to ensure the appropriate response to DNA damage is initiated, such as cell cycle checkpoint arrest, activation of DNA repair pathways or induction of apoptotic processes. These co-ordinated signal transduction pathways have been termed the DNA damage response (DDR). A previous study showed that ATR-dependent damage responses were frequently defective in cell lines from patients with Microcephalic Primordial Dwarfism (MPD) disorders. In this thesis I have further characterised ATR–dependent damage response signalling in several cell lines from patients with various MPD disorders. I have shown that novel mutations in PCNT, which encodes a structural centrosomal protein, result in an MPD disorder and have characterised the associated ATRdependent DNA damage responses. I also contributed to the identification of mutations in ORC1, encoding a component of the DNA replication Origin Recognition Complex, in further MPD patients and examined origin licensing and Sphase progression in the patient derived cell lines. As a novel finding, I observed defects in the ATR-dependent G2/M checkpoint response in these cells. Additionally, I have characterised novel mutations in ATRIP, a gene encoding the obligate partner of ATR, in Seckel Syndrome patients, denoting a novel genetic defect in this condition. Finally, I have explored the role of PLK1 and AurA kinase in ATRdependent G2/M checkpoint control and provided compelling evidence of misregulation of this pathway in various MPD-patient derived cell lines. Collectively these data provide important functional insights into the genetic defects that cause MPD disorders and further explore the link between defective ATR-dependent damage response signalling and microcephaly.

The INO80 complex is a large ATPase chromatin remodeller which contains 15 accessory subunits in S.cerevisiae. Its subunits include the highly conserved ATPases Ruvb1 and Ruvb2, the actin-related proteins Arp5, Arp8, Act1 and Arp4, Actin, and a number of IES (I̱noE̱ighty S̱pecific) subunits Ies1, Ies2, Ies3, Ies4, Ies5 and Ies6, in addition to subunits Nhp10 and Taf14. All 15 of the accessory subunits are assembled around a catalytic core component known as Ino80. The INO80 complex has roles in transcription, DNA repair, replication, and chromosome segregation. These roles are in addition to its traditional nucleosome remodelling activities and the dispacement of H2A.Z from chromatin. Recent studies in S. cerevisiae have identified the subunit Ies6 as a critical component of the INO80 complex. Deletion of IES6, which encodes the small accessory subunit, clearly mimics the deletion f the gene encoding the catalytic subunit, INO80. Surprisingly, only one domain within Ies6 has been formally identified based on sequence analysis. This domain belongs to the L1_C class of domains. Such domains are commonly associated with DNA binding activity and transcription factors. This stud has further characterised the Ies6 subunit both genetically and biochemically. Genetically, it has demonstrated that single point mutations at regions of proposed subunit-subunit interaction between the Arp5 or Rvb2 subunits, or within the YL1_C are not sufficient to disrupt Ies6 function. However, expression of a double point mutation, ies6(K114E/Y125A), in combination with rad50 deletion, caused a sensitivity to replication inhibition, but not chromosome segregation inhibition, indicating a potential separation of function in this utant due to the loss due of only one of the biological functions of Ies6. Biochemically, we have confirmed that DBA binding capacity of Ies6 resides within the YL_C domain. In addition, although it has been demonstrated that the removal of H2A.Z acetylation exacerbates the increase in cellular ploidy observed in ies6 null cells, we found that overall levels of H2A.Z acetylation were not influenced by the loss of Ies6. This indicates that the role of H2A.Z acetylation in chromosome segregation may only affect ploidy status upon the loss of Ies6. In addition, work on the R2TP complex (which contains the INO80 APases Ruvb1/Ruvb2, and subunits Tah1 and Phi1) has revealed the recruitment mechanism for the molecular chaperone, Hsp90, and the telomere length regulation protein, Tel2. Together, the R2TP complex, Hsp90 and Tel2 promote the stabilisation and maturation of multi-protein complexes. These include Phosphatidylinositol 3-kinase-related kinases (PIKKs, a family of kinases involved i Serine and Threonine phosphorylation), subunits of the INO80 complex and subunits of the SWR1 chromatin remodelling complex (a partner comlex to INO80 that incorporates H2A.Z into chromatin).

Over the last decade, major advances in the development and application of microarray-based comparative genomic hybridisation (aCGH) technology have significantly contributed to our understanding of Genomic Disorders. My aims here were to provide insight into the genotype to phenotype relationships of three Genomic Disorders; CUL4B-deleted X-Linked Mental Retardation (XLMR), Wolf-Hirschhorn Syndrome (WHS) and 16p11.2 Copy Number Variant Disorder. CUL4B encodes a structural component of the Cullin-RING-ligase 4-containing class of E3 ubiquitin ligases. CUL4B-deleted XLMR represents a syndromal form of mental retardation whereby patients exhibit other clinical features aside from the MR, such as seizures, growth retardation and disrupted sexual development. I used CUL4B-deleted patient-derived cell lines to investigate the impacts of CUL4B loss on mitochondrial function. I have shown that loss of CUL4B is associated with a distinct set of mitochondrial phenotypes, identifying CUL4B-deleted XLMR as a disorder associated with mitochondrial dysfunction. Furthermore, I have uncovered a reciprocal relationship between CUL4B and Cereblon, providing evidence of a potential role for the CUL4-CRBN E3 ligase complex in maintaining mitochondrial function. Deletion or duplication of the 16p11.2 region is associated with macro-/microcephaly respectively. Here, I have evaluated the cellular consequences of 16p11.2 CNV, specifically with regards KCTD13 expression, DNA replication and checkpoint activation. WHS is typically caused by a small hemizygous telomeric deletion of the 4p16.1 region. Haploinsufficiency of 4p16.1 is associated with microcephaly, growth retardation and complex developmental abnormalities. I investigated the impacts of LETM1 copy number change in WHS patient-derived cells. Here, I have shown that copy number change of LETM1 specifically segregates with mitochondrial dysfunction, likely underlying the seizure phenotype exhibited by the large subgroup of WHS patients whose deletions incorporate LETM1 as well as the rarer instances of the reciprocal duplication. In this thesis I use patient-derived cell lines from three Genomic Disorders as a fundamental tool providing new pathomechanistic insight into the clinical presentation of these conditions.

Mutations are the ultimate source of new genetic information and they can be neutral, harmful or beneficial. The ultimate fate of all mutations is either to be lost or to eventually become fixed in a population. In this thesis I investigate genome wide traces of natural selection in eukaryotes. I focus on the most common type of mutations, point mutations, in protein coding genes. I investigated whether there is adaptive evolution in 11 plant species comparisons by applying an extension of the McDonald Kreitman (MK) test and found little evidence of adaptive evolution. However, most of the investigated plant species have low effective population sizes (Ne) and the rate of adaptive evolution is thought to be correlated to Ne. I therefore extended my study using additional data from mammals, drosophilids and yeast to investigate the relationship between the rate of adaptive evolution and Ne. I found a highly significant correlation between the rate of adaptive evolution relative to the rate of neutral evolution (!a) and Ne. It has been proposed that evidence of adaptive evolution can be an artifact of fluctuating selection. I simulated a model of fluctuating selection, in which the average strength of selection acting upon mutations is zero. Under this model adaptive evolution is inferred using MK-type tests. However, the mutations which become fixed are on average positively selected. The signal of adaptive evolution is therefore genuine. Ne can not only vary between species but also across genomes. However, how much variation there is, and whether this affects the efficiency of natural selection, is unknown. I analysed 10 species and show that variation in Ne is widespread. However, this variation is limited, amounting to a few fold variation in Ne between most genomic regions. This is never-the-less sufficient to cause variation in the efficiency of selection.

Hox genes encode a family of evolutionarily conserved transcription factors involved in the activation of diverse cell differentiation programs along the antero-posterior axis of animals. Hox gene expression is controlled by a complex set of regulatory mechanisms which are still not fully understood. Despite this, misregulation of Hox gene expression can lead to severe developmental abnormalities and various forms of disease. This work addresses the way in which small non-coding RNAs (microRNAs, miRNAs) regulate Hox gene expression and function during development. To do this we use the Drosophila Hox gene Ultrabithorax (Ubx) as a paradigm for Hox gene function. Using a suite of genetic methods we first uncover a novel regulatory interaction between Drosophila Ubx and the miR-310C family of miRNAs during the development of the haltere, a small dorsal appendage involved in flight control. We also show that this miRNA cluster is required to fine tune Ubx expression. Furthermore, our data provides insight into the role played by Ubx during appendage development. Secondly, using a next generation RNA sequencing approach, we identify the full repertoire of miRNAs present in two serially homologous appendages of Drosophila – the wing and haltere. Our results show that these morphologically distinct appendages have divergent miRNA profiles, including miRNAs which display appendage-specific expression patterns. In addition, combining these profiles with available transcriptomic data enabled us to study how miRNAs are integrated into the Ubx gene regulatory networks that govern haltere development. This analysis suggests that haltere miRNAs reinforce the regulatory programmes installed by Ubx during haltere development. Our work therefore contributes to the understanding of the regulatory function of miRNAs during development and sheds light on the ways in which Hox gene expression can contribute to the formation of complex morphological structures.

Maintenance of the genome is essential for life to prosper. Regular insults to the genome are sustained by all cellular life and can foster genetic instability if left unrepaired. The most lethal genetic damage is a double strand break (DSB), the cleavage of the phosphate backbone on both strands of the DNA double helix. Two main pathways exist which provide mechanisms for coping with DSBs; precise repair utilising the identical sister chromatid as a template to recreate the broken segment (homologous recombination; HR), and direct fusion of the broken ends in the absence of an intact template (nonhomologous end joining; NHEJ). NHEJ was first characterised in eukaryotes, and an analogous system has been found to exist in bacteria during the past decade. The bacterial NHEJ pathway is composed of four key proteins; the DNA end binding Ku homodimer, a DNA Ligase, a DNA polymerase and a phosphoesterase (PE). The first results chapter of this thesis details the identification of an orthologous set of proteins in the archaeon Methanocella paludicola, and their subsequent isolation and characterisation. The second results chapter expands on the individual activities of the proteins by combining them, and asserting the ability of archaeal NHEJ to join discontinuous ends in vitro. The role of the PE has been unclear in the bacterial system, but in vitro assays described here suggest that the enzyme plays a role in processing NHEJ intermediates formed by the NHEJ polymerase. The PE is found to optimise repair intermediates for ligation, and to reverse potentially genotoxic DNA strand displacements. The final results chapter investigates the structural aspects of the archaeal NHEJ enzymes. Together these studies establish a functional NHEJ system in an archaeon for the first time, and expand our knowledge of the bacterial system by proposing a standard model of archaeo--‐prokaryotic NHEJ.