Made available in DSpace on 2014-06-12T15:04:39Z (GMT). No. of bitstreams: 2 arquivo4615_1.pdf: 1891643 bytes, checksum: b2ca674f78e6add635447a9d9b7bbb39 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2007 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A contaminação ambiental por metais pesados é uma questão séria e mundial. O aumento das atividades industriais e a deterioração de alguns ecossistemas, devido acumulação de metais têm intensificado a poluição ambiental. O cádmio, metal pesado muito utilizado em processos industriais, é um dos elementos químicos mais tóxicos, o que o torna um relevante contaminante ambiental. Os fungos são organismos de grande importância prática por causa de sua ampla aplicação em diversas áreas da biotecnologia, sobretudo na área ambiental. Neste trabalho, os aspectos fisiológicos do efeito do cádmio concernente à resistência/tolerância a metais, metabolismo do polifosfato, alterações histoquímicas e ultra-estruturais, foram avaliados em Cunninghamella elegans. Os resultados obtidos demonstraram que o cádmio induziu variações na forma, arranjo e distribuição do citoesqueleto de actina, bem como na estrutura fina do fungo associado a mudanças de eletrondensidade, vacuolização, alteração na textura do citoplasma e presença de inclusões e granulações eletrondensas. A citoquímica com vermelho de rutênio revelou, por sua vez, alteração de permeabilidade celular e composição de carboidratos de superfície. Além do que foi observado que C. elegans é capaz resistir, crescer, remover e acumular cádmio em diferentes concentrações e que possivelmente usa o polifosfato como mecanismo de detoxificação, haja vista que seu conteúdo variou muito em função da concentração do metal. Os dados obtidos neste trabalho revelam que C. elegans exibe potencial de uso em biorremediação de metais

Made available in DSpace on 2014-06-12T15:05:32Z (GMT). No. of bitstreams: 2 arquivo4553_1.pdf: 555608 bytes, checksum: 9cb94ebf40323d8ad913699e17a82f12 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2005 / Investigações foram realizadas comparativamente sobre a habilidade de biossorção de cobre e zinco pela biomassa de Phanerochaete chrysosporium UCP 963 e Cunninghamella elegans UCP 596. A remoção dos metais pesados foi avaliada utilizando-se diferentes quantidades de biomassa (120 mg e 240 mg), viva e inativada, adicionadas a solução de cobre e de zinco (4 e 6 mM), pH 5.0 sob agitação de 150 rpm, à temperatura de 28 ºC, por 480 minutos. A amostra de C. elegans demonstrou habilidade de remoção dos metais pesados na concentração de 4 mM com rendimentos de 104 mg.g-1 (55% de remoção) para o cobre, 94,44 mg.g-1 (51% de remoção) de zinco, e na concentração de 6 mM zinco removeu 129,71 mg.g-1 (53%) e cobre 171 mg.g-1 (57%), todos os tratamentos foi utilizado 120 mg da biomassa. A biomassa inativada de P. chrysosporium foi mais eficiente na remoção de cobre na concentração de 6 mM com resultados de 134,18 mg.g-1 (45% de remoção) e em ambas concentrações de zinco (4 e 6 mM) apresentando uma sorção de 73-101 mg.g-1 (59-63 %), respectivamente, durante 480 minutos. Os resultados demonstram que tanto a utilização da biomassa inativada e/ou viva de P. chrysosporium e C. elegans apresentam habilidade no processo de remoção de cobre e zinco, possibilitando uma futura aplicação na sorção de metais pesados em ambientes contaminados

Submitted by Nathália Neves (nathalia.neves@ufpe.br) on 2015-03-05T18:28:51Z No. of bitstreams: 2 Dissertação de Mestrado FINAL 2_2.pdf: 931387 bytes, checksum: 8c678d9ee4559050331bdc307a7e62f9 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-05T18:28:51Z (GMT). No. of bitstreams: 2 Dissertação de Mestrado FINAL 2_2.pdf: 931387 bytes, checksum: 8c678d9ee4559050331bdc307a7e62f9 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2012-02-09 / CAPES / Devido a grande necessidade de reduzir danos ambientais, pelo descarte de resíduos da indústria alimentícia, objetivou-se tratar biologicamente o efluente sólido do efluente de uma indústria de sorvetes, localizada na região metropolitana do Recife, PE, Brasil, utilizando os gêneros Mortierella, Cunninghamella e Mucor, pertencentes à classe Zygomycetes, ordem Mucorales, por possuírem grande capacidade de resistência aos altos índices de contaminação, de acordo com a literatura, produzindo enzimas biocatalíticas. As 19 linhagens pertencem a Coleção de Cultura URM do Departamento de Micologia da Universidade Federal de Pernambuco foram submetidas aos ensaios de capacidade de crescimento nas concentrações 5, 10 e 15% do resíduo; diminuição da Demanda Química de Oxigênio (DQO) e Demanda Bioquímica de Oxigênio (DBO), consumo de nitrogênio, potássio e fósforo e produção de lipases. Inicialmente as linhagens foram selecionadas pela capacidade de crescerem em placas de Petri, contendo meio ágar água destilada, acrescido de 15% (m/v) do resíduo de sorvete. Depois foram realizados os ensaios em caldo resíduo de sorvete (15% m/v), com 17 dias de cultivo, para avaliação da biodegradabilidade e um controle abiótico. As linhagens Cunninghamella elegans URM6017 e URM5780 mostraram-se mais eficientes com a diminuição de 98,98% e 98,09 de DQO e 98,59% e 97,08 de DBO, respectivamente. Enquanto que no controle abiótico ocorreu uma diminuição de 92,49% de DQO e 92,57% de DBO e o nitrogênio de 20,83% e potássio de 98,02%. A linhagem URM 6017 apresentou um consumo de 66,07% de nitrogênio total e 99,85% de potássio. Durante o experimento os valores de pH variaram de 4,97 para o controle abiótico a 7,51 para URM6017 e de 6,92 para URM5780, ao final do processo. A atividade lipolítica das linhagens URM6017 foi de 2,01 U/mL utilizando como substrato o resíduo de sorvete e de 4,61 U/mL com o resíduo de sorvete adicionado de azeite de oliva e para a URM5780 foi de 2,73 U/mL e 3,84 U/mL, respectivamente. Das 19 linhagens estudadas, duas linhagens de Cunninghamella elegans URM6017 e URM5780 mostraram-se eficientes na redução da matéria orgânica contida no resíduo de sorvete.

Abstract Collagen prolyl 4-hydroxylases catalyze the hydroxylation of proline residues in collagens. The vertebrate enzymes are α2β2 tetramers in which the β subunit is identical to protein disulphide isomerase (PDI). Two isoforms of the catalytic α subunit have been identified in vertebrates, forming type I [α(I)]2β2 and type II [α(II)]2β2 collagen prolyl 4-hydroxylase tetramers. This thesis reports on the cloning and characterization of a third vertebrate α subunit isoform, α(III). The recombinant human α(III) isoform associates with PDI to form an active type III collagen prolyl 4-hydroxylase tetramer, and its Km values for the cosubstrates are very similar to those of the type I and II enzymes, those for a peptide substrate and an inhibitor being found to lie between the two. The α(III) mRNA is expressed in all tissues studied but at much lower levels than the α(I) mRNA. A novel mixed tetramer PHY-1/PHY-2/(PDI-2)2 was found to be the main collagen prolyl 4-hydroxylase form produced in the nematode Caenorhabditis elegans in vivo and in vitro. However, mutant nematodes can compensate for the lack of the mixed tetramer by increasing the assembly of PHY-1/PDI-2 and PHY-2/PDI-2 dimers, these forms also being unique. The catalytic properties of the recombinant mixed tetramer were characterized, and it was shown by the analysis of mutant worms that PHY-1 and PHY-2 represent the only catalytic subunits needed for the hydroxylation of cuticular collagens. The roles of the two catalytic sites in a collagen prolyl 4-hydroxylase tetramer were studied by using the C. elegans mixed tetramer and a hybrid C. elegans PHY-1/human PDI dimer. An increase in the chain length of the peptide substrate led to an identical decrease in the Km values in both enzyme forms. It is thus clear that two catalytic sites are not required for efficient hydroxylation of long peptides, and their low Km values most probably result from more effective binding to the peptide-substrate-binding domain. Inactivation of one catalytic site in the mixed tetramer reduced the activity by more than 50%, indicating that the remaining wild-type subunit cannot function fully independently.

Identification of genetic basis of Hybrid Incompatibility (HI) in hybrids between closely related species leads to a comprehensive understanding of speciation. Although model organism C. elegans is well-established in laboratory, it has performed little contribution to this research area, because C. elegans failed to mate with other sister species and produce viable progeny. As a sister species of C. briggsae, which is close to C. elegans, newly discovered C. nigoni made it possible to identify the genetic basis of HI in nematode species. In this study, a new species pair including C. nigoni and C. briggsae was used to study the genetic and molecular bases of HI. 96 GFP markers were randomly integrated into the genome of C. briggsae by biolistic bombardment. Next-Generation Sequencing (NGS) combined with single worm PCR were performed to identify the location of GFP markers. By tracking those markers, the genomic fragments of C. briggsae linked to GFP were backcrossed into C. nigoni. Such process was repeated for at least 15 generations and total 111 strains carrying independent introgressions were generated. Widespread HI loci were identified on a genome-wide scale for the first time in nematode species, which also supported Haldane's Rule and large X-effect theory between the two species. In this study, C. nigoni genome "cn1" was de novo assembled by using a hybrid approach, which combined Illumina synthetic long-read technology and massive parallel sequencing of Fosmid mate-pair library. Two lines of hybrid sterile males each carrying an independent introgression fragment from C. briggsae X chromosome in an otherwise C. nigoni background, demonstrate similar defects in spermatogenesis. A similar pattern of downregulated genes that are specific for spermatogenesis between the two hybrids and wild type control was observed. Importantly, the downregulated genes caused by the X chromosome introgressions are significantly enriched on autosomes, suggesting an epistatic interaction between the X chromosome and autosomes. By measuring small RNAs, the results shows that a subset of 22G RNAs specifically targeting the downregulated spermatogenesis genes are significantly upregulated in hybrids, indicating that perturbation of small RNA-mediated regulation may contribute to the X-autosome interaction.

Sequences coding for three related 16 kd heat shock proteins (hsps) of the nematode Caenorhabditis elegans were isolated and characterized. The extensive accumulation of hsp16 mRNA during heat stress facilitated the identification of two cDNAs, CEHS48 and CEHS41, which encoded hsp16 variants. These plasmids were selected by their ability to hybridize to mRNA which directed the synthesis of hspl6 in vitro, and were further characterized by sequence analysis. Two-dimensional gel electrophoresis of hspl6 synthesized in vitro from mRNA selected by hybridization to either of the cDNAs under conditions of low stringency revealed the existence of at least five electrophoretic variants with significantly different isoelectric points. The above cDNAs were used as specific probes to isolate recombinant bacteriophage containing C. elegans genomic DNA. Overlapping phage clones were used to define a region of approximately 30 kilobases. The genes coding for hsp16-48, previously identified by cDNA cloning, and for another 16 kd hsp designated hspl6-l were characterized by DNA sequencing. These two genes were arranged in a head-to-head orientation. Both the coding and flanking regions of these genes were located within a 1.9 kb region which was duplicated exactly to form a perfect 3.8 kb inverted repeat structure. This structure ended in unusual G + C-rich sequences 24 bp in length. The identity of the two arms of the inverted repeat at the nucleotide sequence level implied that the duplication event may have occurred relatively recently in evolution. Alternatively, gene conversion between the two modules could have maintained homology between the two gene pairs. Comparison of the hsp16-48 gene with its corresponding cDNA revealed the presence of a single, short intron. An intron of comparable length and in an analogous position was also found in the hsp16-1 gene. The introns separated variable and conserved regions within the amino acid sequences of the encoded heat shock proteins. A domain of approximatey 80 amino acids is contained within the conserved second exon and is homologous to a similar region in the small hsps of Drosophila, Xenopus, soybean and man as well as the a-crystallin protein of the vertebrate lens. Each hsp16 gene contained a TATA box upstream of the start of transcription. Promoter sequences, which have been shown to be required for heat inducibility in various systems, were located upstream of either TATA box Northern blot analysis showed that the hsp16-48 and hsp16-1 genes are expressed at levels approximately 20 - 40 fold lower than two closely related genes, hsp16-41 and hsp16-2, upon temperature elevation. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Anesthetics have been used for nearly two centuries, and have proved to be one of the most important tools in surgical interventions, but their methods of action remain mysterious. Previous research has focused on high-level, low-resolution measurements (average activity of many neurons) or low-level, high-resolution measurements (single neurons). The nematode Caenorhabditis elegans provides an excellent model to bridge the gap between these two scales by measuring the activity of many neurons with single neuron resolution. C. elegans display analogous behaviors to humans under anesthesia. Employing confocal imaging of GCaMP, I measured neuronal activity at different isoflurane levels in C. elegans ganglia and in small behavior-controlling circuits. The activity in C. elegans ganglia is similar to that of human ganglia, as assessed using measures that are similar to EEG. Activity in the small behavior-controlling circuit is disrupted, but not suppressed, when dosed with moderate levels of isoflurane. Neural activity in the circuit is randomized resulting in a loss of coordination between neurons that define behavioral states of the system. As such, the onset of the behaviors of anesthesia appears to be the resultant of randomization rather than suppression of individual neuron activity. Employing light sheet microscopy and automated image analysis for neuronal tracking, I expanded the imaging techniques to measure activity of the majority of neurons in the animal’s head. Expansion of these measurements to the whole head region of the nematode confirms these findings, displaying significant decreases in neuron-to-neuron coordination, as well as randomization of individual neuron signals with the onset of anesthesia. These results reveal a new physiological mechanism of action for anesthetics, and provide an avenue forward for investigating the molecular mechanism including specific genetic mutations known to alter susceptibility to anesthetics. / 2021-05-26T00:00:00Z

Digestion and absorption of bioactive free fatty acids have been studied using the nematode Caernohabditis elegans (C. elegans). However, fatty acids mostly occur in foods in the form of triglycerides, which are highly hydrophobic molecules with low water-solubility, thereby making it difficult to study the fate of ingested fatty acids in C. elegans. The purpose of this research was to develop a method to deliver hydrophobic bioactives, including triglycerides, into C. elegans. Nanoemulsions containing triglyceride nanoparticles were prepared by sonication, and nanoparticle ingestion was confirmed by optical and confocal microscopy, and quantified by spectrometry. Changes in fatty acid composition were measured to confirm the absorption of triglycerides delivered as nanoparticles. Nanoparticles with a wide range of particle diameters (40 to 500 nm) were ingested by C. elegans. The ingested triglyceride amount was dependent on the size and concentration of the nanoparticles, but the fatty acid composition of C. elegans was not significantly changed by dietary triglycerides. Nanoemulsion based-delivery systems enable C. elegans to be used for evaluation of hydrophobic bioactives and may provide a useful tool for testing their biological activities.

In C. elegans, the germline is a tightly regulated tissue where silencing pathways regulate genes, allowing expression of “self” while silencing “non-self.” Doublestranded RNAs (dsRNAs), short interfering RNAs (siRNAs), and piwi-associated RNAs (piRNAs) can transmit this regulation across generations via transgenerational epigenetic inheritance (TEI) mechanisms (Bošković and Rando, 2018). Analogously, some pathways can counteract gene silencing to allow sustained expression in the germline. One such example is a non-coding DNA structure called Periodic An/Tn clusters that can prevent the silencing of transgenes in the germline (Frøkjær-Jensen et al., 2016). In this thesis, I developed a novel piRNA-based tool called piRNA interference (piRNAi), where target-specific short “guide” piRNAs (sg-piRNAs) can robustly silence endogenous genes and transgenes. I have used piRNAi to understand the rules for licensing gene expression and transgenerational epigenetic inheritance in the C. elegans germline. Initially, I describe design rules for generating transgenes with PATC-rich introns that resist germline silencing and are robustly expressed from extrachromosomal arrays. PATC-rich transgenes showed more accurate gene expression patterns and did not prevent germline regulation by 3’ untranslated regions (3’ UTRs). Next, I developed the piRNAi technique to understand the role of PATCs in licensing transgene expression and the rules for how endogenous genes can be targeted for piRNA-mediated silencing and TEI. I demonstrate that a PATC-rich gfp transgene and endogenous genes are not resistant to piRNA-mediated silencing. Finally, I used piRNAi to define rules for TEI: 1. I identified two new endogenous targets for TEI (him-5 and him-8) that can inherit silencing for four and six generations respectively, after transient exposure to sg-piRNAs. 2. I demonstrate that an endogenous gene (him-5) can be semi-permanently silenced in the absence of the piRNA/PRG-1 pathway. 3. The duration of TEI was significantly shortened in a transgene that contained PATC-rich introns. Altogether, my thesis shows that an endogenous small RNA pathway can be reprogrammed to silence endogenous genes and transgenes in the germline, which enables novel experimental paradigms for studying inherited and semipermanent silencing.

PNPase, polynucleotide phosphorylase, is a multifunctional exoribonuclease protein with 3` terminal oligonucleotide polymerase activity. Coded by the PNPT1 gene, the protein is associated with mitochondrial homeostasis and functions as a possible target for cancer therapy. In this study, C. elegans was used to investigate the effect of mutation and overexpression of pnpt-1, the gene that encodes PNPase. It was determined that two specific mutations in pnpt-1 did not affect PNPase expression nor did they produce deleterious phenotypes that affected polycistronic transcript accumulation or ROS production. Creation of a stable overexpression model was achieved through Fusion PCR. However, different transgenic strains overexpressing PNPase produced opposite results for polycistronic transcript accumulation while ROS production saw no significant change, suggesting a mosaic overexpression model. In a cancer model, exogenous PNPase was present in the pachytene region of the germline and where expressed the cells were in non-germline cells suggesting differentiation mechanisms associated with overexpression of PNPase. However, further analysis of different mutations in pnpt-1 or optimizations to the overexpression model are necessary to provide a better understanding of PNPase function with mitochondria homeostasis and in a cancer model setting.