The ability to move is essential to an animal’s ability to interact with and respond to its changing environment. The nematode Caenorhabditis elegans is a commonly used organism in the study of the genetic and neural bases of behaviours, yet the mechanistic explanation for its ability to move in a smooth sinusoidal wave remains elusive. Here, I present studies of an uncharacterized gene, sto-6, encoding a stomatin protein that regulates C. elegans motor behaviour. I show that this gene plays a role in two unexplained and fundamental processes to C. elegans locomotion: wave initiation and wave propagation. Furthermore, I examine the genetic interaction between sto-6 and an innexin gene unc-7, providing support for the hypothesis that stomatins regulate gap junction proteins in C. elegans. Together, these studies push forward our understanding of the mechanistic basis of C. elegans locomotion, and open up avenues of further inquiry.

The ability to move is essential to an animal’s ability to interact with and respond to its changing environment. The nematode Caenorhabditis elegans is a commonly used organism in the study of the genetic and neural bases of behaviours, yet the mechanistic explanation for its ability to move in a smooth sinusoidal wave remains elusive. Here, I present studies of an uncharacterized gene, sto-6, encoding a stomatin protein that regulates C. elegans motor behaviour. I show that this gene plays a role in two unexplained and fundamental processes to C. elegans locomotion: wave initiation and wave propagation. Furthermore, I examine the genetic interaction between sto-6 and an innexin gene unc-7, providing support for the hypothesis that stomatins regulate gap junction proteins in C. elegans. Together, these studies push forward our understanding of the mechanistic basis of C. elegans locomotion, and open up avenues of further inquiry.

This thesis seeks to address the critical bottlenecks of current technologies that have slowed the neuroscience research in C. elegans. The objective of this research is to enhance the currently developed systems through the design and construction of simple microdevices and quantitative analytical tools for high-throughput phenotyping C. elegans to investigate functions of nervous systems. First, we developed and used the integrated system combining user-friendly single-layer microfluidics and quantitative analytical tools to study the genetic regulation of target gene expression. We found several putative mutants based on large-scale screens, which would have previously been too labor-intensive to attempt. Second, we developed a simple mathematical model that describes the regulation of a target gene expression. Using the model developed, we simulated phenotypical space of hypothetical mutants to suggest plausible genetic pathways some isolated mutants may affect. Lastly, we developed a high-density multichannel device for rapid trapping, parallel selective stimulating, long-term culturing, and (often repeatedly). We used this integrated system to study the neurodegenerative process based on selective ablation of multiple animals using an optogenetic tool, which would have been taken at least 1 order of magnitude longer. Taken together, we expect that these developments will greatly facilitate a broad range of fundamental, and application studies including aging, neurodegeneration, circuit and behavior.

Little is known about the molecular components and interactions of the planar cell polarity pathway that regulate neuronal polarity. This study uses a prkl-1 induced backwards locomotion defect as an array to perform a prkl-1 suppressor screen in C. elegans looking for new components of the planar cell polarity pathway involved in the neuronal polarization of VC4 and VC5. The screen discovered twelve new alleles of vang-1, one new allele of fntb-1 and five new mutations in unknown polarity genes. fntb-1 encodes for the worm ortholog of Farnesyltransferase β-subunit and is important for neuronal polarization. Acting cell and non-cell autonomously, fntb-1 regulates the function and localization of prkl-1 through the recognition of a CAAX motif. Therefore, fntb-1 modifies prkl-1 to regulate the neuronal polarity of VC4 and VC5.

<p>For all organisms, the ability to sense and react to noxious environments is fundamental to their survival. For multi-celled organisms this process generally involves a nervous system and an extensive network of signal transduction pathways. TRPV ion channels have been shown to participate in signal transduction in response to noxious stimuli. At the cellular level these channels function in sensing of mechanical, thermal, and osmotic stimuli, and at the organismal level they function in homeostasis and nociception. TRPV ion channels participate in nociceptive signal transduction via cation influx, but exactly how these channels function at a mechanistic level and lead to activation of the cell or induction of a specific behavior is elusive. Previous research has shown that the pore-forming unit of an ion channel is critical for channel regulation, gating, ion selectivity, and ion permeation. Various regulatory domains have been identified to date in the pore-forming unit of TRP channels and a clearer picture of channel gating is beginning to emerge, but less is known about ion permeation. </p><p>To better understand the specific domains that are critical to ion capture, selectivity, and permeation in TRPV channels, we investigated the function of these regions using the <italic>C. elegans</italic> TRPV channel OSM-9 <italic>in vivo</italic>, and the mammalian TRPV channel TRPV4 in heterologous cell culture. OSM-9 is the functional ortholog of mammalian TRPV4 and it is likely that critical domains identified in OSM-9 are functionally conserved in TRPV4 and play a similar role in other TRPV channels. OSM-9 is expressed in the ASH neurons and is responsible for all of the behaviors initiated by that cell. The stereotypical avoidance behavior mediated by ASH, in response to noxious stimuli, serves as a model for nociception in vertebrates. As OSM-9 is necessary for all of these behavioral responses, activation of ASH acts as a read-out for OSM-9 function.</p><p>Through targeted mutagenesis of the OSM-9 loop domains and transgenic expression directed to the ASH head sensory neurons in an <italic>osm-9</italic> null background, we discovered a critical role for the amino acids both N- and C- terminal to the pore helix in osmotic avoidance behavior. We confirmed the existence of a selectivity filter C-terminal to the pore helix and revealed that the turret is critical for channel function, possibly as a component of the inactivation gate.</p><p>We first identified the boundaries of the selectivity filter to be M601-F<super>609</super>. We also determined what properties of those residues were critical to Ca<super>2+</super> and Na<super>+</super> selectivity. <italic>In vivo</italic> Ca<super>2+</super> imaging strongly suggested that residues Y<super>604</super>, D<super>605</super>, and F<super>609</super> are critical for Ca<super>2+</super> entry into the cell. Patch-clamp electrophysiology of a chimeric ion channel consisting largely of rat TRPV4, but encompassing transmembranes 5 through 6 of OSM-9, revealed that OSM-9 conducts both Ca<super>2+</super> and Na<super>+</super>. Mutation Y604G disrupted both Ca<super>2+</super> and Na<super>+</super> conductance, whereas mutations Y604F and Y606A increased or maintained Na+ conductance and severely reduced Ca<super>2+</super> conductance, while maintaining avoidance behavior. Homology modeling of OSM-9, based on an alignment of OSM-9 to Kv1.2, suggests that Y<super>604</super> and F<super>609</super> serve structural roles in maintaining filter constraints. Thus, aromatic and negative residues in the OSM-9 selectivity filter are critical to ion permeation and selectivity. </p><p>Our studies involving the selectivity filter support previous research that the selectivity filter is critical for TRP channel function. We also provide evidence that the selectivity filter is critical for nocifensive animal behavior. Fewer studies, however, have investigated the TM5-pore helix linker, known as the turret. The turret is believed to function in the binding of ligands and toxins in K<super>+</super> channels, and more recently was suggested to be critical for temperature sensing in TRPV1. We investigated the function of the turret residues in several sensory submodalities of the OSM-9 channel and found that all deletions tested result in channel defects, including gain- and loss-of-function phenotypes. Several charge reversal mutations in the OSM-9 turret also resulted in partial defects. The discovery of a gain-of-function mutation indicates that the turret functions in gating. When the turret is mutated in this way, the channel is unable to enter into the inactivated state, allowing continued ion influx after repeated stimulation. The loss-of-function phenotypes indicate that the secondary structure of the turret is critical to the function of the channel, and perhaps gating. These findings, combined with the observed charge-reversal defects, support the conclusion that the turret is necessary for transducing conformational changes in response to stimuli.</p><p>Our <italic>in vivo</italic> findings on the external pore forming structures increase the understanding of ion permeation in TRP channels and clarify mechanisms of activation in nociceptor neurons <italic>in vivo</italic>. Furthermore, these studies enhance our insights into evolution of mammalian nociception in view of the established functional orthology of OSM-9 and TRPV4.</p> / Dissertation

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2018-04-02T15:02:43Z No. of bitstreams: 1 LucianaFentanesMouraDeMelo_DISSERT.pdf: 1510669 bytes, checksum: f402172547ca541fafef3500e5ce47a8 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2018-04-05T14:12:02Z (GMT) No. of bitstreams: 1 LucianaFentanesMouraDeMelo_DISSERT.pdf: 1510669 bytes, checksum: f402172547ca541fafef3500e5ce47a8 (MD5) / Made available in DSpace on 2018-04-05T14:12:02Z (GMT). No. of bitstreams: 1 LucianaFentanesMouraDeMelo_DISSERT.pdf: 1510669 bytes, checksum: f402172547ca541fafef3500e5ce47a8 (MD5) Previous issue date: 2018-03-08 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / O uso de plantas medicinais ? uma pr?tica comum da humanidade desde a antiguidade. Tal conhecimento baseado na experi?ncia tem sido repassado por muitas comunidades e grupos ?tnicos, e serve como ponto de partida para estudos bioqu?micos e farmacol?gicos dos princ?pios ativos presentes nas plantas. Para isto, neste trabalho foram avaliados o potencial antioxidante e tamb?m biol?gico, utilizando sistemas in vitro e in vivo dos diferentes extratos obtidos de folhas da planta Coccoloba alnifolia. Foram obtidos inicialmente seis extratos: hexano (EH), clorof?rmio (EC), etan?lico (EE), metan?lico (EM), ?gua final (EAF) e aquoso (EA) a partir das folhas frescas da planta. Posteriormente, foram avaliados o teor de a??car, prote?nas e compostos fen?licos. Os resultados obtidos na triagem fitoqu?mica sugerem a presen?a de fen?is, flavonoides, saponinas, n?cleo triterp?nicos e esteroidais e n?cleo esteroidal insaturado, j? com a metodologia de cromatografia em camada delgada (CCD), foi observado uma abund?ncia de flavonoides. Foram realizados quatro ensaios: Teste do Poder Redutor, Quela??o de ?ons Metais, Quela??o de Cobre e Atividade Sequestradora de Radical Hidroxila. Estes ensaios permitiram verificar que os extratos apresentaram atividade antioxidante nas concentra??es avaliadas de 100, 250 e 500 ?g/mL. Foi verificado principalmente para os extratos EE, EM e EA as atividades antioxidante nos ensaios poder redutor e sequestro do radical hidroxila. A correla??o de Pearson mostrou que estes dados est?o associados aos compostos fen?licos e a??cares. E com o modelo das linhagens celulares foi observado que a maioria dos extratos avaliados neste trabalho n?o mostrou-se citot?xico para as linhagens celulares. Os extratos tamb?m n?o inibiram o crescimento da levedura Candida parapsilosis. Os extratos obtidos da folha de C. alnifolia apresenta um potencial antioxidante interessante, e os resultados obtidos tanto nos modelos in vitro, como as linhagens celulares, refor?am que s?o necess?rios mais estudos para compreender o potencial destes extratos in vivo e identificar quais s?o as biomol?culas associadas com estas atividades e seus respectivos potenciais biotecnol?gicos. / The use of medicinal plants has been a common practice in folk medicine. This knowledge has been based on the experience that it has been passed though many generations. However, many times this knowledge do not have any scientific base. The molecules responsive for these activities are the secondary metabolic produce as a response to environmental conditions (biotic or abiotic stress). The Mata atl?ntica biome has a rich flora, which needs to be studied for its biological activities, and its potential to identify new biomolecules. In order to do this, six extracts (HE, CE, EE, ME, WEE e WE) were obtained from Coccoloba alnifolia leaves and then it was evaluated the antioxidant and the biological potential using different in vitro and in vivo systems. First, it was measured the sugar content, proteins and phenolic compounds. Based on these results, qualitative experiments were carried out to determine the secondary metabolites present in these extracts. It was detected the presence of phenols, flavonoids, saponins, triterpenic, and steroidal nuclei and unsaturated steroidal nuclei. After that, it was evaluated the antioxidant potential by four in vitro assay: Redution Power test, Metal Ion Chelation, Copper Chelation and Hydroxyl Radical Scavenging Activity. These assays allowed us verify that the extracts presented antioxidant activity at extract concentrations: 100, 250 and 500 ?g/mL. It was observed the ethanolic, methanolic and aqueous extracts had activity in reducing power and scavenging of the hydroxyl radical. The Pearson?s correlation showed that these results may be associated to the presence of phenolic compounds and sugars. The third in vitro model used in this work was cell culture. When it was used the 3T3 lineage it was observed that these extracts were not cytotoxic. However, it was not observed any antiproliferative activity in tumor cell lineages. In sumary, the data obtained here showed that using in vitro assays that these extracts in special ethanolic and aqueous had an interesting antioxidant potential and it will be important to go further in this research.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-03-20T17:38:34Z No. of bitstreams: 1 KatiaCristinaBorges_TESE.pdf: 9195885 bytes, checksum: 9e74a9224960e69cb9a7ce42ad92501c (MD5) / Approved for entry into archive by Monica Paiva (monicalpaiva@hotmail.com) on 2017-03-20T17:46:31Z (GMT) No. of bitstreams: 1 KatiaCristinaBorges_TESE.pdf: 9195885 bytes, checksum: 9e74a9224960e69cb9a7ce42ad92501c (MD5) / Made available in DSpace on 2017-03-20T17:46:31Z (GMT). No. of bitstreams: 1 KatiaCristinaBorges_TESE.pdf: 9195885 bytes, checksum: 9e74a9224960e69cb9a7ce42ad92501c (MD5) Previous issue date: 2015-03-09 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Considerado um dos pa?ses com a maior biodiversidade, o Brasil det?m mais de 100.000 mil esp?cies de plantas distribu?das em diferentes biomas, incluindo uma diversidade de frutas tropicais e ex?ticas. Muitas delas ganharam popularidade e atingiram os mercados interno e externo, entretanto uma fra??o permanece pouco estudada. Dentre elas est? a pitanga (Eugenia uniflora), fruta tropical cultivada no Nordeste com sabor, aroma e apar?ncia apreciados, com consider?vel teor de vitamina A e C, al?m de subst?ncias biologicamente ativas, principalmente compostos fen?licos. Contudo, a disponibilidade do fruto apenas em determinadas esta??es do ano e sua alta perecibilidade, dificulta a conserva??o e armazenamento da fruta in natura. Assim, o presente trabalho avaliou a aplica??o de dois processos de secagem (spray drying e liofiliza??o) em duas variedades de pitanga (Eugenia uniflora), visando ? obten??o de um produto com aplica??o funcional. Foram determinadas as caracter?sticas f?sico-qu?micas (composi??o, cor, solubilidade e higroscopicidade), compostos bioativos (compostos fen?licos totais, carotenoides, antocianinas e ?cido asc?rbico), perfil fen?lico (flavonoides, ?cidos fen?licos, proantocianidinas e ?cido el?gico), funcionalidade in vitro (atividades antioxidante, antienzim?tica e antimicrobiana). Tamb?m foi avaliada a funcionalidade in vivo, utilizando o nematoide C. elegans como modelo para investiga??o da longevidade, estresse oxidativo e doen?as neurodegenerativas (Alzheimer e Parkinson). A pesquisa apresenta dados in?ditos sobre a potencialidade funcional e neuroprotetora dessa fruta tropical da biodiversidade brasileira, ainda pouco estudada. / Considerado um dos pa?ses com a maior biodiversidade, o Brasil det?m mais de 100.000 mil esp?cies de plantas distribu?das em diferentes biomas, incluindo uma diversidade de frutas tropicais e ex?ticas. Muitas delas ganharam popularidade e atingiram os mercados interno e externo, entretanto uma fra??o permanece pouco estudada. Dentre elas est? a pitanga (Eugenia uniflora), fruta tropical cultivada no Nordeste com sabor, aroma e apar?ncia apreciados, com consider?vel teor de vitamina A e C, al?m de subst?ncias biologicamente ativas, principalmente compostos fen?licos. Contudo, a disponibilidade do fruto apenas em determinadas esta??es do ano e sua alta perecibilidade, dificulta a conserva??o e armazenamento da fruta in natura. Assim, o presente trabalho avaliou a aplica??o de dois processos de secagem (spray drying e liofiliza??o) em duas variedades de pitanga (Eugenia uniflora), visando ? obten??o de um produto com aplica??o funcional. Foram determinadas as caracter?sticas f?sico-qu?micas (composi??o, cor, solubilidade e higroscopicidade), compostos bioativos (compostos fen?licos totais, carotenoides, antocianinas e ?cido asc?rbico), perfil fen?lico (flavonoides, ?cidos fen?licos, proantocianidinas e ?cido el?gico), funcionalidade in vitro (atividades antioxidante, antienzim?tica e antimicrobiana). Tamb?m foi avaliada a funcionalidade in vivo, utilizando o nematoide C. elegans como modelo para investiga??o da longevidade, estresse oxidativo e doen?as neurodegenerativas (Alzheimer e Parkinson). A pesquisa apresenta dados in?ditos sobre a potencialidade funcional e neuroprotetora dessa fruta tropical da biodiversidade brasileira, ainda pouco estudada.

abstract: Once perceived as an unimportant occurrence in living organisms, cell degeneration was reconfigured as an important biological phenomenon in development, aging, health, and diseases in the twentieth century. This dissertation tells a twentieth-century history of scientific investigations on cell degeneration, including cell death and aging. By describing four central developments in cell degeneration research with the four major chapters, I trace the emergence of the degenerating cell as a scientific object, describe the generations of a variety of concepts, interpretations and usages associated with cell death and aging, and analyze the transforming influences of the rising cell degeneration research. Particularly, the four chapters show how the changing scientific practices about cellular life in embryology, cell culture, aging research, and molecular biology of Caenorhabditis elegans shaped the interpretations about cell degeneration in the twentieth-century as life-shaping, limit-setting, complex, yet regulated. These events created and consolidated important concepts in life sciences such as programmed cell death, the Hayflick limit, apoptosis, and death genes. These cases also transformed the material and epistemic practices about the end of cellular life subsequently and led to the formations of new research communities. The four cases together show the ways cell degeneration became a shared subject between molecular cell biology, developmental biology, gerontology, oncology, and pathology of degenerative diseases. These practices and perspectives created a special kind of interconnectivity between different fields and led to a level of interdisciplinarity within cell degeneration research by the early 1990s. / Dissertation/Thesis / Ph.D. Biology 2013

Alcohol use disorder (AUD) is the fourth leading cause of preventable death in the United States, and the fifth leading risk factor for premature death and disability, globally. There are currently very few treatment options for AUD and there is a need for effective preventive and treatment strategies for this condition. AUD risk has a significant hereditary component, with the contribution of genetic factors being estimated to be about 50%. The Davies-Bettinger laboratory uses C. elegans as a model organism to study the contribution of genetic factors in modulating neuronal responses to ethanol. In this project, we examined the role of mitochondrial beta-oxidation of fatty acids (FA) in altering ethanol responses using loss-of-function (lf) mutants and RNAi-mediated knockdown of specific genes in this pathway. We tested a total of 34 genes and found that lf in 13 genes significantly affected ethanol response phenotypes. We conclude that mitochondrial beta-oxidation of FA is essential for ethanol response behavior in C. elegans. Further experiments need to be conducted to dissect the specific contribution of various components of mitochondrial beta-oxidation in modifying the neuronal responses to ethanol.

Abstract The collagens are a family of extracellular matrix proteins with a widespread tissue distribution. Collagen biosynthesis requires the hydroxylation of a number of proline residues by prolyl 4-hydroxylase. This posttranslational modification is essential for the synthesis of all collagens, as 4-hydroxyproline deficient collagens cannot form stable triple helices at body temperature. The genes for the human and mouse prolyl 4-hydroxylase α(II) subunits were cloned and characterized in this study. The human and mouse genes are 34.6 and 30.3 kb in size, respectively, consisting of 16 exons and 15 introns. The intron sizes vary from 48-49 bp to over 8 kb in both genes. The 5' flanking regions contain no TATA box, but there are several motifs that may act as transcription factor binding sites. A novel mutually exclusively spliced exon 12a was identified in both genes. Both variants of the α(II) subunit were found to be expressed in a variety of tissues and both formed a fully active recombinant tetramer with the β subunit when expressed in insect cells. Tissue distribution of the type I and type II prolyl 4-hydroxylase isoenzymes was studied in developing, mature, and malignant cells and tissues by immunofluorescence and Western blotting. The results indicate that the type I isoenzyme is the main form in many cell types. Skeletal myocytes and smooth muscle cells appeared to have the type I isoenzyme as their only prolyl 4-hydroxylase form, whereas the type II isoenzyme was clearly the main form in chondrocytes. A strong signal for the type II enzyme was detected in cultured umbilical and capillary endothelial cells, whereas the type I isoenzyme could not be detected in these cells by immunostaining or Western blotting. Similar studies on primary chondro- and osteosarcomas and benign bone tumours indicated that the type I isoenzyme is the predominant form in both types of bone sarcoma, whereas the type II isoenzyme was more abundantly expressed in benign tumours. In chondrosarcomas, the type II isoenzyme was expressed in the nonmalignant chondrocytes, whereas their malignant counterparts switched their expression pattern to that of the type I isoenzyme. Two isoforms of the catalytic prolyl 4-hydroxylase α subunit, PHY-1 and PHY-2, have previously been characterized from Caenorhabditis elegans. This study reports the cloning and characterization of a third C. elegans α subunit isoform, PHY-3, which is much shorter than the previously characterized vertebrate and C. elegans α subunits. Nematodes homozygous for a phy-3 deletion were phenotypically wild type and fertile, but the 4-hydroxyproline content of their early embryos was reduced by about 90%. The expression of PHY-3 was found to be restricted to spermatheca of late larvae and adult nematode, indicating that PHY-3 is likely to be involved in the synthesis of collagens of the early embryo egg shells.