Oxygen availability is critical for survival for most organisms. The nematode, C. elegans, has been useful for studying genetic regulation of anoxia tolerance due to the oxygen deprivation response mechanisms shared with other metazoans. Studies examining long-term anoxia (72h, LTA) tolerance have only been conducted at adult day 1. To investigate the effect of aging on anoxia tolerance wild-type and mutant strains were exposed to LTA between adult day 1 and day 9. Wild-type isolates and daf-16(mu86) (FOXO transcription factor regulated by insulin-signaling) and aak-2(gt33) (catalytic subunit of AMP-activated protein kinase) strains were anoxia sensitive at day 1 and displayed increased LTA tolerance with aging correlated with reproductive senescence followed by a decline in survivorhsip through day 9. The daf-2(e1370) (insulin receptor homologue of C. elegans), glp-1(e2141) (a lin-12/Notch receptor) and fog-2(q71) (required for spermatogenesis) strains were LTA-tolerant through day 5. I conclude that aging influences LTA-tolerance in a strain- and age-dependent manner. In addition to being LTA-tolerant the daf-2(e1370) and glp-1(e2141) strains have a longevity phenotype that is suppressed by loss of kri-1 or daf-12. While loss of kri-1 did not suppress the LTA-tolerant phenotype of glp-1(e2141) at day 1 the portion of impaired survivors increased at day 3 and by day 5 tolerance was suppressed. Similarly, when exposed to 4 days of anoxia the glp-1(e2141);daf-12(rh41rh611) double mutant had a reduced survivor rate at all ages analyzed compared to glp-1(e2141) controls. To better understand formation of an anoxia-tolerant physiology I exposed adults to one or more 24h bouts. Recurrent bouts increased LTA tolerance in wild-type hermaphrodites in a dose-dependent manner. Bout-treated daf-16(mu86) animals had increased survival rate compared to controls yet maximum survival remained below age-matched wild-type. Anoxia bouts decreased LTA-tolerance in aak-2(gt33) mutants, indicating the requirement for ATP regulation in establishing an LTA-tolerant phenotype. These data support the idea that anoxia tolerance is multi-factorial and influenced by environment, metabolism, food, reproduction, sex phenotype and likely additional factors.

The multifunctional exoribonuclease protein PNPase is implicated as a potential target for cancer therapy as well as causing mitochondrial disorders in humans, but there has yet to be a whole animal knockdown model created. In this study, C. elegans was used to investigate the effect of knocking down pnpt-1, the gene that encodes PNPase. It was discovered that pnpt-1 knockdown significantly extends lifespan via an increase in superoxide production similar to other known mitochondrial lifespan extension pathways. Additionally, mitochondrial networks, size and respiration are affected indication of other mitochondrial dysfunction.. PNPase is also known to transport small RNAs into the mitochondria which in turn can affect mitochondria RNA splicing and translation of proteins involved in respiration. Further investigation showed a significant accumulation of polycistronic mitochondrial transcripts in knockdown animals. Lastly, this model has shown that PNPase knockdown is functionally comparable across species and is a viable model for future studies.

"Microfluidic technologies are popular for biological research, enabling precise physical and chemical control of the microenvironment surrounding living cells and small organisms. Caenorhabditis elegans, a 1 mm long nematode, is capable of olfactory associative learning using the classical conditioning paradigm of pairing an unconditioned stimulus that elicits an innate response, such as food, with a second stimulus, such as an odor, which then elicits a learned behavioral response to this conditioned stimulus alone. Conventional chemotaxis assays on agar petri-plates have been widely used to observe behavioral changes indicative of associative learning; however, reproducibility of these behavioral assays is a major challenge. Here, we describe a microfluidic system that improves the reproducibility of chemotactic behavioral assays by providing better spatiotemporal control of stimuli, gentler worm handling, and more detailed behavioral quantification. Specifically, the microfluidic designs in this study present a uniform conditioning environment followed by a temporally stable linear odor gradient to assess changes to olfactory preference. Stimuli are presented in an enclosed environment to multiple worm populations whose locomotory patterns are analyzed using machine vision. Furthermore, we established an optimized protocol for a positive associative learning paradigm in which animals increase their preference for an odorant, butanone, when previously paired with bacterial food. We reproduced plate-based learning results in wild-type and learning-deficient genetic mutant animals, and demonstrated how developmental stages and chemicals alter the plasticity of olfactory preference. "

Programmed cell death (PCD or apoptosis) is an evolutionarily conserved, genetically controlled suicide mechanism for cells, which when deregulated, can lead to developmental defects, cancers and degenerative diseases. In C. elegans, DNA damage induces germ cell death by signaling through cep-1/p53 ultimately leading to the activation of the CED-3/caspase. It has been hypothesized that the major regulatory events controlling cell death occur by cell autonomous mechanisms, that is within the dying cell. In support of this, genetic studies in C. elegans have shown that the core apoptosis pathway genes ced-4/APAF1 and ced-3/caspase are required in cells fated to die. However, it is not known whether the upstream signals that activate apoptosis function in a cell autonomous manner. Here I show that two genes, kri-1, an ortholog of KRIT1/CCM1 that is mutated in the human neurovascular disease cerebral cavernous malformations (CCMs) and daf-2, an insulin-like receptor, are required to activate DNA damage-dependent cell death independently of cep-1/p53. Interestingly, I found that both genes can regulate cell death in a non-autonomous manner, revealing a novel role for non-dying cells in eliciting death in response to DNA damage.

Programmed cell death (PCD or apoptosis) is an evolutionarily conserved, genetically controlled suicide mechanism for cells, which when deregulated, can lead to developmental defects, cancers and degenerative diseases. In C. elegans, DNA damage induces germ cell death by signaling through cep-1/p53 ultimately leading to the activation of the CED-3/caspase. It has been hypothesized that the major regulatory events controlling cell death occur by cell autonomous mechanisms, that is within the dying cell. In support of this, genetic studies in C. elegans have shown that the core apoptosis pathway genes ced-4/APAF1 and ced-3/caspase are required in cells fated to die. However, it is not known whether the upstream signals that activate apoptosis function in a cell autonomous manner. Here I show that two genes, kri-1, an ortholog of KRIT1/CCM1 that is mutated in the human neurovascular disease cerebral cavernous malformations (CCMs) and daf-2, an insulin-like receptor, are required to activate DNA damage-dependent cell death independently of cep-1/p53. Interestingly, I found that both genes can regulate cell death in a non-autonomous manner, revealing a novel role for non-dying cells in eliciting death in response to DNA damage.

Investigating the cellular processes anti-ageing compounds interact with can identify genes and pathways involved in ageing. The macrolide lactone FK506 was identified in a phenotypic screen as extending lifespan in yeast and C. elegans through an unknown mechanism. FK506 also ameliorates neurodegeneration and age-related weight gain in rodents. Here, the mechanism of action of FK506 has been investigated in two experimental systems: C. elegans and 3T3-L1 mouse adipocytes. As the general mechanisms of ageing are well conserved between C. elegans and mammals, C. elegans has been used to understand how FK506 acts at an organismal level. Firstly, the result of the phenotypic screen was confirmed. FK506 treatment induced lifespan extension in C. elegans in the presence of population crowding stress, but not in the absence of crowding. FK506 treatment inhibited neither E. coli OP50 growth nor C. elegans pharyngeal pumping, demonstrating that FK506 did not induce dietary restriction to extend lifespan. FK506 treatment increased C. elegans thrashing and pharynx pumping rates in early adulthood and delayed accumulation of gut bacteria, showing that FK506 extended healthspan. A transcriptome analysis of FK506-treated C. elegans allowed the identification of transcripts whose levels change and potential pathways by which FK506 manifests its effect. To explore this and to identify potential targets of FK506, the cellular functions required for FK506 to extend C. elegans lifespan and healthspan were investigated using RNA-seq, RNAi, genetic mutation and co-treatment with small molecule inhibitors and inducers. Interestingly, FK506 was found to have different mechanisms of action on lifespan and healthspan. The mechanism of FK506 on C. elegans thrashing rate was DAF-16 dependent, did not require population crowding stress, had a partial interaction with FUdR and autophagy, and may involve Ca²⁺ flux. The mechanism of FK506-induced C. elegans lifespan extension overlapped with dietary restriction and was dependent on calcineurin, TOR-independent regulation of autophagy and the presence of population crowding stress. FK506 may modulate body weight by influencing metabolism and/or acting on adipocytes directly. FK506-treated aged 3T3-L1 adipocytes accumulated significantly less lipid, indicating that FK506 acts directly on adipocytes. RNA-seq of FK506-treated adipocytes found that translation-associated RNAs were upregulated whilst RNAs associated with lipid metabolism were downregulated. An ER-localised FK506-binding protein was up regulated in both C. elegans and 3T3-L1 adipocytes, fkb-4 and Fkbp2 respectively. In conclusion, FK506 has been confirmed as a potential anti-ageing treatment, through its ability to extend lifespan and healthspan in C. C. elegans. In addition, FK506 has also been shown to act directly on mouse adipocytes, resulting in a reduction in lipid accumulation. This action could explain how FK506 caused weight loss in obese aged rats, restoring body mass to a healthy adult weight.

Submitted by Tania Ivani Rokohl (tania.rokohl@uffs.edu.br) on 2018-03-14T13:09:55Z No. of bitstreams: 1 PHILIPPSEN.pdf: 516265 bytes, checksum: c031eafdd18797ffabec9c1c4bde8d63 (MD5) / Approved for entry into archive by Diego dos Santos Borba (dborba@uffs.edu.br) on 2018-03-14T14:00:02Z (GMT) No. of bitstreams: 1 PHILIPPSEN.pdf: 516265 bytes, checksum: c031eafdd18797ffabec9c1c4bde8d63 (MD5) / Made available in DSpace on 2018-03-14T14:00:02Z (GMT). No. of bitstreams: 1 PHILIPPSEN.pdf: 516265 bytes, checksum: c031eafdd18797ffabec9c1c4bde8d63 (MD5) Previous issue date: 2018-01-18 / Atualmente os produtos orgânicos têm ganhado destaque por sua qualidade nutricional, segurança alimentar e pela sustentabilidade na sua produção. A produção orgânica utiliza fungicidas à base de cobre, como a calda bordalesa, que é composta por sulfato de cobre e cal hidratada. O cobre é um elemento químico natural e fundamental para os organismos vivos, porém há controvérsias em relação aos efeitos causados ao ambiente e à saúde humana. Assim, o objetivo deste estudo foi avaliar os teores de cobre em solos e sucos de laranja e uva de uma propriedade orgânica que utiliza a calda bordalesa como fungicida, e a partir das concentrações encontradas, avaliar os efeitos da exposição aguda e crônica sobre o sistema nervoso de Caenorhabditis elegans. O estudo iniciou a partir da coleta dos solos e das frutas, logo após a colheita foram extraídos os sucos de laranja e uva. Posteriormente, foram analisadas as concentrações de cobre em solos e nos sucos de laranjas e uvas através de Espectrofotometria de Absorção Atômica por Plasma de Argônio. Após a determinação dos teores de cobre nos sucos de laranjas e uvas, os C. elegans foram divididos em cinco grupos, o grupo controle recebeu água ultrapura, e os tratados com cobre receberam as doses de 0,05 mg/L, 0,1 mg/L, 0,3 mg/L e 0,7 mg/L. Após a exposição foram avaliados parâmetros comportamentais e enzimáticos de cepas selvagens de C. elegans. Os ensaios das exposições aguda e crônica dos C. elegans ao cobre demonstraram que houve alterações da atividade da Acetilcolinesterase. Na exposição aguda as doses de 0,05 mg/L, 0,1mg/L e 0,7 mg/L ativaram a enzima, resultado similar foi encontrado após exposição crônica para as doses 0,3 mg/L e 0,7 mg/L. Já as doses de 0,05 mg/L e 0,1 mg/L inibiram a enzima após exposição crônica. Os organismos expostos também mostraram alterações comportamentais, como redução nos batimentos faríngeos à exposição aguda na dose de 0,05 mg/L e 0,7 mg/L em exposição crônica e aumento no ciclo de defecação nas doses de 0,1 mg/L e 0,3 mg/L em exposição aguda e redução em exposição crônica à 0,3 mg/L. Conforme já era esperado os níveis de cobre estavam aumentados nos solos que receberam o fungicida a base de cobre, o mesmo foi encontrado para os sucos. Porém, observou-se que as doses encontradas nos sucos promoveram significativas alterações no sistema colinérgico o que repercutiu no comportamento dos vermes. Assim, recomenda-se maior critério para aplicação de calda bordalesa, considerando que o cobre por ser um metal tem potencial de bioacumulação nos sistemas biológicos. / Currently, organic products have gained attention for its nutritional quality, food safety and sustainability in its production. Organic farming uses copper-based fungicides such as a Bordeaux mixture, which is composed of hydrated lime and copper sulfate. Copper is a natural and essential chemical element for living organisms, but there are controversies regarding the effects on the environment and human health. Thus, the objective of this study was to evaluate the copper content in soils, orange and grape juice of an organic property that uses the Bordeaux mixture as fungicide, and from the concentrations found, to evaluate the effects of acute and chronic exposure on Caenorhabditis elegans nervous system. The study started from the collection of soil and fruit, after harvest were extracted the orange and grape juices. Subsequently, the copper concentrations in soil and in the juice were analysed by Atomic Absorption Spectrometry Plasma Argon. After determination of the copper concentration in the juices, the C. elegans were divided into five groups, the control group received ultrapure water, and treated with copper received the 0.05 mg/L, 0.1 mg/L, 0.3 mg/L and 0.7 mg/L. After the exposure, behavioral and enzymatic parameters of wild strains of C. elegans were evaluated. The trials of acute and chronic exposures of C. elegans to copper showed that there were changes in acetylcholinesterase activity. In the acute exposure doses of 0.05 mg/L, 0.1 mg/L and 0.7 mg/L activated the enzyme, a similar result was found after chronic exposure at doses 0.3 mg/L and 0.7 mg/L. Doses of 0.05 mg/L and 0.1 mg/L inhibited the enzyme after chronic exposure. Exposed organisms also showed behavioral changes, such as reduction in pharyngeal beats to acute exposure at a dose of 0.05 mg/L and 0.7 mg/L on chronic exposure and increase in the defecation cycle at doses of 0.1 mg/L and 0.3 mg/L on acute exposure and reduction on chronic exposure at 0.3 mg/L. As expected, copper levels were increased in soils that received the copper-based fungicide, the same was found for juices. However, it was observed that the doses found in the juices promoted significant changes in the cholinergic system, which had repercussions on the behavior of the worms. Thus, it is recommended major criterion for the application of Bordeaux mixture, considering that copper metal has to be a potential for bioaccumulation in biological systems.

No description available.

Alterations in the microbiota have been observed in many human diseases, including diseases of neurodegeneration. However, specific microbiome factors that either promote or protect against neurodegeneration are largely unknown. We examined the effects of human microbiota in tauopathies, a class of age-associated neurodegenerative diseases that are characterized by the accumulation of tau protein inclusions. Using a Caenorhabditis elegans model expressing an aggregate prone human tau protein, we examined the influence of specific bacteria present in the human respiratory tract on neurodegeneration. We identified a bacterial species, Rothia aeria, that is neuroprotective in a C. elegans model of tauopathy. We determined that the R. aeria induced neuroprotection observed in PLM neurons is fat-3 dependent. fat-3 encodes the protein Delta (6)-fatty-acid desaturase. We also showed that a lipid(s) produced by R. aeria decreases neurodegeneration in C. elegans. Further investigation is needed to identify the lipid and the underlying mechanism. / Dissertation / Doctor of Philosophy (PhD) / The human body hosts trillions of microbes, including bacteria, viruses, and fungi. These components make up the human microbiota. Gut microbiota have an essential role in human health. In correlative studies, alterations in gut microbiota have been observed in many human diseases and conditions, including Alzheimer’s disease (AD). Diet, antibiotics, and probiotics change the composition of the microbiota and could decrease or increase the risk of developing AD. Modulation of the microbiome through diet and other interventions could be beneficial for AD. AD, the most common type of dementia, is a progressive brain disorder that slowly destroys memory and mental skills. The Alzheimer’s Society of Canada estimates that over half a million Canadians live with dementia, increasing significantly by 2031. The total estimated indirect and direct costs of dementia in 2016 in Canada was $10.4 billion. Currently, no treatments slow or stop Alzheimer’s disease and available medications can only improve the symptoms in some patients. One of the main characteristics of AD is the accumulation of a protein called tau in neurons. In this research, we look at the impact of microbiota on AD using Caenorhabditis elegans as a model organism. C. elegans is a free-living nematode. These nematodes are small, transparent, feed on bacteria and have a simple nervous system. All these criteria make C. elegans a perfect model for studying the impact of microbiota on AD. We exposed the C. elegans model of AD with high tau aggregation in their neurons to different bacteria collected from human microbiota and measured their neuronal health. We have found several species of bacteria that decreased neurodegeneration in this model. Currently, we are investigating how these bacteria improve neuronal health. Our findings suggest the involvement of human microbiota in AD. This suggests future treatment and preventative measures for AD should also consider microbiota composition. The result of this research will expand our knowledge of AD development and progression.

The nematode <i>Caenorhabditis elegans</i> is a small invertebrate whose nervous system, general anatomy, and normal development are all (comparatively) extremely simple and reproducible, and have all been well characterised. This dissertation describes work based on two different approaches to the study of the control of neural development in <i>C. elgans</i>. In the first part the course of neural outgrowth in the region of the ventral nerve cord was followed from electron microscope reconstructions of a series of fixed embryos. Following this, neurons whose processes grew out early were removed by laser ablation of their parent cells and the effect on subsequent nerve outgrowth was assayed by electron microscope reconstruction. The first process to grow along the ventral cord is that of AVG, and its presence is required for the normal, highly asymmetrical structure of the cord. Two more examples of dependency on particular nerve processes for correct guidance can be deduced from experiments in which cells at the back of the animal were removed. The observations of normal development and the ablation experiments can in some cases be related to defects seen in <i>uncoordinated</i> mutants with defective nerve process organisation. The second approach was to establish and analyse a computer data base containing all the synaptic connectivity data obtained by White et al. (1986), who reconstructed at an electron microscope level the entire central nervous system regions of two <i>C. elegans</i> specimens. Since the circuitry is highly reproducible, comparisons of connections between equivalent pairs of cells can be used to infer properties of synapse formation. Overall, the <i>C. elegans</i> circuitry is anatomically highly directional, and what little chemical synaptic feedback that is seen is mostly part of reciprocal synaptic connections. There is also evidence for physical organisation of the nerve processes in subbundles of the main process tract in the central nervous system.