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Biochemical Mechanism of RNA Interference in Higher Organisms: A DissertationSchwarz, Dianne S. 26 August 2005 (has links)
RNA interference (RNAi) is an evolutionarily conserved, sequence-specific gene silencing pathway found in eukaryotes, in which 21-nucleotide, small interfering RNAs (siRNAs) guide destruction of a corresponding target mRNA. RNAi is a natural mechanism for both genome surveillance and gene regulation. Moreover, siRNAs can be transfected into cultured mammalian cells, causing the sequence-specific ‘knock down’ of an mRNA. My work in the Zamore lab has centered around the Drosophilain vitro system and cultured mammalian cells to study the RNA interference (RNAi) pathway. small interfering RNAs (siRNAs) are incorporated into the RNA-induced silencing complex (RISC), which culminates in the cleavage of a complementary target mRNA. Previous work proved that certain structural features of siRNAs are essential for RNAi in flies, including the requirement for 5´ phosphates and 3´ hydroxyl groups. In cultured mammalian cells, the requirement for a 5´ phosphate also holds true, but we found no evidence to support the necessity for 3´ hydroxyls in either system. In addition, siRNAs can act as single strands entering the pathway downstream of double-stranded siRNAs, both of which are competent in directing the cleavage of its cognate mRNA at a single site.
While these key features are a requirement for functional siRNAs, alone they do not determine the efficiency to which an siRNA can enter the RISC. In fact, both strands of an siRNA can enter RISC to a different degree as determined by the stabilities of the 5´ ends of the siRNA strand, a phenomenon termed ‘functional asymmetry’. This characteristic is also reflected in another class of small RNAs involved in gene silencing known as microRNAs (miRNAs), which are processed from long hairpin RNA structures into mature, single-stranded non-coding RNAs. The asymmetric loading of siRNAs suggests that miRNAs are initially generated from siRNA-like duplexes cleaved from the stem of the hairpins. The strand whose 5´ end is less tightly paired will be processed into the mature miRNA, while the other strand is destroyed. By applying the rules of siRNA asymmetry it is possible to predict which side of the stem will be processed into the mature miRNA, a finding verified experimentally by our lab and others. This discovery also has additional implications in designing highly effective siRNAs and in reducing siRNA off-target effects.
We used these results to design siRNAs that target the single nucleotide polymorphism in superoxide dismutase that causes the familial form of amyotrophic lateral sclerosis (ALS), but leave the wild-type mRNA intact and functional. Our experiments have helped define the ‘rules’ for creating SNP-specific siRNAs. In particular, we found that only siRNAs with a purine:purine mismatch to the allele not intended for destruction show good discrimination. The placement of the mismatch in a tiled set of siRNAs shows that mismatches located in the 5´ region of the siRNA, a region shown to be responsible for siRNA binding, can not discriminate between alleles. In contrast, mismatches in the 3´ region of the siRNA, the region contributing to catalysis, discriminate between wild-type and mutant alleles. This work is an important step in creating allele-specific siRNAs as therapeutics for dominant negative genetic diseases.
But how does RISC cleave its target? By isolating both the 5´ and 3´ cleavage products produced by RISC in the Drosophila in vitro system, we discovered that RISC acts as a Mg2+-dependent endonuclease that cleaves a single phosphodiester bond in the mRNA target, leaving 5´ phosphate and 3´ hydroxyl groups. These findings were a critical step in the demonstration that Argonaute, a protein known to be a component of RISC, is the RNAi endonuclease.
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Characterization of JNK Binding Proteins: A DissertationRogers, Jeffrey Scott 27 July 2005 (has links)
The JNK signal transduction pathway mediates a broad, complex biological process in response to inflammatory cytokines and environmental stress. These responses include cell survival and apoptosis, proliferation, tumorigenesis and the immune response. The divergent cellular responses caused by the JNK signal transduction pathway are often regulated by spatial and cell type contexts, as well as the interaction with other cellular processes. The discovery of additional components of the JNK signal transduction pathway are critical to elucidate the stress response mechanisms in cells.
This thesis first discusses the cloning and characterization of two novel members of the JNK signal transduction pathway. JIP1 and JMP1 were initially identified from a murine embryo library through a yeast Two-Hybrid screen to identify novel JNK interacting proteins. Full length cDNAs of both genes were cloned and analyzed. JIP1 represents the first member of the JIP group of JNK scaffold proteins which were characterized. The JNK binding domain (JBD) of JIP1 matches the D-domain consensus of other JNK binding proteins, and it demonstrates JNK binding both in vitro and in vivo. This JNK binding was demonstrated to inhibit JNK signal transduction and over-expression of JIP1 inhibits the JNK mediated pre-B cell transformation by bcr-abl. Over-expressed JIP1 also sequesters JNK in the cytoplasm, which may be a mechanism of the inhibition of JNK signaling. A new, high-resolution digital imaging microscopy technique using deconvolution demonstrated the absence of JNK1 in the nucleus of co-transfected JIP1 and JNK1 cells.
The other protein discussed in this thesis is JMP1, a novel JNK binding, microtubule co-localized protein. There is a JBD in the JMP1 carboxyl end and a consensus D-domain within this region. The JMP1 JBD demonstrates an increased association with phospho-JNK from UV irradiated cells compared to un-irradiated cells in vivo. JMP1 also has 12 WD-repeat motifs in its amino terminal end which are required for microtubule co-localization. JMP1 demonstrates a cell cycle specific localization at the mitotic spindle poles. This co-localization is dependent on intact microtubules and the amino-terminal WD-repeats are required for this localization. JMP1 mRNA is highly expressed in testis tissues. Immunocytochemistry on murine testis sections using an affinity purified anti-JMP1 antibody demonstrates JMP1 protein in the lumenal compartment of the seminiferous tubules. JMP1 protein is expressed in primary and secondary spermatocytes, cells which are actively undergoing meiosis.
The results obtained from the localization of JMP1 in meiotic spermatocytes led to an investigation of the roles of JNK signal transduction in the testis. The testis is an active region of cellular proliferation, apoptosis and differentiation, which make it an appealing model for studying JNK signal transduction. However, the roles JNK signaling have in the testis are poorly understood. I investigated the reproduction capability of Jnk3-/- male mice and discovered older Jnk3-/- males had a reduced capacity to impregnate females compared to younger animals and age-matched wild type controls. The testis morphology and sperm motility of these animals were similar to wild-type animals, and there was no alteration of apoptosis in the testis. The final section of this thesis involves the study of this breeding defect and investigating for cellular defects that might account for this age-related Jnk3-/- phenotype.
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RNA Silencing Pathways in <em>Schizosaccharomyces pombe</em> and <em>Drosophila melanogaster</em>: A DissertationSigova, Alla A. 03 November 2006 (has links)
RNA silencing is an evolutionary conserved sequence-specific mechanism of regulation of gene expression. RNA interference (RNAi), a type of RNA silencing in animals, is based on recognition and endonucleolytic cleavage of target mRNA complimentary in sequence to 21-nucleotide (nt) small RNA guides, called small interfering RNAs (siRNAs). Another class of 21-nt small RNAs, called micro RNAs (miRNAs), is endogenously encoded in eukaryotic genomes. Both production of siRNAs from long double-stranded RNA (dsRNA) and biogenesis of miRNAs from hairpin structures are governed by the ribonuclease III enzyme Dicer. Although produced as duplex molecules, siRNAs and miRNAs are assembled into effector complex, called the RNA-induced silencing complex (RISC), as single-strands. A member of the Argonaute family of small RNA-binding proteins lies at the core of all known RNA silencing effector complexes. Plants and animals contain multiple Argonaute paralogs. In addition to endonucleolytic cleavage, Argonaute proteins can direct translational repression/destabilization of mRNA or transcriptional silencing of DNA sequences by the siRNAdirected production of silent heterochromatin.
The Schizosaccharomyces pombe genome encodes only one of each of the three major classes of proteins implicated in RNA silencing: Dicer (Dcr1), RNA-dependent RNA polymerase (RdRP; Rdp1), and Argonaute (Ago1). These three proteins are required for silencing at centromeres and for the initiation of transcriptionally silent heterochromatin at the mating-type locus. That only one Dicer, RdRP and Argonaute is expressed in S. pombe might reflect the extreme specialization of RNA silencing pathways regulating targets only at the transcriptional level in this organism. We decided to test if classical RNAi can be induced in S. pombe. We introduced a dsRNA hairpin corresponding to a GFP transgene. GFP silencing triggered by dsRNA reflected a change in the steady-state concentration of GFP mRNA, but not in the rate of GFP transcription. RNAi in S. pombe required dcr1, rdp1, and ago1, but did not require chp1, tas3, or swi6, genes required for transcriptional silencing. We concluded that the RNAi machinery in S. pombecould direct both transcriptional and posttranscriptional silencing using a single Dicer, RdRP, and Argonaute protein. Our findings suggest that, in spite of specialization in distinct siRNA-directed silencing pathways, these three proteins fulfill a common biochemical function.
In Drosophila, miRNA and RNAi pathways are both genetically and biochemically distinct. Dicer-2 (Dcr-2) generates siRNAs, whereas the Dicer-1 (Dcr-1)/Loquacious complex produces miRNAs. Argonaute proteins can be divided by sequence similarity into two classes: in flies, the Ago subfamily includes Argonaute1 (Ago1) and Argonaute2 (Ago2), whereas the Piwi subfamily includes Aubergine, Piwi and Argonaute 3. siRNAs and miRNAs direct posttranscriptional gene silencing through effector complexes containing Ago1 or Ago2. The third class of small RNAs, called repeat-associated small interfering RNAs (rasiRNAs), is produced endogenously in the Drosophilagerm line. rasiRNAs mediate silencing of endogenous selfish genetic elements such as retrotransposons and repetitive sequences to ensure genomic stability.
We examined the genetic requirements for biogenesis of rasiRNAs in both male and female germ line of Drosophilaand silencing of 8 different selfish elements, including tree LTR retrotransposons, two non-LTR retrotransposons, and three repetitive sequences. We find that biogenesis of rasiRNAs is different from that of miRNAs and siRNAs. rasiRNA production appears not to require Dicer-1 or Dicer-2. rasiRNAs lack the 2´,3´ hydroxy termini characteristic of animal siRNA and miRNA. While siRNAs derive from both the sense and antisense strands of their dsRNA precursors, rasiRNAs accumulate in antisense polarity to their corresponding target mRNAs. Unlike siRNAs and miRNAs, rasiRNAs function through the Piwi, rather than the Ago, Argonaute protein subfamily. We find that rasiRNAs silence their target RNAs posttranscriptionally: mutations that abrogate rasiRNA function dramatically increase the steady-state mRNA level of rasiRNA targets, but do not alter their rate of transcription, measured by nuclear run-on assay.
Our data suggest that rasiRNAs protect the fly germ line through a silencing mechanism distinct from both the miRNA and RNAi pathways.
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Epigenetic Telomere Protection by Drosophila DNA Damage Response Pathways: A DissertationOikemus, Sarah R. 08 September 2006 (has links)
Several aspects of Drosophila telomere biology indicate that telomere protection can be regulated by an epigenetic mechanism. First, terminally deleted chromosomes can be stably inherited and do not induce damage responses such as apoptosis or cell cycle arrest. Second, the telomere protection proteins HP1 and HOAP localize normally to these chromosomes and protect them from fusions. Third, unprotected telomeres still contain HeT-A sequences at sites of fusions. Taken together these observations support a model in which an epigenetic mechanism mediated by DNA damage response proteins protects Drosophilatelomeres from fusion.
Work presented in this thesis demonstrates that the Drosophila proteins ATM and Nbs are required for the regulation of DNA damage responses similar to their yeast and mammalian counterparts. This work also establishes a role for the ATM and ATR DNA damage response pathways in the protection of both normal and terminally deleted chromosomes. Mutations that disrupt both pathways result in a severe telomere fusion phenotype, similar to HP1 and HOAP mutants. Consistent with this phenotype, HOAP localization at atm,atr double mutant telomeres is completely eliminated. Furthermore, telomeric sequences are still present, even at the sites of fusions. These results support a model in which an epigenetic mechanism mediated by DNA damage response proteins protects Drosophila telomeres from fusion.
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The Molecular Mechanisms of Activity-Dependent Wingless (Wg)/Wnt Signaling at a Drosophila Glutamatergic Synapse: a DissertationAtaman, Bulent 01 February 2008 (has links)
Synaptic plasticity, the ability of synapses to change in strength, underlies complex brain functions such as learning and memory, yet little is known about the precise molecular mechanisms and downstream signaling pathways involved. The major goal of my doctoral thesis was to understand these molecular mechanisms and cellular processes underlying synaptic plasticity using the Drosophilalarval neuromuscular junction (NMJ) as a model system.
My work centered on a signaling pathway, the Wg/Wnt signaling pathway, which was found to be crucial for activity-driven synapse formation. The Wg/Wnt family of secreted proteins, besides its well-characterized roles in embryonic patterning, cell growth and cancer, is beginning to be recognized as a pivotal player during synaptic differentiation and plasticity in the brain. At the DrosophilaNMJ, the Wnt-1 homolog Wingless (Wg) is secreted from presynaptic terminals and binds to Frizzled-2 (DFz2) receptors in the postsynaptic muscle. Perturbations in Wg signaling lead to poorly differentiated NMJs, containing synaptic sites that lack both neurotransmitter release sites and postsynaptic structures. In collaboration with other members of the Budnik lab, I set out to unravel the mechanisms by which Wg regulates synapse differentiation. We identified a novel transduction pathway that provides communication between the postsynaptic membrane and the nucleus, and which is responsible for proper synapse development. In this novel Frizzled Nuclear Import (FNI) pathway, the DFz2 receptor is internalized and transported towards the nucleus. The C-terminus of DFz2 is subsequently cleaved and imported into the postsynaptic nucleus for potential transcriptional regulation of synapse development (Mathews, Ataman, et al. Science (2005) 310:1344).
My studies also centered on the genetic analysis of Glutamate Receptor (GluR) Interacting Protein (dGRIP), which in mammals has been suggested to regulate the localization of GluRs and more recently, synapse development. I generated mutations in the gene, transgenic strains carrying a dGRIP-RNAi and fluorescently tagged dGRIP, and antibodies against the protein. Remarkably, I found dgrip mutants had synaptic phenotypes that closely resembled those in mutations altering the FNI pathway. Through the genetic analysis of dgrip and components of the FNI pathway, immunoprecipitation studies, electron microscopy, in vivotrafficking assays, time-lapse imaging, and yeast two-hybrid assays, I demonstrated that dGRIP had a hitherto unknown role as an essential component of the FNI pathway. dGRIP was found in trafficking vesicles that contain internalized DFz2. Further, DFz2 and dGRIP likely interact directly. Through the use of pulse chase experiments I found that dGRIP is required for the transport of DFz2 from the synapse to the nucleus. These studies thus provided a molecular mechanism by which the Wnt receptor, DFz2, is trafficked from the postsynaptic membrane to the nucleus during synapse development and implicated dGRIP as an essential component of the FNI pathway (Ataman et al. PNAS (2006) 103:7841).
In the final part of my dissertation, I concentrated on understanding the mechanisms by which neuronal activity regulates synapse formation, and the role of the Wnt pathway in this process. I found that acute changes in patterned activity lead to rapid modifications in synaptic structure and function, resulting in the formation of undifferentiated synaptic sites and to the potentiation of spontaneous neurotransmitter release. I also found that these rapid modifications required a bidirectional Wg transduction pathway. Evoked activity induced Wg release from synaptic sites, which stimulated both the postsynaptic FNI pathway, as well as an alternative presynaptic Wg pathway involving GSK-3ß/Shaggy. I suggest that the concurrent activation of these alternative pathways by the same ligand is employed as a mechanism for the simultaneous and coordinated assembly of the pre- and postsynaptic apparatus during activity-dependent synapse remodeling (Ataman et al. Neuron (2008) in press).
In summary, my thesis work identified and characterized a previously unrecognized synaptic Wg/Wnt transduction pathway. Further, it established a mechanistic link between activity-dependent synaptic plasticity and bidirectional Wg/Wnt signaling. These findings provide novel mechanistic insight into synaptic plasticity.
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Regulation of Life Span by <em>DAF-16</em>/Forkhead Transcription Factor in <em>Caenorhabditis elegans</em>: A DissertationOh, Seung Wook 01 October 2005 (has links)
The insulin/IGF-1 signaling pathway plays a pivotal role in life span regulation in diverse organisms. In Caenorhabditis elegans, a PI 3-kinase signaling cascade downstream of DAF-2, an ortholog of the mammalian insulin and insulin-like growth factor-1 (IGF-1) receptor, negatively regulates DAF-16/forkhead transcription factor. DAF-16 then regulates a wide variety of genes involved in longevity, stress response, metabolism and development. DAF-16 also receives signals from other pathways regulating life span and development. However, the precise mechanism by which DAF-16 directs multiple functions is poorly understood.
First, in Chapter II, we demonstrate that JNK is a novel positive regulator of DAF-16 in both life span regulation and stress resistance. Our genetic analysis suggests that the JNK pathway acts in parallel with the insulin-like signaling pathway to regulate life span and both pathways converge onto DAF-16. We also show that JNK-1 directly interacts with and phosphorylates DAF-16. Moreover, in response to heat stress, JNK-1 promotes the translocation of DAF-16 into thc nucleus. Our findings define a novel interaction between the stress response pathway (JNK) and the master regulator of life span (DAF-16), and provide a mechanism by which JNK regulates longevity and stress resistance.
Next, in Chapter III, we focus on the downstream targets of DAF-16. Here, we used a modified chromatin immunoprecipitation (ChIP) method to identify direct target promoters of DAF-16. We cloned 103 target sequences containing consensus DAF-16 binding sites and randomly selected 33 targets for further analysis. The expression of majority of these genes is regulated in a DAF-16-dependent manner. Moreover, inactivation of more than 50% of these genes significantly altered DAF-16-dependent functions such as longevity, fat storage and dauer diapause. Our results show that the ChIP-based cloning strategy leads to greater enrichment of DAF-16 target genes, compared to previous studies using DNA micro array or bioinformatics. We also demonstrate that DAF-16 is recruited to multiple promoters to coordinate regulation of its downstream target genes.
In summary, we identified the JNK signaling pathway as a novel input into DAF-16 to adapt animals to the environmental stresses. We also revealed a large number of novel outputs of DAF-16. Taken together, these studies provide insight into the complex regulation by DAF-16 to control diverse biological functions and eventually broaden our understanding of aging.
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Activation and Role of Memory CD8 T Cells in Heterologous Antiviral Immunity and Immunopathology in the Lung: A DissertationChen, Hong 09 December 2002 (has links)
Each individual experiences many sequential infections throughout the lifetime. An increasing body of work indicates that prior exposure to unrelated pathogens can greatly alter the disease course during a later infection. This can be a consequence of a phenomenon known as heterologous immunity. Most viruses invade the host through the mucosa of a variety of organs and tissues. Using the intranasal mucosal route of infection, the thesis focused on studying modulation of lymphocytic choriomeningitis virus (LCMV)-specific memory CD8 T cells upon respiratory vaccinia virus (VV) infection and the role of these memory CD8 T cells in heterologous immunity against VV and altered immunopathology in the lung.
The VV infection had a profound impact on memory T cells specific for LCMV. The impact included the up-regulation of CD69 expression on LCMV-specific CD8 memory T cells and the activation of their in vivoIFN-γ production and cytotoxic function. Some of these antigen-specific memory T cells selectively expanded in number, resulting in modulation of the original LCMV-specific T cell repertoire. In addition, there was a selective organ-dependent redistribution of these LCMV-specific memory T cell populations in secondary lymphoid tissue (the mediastinal lymph node and spleen) and the non-lymphoid peripheral (the lung) organs. The presence of these LCMV-specific memory T cells correlated with IFN-γ-dependent enhanced VV clearance, decreased mortality and marked changes in lung immunopathology. Thus, the participation of pre-existing memory T cells specific for unrelated agents can alter the dynamics of mucosal immunity. This is associated with an altered disease course in response to a pathogen.
The roles for T cell cross-reactivity and cytokines in the modulation of memory CD8 T cells during heterologous memory CD8 T cell-mediated immunity and immunopathology were investigated. Upon VV challenge, there were preferential expansions of several LCMV-specific memory CD8 T cell populations. This selectivity suggested that cross-reactive responses played a role in this expansion. Moreover, a VV peptide, partially homologous to LCMV NP 205, stimulated LCMV-NP205 specific CD8 T cells, suggesting that NP205 may be a cross-reactive epitope. Poly I:C treatment of LCMV-immune mice resulted in a transient increase but no repertoire alteration of LCMV-epitope-specific CD8 T cells. These T cells did not produce IFN-γ in vivo. These results imply that poly I:C, presumably through its induced cytokines, was assisting in initial recruitment of LCMV-specific memory CD8 T cells in a nonspecific manner. VV challenge of LCMV-immune IL-12KO mice resulted in activation and slightly decreased accumulation of LCMV-specific CD8 T cells. Moreover, there was a dramatic reduction of in vivoIFN-γ production by LCMV-specific IL-12KO CD8 T cells in the lung. I interpreted this to mean that IL-12 was important to augment IFN-γ production by memory CD8 T cells upon TCR engagement by antigens and to induce further accumulation of activated memory CD8 T cells during the heterologous viral infection.
This thesis also systematically examined what effect the sequence of two heterologous virus challenges had on viral clearance, early cytokine profiles and immunopathology in the lung after infecting mice immune to one virus with another unrelated viruses. Four unrelated viruses, [LCMV, VV, influenza A virus or murine cytomegalovirus (MCMV)], were used. There were many common changes observed in the acute response to VV as a consequence of prior immunity to any of three viruses, LCMV, MCMV or influenza A virus. These included the enhanced clearance of VV in the lung, associated with enhanced TH1 type responses with increased IFN-γ and suppressed pro-inflammatory responses. However, immunity to the three different viruses resulted in unique pathologies in the VV-infected lungs, but with one common feature, the substitution of lymphocytic and chronic mononuclear infiltrates for the usual acute polymorphonuclear response seen in non-immune mice. Immunity to influenza A virus appeared to influence the outcome of subsequent acute infections with any of the three viruses, VV, LCMV and MCMV. Most notably, influenza A virus-immunity protected against VV but it actually enhanced LCMV and MCMV titers. This enhanced MCMV replication was associated with enhanced TH1 type response and pro-inflammatory cytokine responses. Immunity to influenza A virus appeared to dramatically enhance the mild lymphocytic and chronic mononuclear response usually observed during acute infection with either LCMV or MCMV in non-immune mice, but LCMV infection and MCM infection of influenza A virus-immune mice each had its own unique features. Thus, the specific sequence of virus infections controls the outcome of disease.
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Molecular and Behavioral Analysis of <em>Drosophila</em> Circadian Photoreception and Circadian Thermoreception: A DissertationBusza, Ania 23 May 2007 (has links)
Circadian clocks are biological timekeepers that help maintain an organism’s behavior and physiological state optimally timed to the Earth’s day/night cycle. To do this, these internal pacemakers must accurately keep track of time. Equally importantly, they must be able to adjust their oscillations in response to external time cues to remain properly synchronized with the environment, and correctly anticipate environmental changes. When the internal clock is offset from its surrounding day/night cycle, clinically relevant disruptions develop, ranging from inconveniences such as jet-lag to more severe problems such as sleep disorders or mood disorders. In this work, I have used the fruit fly, Drosophila melanogaster, as a model organism to investigate how light and temperature can synchronize circadian systems.
My initial studies centered on an intracellular photoreceptor, CRYPTOCHROME (CRY). CRY is a blue light photoreceptor previously identified as a major component of the primary light-input pathway into the Drosophila circadian clock. We used molecular techniques to show that after light-activation, CRY binds to the key circadian molecule TIMELESS (TIM). This interaction irreversibly targets TIM, but not CRY, for degradation. Further studies characterizing a newly isolated cry mutant, crym, showed that the carboxyl-terminus of CRY is not necessary for CRY’s ability to impart photic information to the molecular clock. Instead, the C-terminus appears to be necessary for normal CRY stability and protein-protein interactions. Thus, we conclude that in contrast to previous reports on CRYs of other species, where the C-terminal domain was required for transduction of photic information, the C-terminus of DrosophilaCRY has a purely modulatory function.
During the second part of my dissertation work, I focused my studies on circadian thermoreception. While the effects of light in synchronization of the Drosophilaclock to environmental cycles have been extensively characterized, significantly less is known about temperature input pathways into the circadian pacemaker. I have used two approaches to look at how temperature affects the circadian system. First, I conducted a series of behavioral analyses looking at how locomotor rhythms can be phase-shifted in response to temperature cycles. By examining the behavior of genetically ablated flies, we determined that the well-characterized neurons controlling morning and evening surges of activity during light/dark cycles are also implicated in morning and evening behaviors under temperature cycles. However, we also find evidence of cells that contribute to modulating afternoon and evening behavior specifically under temperature cycles. These data contribute to a growing number of studies in the field suggesting that pacemaker cells may play different roles under various environmental conditions. Additionally, we provide data showing that intercellular communication plays an important role in regulating circadian response to temperature cycles. When the morning oscillator is absent or attenuated, the evening cells respond abnormally quickly to temperature cycles. My work thus provides information on the roles of different cell groups during temperature cycles, and suggests that beyond simply synchronizing individual oscillating cells, intercellular network activity may also have a role in modulating proper response to environmental time cues.
Finally, I present some preliminary work looking at effects of temperature on known circadian molecules. Using a combination of in vivo and cell culture techniques, I have found that TIM protein levels decrease at higher temperatures. My cell culture data suggest that this is a proteasome-independent degradation event. As TIM is also a key molecule in the light-input pathway, the stability of TIM proteins may be a key point of integration for light and temperature input pathways. While additional research needs to be conducted to confirm these effects in vivoin wild-type flies, these preliminary results identify a possible avenue for further study.
Taken together, my work has contributed new data on both molecular and neuronal substrates involved in processing light and temperature inputs into the Drosophila circadian clock.
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Molecular and Neuronal Analysis of Circadian Photoresponses in <em>Drosophila</em>: A DissertationMurad, Alejandro D. 25 October 2007 (has links)
Most organisms, from cyanobacteria to humans are equipped with circadian clocks. These endogenous and self-sustained pacemakers allow organisms to adapt their physiology and behavior to daily environmental variations, and to anticipate them. The circadian clock is synchronized by environmental cues (i.e. light and temperature fluctuations).
The fruit fly, Drosophila melanogaster, is well established as a model for the study of circadian rhythms. Molecular mechanisms of the Drosophilacircadian clock are conserved in mammals. Using genetic screens, several essential clock proteins (PER, TIM, CLK, CYC, DBT, SGG and CK-II) were identified in flies. Homologs of most of these proteins are also involved in generating mammalian circadian rhythms. In addition, there are only six neuronal groups in the adult fly brain (comprising about 75 pairs of cells) that express high levels of clock genes. The simplicity of this system is ideal for the study of the neural circuitry underlying behavior.
The first half of this dissertation focuses on a genetic screen designed to identify novel genes involved in the circadian light input pathway. The screen was based on previous observations that a mutation in the circadian photoreceptor CRYPTOCHROME (CRY) allows flies to remain rhythmic in constant light (LL), while wild type flies are usually arrhythmic under this condition. 2000 genes were overexpressed and those that showed a rhythmic behavior in LL (like crymutants) were isolated. The candidate genes isolated in the screen present a wide variety of biological functions. These include genes involved in protein degradation, signaling pathways, regulation of transcription, and even a pacemaker gene. In this dissertation, I describe work done in order to validate and characterize such candidates.
The second part of this dissertation focuses on identifying the pacemaker neurons that drive circadian rhythms in constant light (LL) when the pacemaker gene period is overexpressed. We found that a subset of pacemaker neurons, the DN1s, is responsible for driving rhythms in constant light. This attractive finding reveals a novel role for the DN1s in driving behavioral rhythms under constant conditions and suggests a mechanism for seasonal adaptation in Drosophila.
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Procedimentos de biossegurança para as novas instalações do Laboratório de Experimentação Animal (Laean) de Bio-ManguinhosMajerowicz, Joel January 2005 (has links)
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Previous issue date: 2005 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / Os biotérios de experimentação são uma extensão doslaboratórios de pesquisa. Em
unidade de produção de imunobiológicos, são partes do controle de qualidade e fundamentais ao desenvolvimento de novos produtos. Instalações adequadas e procedimentos operacionais padrão são exigências legais e recomendadas para que se obtenham resultados confiáveis.
No caso específico do Instituto de Tecnologia em Imunobiológicos da Fiocruz (Bio-Manguinhos), devem ser incorporados, obrigatoriamente, às atividades, as Boas Práticas de Fabricação (BPF) e Boas Práticas de Laboratório (BPL), atendendo à regulamentação das agências governamentais.
As atividades com animais de laboratórios são especiais, visto as particularidades que
só são encontradas em biotérios, como por exemplo, os riscos inerentes aos animais,
especialmente os físicos, que compreendem aqueles em que o profissional é expostoa
mordidas, arranhões ou outra forma de defesa; os biológicos próprios da sua biota, zoonótica ou experimental e a produção de alérgenos; os químicos, tais como, os produtos de limpeza e desinfecção e os relacionados aos trabalhos experimentais.
Apresenta-se, nesta dissertação, uma comparação entre as atuais e as futuras
instalações do Laboratório de Experimentação Animalde Bio-Manguinhos (Laean), descrevendo suas características e procedimentos operacionais relativos a biossegurança.
O objetivo deste trabalho é expor, de modo ordenado e sistemático os procedimentos
de biossegurança para o acesso e saída de técnicos,de materiais e animais; de esterilização e descontaminação de produtos, insumos e descartes; de desinfecção de materiais e ambientes e, aqueles relativos ao manuseio de animais envolvidoscom risco biológico. / Animal housing is an extension of the research laboratories. In an immunobiological production unit, they are part of the quality control and the development of new products.
Adequate facilities and standardized operational procedures are legal and recommended requirements for the reliability of results. Specifically speaking about the Institute of Technology on Immunobiologicals of Fiocruz (Bio-Manguinhos) Good Manufacturing Practices (GMP) and Good Laboratory Practices (GLP) must become part of the activities in order to meet governmental agencies regulations.
Activities with laboratory animals are special dueto their peculiarities that are found in animal housings only, as for example, animals inherent risks to include those which professionals are exposed: physical like bites, scratches or any other defense actions; biological pertaining to biota, zoonotic or experimental and allergen production; chemical like cleaning products and those related to experimentalworks.
In this dissertation, a comparison is made between the current and future facilities of
the Laboratory of Animal Experimentation in Bio-Manguinhos (Laean). It describes the
characteristics and operational procedures related to biosafety.
The objective of this work is to expose order and systematically biosafety procedures for entrance and exit of personnel, materials and animals, sterilization and decontamination of
products, input and disposal, disinfection of materials and environments, and those related to handling of animals involved in biological risks.
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