The purpose of this study was to investigate the mechanism of action of lipolysis by growth hormone in rat adipocytes. GH-induced lipolysis, in contrast to that of isoproterenol (ISO), is slow in onset (lag time >1h), small in magnitude (~2X basal). and requires corticosteroid. Evidence for direct coupling between GH receptors and adenylyl cyclase or G-proteins is lacking, and although we could detect no measurable change in cAMP content after treatment with GH + dexamethasone (Dex), it is likely that cAMP activation of protein kinase A is a central event in GH-induced lipolysis. Rp-cAMPS, a competitive antagonist of cAMP was equally effective in decreasing lipolysis in tissues treated with GH/Dex or a comparably lipolytic dose of ISO. Incorporation of 32P from γ-32P-ATP into kemptide, a synthetic oligopeptide substrate for protein kinase A, was increased in homogenates of GH/Dex-treated tissue. This increase was correlated with increased lipolysis. Earlier estimates based upon 32P-ribosylation of Gi catalysed by pertussis toxin (PTx) suggested that the abundance of Gi in adipocyte membranes was decreased 4h after treatment of hypophysectomized rats with GH. We therefore examined the possibility that changes in amount or distribution of G-proteins in adipocyte membranes might account for the lipolytic action of GH. Homogenates of GH/Dex-treated and control adipocytes were subjected to differential centrifugation and the abundance of G-proteins in low speed, l6k x g (16k), pellets and high speed, 100k x g (100k), pellets were determined by quantitative Western analysis with densitometry. A 35% loss of Giα2 from the l6k pellet compared from tissues treated with GH/Dex was associated with a 70% increase of Giα2 in the 100k pellet. No change in Gsα was observed in the l6k pellet but a 35% loss of Gsα was seen in the 100k pellet. The G proteins in the l6k pellet were fractionated on a continuous sucrose gradient followed by quantitation with Western analysis or autoradiography after 32P-NAD ribosylation. Giα2 was consistently shifted from heavier to lighter fractions of the l6k pellet after treatment with GH/Dex. Similar shifts of Gsα were not seen. The distribution of 32P-labelled proteins was comparably altered after incubation of homogenates of control and GH/Dex treated adipocytes with PTx and 32P-NAD. These shifts were blocked by treatment of adipocytes with 100μM colchicine which also blocked the lipolytic action of GH/Dex. We propose that an action of GH/Dex on the cytoskeleton of fat cells may change the cellular distribution of G-proteins in a manner that produces a relative decrease in the tonic inhibitory influence of Gi on adenylyl cyclase.

RNA interference (RNAi) in the nematode Caenorhabditis elegans is a type of homology-dependent post-transcriptional gene silencing induced by dsRNA. This dissertation describes the genetic analysis of the RNA interference pathway and inheritance properties associated with this phenomenon. We demonstrate that the RNAi effect can be observed in the progeny of the injected animal for at least two generations. Transmission of the interference effect occurs through a dominant extragenic agent. The wild-type activities of the RNAi pathway genes rde-l and rde-4 are required for the formation of this interfering agent but are not needed for interference thereafter. In contrast, the rde-2 and mut-7 genes are required downstream for interference. These findings provide evidence for germline transmission of an extragenic sequence-specific silencing factor and implicate rde-l and rde-4in the formation of the inherited agent. Other forms of homology-dependent silencing in C. elegansinclude co-suppression and transcriptional silencing of transgenes in the germline. We demonstrate that silencing of a germline transgene can be initiated by injected dsRNA, via the RNAi pathway, and then maintained on a different level. This observation indicates that post-transcriptional and transcriptional silencing of homologous genes could be connected. This dissertation also describes the connection between RNAi and developmental pathways of gene regulation in C. elegans. We show that inactivation of genes related to RNAi pathway genes, a homolog of Drosophila Dicer (dcr-l), and two homologs of rde-1 (alg-l and alg-2) cause heterochronic phenotypes similar to lin-4 and let-7 mutations. Further we show that dcr-l, alg-l, and alg-2 are necessary for the maturation and activity of the lin-4 and let-7small temporal RNAs that regulate stage-specific development. Our findings suggest that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation. Finally, this study illustrates the detection of small interfering RNAs (siRNAs), intermediates in the RNAi process, and describes requirements for their accumulation. We show that, in the course of RNAi induced by feeding dsRNA, C. elegans accumulate only siRNAs complementary to the target gene. This accumulation depends on the presence of the target sequence and requires activities of several RNAi-pathway genes. We show that selective retention or amplification of RNAi-active molecules can create a reservoir of memory antisense siRNAs that prevent future expression of the genes with complementary sequence. This suggests a parallel at the molecular level with the clonal selection of antibody forming cells and in the vertebrate immune system.

Understanding how memory is formed requires looking beyond the genes involved to the neural circuitry and temporal aspects of memory. In this dissertation I have focused my investigation on Dorsal Paired Medial (DPM) neurons, two modulatory neurons essential for memory in Drosophila. DPM neurons highly express the amnesiac (amn) gene, which encodes for a putative pre-pro-neuropeptide. amn function in DPM neurons is required for memory. Here I provide evidence that DPM neurons are cholinergic and that acetylcholine (ACh) and AMN act as co-transmitters essential for DPM function. In order to investigate the temporal requirements of DPM output I blocked transmitter release during discrete intervals in the memory process using shibirets1 and tested flies for shock and sugar-reinforced memory. These experiments demonstrated that stable memory requires persistent transmitter release from DPM neurons. Furthermore these results suggest AMN and DPM neurons act as general stabilizers of mushroom body dependent memory. To further investigate the neural circuitry underlying DPM function I disrupted DPM projections onto the mushroom body lobes by ectopically expressing DScam17-2::GFP in DPM neurons. Flies with DPM neurons that predominantly project to the mushroom body α´/β´ lobes exhibit normal memory, and blocking transmitter release from the mushroom body prime lobes neurons themselves abolishes memory indicating DPM neuron-mushroom body α´/β´ neuron interaction that are critical for memory. Taken together, the experimental evidence presented here are used to provide a rudimentary model of the neural circuitry involved in memory stability, where DPM neurons form a recurrent feedback loop with the mushroom body α´/β´ lobe neurons and act to stabilize odorspecific conditioned memories at Kenyon cell synapses.

The fusion protein of the Newcastle Disease Virus (NDV) contains three hydrophobic domains. To explore the topogenic signals of these domains, mutants were constructed in which each of the hydrophobic domains was deleted. The membrane insertion and topology of these proteins was characterized in a wheat germ cell-free translation system supplemented with canine microsomal membranes. The results indicated that the first 13 amino acids of the fusion protein are necessary to confer translation inhibition by SRP. Translocation of the nascent chains containing all or part of the first hydrophobic sequence resulted in the appearance of a species of higher molecular weight consistent with glycosylation of at least four of the five potential N-linked glycosylation sites. When glycosylation was inhibited with a glycosylation competitor peptide, signal sequence cleavage was detected. Protease digestion of mutants missing the C-terminal hydrophobic domain indicated that the C-terminus has stop transfer activity. A comparison of membrane insertion of the wild-type fusion protein to that of a mutant missing the second hydrophobic domain, the fusion sequence, indicated that the fusion domain has stop-transfer activity when synthesized in vitro. Furthermore, the fusion domain shows little signal sequence activity when positioned near the amino terminus of the fusion protein. The fusion protein has a highly conserved leucine zipper motif immediately upstream from the transmembrane domain of the F1 subunit. In order to determine the role that the conserved leucines have for the oligomeric structure and biological activity of the NDV fusion protein, the heptadic leucines at positions 481,488, and 495 were changed individually and in combination to an alanine residue. Whereas single amino acid changes had little effect on fusion, substitution of two or three leucine residues abolished the fusogenic activity of the protein although cell surface expression of the mutants and sedimentation in sucrose gradients was similar to that of the wild type. Furthermore, deletion of the C-terminal 91 amino acids, including the leucine zipper motif and transmembrane domain resulted in secretion of an oligomeric structure. These results indicate that the conserved leucines do not play a role in oligomer formation but are required for the fusogenic ability of the protein. When the polar face of the potential alpha helix was altered by nonconservative substitutions of a serine-to-alanine (position 473), glutamic acid-to-lysine (position 482) or an asparagine-to-lysine (position 485), the fusogenic ability of the protein was not significantly disrupted. A phenylalanine residue is at the amino terminus of the F1 protein of all paramyxovirus fusion proteins with the exception of the avirulent strains which have a leucine residue in this position. To explore the role of this phenylalanine in the fusion activity of the protein, this residue was changed to leucine (F117L) or to glycine (F117G) by site-specific mutagenesis while maintaining the cleavage site sequence of virulent strains of NDV. Whereas both the wild-type and the F117G proteins were proteolytically cleaved and F1 was detected, the leucine subsitution abolished cleavage. When co-expressed with the HN protein, the fusion protein with either a phenylalanine and glycine residue at position 117, but not a leucine, was shown to stimulate membrane fusion. However, incubation in trypsin activated the fusion activity of the F117L protein. Thus the presence of a leucine at position 117 of the precursor sequence blocks cleavage, but not fusion acitivity, and indicated that the phenylalanine at the amino terminus of the F1 subunit is not conserved for the fusion activity of the protein.

This thesis examines the structure and regulated expression of the gene encoding the neuroendocrine peptides neurotensin and neuromedin N (NT/N gene). Previous studies have shown that expression of NT/N mRNA and NT peptide in PC12 cells are strictly dependent on simultaneous exposure to combinations of nerve growth factor (NGF), glucocorticoids, activators of adenylate cyclase, and lithium ion. My objective was to characterize the cis-regulatory DNA sequences involved in regulated expression of this gene. The initial focus of this study was an analysis of the structure, tissue-specific expression, and exon evolution of the rat NT/N gene. Nucleotide sequence comparisons between the rat gene and the canine and bovine cDNA sequences indicated that the predicted structure of a 170 amino acid precursor protein is highly conserved. Furthermore, the close similarity between the two cDNAs suggested that identical precursor proteins are expressed in neural and endocrine tissues. RNA analysis revealed that the gene is transcribed to yield two distinct mRNAs, 1.0 kb and 1.5 kb in size. The two mRNA species differ only in the size of their 3' untranslated regions. Interestingly, the smaller mRNA is predominant in the gastrointestinal tract, while both mRNAs are equally abundant in all neural tissues examined, except the cerebellum, where no expression was observed. Transient transfection assays were used to delineate the rat NT/N gene cis-regulatory DNA sequences. Progressive deletion of the NT/N 5' flanking region revealed that sequences between -216 and +56 of the NT/N gene are sufficient to confer the full spectrum of responses of the endogenous gene to either of two reporter genes. A detailed mutational analysis of the NT/N control region indicated that it is composed of an array of inducible cis-regulatory elements, including an AP-1 site, two cAMP-responsive elements (CREs), and a glucocorticoid-responsive element (GRE). Specific mutations to the AP-1 site and either CRE suggested that these elements are functionally interdependent. I propose that this array of cis-regulatory sequences in the NT/N transcriptional control region serves to integrate multiple environmental stimuli into a unified transcriptional response. To further examine the role of the AP-1 site and CREs in the NT/N promoter, reporter genes containing either a single or multiple AP-1 or CRE sites were expressed in PC12 cells and protein kinase A-deficient PC12 cells treated with forskolin, NGF, and lithium, either individually, or in combination. The results indicated that lithium and NGF markedly activate promoters containing multiple AP-1 sites, but not a single site, and that these effects were additive. Both agents potentiated forskolin-induced activation of promoters containing a single or multiple CREs, but had no effect, individually. Also, in contrast to the activation of multiple AP-1 sites by lithium and NGF, activation of the NT/N promoter and promoters containing CREs is absolutely dependent on protein kinase A activity. These results suggested that promoters containing multiple AP-1 sites, or a single AP-1 site in the context of nearby active CREs, are selectively activated by lithium and NGF in PC12 cells. Based on the results of this thesis I have proposed a model to account for the complex transcriptional regulation of the NT/N gene in PC12 cells. I have also addressed the relevance of these findings to the mechanisms of phenotypic plasticity of embryonic neural crest cells, NGF-induced neuronal differentiation, and the pharmacological actions of lithium.

In diverse eukaryotic organisms, double-stranded RNA (dsRNA) induces robust silencing of cellular RNA cognate to either strand of the input dsRNA; a phenomenon now known as RNA interference (RNAi). Within the RNAi pathway, small, 21 nucleotide (nt) duplexed RNA, dubbed small interfering RNAs (siRNAs), derived from the longer input dsRNA, guide the RNA induced silencing complex (RISC) to destroy its target RNA. Due to its ability to silence virtually any gene, whether endogenous or exogenous, in a variety of model organisms and systems, RNAi has become a valuable laboratory tool, and is even being heralded as a potential therapy for an array of human diseases. In order to understand this complex and unique pathway, we have undertaken the biochemical characterization of RNAi in the model insect, Drosophila melanogaster. To begin, we investigated the role of ATP in the RNAi pathway. Our data reveal several ATP-dependent steps and suggest that the RNAi reaction comprises as least five sequential stages: ATP-dependent processing of double-stranded RNA into siRNAs, ATP-independent incorporation of siRNAs into an inactive ~360 kDa protein/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active complex, ATP-dependent activation of RISC following siRNA unwinding, and ATP-independent recognition and cleavage of the RNA target. In addition, ATP is used to maintain 5´ phosphates on siRNAs, and only siRNAs with these characteristic 5´ phosphates gain entry into the RNAi pathway. Next, we determined that RISC programmed exogenously with an siRNA, like that programmed endogenously with microRNAs (miRNAs), is an enzyme. However, while RISC behaves like a classical Michaelis-Menten enzyme in the presence of ATP, without ATP, multiple rounds of catalysis are limited by release of RISC-produced cleavage products. Kinetic analysis of RISC suggests that different regions of the siRNA play distinct roles in the cycle of target recognition, cleavage and product release. Bases near the siRNA 5´ end disproportionately contribute to target RNA-binding energy, whereas base pairs formed by the central and 3´ region of the siRNA provide helical geometry required for catalysis. Lastly, the position of the scissile phosphate is determined during RISC assembly, before the siRNA encounters its RNA target. In the course of performing the kinetic assessment of RISC, we observed that when siRNAs are designed with regard to 'functional asymmetry' (by unpairing the 5´ terminal nucleotide of the siRNA's guide strand, i.e. the strand anti-sense to the target RNA), not all of the RISC formed was active for target cleavage. We observed, somewhat paradoxically, that increased siRNA unwinding and subsequent accumulation of single-stranded RNA into RISC led to reduced levels of active RISC formation. This inactive RISC did not act as a competitor for the active fraction. In order to characterize this non-cleaving complex, we performed a series of protein-siRNA photo-crosslinking assays. From these assays we found that thermodynamic stability and termini structure plays a role in determining which proteins an siRNA will associate with, and how association occurs. Furthermore, we have found, by means of the photo-crosslinking assays, that siRNAs commingle with components of the miRNA pathway, particularly Ago1, suggesting overlapping functions or crosstalk for factors thought to be involved in separate, distinct pathways.

Transcription of the osteocalcin gene, which encodes a bone-specific 10 kDa protein, is controlled by the coordinated utilization of modularly organized basal and hormone-responsive enhancer elements. Activation of these sequences involves the interaction of specific transcription factors to these promoter elements. It is becoming increasingly accepted that nuclear architecture provides a basis for support of tightly regulated modulation of cell growth and tissue-specific transcription which is required for the onset and progression of differentiation. Thus packaging of DNA as chromatin can facilitate the cooperative interaction between activities of independent regulatory elements that contribute to the level of transcription. Here, we show that a specific nucleosomal organization supports the constitutive expression of the osteocalcin gene in ROS 17/2.8 rat osteosarcoma cells and that chromatin remodeling directly correlates with the developmentally regulated transcriptional activation of this gene in normal diploid osteoblasts. By combining DNase I, micrococcal nuclease, and specific restriction endonuclease digestion analysis, we observed that the presence of DNase I hypersensitive sites (proximal: -170 to -70, and distal: -600 to -400) and a selective nucleosome positioning over the osteocalcin gene promoter are directly associated with developmentally stage-specific transcriptional activation in bone-derived cells. In addition, we found that chromatin hyperacetylation prevents a key transition in the chromatin structure which is required for the formation of the distal DNase I hypersensitive site. This transition involves the interaction of specific nuclear factors and is necessary for the subsequent ligand-dependent binding of the vitamin D receptor complex. Finally, we have established a requirement for sequences residing in the proximal region of the osteocalcin gene promoter for both formation of the proximal hypersensitive site and basal transcriptional activity. Our approach was to assay nuclease accessibility in ROS 17/2.8 cell lines stably transfected with promoter deletion constructs driving expression of a CAT reporter gene.

Este trabalho tem como objetivo descrever as principais doenças diagnosticadas em caprinos no Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul no período de 2000 a 2016. No primeiro artigo foi realizado um estudo retrospectivo das causas de morte em caprinos diagnosticadas de 2000 a 2016. Do total de 322 caprinos necropsiados neste período, 290 (90%) obtiveram um diagnostico conclusivo. Dos casos conclusos, 167 (57,6%) corresponderam a enfermidades de origem infecciosa e toxi-infecciosas e 123 (42,4%) enquadrados em causas não infecciosas. Entre as doenças infecciosas foram contabilizados 54 casos de origem bacteriana, 60 casos com envolvimento parasitário, 14 casos de origem viral, além de 39 casos toxi-infecciosos. As doenças de caráter não infeccioso foram ainda agrupadas em doenças metabólicas (44 casos), intoxicações por plantas ou substâncias tóxicas (36), deficiências minerais e nutricionais (20), neoplasias e distúrbios no desenvolvimento (5). A hemoncose, eimeriose, pleuropneumonias e a enterotoxemia foram as doenças mais frequentemente diagnosticadas neste período. O segundo artigo descreve um surto de tuberculose em caprinos jovens. Onze de um total de 15 caprinos, de cinco a 15 dias de idade, foram positivos ao teste de tuberculina. Na necropsia, o parênquima pulmonar de todos os caprinos positivos apresentavam nódulos de 0,3 a 10 cm de diâmetro, coloração brancacenta a amarelada, ocasionalmente, também observados no fígado e baço Os linfonodos retrofaríngeos, mediastínicos e traqueobrônquicos apresentavam-se acentuadamente aumentados de tamanho e aspecto caseoso. Na avaliação histológica, a lesão era caracterizada por intensa necrose caseosa, com áreas de mineralização distrófica, associados a acentuado infiltrado inflamatório granulomatoso. A coloração de Ziehl-Neelsen e a marcação por imuno-histoquímica anti-complexo Micobacterium tuberculosis evidenciou discreta a moderada quantidade de bacilos álcool-ácido resistentes. O cultivo microbiológico e a análise molecular confirmaram o agente etiológico M. bovis. O terceiro artigo descreve dermatite e hepatopatia tóxica crônica natural e experimental em caprinos associadas ao consumo de farelo de arroz desengordurado. Caprinos jovens, de um a quatro meses de idade, apresentavam alopecia e formações crostosas na pele, apatia, emagrecimento, prurido discreto e, vinham a óbito em um período de 30-40 dias. À necropsia, o fígado apresentava irregularidades na superfície capsular, coloração alaranjada a avermelhada, além de rins com múltiplas áreas circulares brancacentas na superfície capsular. À análise microscópica, acentuada atrofia de hepatócitos em região periportal hepática e moderada degeneração hepatocelular microvacuolar. No estudo experimental comprovou-se a etiologia dos casos, através da manifestação de lesões de pele, hepática e renais similares ao dos casos naturais, entretanto em menor intensidade. / This work aims to describe the main diseases diagnosed in goats in the Sector of Veterinary Pathology of the Federal University of Rio Grande do Sul from 2000 to 2016. The first article describes the main causes of death in goats diagnosed between 2000 and 2016. A conclusive diagnosis was obtained in 290 (90%) cases from a total of 322 goats necropsied. Of these cases, 167 (57.6%) corresponded to infectious and toxi-infectious diseases, and 123 (42.4%) included non-infectious causes. Among the infectious diseases 54 cases were of bacterial origin, 60 cases were caused by parasite agents, 14 cases of viral origin, and 39 toxi-infectious cases. Non-infectious diseases were also grouped into metabolic diseases (44 cases), poisoning by plants or toxic substances (36), mineral and nutritional deficiencies (20), neoplasms and developmental disorders (5). Haemonchosis, eimeriosis, pleuropneumonia and enterotoxemia remain as one of the major control obstacles in goat farms. The second article describes an outbreak of tuberculosis in goat kids. Eleven of a total of 15 kids, from 5 to 15 days old, were positive to tuberculin. At necropsy, the pulmonary parenchyma of all positive goats had white to yellowish nodules of 0.3 to 10 cm in diameter, that were occasionally also observed in the liver and spleen The retropharyngeal, mediastinal and tracheobronchial lymph nodes were markedly enlarged and with a caseous aspect. Histologically, the lesion was characterized by an intense caseous necrosis, with areas of dystrophic mineralization, associated to a marked granulomatous inflammatory infiltrate. Ziehl-Neelsen histochemistry exam and immunohistochemical anti-Micobacterium tuberculosis complex evidenced mild to moderate amount of bacilli. Microbiological culture and molecular analysis confirmed M. bovis as the etiological agent. The third article describes a natural and an experimental toxic liver disease associated with the consumption of defatted rice bran in goats. These presented with alopecia and crusted formations on the skin, apathy, weight loss, mild pruritus, and death within a period of 30-40 days. At necropsy, the liver presented multifocal to coalescing orange to reddish irregular areas on the capsular surface, and the kidneys presented multiple white circular areas on the capsular surface. Microscopic analysis revealed a marked hepatocyte atrophy at the hepatic periportal region, and a moderate microvacuolar hepatocellular degeneration. In the experimental study, the etiology of the cases was demonstrated through the manifestation of lower intensity skin, liver and kidney lesions similar to those of the natural cases.

Bile acid metabolism is integrally involved in cholesterol homeostasis in mammals because it is the major means by which cholesterol is eliminated from the body. We have undertaken an effort to study the molecular mechanisms underlying the regulation of bile acid metabolism by isolating and characterizing the cDNA and gene for an enzyme that hydroxylates lithocholic acid (LCA) at position 6β, lithocholic acid 6β-hydroxylase; the first bile acid-induced gene reported. LCA is a very hydrophobic, toxic bile acid formed from chenodeoxycholic acid in the gut lumen upon reduction of the 7α-hydroxy group by microbial enzymes. The proper elimination of LCA is essential for maintenance of the bile acid pool and for prevention of cholestasis which results from LCA precipitating in the cannaculi of the liver when its concentration is high. The LCA 6β-hydroxylase cDNA was isolated by differential hybridization of hamster liver libraries prepared from animals fed either a cholic acid enriched diet or a cholestipol-rich chow and was named CYP3A10 based on its homology with other cytochrome P450s (P450) in family 3A. We found that CYP3A10 was essentially expressed only in males. A statistical analysis of RNA from young males fed with cholic acid and normal chow showed that the cholic acid induction was about 50% at the RNA level. We determined the biological nature of the protein encoded by CYP3A10 by expression of the cDNA in COS cells. Microsomes prepared from transfected cells were assayed with LCA as a substrate and found to hydroxylate LCA predominantly at position 6β. We examined whether CYP3A10 could hydroxylate other steroid compounds by assays with testosterone, progesterone and androstenedione and found that, although 6β-hydroxylase (as well as others) activity was observed with all three, LCA was the preferred substrate based on kinetic analysis. A developmental time course of CYP3A10 expression in males showed little expression before puberty, a striking induction of expression at puberty and a fourfold induction thereafter through adulthood. We then examined the male-specific expression of CYP3A10 in hamster liver. We disrupted the pattern of GH secretion in male hamsters by hypophysectomy, neonatal glutamate treatment and by continuous infusion of GH via osmotic minipumps (to mimic the female pattern of GH secretion) and found no significant effect on CYP3A10 expression. Conversely, in females, hypophysectomy and neonatal glutamate treatment significantly induced CYP3A10 expression 5- to 10-fold. Additionally, when females treated neonatally with glutamate were injected twice daily with GH as adults (to mimic the male pattern of GH secretion), the levels of CYP3A10 expression were not significantly different from those of normal males. These results led us to conclude that the pattern of GH secretion in males does not control the male-specific expression of CYP3A10 but that in females expression can be induced by altering the tonic secretion of GH. No significant effect on CYP3A10 expression was observed by castration of adult males, indicating that circulating androgens were not required for expression. We found that gonadal hormones (e.g. estrogen and progesterone) do not have a suppressive effect on CYP3A10 expression in females since ovariectomy did not induce expression. Many genes are "imprinted" neonatally by exposure to a given effector for developmental-, tissue- or sexually regulated expression. We investigated whether neonatal androgen exposure was required for male-specific expression of CYP3A10 by castrating hamsters neonatally and determining the level of CYP3A10 expression in adulthood. Our results indicate that androgens are required neonatally for CYP3A10 expression since no expression was observed in neonatally castrated hamsters. We were unable to induce expression in neonatally castrated hamsters by either GH or testosterone injections. These results suggest several notable points 1) that CYP3A10 expression is programmed neonatally by androgen exposure; 2) that androgens exert their effect directly on the liver and not via the hypothalamus; 3) that neither testosterone nor GH can restore CYP3A10 expression when males have not been exposed to androgens neonatally; and 4) that in experimental conditions, females can be induced to express CYP3A10, which indicates that there are two modes for regulating expression: by "imprinting" in males and by GH and testosterone in females. We are now studying the molecular mechanisms involved in the bile acid-mediated induction and the male-specific expression of CYP3A10. We have cloned approximately 8 kb of 5' flanking DNA from a hamster genomic library and sequenced about 1 kb of proximal DNA. Primer extension and S1 digestion analyses indicate that the mRNA for CYP3A10 has multiple transcription initiation sites clustered about 90 bp from the initiator methionine codon. We have also prepared CYP3A10 promoter/lacZ chimeric constructs to begin delineating the cis-acting elements controlling CYP3A10 expression and regulation. We used H2.35 cells as recipients because they are a mouse hepatocyte cell line that has been transformed with a temperature sensitive SV40. These cells can be grown at the permissive temperature and can be induced to behave like liver cells, the differentiated condition, by switching to a nonpermissive temperature. We have found that the construct with 1 kb of proximal CYP3A10 5' flanking DNA was able to express the reporter gene at higher levels under differentiated conditions, which were consistent with higher expression of an albumin promoter/lacZconstruct, upon switching the cells to the more liver phenotype. The system characterized and described here is ideally suited for dissecting the molecular details governing bile acid-mediated regulation and sexually dimorphic expression of liver genes. Very little is known about both these very important biological phenomena. Much could be learned about transcriptional regulation of liver genes by investigating the cis-elements and trans-acting factors mediating regulation of CYP3A10 expression.

During embryonic development of the nematode Caenorhabditis elegans, cell fates are specified by asymmetric segregation of cell fate determinants and via cell-cell signaling events. Specification of the eight-cell stage blastomere E, the endoderm progenitor cell, requires both cell signaling and asymmetric cell division. At the four-cell stage, a polarity-inducing signal from the P2 cell is required for the EMS cell to divide asymmetrically to produce an anterior daughter MS, and posterior daughter E. In the absence of signal, the EMS cell divides symmetrically to produce two daughters that adopt the MS fate. This thesis describes the identification and analyses of seven genes required to tranduce this polarity-inducing signal and specify endoderm formation. The mom-1, mom-2, mom-5, apr-1, and wrm-1 genes are homologous to components of the Wnt/Wingless signal transduction pathway, and the mom-4, and lit-1 genes are related to components of the mitogen-activated protein kinase pathway. Biochemical analysis of these signaling molecules reveal a novel convergence of these pathways at the level of the LIT-1 and WRM-1 proteins, which appear to function as a kinase complex and are required for the downregulation of POP-1. Together these genes constitute components of a complex genetic pathway required for specification of the E cell fate.