Thesis (Ph. D.)--University of Missouri-Columbia, 2006. / Title from title screen of research.pdf file (viewed on December 22, 2006). The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "May 2006" Includes bibliographical references.

In order to identify new players in fruit development, a forward genetic screen was performed on EMS mutagenized plants. A mutant named elongated gynophore (egy) was identified in the M2 population based on altered fruit morphology. Genetic analysis established that the egy phenotype is due to a monogenic and recessive mutation. The egy plants show additional developmental defects including shorter root, narrower cotyledons and malformed leaf lamina. Molecular mapping and whole genome sequencing analyses showed a G/C deletion at the position 4414980 on the AT5G13680 gene locus which is predicted to encode the ELONGATA2 (ELO2) protein. ELO2 is a constituent member of the elongator complex which helps in transcriptional elongation in association with the phosphorylated form of RNA polymerase II. This complex has been implicated in controlling development, abiotic stress and biotic stress. Genetic complementation test confirmed that egy is indeed allelic to elo2-3. Surprisingly, the EGY overexpression line 35S::EGY showed loss-of-function phenotype, suggesting transgene silencing. In angiosperms, fruit is derived from the fertilized ovary. The initiation of the female reproductive organ commences with a lump of cells which eventually develops into the gynoecium with a stigma, a style, two fused ovaries and a gynophore, arranged from the apical to basal axis in that order. Genetic networks faithfully shapes up the carpel primordium into predetermined gynoecium shape. Following fertilization, siliques elongate concomitantly with developing embryos. Here we show that the egy mutant has apical basal patterning defect with longer gynophore at the base. This gynophore phenotype resembles the phenotype found in the mutants with altered auxin and cytokinin levels/signaling. We show that egy is hypersensitive to cytokinin treatment; egy fruits treated with cytokinin display phenotype similar to the plants expressing IPT7 under fruit-specific promoter. These results suggest that broadened shoulders at the apical region of egy gynoecium possibly results from higher cytokinin level/response. Genetic interaction studies have shown that EGY act independent of AGAMOUS and PEAPOD to suppress the medio-lateral growth of the apical gynoecium region. Genetic and expression studies suggest that PINOID and TMO5/T5L1 work downstream to EGY, while ETTIN acts in parallel to EGY. We also observed larger seeds in the egy mutant and show that this is controlled maternally. Thus, the gametic lethality in egy can possibly be accounted for by the defective ovules. We show that egy primary roots are shorter compared to Col-0, though egy seeds have longer embryonic root to begin with, suggesting a defect in cell division. The root cells are arranged radially in a stereotypic pattern in root meristem from the outer epidermis to the inner stele specific the vascular bundles. The four QC cells are also surrounded by stem cells of various identities. This stereotypic pattern of cell arrangement is perturbed in the egy root. The stele, composed of pericycle and vascular bundles is reduced in the egy mutant, suggesting a positive role of EGY in vascular cell division. Confocal microscopic studies and real-time PCR data suggest that TMO5/T5L1 work downstream to EGY. Thus, the Arabidopsis „ELONGATOR‟ complex regulates the transcription of target genes that are necessary for plant growth and development. A proposed genetic network for the role of EGY in fruit and root development. Based on the genetic interaction studies and expression analysis, we have placed EGY in the existing molecular network that control fruit and root vascular development in Arabidopsis

Made available in DSpace on 2015-03-26T13:36:37Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1316896 bytes, checksum: 78d0e7e529325c1fb43cef3913f21394 (MD5) Previous issue date: 2008-06-28 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The purpose of this work was to evaluate the physiological characteristics of ripening and to describe the potential conservation of fresh-cut Firme and Alambra mutant tomatoes (long life), in relation to Santa Clara (normal) and F1 (Firme x Santa Clara) tomatoes. Fruits were harvested at ripening stage 4 and kept for 15 days at 12 ºC, except for Alambra, harvested at stage 3 and kept at room temperature until reaching stage 4, prior to conservation at 12°C. Color, carotenoids and firmness analyses showed that all fruits acquired the red color and ripened simultaneously during the conservation period. Firme tomatoes presented lower percentage of total soluble solids and higher carotenoids content than the other genotypes. Processing of tomatoes was as follows: fruits were sanitized, cut in slices 5 mm thickness, drained, placed in polypropylene stoppered jars, and kept at 5°C for 15 days. Initial firmness of the slices was low as compared to whole fruits, and there were small changes in slices during conservation. Carotenoids and color increase indicated fresh-cut genotypes ripened during the conservation period at 12°C. Alambra and F1 sliced tomatoes presented higher percentages of exudation than sliced Santa Clara and Firme tomatoes. Exudation showed to be the main factor responsible for the loss of fresh mass, and seemed to have contributed to the rapid loss of desirable appearance. Tests of acceptance for color, texture, aroma and flavor, indicated acceptance of Santa Clara and Firme slices until the sixth day of conservation. However, loss in visual appearance of slices was faster and more remarkable than quality losses observed by means of acceptance tests. Microbial counts during conservation of fresh-cut tomatoes did not exceed 104 CFU.gFW-1 of psychrotrophic bacteria and fungi. Thus, microbial growth was not the cause for freshcut tomatoes deterioration. Under the present experimental conditions, the studied genotypes did not differ in their potential conservation as fresh-cut products. Alambra and F1 seemed to be less suitable for fresh-cut processing. Comparatively, Firme seems to have some potential for use as fresh-cut tomato; however, this needs improvement in the processing and conservation techniques. / O objetivo do trabalho foi avaliar características fisiológicas como ferramenta para descrever o potencial de conservação dos tomates mutante Firme e Alambra (longa vida) minimamente processados, em relação aos genótipos Santa Clara (normal) e F1 (Firme x Santa Clara). Frutos dos genótipos foram colhidos no estádio 4 de amadurecimento e conservados inteiros por 15 dias a 12ºC, exceto para Alambra, que foram colhidos no estádio 3 e mantidos em temperatura ambiente até atingir o estádio 4. Análises de cor, carotenóides e firmeza mostraram que todos os genótipos inteiros adquiriram cor vermelha e amadureceram simultaneamente ao longo dos 12 dias de conservação. O Firme apresentou menores porcentagens de sólidos solúveis totais e maiores teores de carotenóides totais em relação aos demais genótipos. Tomates inteiros foram sanitizados, fatiados em rodelas de 5 mm de espessura, drenados, acondicionados em potes de polipropileno e mantidos a 5°C, por 15 dias. A firmeza inicial das fatias foi menor em relação àquela apresentada pelos frutos inteiros e houve pequena alteração subseqüente na firmeza. O aumento nos teores de carotenóides e da cor indicou que todos os genótipos processados amadureceram ao longo dos 12 dias de conservação. Os tomates Alambra e F1 apresentaram elevadas porcentagens de exsudação em relação aos genótipos Santa Clara e Firme. A exsudação foi o principal fator que contribuiu para perda de massa fresca, e parece ter contribuído também para rápida perda da aparência satisfatória. Testes de aceitação para cor, textura, aroma e sabor indicaram aceitação do Santa Clara e Firme até o sexto dia de conservação. Todavia, mudanças na aparência das fatias foram mais rápidas e notáveis do que àquelas observadas nos testes de aceitação. A contaminação dos tomates minimamente processados não ultrapassou 104 UFC g MF-1 de psicrotróficos e fungos e, portanto, o crescimento microbiano não foi a causa da deterioração de tomates minimamente processados. Nas condições experimentais, os genótipos estudados não diferiram quanto à conservação, contudo, Alambra e F1 mostraram-se menos adequados para o processamento mínimo. Firme tem potencial para utilização na forma minimamente processada, o que, entretanto necessita de avanços nas técnicas de processamento e conservação.

Resumo: Este trabalho objetivou avaliar a anatomopatologia, o hemograma e o perfil bioquímico sérico de aves de postura inoculadas por Salmonella Gallinarum (SG) contendo os genes cobS e cbiA inoperantes (SGcobScbiA) que mostrou ser avirulenta em trabalhos anteriores, comparando-a com cepas virulentas SGNalr e SGcobS, para mostrar se SGcobScbiA pode ser componente de vacina contra cepas selvagens de SG e S.Enteritidis. 280 pintainhas foram distribuídas em 4 grupos (G); G1 (SGcobS), G2 (SGNalr), G3 (SGcobScbiA) e G4 (controle). Com exceção do G4, os grupos receberam 0,2 mL de suas respectivas cepas contendo aproximadamente 108 UFC/mL de inóculo, aos 5 dias de idade. A eutanásia foi realizada 24h antes (1DAI) e após a inoculação (1DPI), e 3 (3DPI), 5 (5DPI), 7 (7DPI), 10 (10DPI) e 15 (15DPI) dias após a administração do inóculo, sacrificando-se, em cada momento, dez aves de cada grupo. As aves foram sacrificadas, obtendo-se amostras de sangue utilizadas para os exames hematológicos e bioquímicos. Fragmentos de fígado, baço, timo, bursa de Fabricius, rins e coração foram destinados aos exames histológicos. As aves inoculadas com a cepa SGcobS tiveram comportamento semelhante às aves inoculadas por SGNalr, porém com algumas respostas diferentes nos exames hematológicos e bioquímicos. As aves inoculadas com a cepa SGcobScbiA tiveram comportamento semelhante ao grupo controle, entretanto foi verificado alterações brandas em alguns parâmetros, mostrando que estudos futuros devem ser feitos, verificando se as alterações constatadas não irão interferir no desempenho de aves vacinadas com a cepa SGcobScbiA. / Abstract: The aim of the present study was to evaluate anatomopathology, hemogram and blood serum components of commercial layers experimentally inoculated with SGcobScbiA, which is a Salmonella Gallinarum (SG) strain it shows attenuation of the virulence in previous research and it was compared with high virulence SGNalr and SGcobS strains in order to show if SGcobScbiA has potential to be use as a vaccine against SG and S. Enteritidis wild strains. 280 commercial layers were divided into 4 groups (G); G1 (SGcobS), G2 (SGNalr), G3 (SGcobScbiA) and G4 (control group). With exception of G4, all the other groups received 0,2 mL of their respective strain containing about 108 CFU/mL of the inoculum with five days of age. Birds were sacrificed 24 hours before (1DBI) and 24 hours after the inoculation (1DAI), and three (3DAI), five (5DAI), seven (7DAI) ten (10DAI), and fifteen (15DAI) days after the administration of the inoculum, slaughtering ten birds at a time in each group. Birds were submitted to euthanasia and blood samples were collected in order to make the hematological and blood serum components test. Samples of liver, spleen, thymus, bursa of Fabricius, kidneys and heart were collected for the histological test. The birds inoculated with SGcobS strain had similar behavior when compared with that ones who received SGNalr strain, however some different responses in the hematological and blood serum components were found. On the other hand, the birds inoculated with SGcobScbiA strain had similar behavior when compared with the control group, however, lower alterations in some parameters were found. Further studies must be done to verify if these alterations will not interfere in the performance of the vaccinate birds with SGcobScbiA strain. / Orientador: Ângelo Berchieri Júnior / Coorientador: José Jurandir Fagliari / Banca: Antonio Carlos Alessi / Banca: Raimundo Souza Lopes / Mestre

A major challenge in biology is to understand and predict the effect of mutations on protein structure, stability and function. Chapter 1 provides a general introduction on protein sequence-structure relationships and use of the CcdAB toxin-antitoxin system as a model to study molecular determinants of mutant phenotypes. In Chapter 2, we describe the use of saturation mutagenesis combined with deep sequencing to determine phenotypes for 1664 single-site mutants of the E. coli cytotoxin, CcdB. We examined multiple expression levels, effects of multiple chaperones and proteases and employed extensive in vitro characterization to understand how mutations affect these phenotypes. While general substitution preferences are known, eg polar residues preferred at exposed positions and non-polar ones at buried positions, we show that depth from the surface is important and that there are distinctly different energetic penalties for each specific polar, charged and aromatic amino acid introduced at buried positions. We also show that over-expression of ATP independent chaperones can rescue mutant phenotypes. Other studies have primarily looked at effects of ATP dependent chaperone expression on phenotype, where it is not possible to say whether mutational effects on folding kinetics or thermodynamic stability are the primary determinant of altered phenotypes, since there is energy input with these chaperones. The data suggest that mutational effects on folding rather than stability determine the in vivo phenotype of CcdB mutants. This has important implications for efforts to predict phenotypic effects of mutations and in protein design. While looking at the mutational landscape of a given gene from an evolutionary perspective, it is important to establish the genotype-phenotype relationships under physiologically relevant conditions. At the molecular level, the relationship between gene sequence and fitness has implications for understanding both evolutionary processes and functional constraints on the encoded proteins. Chapter 3 describes a methodology to test the fitness of individual CcdB mutants in E.coli over several generations by monitoring the rate of plasmid loss. We also propose a methodology for high throughput analysis of a pool of CcdB mutants using deep sequencing to quantitate the relative population of each mutant in a population of E.coli cells, grown for several generations and build the fitness landscape. While the F-plasmid based CcdAB system is known to be involved in plasmid maintenance through post-segregational killing, recent identification of ccdAB homologs on the chromosome, including in pathogenic strains of E.coli and other bacteria, has led to speculations on their functional role on the chromosome. In Chapter 4, we show that both the native ccd operon of the E.coli O157 strain as well as the ccd operon from the F- plasmid when inserted on the E.coli chromosome lead to protection from cell death under multiple antibiotic stress conditions through formation of persisters. Both the ccdF and ccdO157 operons may share common mechanisms for activation under stress conditions and also display weak cross activation. The chromosomal toxin shows weaker activity as compared to the plasmidic counterpart and is therefore less efficient in causing cell death. This has important implications in generation of potential therapeutics that target these TA systems. Chapter 5 describes the use of site-saturation mutagenesis coupled with deep sequencing to infer mutational sensitivity for the intrinsically disordered antitoxin, CcdA. The data allows us to make comparisons between overall as well as residue specific mutational sensitivity patterns with that of globular proteins, like CcdB (described in Chapter 2) and study toxin- antitoxin interaction and regulation through saturation suppressor mutagenesis. Interestingly, we found several examples of synonymous point mutations in CcdA that lead to loss of its activity. In Chapter 6 we attempt to explore the molecular bases for some of these synonymous mutations. In most cases the mutated codon has a similar overall codon preference to the WT one. Initial findings suggest a change in mRNA structure leading to change in CcdB: CcdA ratio, thereby causing cell death. These observations have important implications, because TA systems are ubiquitous, highly regulated and are known to be involved in multiple functions including drug tolerance. However a role for RNA structure in their regulation has not been shown previously. Appendix–I lists the mutational sensitivity scores for the CcdB mutants. Phenotypes for CcdA mutants obtained through deep sequencing have been tabulated in Appendix-II. Overall, we provide extensive datasets for mutational sensitivities of a globular (CcdB) and an intrinsically disordered protein (CcdA). Exploration of the molecular determinants of these mutant phenotypes not only provides interesting insights into CcdAB operon function but is also useful in understanding various aspects of protein stability, folding and activity as well as regulation of gene expression in bacteria.

Mutation testing is a powerful, but resource intense technique for asserting software quality. This report investigates two claims about one of the mutation operators on procedural logic, the relation operator replacement (ROR). The constrained ROR mutant operator is a type of constrained mutation, which targets to lower the number of mutants as a “do smarter” approach, making mutation testing more suitable for industrial use. The findings in the report shows that the hypothesis on subsumption is rejected if mutants are to be detected on function return values. The second hypothesis stating that a test case can only detect a single top-level mutant in a subsumption graph is also rejected. The report presents a comprehensive overview on the domain of mutation testing, displays examples of the masking behaviour previously not described in the field of mutation testing, and discusses the importance of the granularity where the mutants should be detected under execution. The contribution is based on literature survey and experiment. The empirical findings as well as the implications are discussed in this master dissertation.

Le système endocannabinoïde est un important modulateur des fonctions physiologiques. Dans le cerveau, son contrôle s’exerce essentiellement par les récepteurs cannabinoïdes de type 1 (CB1). Les récepteurs CB1 sont abondamment exprimés sur les neurones excitateurs glutamatergiques et les interneurones inhibiteurs GABAergiques et leur stimulation inhibe la libération du glutamate et du GABA. Récemment, l’activité des récepteurs CB1 sur les astrocytes a été proposée comme facilitant la transmission excitatrice. Par ce contrôle général de la neurotransmission, l’activité des récepteurs CB1 induit différents phénomènes de plasticté synaptique associés aux processus de mémoire. Les récepteurs CB1 jouent un rôle complexe dans les fonctions de mémoire. En particulier, la stimulation exogène des récepteurs CB1 perturbe la mémoire de travail. D’autre part, la signalisation endogène des récepteurs CB1 est nécessaire à l’adaptation des réponses de peur apprises. Cependant, les mécanismes par lesquels les récepteurs CB1 régulent ces processus de mémoire n’ont été que peu analysés. L’objectif de ce travail fut de caractériser les mécanismes cellulaires par lesquels les récepteurs CB1 contrôlent la mémoire de travail et les réponses de peur apprises. Nous avons utilisé les modèles de mutation constitutive et conditionnelle des récepteurs CB1 chez la souris afin d’analyser les conséquences de la délétion de ces récepteurs sur des types cellulaires particuliers. Dans une première étude, nous avons montré que les cannabinoïdes exogènes tels que le Δ9-tetrahydocannabinol (THC, principal composé psychoactif du cannabis) induisent des déficits de mémoire de travail spatiale par la stimulation des récepteurs CB1 exprimés sur les astrocytes. Les cannabinoides induisent une forme de dépression à long-terme dans l’hippocampe dont plusieurs mécanismes cellulaires sont similaires à ceux supportant les déficits de mémoire mis en évidence par l’analyse comportementale. Ces résultats suggèrent que les cannabinoïdes altèrent la mémoire de travail spatiale par une modification de la plasticité synaptique de l’hippocampe induite par la stimulation des récepteurs CB1 astrogliaux. Dans une seconde étude, nous avons mis en évidence que les récepteurs CB1 localisés sur les neurones GABAergiques et glutamatergiques exercent un contrôle opposé sur le type de réponse élicité par un stimulus conditioné aversif. La ré-expression sélective des récepteurs CB1 dans l’amygdale des souris mutantes constitutives a permis de préciser l’implication de cette structure dans la régulation des réponses de peur conditionnées par les récepteurs CB1.L’ensemble de ces travaux indiquent que le système endocannabinoïde contrôle les fonctions de mémoire par une régulation de l’activité de cellules spécifiques dans le cerveau. L’implication des astrocytes dans les effets des cannabinoïdes sur la mémoire souligne l’importance de ces cellules dans les processus cognitifs et suggère que les récepteurs CB1 astrogliaux jouent un rôle dans d’autres fonctions cérébrales. Nos résulats révèlent également l’importance de l’évaluation de différents comportements dans le cadre des modèles expérimentaux d’adaptation à la peur. / The endocannabinoid system is an important regulator of physiological functions. In the brain, this control is mainly exerted through the type-1-cannabinoid (CB1) receptors. CB1 receptors are abundant at excitatory glutamatergic and inhibitory GABAergic neuron terminals where their stimulation inhibits neurotransmitter release. The activity of CB1 receptors on astrocytes has been recently proposed as facilitating excitatory transmission. Through this general control on brain neurotransmission, CB1 receptors mediate distinct forms of synaptic plasticity that are associated with memory processing. Indeed, CB1 receptors control memory functions. In particular, the exogenous stimulation of CB1 receptors impairs working memory. Moreover, the endogenous CB1 receptor signalling ensures the adaptation of learned fear responses. However, the brain mechanisms of this CB1-mediated control of memory functions are poorly characterized. The goals of this research work were to dissect the cellular mechanisms by which CB1 receptors control both working memory and learned fear responses. We used constitutive and conditional mutagenesis in mice to address the roles of CB1 receptors on particular cell types in these functions. We first showed that exogenous cannabinoids, including Δ9-tetrahydocannabinol (THC, the main psychoactive constituent of cannabis), impairs spatial working memory through the stimulation of astroglial CB1 receptors. Cannabinoids also induce a form of in vivo long-term depression in the hippocampus that shares several cellular mechanisms with the cannabinoid-induced working memory impairments. These results suggest that cannabinoids disrupt spatial working memory by altering hippocampal synaptic plasticity through astroglial CB1 receptor stimulation. We then showed that CB1 receptors expressed on GABAergic and glutamatergic neurons oppositely control fear coping strategies in the presence of fear conditioned stimuli. The selective and local re-expression of CB1 receptors in the amygdala of constitutive CB1 mutant mice allowed to precise the involvement of this brain structure in the regulation of conditioned fear responses by CB1 receptors. Altogether, these studies indicate that the endocannabinoid system differentially controls memory functions through its distinct modulation of the activity of specific brain cells. The involvement of astrocytes in the effects of cannabinoids on memory highlights their key roles in cognitive processes and further suggests that astroglial CB1 receptors might play a role in other high order brain functions. Our results also point the importance of performing thorough behavioral analyses in the experimental models of fear adaptation.

O mutante redA de Dictyostelium discoideum, obtido por inativação gênica aleatória, tem crescimento aparentemente normal, porém seu ciclo de desenvolvimento não progride além do estágio de agregados compactos. Neste trabalho relatamos a caracterização deste mutante, cujo gene defeituoso codifica a enzima NADPH citocromo P450 redutase (NCPR). O principal papel desta enzima é transportar elétrons do NADPH para as várias isoformas do citocromo P450. Um cDNA de 2094 pb que codifica a NCPR de D. discoideum (DdNCPR) foi isolado através da varredura de uma biblioteca de cDNA com uma sonda derivada de um fragmento do gene inativado no mutante redA. A análise da seqüência de aminoácidos deduzida do cDNA DdNCPR revelou que esta codifica uma proteína de 631 aminoácidos com 31% de identidade e 50% de similaridade com a NCPR humana. Verificamos o acúmulo do mRNA da DdNCPR durante fase de crescimento mas durante as fases iniciais do desenvolvimento ocorre significativa diminuição em seus níveis até a formação dos agregados compactos onde o mRNA da NCPR não é detectável. Demonstramos que o gene que codifica a NCPR aparentemente está presente em uma única cópia no genoma de Dictyostelium. Ademais, a análise de outras linhagens mutantes nocautes do gene da NCPR confirmaram que a inativação deste gene está diretamente relacionada ao fenótipo exibido pelo mutante redA-. Contudo, é provável que um ou mais produtos gênicos possam complementar a ausência desta enzima, uma vez que nem a linhagem redA nem as outras linhagens nocautes do gene da NCPR apresentaram alteração na taxa de crescimento e, em algumas circunstâncias experimentais, não exibiram qualquer alteração no ciclo de desenvolvimento. Nossos resultados sugerem, ainda que o bloqueio do desenvolvimento eventualmente observado no mutante redA pode ser devido a um provável papel da NCPR no metabolismo de DIF-1 (fator indutor de diferenciação-1), que parece desempenhar um papel primordial no controle da diferenciação de células pré-talo e células pré-esporo durante o desenvolvimento de D. discoideum. / The Dictyostelium discoideum redA mutant, obtained by random gene inactivation, exhibits normal growth but has its developmental cycle impaired at tight mound stage. In this study we describe the characterization of this mutant whose defective gene encodes the enzyme NADPH cytochrome P450 reductase (NCPR). NCPR is known to play an essential role in the transfer of reducing equivalents from NADPH to various cytochrome P450 isoforms. We isolated a 2094 bp cDNA that encodes D. discoideum NCPR (DdNCPR) by screening a cDNA library using as probe the mutated gene fragment rescued from redA cells. Analysis of the deduced aminoacid sequence of DdNCPR cDNA shows that it encodes a 631 aminoacid protein with 31% of identity and 50% of similarity with human NCPR. Northern blot analysis showed that DdNCPR mRNA levels is maximum during growth phase and decreases at early stages of the development. After slug stage this mRNA is not detectable. D. discoideum has a single copy of NCPR gene and, as shown by analysis of other NCPR knockout mutants, inactivation of this gene is strongly correlated to the redA phenotype. However, redA, as well as the other NCPR knockout strains, do have growth alterations and in some circumstances they do not show the described developmental defects. Thus, it is possible that one or more proteins be able to compensate for the lack of NCPR in these mutants. Our results also suggest that the redA developmental phenotype might play a role of NCPR on the metabolism of DIF-1, a prime candidate for controlling prestalk and prespore cell differentiation during D. discoideum development.

Osteopetrosis is a metabolic bone disease in mammals characterized by a generalized skeletal sclerosis caused by reduced bone resorption. This reduced bone resorption is manifested in afflicted animals by abnormal bone shape, reduced or absent marrow cavities, extramedullary hemopoiesis, abnormal mineral homeostasis and absent or delayed tooth eruption. The available osteopetrotic animal mutations have been a constant source of fruitful investigations concerning the systemic regulation of osteoclastogenesis and bone metabolism. Tooth eruption, on the other hand, is a localized manifestation of the timely activation of bone resorption and bone formation on opposite sides of an erupting tooth. Its rate-limiting step is the speed of bone resorption to form the eruption pathway. In this dissertation, we used two osteopetrotic rat mutations, toothless (tl) and microphthalmia blanc (mib), to investigate the abnormal development of osteoclasts and tooth eruption in mutant rats with an emphasis on the role of systemic and local factors. The significant contributions to this work are listed below. 1. In the toothless rat, a mutation lacking erupted dentition due to severely reduced bone resorption, colony-stimulating factor-1 (CSF-1) promoted tooth eruption but this was delayed compared to normal rats. Eruption was accompanied by changes in the populations of tartrate-resistant acid phosphatase-positive (TRAP+) mononuclear cells in the dental follicle and TRAP+ osteoclasts on adjacent alveolar bone surfaces. These cell populations were dramatically increased in treated mutants compared to untreated tl rats, but the timing of their appearance was delayed compared to normal littermates. This lag in the appearance of osteoclasts and their precursors corresponded to the delay in eruption of first molars in treated tl rats. 2. CSF-1 also accelerated the eruption of molars in normal rats. CSF-1 increased the number of TRAP+ mononuclear cells in the dental follicle and TRAP+ osteoclasts on adjacent alveolar bone surfaces, but had no effect on the timing of their appearance in normal rats. 3. Our data revealed a differential effect on tooth eruption of the growth factors CSF-1 and epidermal growth factor (EGF). CSF-1 accelerated eruption of molars in normal rats, but had no effect on incisor eruption. On the other hand, EGF accelerated incisor eruption; but did not affect molar eruption in normal rats. 4. We have described the mechanism for the transient, mild form of osteopetrosis inherited by mib rats. Mutant animals possess a typical sclerosis at birth, which diminished--but was not resolved--during the first postnatal month. These characteristics are caused by early reductions in osteoclast number and function which improve to normal levels by 4 weeks. Osteoclast numbers were severely reduced in mib rats between birth and 2 weeks, but improved to near normal levels by 4 weeks. Neonatal abnormalities in osteoclast function included reduced staining for the functional enzymes TRAP and TrATPase, decreased levels of mRNA for both TrATPase and CAll, and inability to form a well-developed ruffled border. None of these defects were apparent after the first postnatal month. 5. Finally, we have shown that the dental abnormalities caused by the mild, transient form of osteopetrosis in mib rats are limited to incisor defects and delayed eruption of all teeth. Histologic and radiographic examination of mutant incisors revealed that, contrary to the situation in normal rats, the apex of the incisors of mib rats failed to extend past the first molar region to the third molar. The incisor apex of newborn mib rats was misshaped due to ankylosis of incisor matrices with alveolar bone. This ankylosis was temporary, being resolved by the third postnatal day. The delayed eruption of incisors in mib rats and abnormal shape and occlusion of these teeth in older animals is a consequence of the temporary ankylosis in newborn rats.

MSCAGR ( Plant Production) / Department of Plant Production / See the attached abstract below