Indiana University-Purdue University Indianapolis (IUPUI) / External apical root resorption (EARR) is a common complication of orthodontic treatment. Genetic factors account for approximately 50% of the variation in EARR. Data have indicated variation in histological root resorption associated with orthodontic force (RRAOF) among different inbred strains of mice. Differences in expression of RANKL and OPG were investigated in two strains of mice with different susceptibility to RRAOF using irnmunohistochemistry. Increased localization of RANKL was detected in the tissues surrounding the root of the susceptible strain compared to the resistant strain and the controls. In contrast, increased localization of OPG was found in the tissues surrounding the roots in the resistant A/J strain compared to the susceptible DBA/2J strain. We conclude that differences in the expression of these key bone resorption mediators play a role in determining RRAOF susceptibility. Changes in serum TRAP 5b level in response to orthodontic force were investigated among female A/J, DBA/2J and BALB/cJ mice. The three strains differed in their TRAP positive cell numbers as well as their serum TRAP 5b level at baseline and when treated. A significant increase in the serum TRAP 5b level with treatment was only detected in the RRAOF susceptible DBA/2J strain, and not in RRAOF resistant strains. Our analysis indicates that differences in osteoclast/odontoclast activity play a role in susceptibility to RRAOF that is genetically determined. Serum TRAP 5b levels have a potential role in screening for individuals with greater susceptibility to root resorption. RRAOF was determined for male and female mice of the A/J, DBA/2J and BALB/cJ strains, as well as A/J x DBA/2J and A/J x BALB/cJ crosses. Sex differences were observed among the BALB/cJ strain only, with females more resistant to RRAOF when compared to males. Fis from the A/J x BALB/cJ cross were resistant suggesting that the A/J have dominant resistance alleles, while Fis from the A/J x DBA/2J cross had RRAOF intermediate between their parental A/J and DBA/2J mice, suggesting a polygenic trait. We concluded that the mode of inheritance of RRAOF in mice was polygenic in nature.

Ph.D. / Behavioral Neuroscience / The purpose of the research described in this dissertation was to determine the neural circuits involved with baseline ethanol consumption and increases in ethanol consumption seen in our animal model of ethanol dependency (further described below). The brain region of focus was the central extended amygdala (cEA) since this region has been shown to be involved in baseline consumption and self-administration of ethanol in rats (Hyytia & Koob, 1995; Eiler et al., 2002) and the changes in ethanol consumption induced by chronic intermittent ethanol vapor exposure seen in rats and mice (Funk et al., 2006; Finn et al., 2007). To determine if the cEA is involved in these behavioral phenotypes, the components of the cEA were lesioned separately. These components included the lateral posterior portion of the bed nucleus of the stria terminalis (BNSTLP), the central nucleus of the amygdala (CeA) and the nucleus accumbens shell (NAc shell). Chapter 2 illustrates that lesions of the BNSTLP decreased baseline ethanol consumption in a 2 hr limited access procedure, but not in a continuous access procedure. Chapter 3 and chapter 4 illustrate that the CeA and NAc shell are involved in baseline ethanol consumption in a limited access procedure, since lesions of these nuclei decreased ethanol consumption. To determine if these nuclei were involved in increases in ethanol consumption, a murine model of ethanol dependency was used. In this procedure C57BL/6J (B6) mice are first acclimated to a limited access two-bottle choice preference procedure. The access period begins 3 hrs into the dark-cycle and continues for 2 hrs. Once acclimated, mice undergo chronic exposure to and intermittent withdrawal from ethanol vapor. Results from chapter 4 indicate that intermittent vapor exposure, as opposed to continuous ethanol vapor exposure, optimizes the increased ethanol x consumption response. As indicated in chapter 2, 3, and 4, lesions of these three components of the cEA did not block the intermittent ethanol vapor induced increase in ethanol consumption. In chapter 4, to determine the brain regions that activate in response to increases in ethanol consumption, a c-fos immunoreactivity study was carried out. The results suggest that the NAc shell and NAc core are the two main brain regions that activate as a result of ethanol consumption specifically in the mice that have been exposed to the intermittent ethanol vapor exposure that show the increase in ethanol consumption. Thus the results suggest that while the NAc shell activates in response to heightened levels of ethanol consumption, it is not necessary to see this increase in ethanol consumption. Overall, the results from these three chapters suggest that while the components of the cEA are involved in baseline ethanol consumption, and are responsive to changes in ethanol consumption (as was the case with the NAc shell), they are not necessary to see the ethanol vapor induced increase in ethanol consumption. These results have implications for understanding the neural circuitry involved in ethanol dependence.

Malaria, an infectious disease caused by Plasmodium parasites, is one of the major world-scale health problems. Despite the efforts aimed at finding an effective way to control the disease, the success has been thwarted by the emergence of parasite drug resistance and mosquito resistance to insecticides. This thesis focuses on the genetic analysis of resistance to murine malaria induced by the lethal Plasmodium berghei ANKA using a wild-derived-inbred strain (WDIS). The aim of this thesis was to exploit the genetic diversity represented among WDIS for identifying loci contributing to resistance/susceptibility to murine malaria. The work included a genome-wide polymorphism survey using microsatellite markers performed on 10 WDIS. Comparisons of these strains to laboratory inbred strains confirmed a higher rate of polymorphism among the WDIS. We conclude that these WDIS represent repositories of unique naturally occurring genetic variability that may prove to be invaluable for the study of complex phenotypes. Next, we used the WDIS to search for novel phenotypes related to malaria pathogenesis. Whereas most laboratory strains were susceptible to experimental cerebral malaria (ECM) after infection with P. berghei ANKA, several WDIS were found to be resistant. To study the genetic inheritance of resistant/susceptibility to P. berghei ANKA infection we analysed backcross and F2 cohorts derived from crossing the WLA wild-derived strain with a laboratory mouse strain (C57BL/6). A novel phenotype represented by the cure of infection, clearance of parasitaemia and establishment of immunological memory was observed in the F2 progeny. The backcross progeny was used to genetically map one locus on chromosome 1 (Berr1) and one locus on chromosome 11 (Berr2) that mediate control of resistance to ECM induced by P. berghei ANKA. Genetic mapping using the F2 progeny showed that a locus on chromosome 1 (Berr1) and a locus on chromosome 9 (Berr3) were contributing to control survival time after infection with lethal Plasmodium. Finally, we identified, a locus on chromosome 4 (Berr4) that appears to control time of death due to hyperparasitaemia. This thesis underlines the value of using WDIS to reveal genetic factors involved in the aetiology of disease phenotypes. The characterisation of the genetic factors represented by the malaria resistance loci identified here are expected to provide a better understanding of the malaria pathology.

Interferon regulatory factor-8 (Irf-8), a hematopoietic transcriptional regulator, controls myeloid-cell proliferation and coordinates innate and adaptive host immune responses. Mice from the BXH-2 recombinant inbred strain carry an endogenous R294C mutation in Irf-8. This loss-of-function mutation induces clonal infiltration of undifferentiated Mac-1+/Gr-1 + granulocytic precursors in BXH-2 mice, extramedullary hematopoiesis, and splenomegaly similar to those seen in human chronic myeloid leukemia. It also renders the host permissible to the otherwise avirulent Mycobacterium bovis (BCG), and negatively affects survival or recovery of these mice to other infectious pathogens. Here, we generated a polyc1onal anti-Irf-8 antibody to better characterize the effects of the R294C mutation on Irf-8 protein expression, stability, and inducibility in hematopoietic and non-hematopoietic tissues. We found that mutant Irf-8C294-expressing tissues consistently displayed reduced Irf-8 abundance compared to their wild-type counterparts in both primary splenocytes and following transfection into heterologous cells, presumably due to decreased stability or increased rate of degradation of the mutant isoform. Results also indicate that native Irf-8 is also expressed in the heart, and to a lesser extent, in the kidneys. Since neither of these organs is well-known to be associated with hematopoietic or immune functions, this finding strengthens the possibility that Irf-8 may exert additional regulatory functions in other cellular contexts. Taken together, our study provides a better understanding about the molecular features of the mutant Irf-8 C294 protein and contributes to a growing body of evidence in support of Irf-8 expression in non-hematopoietic tissues.

No description available.

17 beta-Hydroxy-11 beta(4-dimethylaminophenyl)-17 alpha-(1-propynyl)estra-4, 9-dien-3-one (RU486) inhibited the in vitro development of different stages of mouse preimplantation embryos under study. Two-celled embryos, morulae, and early blastocysts were obtained from B6D2F1 mice. The embryos were grown in Ham F-10 nutrient mixture (with glutamine) supplemented with sodium bicarbonate (2.1 g/L), calcium lactate (282 mg/L), and bovine serum albumin (fraction V, 3 mg/mL) at 37 degrees C in a humidified incubator supplied with 5% CO2 in air. RU486 was added to the culture medium at concentrations of 1, 5, 10, and 20 micrograms/mL. Culture medium with 0.05% ethanol served as the control. In vitro growth of embryos was assessed by the following criteria: (i) two-celled stage embryo development to blastocyst stage after 72 h, (ii) morula stage grown to blastocyst stage after 24 h, and (iii) early blastocyst stage development to hatching blastocyst after 12 h, in culture. RU486 inhibited the in vitro development of two-celled embryos, morulae, and early blastocysts at concentrations of 5, 10, and 20 micrograms/mL culture medium (p less than 0.001). The inhibitory effect of RU486 at these concentrations on the development of all the stages of embryos under study was irreversible. However, RU486 did not affect embryo development at 1 microgram/mL culture medium. The study indicates the direct adverse effect of RU486 at 5 micrograms/mL and higher concentrations in culture medium on the development of mouse preimplantation embryos in vitro, and it encourages its further investigation as a postcoital contraceptive in animal models and humans.

Repeated exposure of the rat vas deferens to the imidazoline oxymetazoline (OXY) results in a progressive loss of response which can appear selective for imidazoline agonists. The present study tests the hypothesis that imidazolines produce desensitization through prolonged blockade or inactivation of alpha-1 adrenoreceptors. Repeated exposure to OXY, naphazoline (NPZ) or tetrahydrozoline (THZ) produces a concentration- and time-dependent rightward shift and depression of the (-)-epinephrine concentration-effect curve, suggesting a mechanism of prolonged receptor blockade or inactivation. (-)-Epinephrine Kd values were similar when estimated after either receptor inactivation with phenoxybenzamine or repeated exposure to imidazolines. The differences in the ability of individual imidazolines to produce desensitization (order of potency: OXY greater than NPZ greater than or equal to THZ) do not follow their intrinsic activity (NPZ approximately THZ approximately OXY) or affinity (OXY greater than or equal to NPZ greater than THZ). The ability of individual imidazoline and phenethylamine agonists to produce a response in imidazoline-desensitized rat vas deferens reflects agonist intrinsic efficacy. Desensitization by imidazoline exposure does not affect contraction produced by either KCl or neurokinin A. Imidazolines produce effects similar to receptor inactivation and their desensitization in vas deferens can be explained without invoking an imidazoline subtype of alpha-1 adrenoreceptor.

Bone morphogenetic proteins (BMPs) play essential roles in the control of skin development, postnatal tissue remodelling and tumorigenesis. To explore whether some of the effects of BMP signalling are mediated by microRNAs, we performed genome-wide microRNA (miRNA) screening in primary mouse keratinocytes after BMP4 treatment. Microarray analysis revealed substantial BMP4-dependent changes in the expression of distinct miRNAs, including miR-21. Real-time PCR confirmed that BMP4 dramatically inhibits miR-21 expression in the keratinocytes. Consistently, significantly increased levels of miR-21 were observed in transgenic mice overexpressing the BMP antagonist noggin under control of the K14 promoter (K14-noggin). By in situ hybridization, miR-21 expression was observed in the epidermis and hair follicle epithelium in normal mouse skin. In K14-noggin skin, miR-21 was prominently expressed in the epidermis, as well as in the peripheral portion of trichofolliculoma-like hair follicle-derived tumours that contain proliferating and poorly differentiated cells. By transfecting keratinocytes with a miR-21 mimic, we identified the existence of two groups of the BMP target genes, which are differentially regulated by miR-21. These included selected BMP-dependent tumour-suppressor genes (Pten, Pdcd4, Timp3 and Tpm1) negatively regulated by miR-21, as well as miR-21-independent Id1, Id2, Id3 and Msx2 that predominantly mediate the effects of BMPs on cell differentiation. In primary keratinocytes and HaCaT cells, miR-21 prevented the inhibitory effects of BMP4 on cell proliferation and migration. Thus, our study establishes a novel mechanism for the regulation of BMP-induced effects in the skin and suggests miRNAs are important modulators of the effects of growth factor signalling pathways on skin development and tumorigenesis.

A alteração do tráfego mitocondrial em neurônios leva ao aumento do estresse oxidativo, privação de energia, deficiência da comunicação intercelular e neurodegeneração. Há evidências de que essas alterações de tráfego antecedem a morte neuronal associada à agregação proteica. Portanto, conhecer a relação entre a mobilidade mitocondrial e a formação de agregados proteicos pode ser um passo importante para o melhor entendimento dos mecanismos da neurodegeneração. Com isso, o objetivo do presente estudo é analisar a mobilidade das mitocôndrias em culturas de células do hipocampo, substância negra e locus coeruleus expostas a rotenona e MPTP, como agentes neurodegenerativos, e à rapamicina como ativador da autofagia. Um outro objetivo do estudo é avaliar o papel do cálcio (através do emprego de EGTA e ionomicina) no modelo experimental. Os resultados mostraram aumento da mobilidade mitocondrial no hipocampo e diminuição na substância negra, já no locus coeruleus houve aumento seguido de diminuição da mobilidade mitocondrial dependendo da concentração de rotenona. O emprego do EGTA e ionomicina mostra que a ação da rotenona sobre o tráfego mitocondrial envolve o cálcio, mas não se relaciona com uma possível alteração da integridade mitocondrial, já que não foi observada alteração no potencial de membrana mitocondrial. Foram também realizados experimentos a fim de avaliar a mobilidade mitocondrial em modelo utilizando rapamicina para ativar a autofagia e MPTP como indutor da neurodegeneração em culturas de células, onde foi observado aumento da mobilidade no hipocampo e no locus coeruleus quando exposto a rapamicina e aumento da mobilidade mitocondrial em cultura de células do hipocampo exposto a MPTP já no locus coeruleus houve uma diminuição significativa da mobilidade mitocondrial. Os resultados permitem concluir que o tráfego mitocondrial está alterado antes da agregação proteica podendo contribuir com a neurodegeneração / Altered mitochondrial traffic in neurons can lead to increased oxidative stress, energy deprivation, impaired intercellular communication and neurodegeneration. There are evidences mitochondria disturbing precedes neuronal death associated with protein aggregation. Therefore, the study of mitochondrial traffic and protein aggregation can be an important step towards a better understanding of the mechanisms of neurodegeneration. Thus, the aim of this study is to analyze mitochondria mobility in cultured cells of the hippocampus, substantia nigra and locus coeruleus exposed to rotenone and MPTP, as neurodegeneration-promoting agents, and rapamycin to activate autophagy. The other objective of the study was to analyze the role of calcium (through EGTA and ionomycin) in the experimental model. The results showed increased and decreased mobility mitochondrial in cells from hippocampus and substantia nigra, respectively, while the locus coeruleus cell culture has increased followed by decreased mitochondrial mobility depending upon rotenone concentration. The use of EGTA and ionomycin showed that alteration of mitochondrial traffic is associated with calcium, however it is not related with changes in mitochondrial membrane potential. Additional experiments were also conducted to assess mitochondrial mobility in a model using rapamycin to activate autophagy and MPTP to induce neurodegeneration in cell cultures. The results of these experiments showed increased mitochondrial mobility in the hippocampus and locus coeruleus when exposed to rapamycin; while MPTP also increased mitochondria mobility in hippocampal cell cultures, but decreased it in locus coeruleus. Results suggest that mitochondrial traffic is altered before protein aggregation, which may contribute to neurodegeneration

A alteração do tráfego mitocondrial em neurônios leva ao aumento do estresse oxidativo, privação de energia, deficiência da comunicação intercelular e neurodegeneração. Há evidências de que essas alterações de tráfego antecedem a morte neuronal associada à agregação proteica. Portanto, conhecer a relação entre a mobilidade mitocondrial e a formação de agregados proteicos pode ser um passo importante para o melhor entendimento dos mecanismos da neurodegeneração. Com isso, o objetivo do presente estudo é analisar a mobilidade das mitocôndrias em culturas de células do hipocampo, substância negra e locus coeruleus expostas a rotenona e MPTP, como agentes neurodegenerativos, e à rapamicina como ativador da autofagia. Um outro objetivo do estudo é avaliar o papel do cálcio (através do emprego de EGTA e ionomicina) no modelo experimental. Os resultados mostraram aumento da mobilidade mitocondrial no hipocampo e diminuição na substância negra, já no locus coeruleus houve aumento seguido de diminuição da mobilidade mitocondrial dependendo da concentração de rotenona. O emprego do EGTA e ionomicina mostra que a ação da rotenona sobre o tráfego mitocondrial envolve o cálcio, mas não se relaciona com uma possível alteração da integridade mitocondrial, já que não foi observada alteração no potencial de membrana mitocondrial. Foram também realizados experimentos a fim de avaliar a mobilidade mitocondrial em modelo utilizando rapamicina para ativar a autofagia e MPTP como indutor da neurodegeneração em culturas de células, onde foi observado aumento da mobilidade no hipocampo e no locus coeruleus quando exposto a rapamicina e aumento da mobilidade mitocondrial em cultura de células do hipocampo exposto a MPTP já no locus coeruleus houve uma diminuição significativa da mobilidade mitocondrial. Os resultados permitem concluir que o tráfego mitocondrial está alterado antes da agregação proteica podendo contribuir com a neurodegeneração / Altered mitochondrial traffic in neurons can lead to increased oxidative stress, energy deprivation, impaired intercellular communication and neurodegeneration. There are evidences mitochondria disturbing precedes neuronal death associated with protein aggregation. Therefore, the study of mitochondrial traffic and protein aggregation can be an important step towards a better understanding of the mechanisms of neurodegeneration. Thus, the aim of this study is to analyze mitochondria mobility in cultured cells of the hippocampus, substantia nigra and locus coeruleus exposed to rotenone and MPTP, as neurodegeneration-promoting agents, and rapamycin to activate autophagy. The other objective of the study was to analyze the role of calcium (through EGTA and ionomycin) in the experimental model. The results showed increased and decreased mobility mitochondrial in cells from hippocampus and substantia nigra, respectively, while the locus coeruleus cell culture has increased followed by decreased mitochondrial mobility depending upon rotenone concentration. The use of EGTA and ionomycin showed that alteration of mitochondrial traffic is associated with calcium, however it is not related with changes in mitochondrial membrane potential. Additional experiments were also conducted to assess mitochondrial mobility in a model using rapamycin to activate autophagy and MPTP to induce neurodegeneration in cell cultures. The results of these experiments showed increased mitochondrial mobility in the hippocampus and locus coeruleus when exposed to rapamycin; while MPTP also increased mitochondria mobility in hippocampal cell cultures, but decreased it in locus coeruleus. Results suggest that mitochondrial traffic is altered before protein aggregation, which may contribute to neurodegeneration