Many Oenothera species are complete translocation heterozygotes and arose by hybridization. Since complete translocation heterozygosity suppresses nuclear genetic recombination, examination of any chloroplast gene effects on hybridization is particularly suitable in these flowering plants. To verify translocation status in Oenothera villaricae, Oe. picensis ssp. picensis and lines of their interspecific hybrids, meiotic figures from parents and hybrids were obtained using confocal optical sectioning (a new application of this technique). These data confirm published results, and are consistent with a bivalent-forming common ancestor which underwent translocations to produce each of the parent species. The full translocation ring appeared in only one of the four possible hybrid nuclei. Oenothera species of hybrid origin form translocation rings at meiosis; progenitor species are structurally homozygous and form seven bivalents. Comparison of ring- and bivalent-forming species revealed hollow, abortive pollen grains in the ring-forming species (25-30% relative to 0-3% abortive pollen abortion in bivalent-forming species). F$\sb1$ hybrids of the picensis maternal lineage may abort up to 85% of pollen; nonreciprocality in the corresponding villaricae hybrid indicates nucleocytoplasmic incompatibility. Statistical analysis shows a high correlation between pollen stainability and meiotic disjunction frequency in Oe. villaricae, Oe. picensis and hybrids, suggesting that the abortive grains are the products of nondisjunctional meiosis. Significant effects (P $<$.001) on disjunction frequency and pollen stainability were noted for nucleus and cytoplasm alone and in combination, revealing an interaction or combined effect of nucleus and cytoplasm. This combined effect influences the chromosomal pairing behavior at meiosis; hence also the pollen viability. The fitness consequences of cytoplasmic hybridization intolerance (e.g., chlorosis, pollen abortion, lethality) probably affect the relative success of incipient species. Of the two cytoplasmically inherited genetic systems, the plastid genome (plastome) is the more accessible to study. Five discrete European plastid genotypes (typified by Oe. hookeri, biennis, Lamarckiana, parviflora and argillicola), as well as those of South American Oe. villaricae and picensis, have been identified by restriction fragment length polymorphisms (RFLPs) using endonucleases PvuII, SalI, KpnI and PstI. The present RFLP study employed PvuII and KpnI to analyze plastid DNAs from across subsection Munzia, the South American taxonomic group which includes Oe. villaricae and picensis, in comparison with known European plastomes. Subsect. Munzia is divided into three series: Ancestral Renneria, the younger Allochroa, and the most recently evolved, Clelandia, composed entirely of hybrids between members of Renneria and Allochroa. At least ten new plastid genotypes are noted among 28 Munzia species examined. All three series were to some degree polymorphic. In certain hybrids of known origin, comparison of the cpDNA genetic fingerprint with those of the parent species can reveal the maternal and paternal lineage, i.e. the direction of hybridization. One particular cpDNA fingerprint which predominates throughout the subsection is presumed to represent the ancestral plastome type.

While various chronic diseases and medication use have been associated with an increased risk of falling, neuromuscular deficits, due to aging or pathology, have been strongly implicated in the etiology of falls. In particular, it has been suggested that inadequate pre-innervation can result in impact forces large enough to cause femoral neck fractures, thereby leading to a fall. Less dramatically, inadequate pre-innervation may lead to an inability to compensate for potentially destabilizing forces occurring on impact. Unfortunately, little is known about the neuromuscular activation patterns present among fallers. Neuromuscular patterns were examined between 12 elderly male fallers (mean age = 74.75) and 12 elderly males (mean age = 72.75), with no history of falling. Falling status was verified through hospital records, physician's records, or spousal interview. Fallers had experienced at least one fall during the prior year, while nonfallers had no history of falling during the prior year. All subjects were free from any known neurological disorders. Neuromuscular patterns were assessed, via EMG, over three days. A step-down task was performed from heights of 10, 20, and 30 centimeters, during sighted and unsighted conditions. In addition, balance was assessed via the stork stand, tandem walk, Romberg, and get-up-and-go tests; while various strength measurements were assessed via dynamometer or EMG. The results indicate the following: (1) Fallers had significantly slower movement time than nonfallers and were more likely to display a polyphasic pre-innervation EMG pattern; (2) Fallers had significantly shorter pre-innervation times during step-downs from 10 and 20 centimeter, but this distinction failed to hold during step-downs from 30 centimeters; (3) Fallers performed more poorly on all tests of balance; (4) No statistically significant group difference could be found on any strength measure or anthropometric assessment. Consequently, in the present study population, falling status appears to be related to a slowing of the neuromuscular system. Unfortunately, the specifics or nature of that slowing remain obscure.

Animals exhibit physiological and behavioral changes in anticipation of and response to changing environmental conditions. One example of this is the influence of photoperiod on the reproductive cycle of temperate zone seasonal breeders, such as Syrian hamsters. In addition to physiological changes which prevent ovulation and reduce gonadal steroid secretion, photoperiod can influence behavioral responses to gonadal steroid replacement, suggesting that photoperiods which inhibit reproduction alter neural responsiveness to estradiol and progesterone. The experiments described here were designed to address possible mechanisms by which photoperiod might produce these changes. In Experiment 1, immunocytochemistry was used as a semi-quantitative and anatomically specific technique to explore the hypothesis that reduced neural responsiveness to gonadal steroids was due to changes in the number of estrogen and progestin receptors in nuclei mediating hormonal effects on reproductive behavior and physiology. Exposure to a short photoperiod was associated with decreased progestin receptor immunoreactivity (Experiment 1B) but not with changes in estrogen receptor immunoreactivity (Experiments 1A, 1D), suggesting that neural responsiveness to estradiol is reduced in short photoperiods but that this effect is not due to changes in estrogen receptor number. Changes in responsiveness to estradiol are not dependent on peripheral effects of photoperiod on estradiol metabolism, as progestin receptor-immunoreactivity was reduced in short days even when estradiol was implanted directly into the mediobasal hypothalamus (Experiment 1C). Experiment 2 utilized Fos as a marker of neuronal activity to identify specific neural sites where photoperiod might act to influence neural responsiveness to steroid hormones and sociosexual cues responsible for induction of lordosis. Effects of photoperiod on lordosis and Fos were highly variable among experiments, but exposure to a short photoperiod was associated in three of the four experiments (Experiment 2A, 2B, and 2D) with increased Fos immunoreactivity in the ventromedial hypothalamus. Exposure to a short photoperiod may increase the responsiveness of the ventromedial hypothalamus to one or more hormonal or sociosexual cues to which hamsters were exposed.

Several genes implicated in Parkinson's disease (PD) encode components of the ubiquitin-proteasome pathway. In a specific form of PD (human Autosomal Recessive Juvenile Parkinsonism, AR-JP), loss of functional Parkin (ubiquitin E3 ligase) results in a selective loss of midbrain dopaminergic neurons and a absence of Lewy bodies (LB) from the surviving dopaminergic neurons. Since cells in patients with AR-JP do not express functional Parkin, it is unclear why most neuronal and non-neuronal populations remain unaffected. One possible explanation is that most cells express a redundant ubiquitin E3 ligase(s) that is absent from dopaminergic neurons. Such candidate(s) redundant E3-ligase would be expected to fulfill several criteria: (1) bind similar E2 Ubiquitin conjugating enzymes; (2) interact with the same cellular substrates; (3) facilitate the formation of aggresome/lewy bodies with similar properties of those induced by Parkin; (4) be expressed in the nervous system but presumably absent (or largely absent) from dopaminergic neurons. In this thesis I have demonstrated that the Human Homolog of Drosophila Ariadne-1 (HHARI) is a candidate for such a redundant E3 ligase. In addition I have shown that even though HHARI induces the formation of LB like aggresomes in cell culture with properties similar to those produced by Parkin, these aggresomes differ in their detergent solubility properties. Using mouse C2C12 primary skeletal muscle cells with altered expressions of Ariadne-1 or Parkin, I determined if HHARI and Parkin may serve redundant protective roles. Using cell viability assays I have shown that HHARI does not confer protection to cells treated with toxic insults like those implicated in PD. On the contrary, using RNA silencing, I have shown that reduced Ariadne-1 expression appears to confer some benefit. Finally, based on phenotypes reported for Ariadne-1-/- escaper and Parkin-/- flies as well as our protein interaction data, I investigated the roles of Parkin and HHARI during myogenesis. Using engineered C2C12 cells I have shown that Ariadne-1 levels are tightly regulated in proliferating and differentiating C2C 12 cells and that increased cellular abundance of Ariadne-1 affects muscle terminal differentiation downstream of myogenin, strongly highlighting the importance of Ariadne-1 and perhaps the Ubiquitin Proteasome Pathway in myogenesis.

Breast cancer is the most frequent tumor type among women. Heightened susceptibility of the breast to tumor development has been associated with early menarche, nulliparity, exposures to ionizing radiation, and family history, but the underlying molecular mechanisms are poorly understood. Unfortunately, the etiology of breast cancer is complex and is complicated by the fact that it is a heterogeneous disease. The p53 tumor suppressor gene was altered in a large proportion of these spontaneous breast tumors implicating its involvement in the progression of breast cancer development. The aim of this dissertation was to determine the regulation of p53 in the normal mammary gland and whether it is involved in suppressing the development of mammary tumors. To evaluate the effect of p53 on mammary tumor formation, the first component of this work involved the characterization of BALB/c- p53-deficient mice. BALB/c-p53+/− and p53−/− mice were examined for tumor spectrum and mammary abnormalities. Mammary transplants were performed to evaluate the role of p53 in tumor suppression in the mammary gland. This work demonstrated that p53 is critical in suppressing mammary tumorigenesis in the mammary gland as BALB/c mice deficient in p53 readily develop mammary carcinomas. The second element of this project examined the expression, localization and activity of p53 in normal mammary tissues. Since the mammary gland is a tissue that is sensitive and responsive to local and systemic hormones, the last chapter of this dissertation focused on the hormonal effects on p53 activity. Results from these experiments demonstrated that p53 was expressed at high levels localized to the cytoplasm of the ductal epithelium of the quiescent mammary gland. P53 was not responsive to radiation-induced DNA damage suggesting its function is compromised in the nulliparous mammary gland. Further experiments demonstrated that the functional state of wild type p53 in the mammary epithelium could be regulated by hormonal stimuli.

The development and maintenance of multicellular organisms depends fundamentally on cell division, a series of events largely mediated by the mitotic spindle. Errors in spindle formation and/or function are often associated with severe consequences, most notably cancer. In order to elucidate the cause of such errors and the potential for therapeutic intervention, it is imperative to attain a clear understanding of how cell division normally operates. In this regard, this dissertation focuses on the activity of two microtubule-based motor proteins, Eg5 and dynein, prior to and immediately following nuclear envelope breakdown during mitosis. I show that prophase microtubules are remarkably more dynamic than their metaphase counterparts, moving both toward and away from centrosomes across a wide distribution of rates. Inhibition of Eg5, dynein and Kif2a revealed that a subset of this motion is consistent with microtubule flux, a well-established phenomenon temporally limited to metaphase and anaphase spindles by the preceding literature. My data indicates that flux is operational throughout all of mitosis, possibly functioning at early stages to collect centrosomal components. Immediately following prophase, cells begin assembling bipolar spindles. While the establishment of spindle bipolarity fails in the physical or functional absence of Eg5, I show that co-inhibition of dynein restores a cell's ability to organize microtubules into a bipolar structure. Despite inhibition of both Eg5 and dynein, these spindles are morphologically and functionally equivalent to controls. Together, these data suggest that Eg5 and dynein share an antagonistic relationship and that a balance of forces, rather than a definitive set of players, is important for spindle assembly and function. To determine how Eg5- and dynein-mediated forces functionally coordinate to bring about antagonism during spindle assembly, I utilize a nocodazole washout assay. I show, via in vivo imaging and in silico modeling, that spindle collapse in the absence of functional Eg5 requires dynein activity and an initial intercentrosomal distance of less than 5.5μm. These data are consistent with a model in which dynein antagonizes Eg5 by crosslinking and sliding antiparallel microtubules, a novel role for dynein within the framework of spindle assembly.

Utilizing the diverse functionality of amino acids, a new class of amphiphilic graft copolymers has been synthesized, characterized, and explored for applications in biomaterials and nucleic acid delivery. This thesis research focused on the syntheses of oligopeptide-functionalized polyesters and polyolefins. Polyester functionalization was geared towards applications in biomaterials, tissue engineering, and drug delivery by incorporating sequences that promote cell-adhesion. These polyester-graft-oligopeptide materials were prepared by a 1,3-Huisgen cycloaddition reaction, “click” chemistry, of an azide-terminated oligopeptide (prepared by Fmoc-based solid phase peptide synthesis (SPPS)) and alkyne-containing polyester (synthesized by ring-opening polymerization). Following the syntheses of these materials, they were analyzed by nuclear magnetic resonance (NMR) and organic gel permeation chromatography (GPC). The oligopeptide-functionalized polyolefins were designed for nucleic acid complexation, and therefore the oligopeptide sequences were intended to incorporate positively-charged moieties (e.g., oligolysine) for DNA and short interfering RNA (siRNA) complexation. These graft copolymers, prepared by SPPS followed by ring-opening metathesis polymerization, have highly tunable structures that enable control over charge density and polymer backbone rigidity. Moreover, non-ionic hydrophilic grafts such as polyethylene glycol were integrated into these polyelectrolytes such that the charges along the polymer backbone are spaced accordingly while maintaining the hydrophilicity of the polymer. While numerous applications for such charged, “bio-tailored” materials can be envisioned, this work is geared towards positively-charged polyelectrolytes for their potential application in nucleic acid therapy, specifically the delivery of plasmid DNA and siRNA. These graft copolymers were characterized (1H, 13C NMR, organic and aqueous GPC), studied for their solution properties (static and dynamic light scattering), and investigated as polyplexes with plasmid DNA.

Chloroplast biogenesis relies on the import of thousands of nuclear encoded proteins into the organelle and proper sorting to their sub-organellar compartment. The majority of nucleus-encoded chloroplast proteins are synthesized in the cytoplasm and imported into the organelle via the Toc-Tic translocation systems of the chloroplast envelope. In many cases, these proteins are further targeted to subcompartments of the organelle (e.g. the thylakoid membrane and lumen or inner envelope membrane) by additional targeting systems that function downstream of the import apparatus. The inner envelope membrane (IEM) plays key roles in controlling metabolite transport between the organelle and cytoplasm, and is the major site of lipid and membrane biogenesis within the organelle. In contrast to the protein import and thylakoid targeting systems, our knowledge of the pathways and molecular mechanisms of protein targeting and integration at the IEM are very limited. Previous reports have led to the conclusion that IEM proteins are transferred to the IEM during protein import via a stop-transfer mechanism. Recent studies have shown that at least two components of the Tic machinery (AtTic40 and AtTic110) are completely imported into the stroma and then re-inserted into the IEM in a post-import mechanism. This led me to investigate the mechanisms and pathways involved in the integration of chloroplast IEM proteins in more detail. I selected candidates (AtTic40 for post-import and IEP37 for stop-transfer) that are predicted to have only one membrane-spanning helix and adopt the same IEM topology to facilitate my analysis. My studies confirm the existence of both stop-transfer and post-import mechanisms of IEM protein targeting. Furthermore, I conclude that the IEP37 transmembrane domain (TMD) is a stop-transfer signal and is able of diverting AtTic40 to this pathway in the absence of AtTic40 IEM targeting information. Moreover, the IEP37 TMD also functions as a topology determinant. I also show that the AtTic40 targeting signals are context dependent, with evidence that in the absence of specific information in the appropriate context, the AtTic40 TMD behaves as a stop-transfer signal. This is an indication that the stop-transfer pathway is the default mechanism of protein insertion in the IEM.

Wnt ligands are a family of highly conserved glycoproteins that act as morphogens to regulate development in many organisms. Drosophila Wnt-1 (Wingless) is involved in directing cell fate decisions and pattern formation during differentiation. Wnt signaling are of high interest of many developmental biologists due to their important functions, yet little is known about how these ligands function on a biochemical level. Previously it was found that Porcupine, an ER-membrane-bound acyltransferase is required for Wingless secretion. But it is unclear how a secreted morphogen requires an acyltransferase to function. Studies reported here demonstrated that Wingless undergoes a hydrophobic modification, in which a lipid moiety containing a palmitate group is covalently attached to the polypeptide through an ester linkage. And it partitions with the specialized detergent insoluble lipid raft microdomains in the plasma membrane. Porcupine is required for the modification and the raft targeting of Wingless. Blocking Wingless modification with a specific inhibitor results in the loss of rafts-association as well as loss of protein secretion. Disrupting raft microstructures by cholesterol depletion reagents also impaired Wingless secretion, indicating that the ligand secretion is dependent on its specific association with the plasma membrane. This work provided the first insight on the function of Porcupine and the important biochemical evidence on the role of specialized membrane microdomains in Wnt signaling.

In mammalian eggs, the fertilizing sperm evokes intracellular Ca 2+ ([Ca2+]i) oscillations that are essential for initiation of egg activation and embryonic development. Although the exact mechanism leading to initiation of [Ca2+]i oscillations still remains unclear, accumulating studies suggest that a presently unknown substance, termed sperm factor, is delivered from the fertilizing sperm into the ooplasm and triggers [Ca2+]i oscillations. Recent evidence shows that sperm/sperm factor stimulates persistent production of inositol 1,4,5-trisphosphate (IP3) and that a sperm-specific phosphoinositide-specific phospholipase C (PLC), PLCζ, may be the long sought-after [Ca 2+]i oscillation-inducing molecule in sperm. In the first study, we show that subtle modification of the sequence of events that lead to fertilization, such as that seen in fertilization achieved by intracytoplasmic sperm injection (ICSI), alters the pattern of [Ca2+]i oscillations and embryonic development, and this alternation is possibly due to the non-physiological release of sperm factor (Chapter 3). Thereafter, we demonstrate that activation of a Src family kinase, which is essential for fertilization-associated Ca2+ release in non-mammalian species, is neither necessary nor sufficient for triggering fertilization-induced [Ca2+]i oscillations in mouse (Chapter 4). Finally (Chapter 5), we demonstrate the complete extraction of the [Ca2+] i oscillation-inducing activity from porcine sperm, and show that regardless of the cellular location or solubility, the same molecule/complex appears to be responsible for [Ca2+]i oscillations induced by injection of porcine sperm extracts (pSEs). Moreover, our results suggest that PLCζ may not be the only active molecule in pSEs. We demonstrate that biotin-labeled peptide A7 (B-A7), a Src kinase inhibitory peptide, specifically binds to and precipitates the active component of sperm factor with avidin beads. Although the proteins that we have isolated and identified thus far are, however, unlikely to be involved in activation of the phosphoinositide pathway when introduced into mouse eggs, future studies using B-A7 should lead to identification of the putative sperm factor molecule.