<p> This study examined various hybrid peptides that possess both collagen [(POG)_n] and cell penetrating peptides (CPP) [(RRG)_n or R_n] sequences. The hybrid peptides were able to fold into triple helical conformation when the surrounding temperature was lower than their transition temperature (T_m) which resulted in cellular internalization. The peptide that lacked collagen [(POG)_n] domain failed to penetrate the cell. The hybrid peptide under study, FL7V1, was shown to have the ideal T_m (17.3&deg;C) for the potential purpose as a drug carrier. <i>In vitro</i> study of FL6V1 with temperature gradient showed cellular internalization at low temperatures (10&deg;C-20&deg;C) while no uptake was achieved at high temperatures (24&deg;C-32&deg;C). <i> In vivo</i> study of FL7V1 with <i>P. leidyi</i> corresponded with the results of <i>in vitro</i> study at constant and gradient temperature.</p>

Fluorescent ratiometric imaging of Lilium longiflorum pollen tubes loaded with the Ca$\sp{2+}$ indicator fura-2 dextran has revealed a tip-focused gradient of free intracellular calcium ions in actively growing pollen tubes which extends from above 3.0 $\mu$M at the apex to a uniform basal level of $\sim$0.2 $\mu$M within 20 $\mu$m from the tip. Application of the Ca$\sp{2+}$ specific vibrating electrode revealed a tip-directed extracellular Ca$\sp{2+}$ influx between 1.4 and 14 pmol cm$\sp{-2}$ sec$\sp{-1}$. The relationship between these phenomena and their role in tube growth was examined using different 1,2-bis (o-aminophenoxy)ethane $N,N,N\sp1,N\sp1$-tetraacetic acid (BAPTA)-type buffers, and hypertonic media. Injection of active BAPTA-type buffers or application of elevated sucrose levels reversibly inhibited growth, destroyed tip zonation of organelles, and modified normal patterns of cytoplasmic streaming. Simultaneously, these treatments dissipated both the intracellular tip-focused gradient and the extracellular Ca$\sp{2+}$ flux providing evidence that growing pollen tubes have open Ca$\sp{2+}$ channels in their tip which become inactivated in nongrowing tubes. The elevated sucrose studies support the view that stretching of the apical plasma membrane contributes to the maintenance of the Ca$\sp{2+}$ signal. The presence and localization of actin and myosin have been examined in Lilium longiflorum and Nicotiana alata pollen tubes in order to determine their roles in pollen tube growth. Immunoblot analysis of pollen tube extracts with antibodies to actin, myosins IA and IB, myosin II, and myosin V reveals the presence of these contractile proteins. Immunofluorescence microscopy reaffirmed longitudinal actin localization. Myosin I was localized to the plasma membrane, larger organelles, the surface of the generative cell and the vegetative nucleus. Myosin V and Myosin II were found in the vegetative cytoplasm in a punctate fashion representing smaller and larger organelles, respectively. These results suggest the presence of three classes of myosins in pollen and lead to the following hypothesis: Myosin I may move the generative cell and vegetative nucleus unidirectionally toward the tip, while myosin V moves the smaller organelles and myosins I and II move the larger organelles (bidirectionally).

As an extension of the previous work on the pH-dependent reorganization of artificial lipid vesicles by poly(2-ethylacrylic acid) (PEAA), the effect of PEAA on living cells are examined by utilizing the acidification process of the epidermal growth factor (EGF) endocytic pathway in A431 cells. Fluoresceinated EGF (f-EGF) was used in conjunction with two-wavelength frame transfer fluorescence imaging techniques to measure the pH's of endocytic vesicles of individual cells. Preliminary results, including feasibility study and PEAA toxicity to A431 cells, are presented in chapter 3. Two projects branched out of this initial study; one involves the recording of PEAA channel activity using single-channel recording techniques (chapter 2), and another the measurement of binding kinetics of f-EGF to A431 cells at the single cell level (chapter 1). The synthetic anionic polyelectrolyte PEAA has been shown to reorganize lipid vesicles under acidic conditions. Single-channel recording techniques are used to further characterize the interactions between PEAA and artificial phospholipid bilayers. PEAA is shown to induce pore formation in bilayers at low (PEAA) ($<$ 20 $\mu$g/ml) in a pH-dependent manner. Discrete events resembling those characteristic of biological channels are observed. PEAA channels are found to be cation-selective, as demonstrated by comparing the relative Na$\sp+$ and Cl$\sp-$ permeabilities of the channels, but the Na$\sp+$/Cl$\sp-$ permeability ratio spans a wide range, indicating the existence of channels of different ion selectivities. Point amplitude histogram analysis of one type of channel event reveals at least four separate single-channel conductance states. The interpretation of ligand-receptor equilibrium binding data collected from a population of cells is often ambiguous. A method using quantitative fluorescence microscopy to measure EGF/receptor binding kinetics at the single cell level is developed. The binding of f-EGF to single cells is recorded over time and fitted to models with photobleaching correction and either one monovalent binding site or two independent monovalent binding sites. The results disprove the one-site model; therefore, the reported EGF receptor affinity heterogeneity exists at the single cell level.

The neuromuscular junction is the site where signals are transmitted from a nerve to a target muscle fiber. The mechanisms responsible for the maintenance of motor nerve terminals at synaptic sites are not understood. Here, I investigated the role of target-muscle fibers in the maintenance of frog motor nerve terminals. Cutaneous pectoris muscle fibers were selectively removed and prevented from regenerating while leaving the motor innervation intact. The role of target muscle fibers in nerve terminal structure and function was examined. First, the maintenance of presynaptic activity in the absence of target was assayed with the activity-dependent dye FM1-43. I found that target-deprived nerve terminals maintain their presynaptic function of synaptic vesicle recycling for up to 5 months of target deprivation. These results indicated that the molecular machinery required for vesicular release is maintained in a functional state for long periods of target deprivation. Second, I quantified the stability of target-deprived nerve terminals using in vivo repeated imaging. I found that most target-deprived nerve terminals were remarkably well maintained for several months after muscle fiber removal. These data indicate that the cues that confer stability to frog motor nerve terminals reside outside the muscle fibers such as in the synaptic basal lamina or terminal Schwann cells. Destabilization observed at some nerve terminals after extended target-deprivation, could result from the turning over of the stabilizing cues. Finally, the molecular organization of target-deprived nerve terminals was analyzed. I found that the levels of two synaptic vesicle proteins, SV-2 and synaptotagmin were reduced in target-deprived nerve terminals when compared to intact neuromuscular junctions. Analysis of cytoskeletal proteins revealed that F-actin was located at discrete bands along synaptic sites that do not colocalize with synaptic vesicle clusters. F-actin is suggested to be located at either the Schwann cell processes and/or the nerve terminal immediately above them. A possible adhesion between nerve terminals and Schwann cell processes, could contribute to the maintenance of the frog nerve terminal at the synaptic site. Finally, all target-deprived synaptic sites were found to be associated with variable levels of agrin immunoreactivity, implicating agrin as a possible maintenance molecule.

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.