No description available.

Collagen is one of the most abundant human proteins. It exists as a right-handed superhelix called the triple helix. The triple helix consists of three left-handed polyproline type II (PPII helices) that intertwine around a common axis. Each PPII helix has the repeating peptide sequence (Gly–Xaa–Yaa)n with a high content of (2S)-proline (Pro) in the Xaa position (ca. 28%) and (2S,4R)-hydroxyproline (Hyp) in the Yaa position (ca. 38%). Unique to the prolyl amide is the ease of cis-trans isomerization. Since the triple helix necessitates that all peptide bonds be in the trans conformation, isomerization is the rate-limiting step in collagen folding. However, eliminating isomerization with a trans-locked alkene isostere destabilizes collagen-like peptides. Collagen is stabilized by electronic interactions, namely the n→π* interaction. Halo-alkene isosteres may be used to recapture these electronic interactions and stabilize a collagen-like peptide. An in-depth conformational analysis was conducted at the MP2/6-311+G(2d,p) level of theory to determine the viability of conformationally-locked halo-alkene isosteres. Fluoro-alkenes and chloro-alkenes were modeled at both the Gly–Pro and Pro–Pro (as a Pro–Hyp mimic) amide positions. Compared to the collagen crystal structure PDB ID: 1K6F, we found the fluoro-alkenes were closer geometric matches to both Gly–Pro and Pro–Pro than the corresponding chloro-alkenes. The chloro-alkene was predicted to have stronger n→π* interactions. The trans-locked proteo-alkene was also analyzed to understand why it destabilized the triple helix. We found that these models had other local minima close to the desired PPII geometry, likely leading to enhanced backbone flexibility. This deleterious flexibility was not predicted for either fluoro-alkene or chloro-alkene models. The conformationally-locked halo-alkene isostere Fmoc–Gly–Ψ[(Z)CF=C]-Pro–Hyp(tBu)–OH was designed and synthesized as a (Z)-fluoro-alkene Gly–Pro isostere. We used the chiral catalyst, L-Thr, for asymmetric aldol addition to cyclopentanone, which inadvertently enhanced the yield of the wrong enantiomer, in contrast with aldol addition to cyclohexanone. A Mg2+-promoted Horner-Wadsworth-Emmons reaction afforded the (Z)-fluoro-alkene over the (E)-fluoro-alkene in about a 2:1 ratio. The two diastereomers, Fmoc–Gly–Ψ[(Z)CF=C]-L-Pro–Hyp(tBu)–OH and Fmoc–Gly–Ψ[(Z)CF=C]-D-Pro–Hyp(tBu)–OH were separated by supercritical CO2 chromatography. The collagen-like peptides Ac–(Gly–Pro–Hyp)3–Gly–Ψ[(Z)CF=C]-L-Pro–Hyp–(Gly–Pro–Hyp)4–Gly–Gly–Tyr–NH2, Ac–(Gly–Pro–Hyp)3–Gly–Ψ[(Z)CF=C]-D-Pro–Hyp–(Gly–Pro–Hyp)4–Gly–Gly–Tyr–NH2, and the control peptide Ac–(Gly–Pro–Hyp)8–Gly–Gly–Tyr–NH2 were synthesized on solid-phase resin. The CD spectra of all three peptides showed the characteristic collagen triple-helix signature. The folding stability was determined by thermal melting (Tm). The peptide with the fluoro-alkene guest, Gly–Ψ[(Z)CF=C]-L-Pro–Hyp, was found to have a Tm value of 42.2 °C. The Tm of the control peptide was found to be 49.0 °C, a difference in stability of only ΔTm –6.8. Thus, the (Z)-fluoro-alkene as a Gly–Pro isostere forms a relatively stable triple helix. The peptide with the Gly–Ψ[(Z)CF=C]-D-Pro–Hyp guest was shown to have a linear relationship between ellipticity and temperature, indicating that a stable triple helix did not form. The enhanced stability of the (Z)-fluoro-alkene compared to the (E)-alkene Gly–Pro isostere (Tm = 28.3 °C) may be due to a stabilizing n→π* interaction, as determined by NMR deshielding of the 19F nucleus in the collagen-like peptide. In biological systems, isomerization of the prolyl amide is catalyzed by enzymes called PPIases. The PPIase Pin1 specifically catalyzes isomerization of the pSer–Pro sequence from the cis-conformation to the trans-conformation. Pin1 plays a crucial role in the G2→M transition of the cell cycle, implying the importance of cis-trans isomerization. The dipeptides H–Ser–Ψ[(Z)CH=C]-Pro–OH, H–Ser–Ψ[(E)CH=C]-Pro–OH and native H–Ser–Pro–OH were synthesized by literature methods, and activated for aminoacylation of tRNACUA for in vitro transcription-translation. Aminoacylation by chemical methods required the synthesis of a pdCpA dinucleotide. Formation of the dipeptide-dinucleotide complex was not completed because protection of the Ser side chain was problematic. On the other hand, conversion of the dipeptide into the 3,5-dinitrobenzyl ester conjugate allowed for enzymatic aminoacylation using the dFx flexizyme, an RNA enzyme. The native dipeptide was successfully coupled to tRNACUA and is ready for incorporation into a full-length Bora protein by in vitro transcription-translation. Both cis- and trans-locked alkene mimics have been converted to their respective 3,5-dinitrobenzyl ester conjugates. / Doctor of Philosophy / The proline amide (Xaa–Pro) in peptides and proteins is unique in that it allows for cis-trans isomerization. The triple-helix region of human collagen consists mostly of the repeating sequence (Gly–Pro–Hyp)n. Xaa–Pro amide-bond isomerization is rate-limiting for triple-helix formation. We eliminated isomerization at one position in a collagen-like peptide with a locked alkene mimic of Gly–Pro to attempt to stablize the triple-helix. Our computational results predicted that a fluoro-alkene Gly–Pro isostere would be a close geometric match for the native amide. Experimental results showed that a collagen-like peptide with a fluoro-alkene Gly–Pro isostere has an unfolding temperature that is 6.9 °C lower than the native control peptide. 19F NMR data of the collagen-like peptide shows a surprising deshielding of the fluorine nucleus, suggesting its participation in a stabilizing n→π* electronic interaction, similar to the native amide. Isomerization also plays a key role in proper cell division. We followed established methods to synthesize the cis- and trans-locked alkene mimics of Boc–Ser–Pro–OH and converted them into the 3,5-dinitrobenzyl ester conjugates. The 3,5-dinitrobenzyl ester is recognized by the dinitrobenzyl flexizyme (dFx) for enzymatic aminoacylation of tRNA. Once the alkene isosteres are aminoacylated, they will be incorporated into a full-length cell cycle regulatory protein called Bora to determine whether the cis- or trans-Pro state is necessary for healthy human mitosis, and which results in cancerous human mitosis.

During mitosis, adherent animal cells undergo a drastic shape change, from essentially flat to round, in a process known as mitotic cell rounding (MCR). The aim of this thesis was to critically examine the physical and biological basis of MCR. The experimental part of this thesis employed a combined optical microscope-atomic force microscope (AFM) setup in conjunction with flat tipless cantilevers to analyze cell mechanics, shape and volume. To this end, two AFM assays were developed: the constant force assay (CFA), which applies constant force to cells and measures the resultant height, and the constant height assay (CHA), which confines cell height and measures the resultant force. These assays were deployed to analyze the shape and mechanical properties of single cells trans-mitosis. The CFA results showed that cells progressing through mitosis could increase their height against forces as high as 50 nN, and that higher forces can delay mitosis in HeLa cells. The CHA results showed that mitotic cells confined to ~50% of their normal height can generate forces around 50-100 nN without disturbing mitotic progression. Such forces represent intracellular pressures of at least 200 Pascals and cell surface tensions of around 10 nN/µm. Using the CHA to compare mitotic cell rounding with induced cell rounding, it was observed that the intracellular pressure of mitotic cells is at least 3-fold higher than rounded interphase cells. To investigate the molecular basis of the mechanical changes inherent in mitotic cell rounding, inhibitors and toxins were used to pharmacologically dissect the role of candidate cellular processes. These results implicated the actomyosin cortex and osmolyte transporters, the most prominent of which is the Na+/H+ exchanger, in the maintenance of mechanical properties and intracellular hydrostatic pressure. Observations on blebbing cells under the cantilever supported the idea that the actomyosin cortex is required to sustain hydrostatic pressure and direct this pressure into cell shape changes. To gain further insight into the relationship between actomyosin activity and intracellular pressure, dynamic perturbation experiments were conducted. To this end, the CHA was used to evaluate the pressure and volume of mitotic cells before, during and after dynamic perturbations that included tonic shocks, influx of specific inhibitors, and exposure to pore-forming toxins. When osmotic pressure gradients were depleted, pressure and volume decreased. When the actomyosin cytoskeleton was abolished, cell volume increased while rounding pressure decreased. Conversely, stimulation of actomyosin cortex contraction triggered an increase in rounding pressure and a decrease in volume. Taken together, the dynamic perturbation results demonstrated that the actomyosin cortex contracts against an opposing intracellular pressure and that this relationship sets the surface tension, pressure and volume of the cell. The discussion section of this thesis provides a comprehensive overview of the physical basis of MCR by amalgamating the experimental results of this thesis with the literature. Additionally, the biochemal signaling pathways and proteins that drive MCR are collated and discussed. An exhaustive and unprecedented synthesis of the literature on cell rounding (approx. 750 papers as pubmed search hits on “cell rounding”, April 2012) reveals that the spread-to-round transition can be thought of in terms of a surface tension versus adhesion paradigm, and that cell rounding can be physically classified into four main modes, of which one is an MCR-like category characterized by increased actomyosin cortex tension and diminution of focal adhesions. The biochemical pathways and signaling patterns that correspond with these four rounding modes are catalogued and expounded upon in the context of the relevant physiology. This analysis reveals cell rounding as a pertinent topic that can be leveraged to yield insight into core principles of cell biophysics and tissue organization. It furthermore highlights MCR as a model problem to understand the adhesion versus cell surface tension paradigm in cells and its fundamentality to cell shape, mechanics and physiology.

During mitosis, adherent animal cells undergo a drastic shape change, from essentially flat to round, in a process known as mitotic cell rounding (MCR). The aim of this thesis was to critically examine the physical and biological basis of MCR. The experimental part of this thesis employed a combined optical microscope-atomic force microscope (AFM) setup in conjunction with flat tipless cantilevers to analyze cell mechanics, shape and volume. To this end, two AFM assays were developed: the constant force assay (CFA), which applies constant force to cells and measures the resultant height, and the constant height assay (CHA), which confines cell height and measures the resultant force. These assays were deployed to analyze the shape and mechanical properties of single cells trans-mitosis. The CFA results showed that cells progressing through mitosis could increase their height against forces as high as 50 nN, and that higher forces can delay mitosis in HeLa cells. The CHA results showed that mitotic cells confined to ~50% of their normal height can generate forces around 50-100 nN without disturbing mitotic progression. Such forces represent intracellular pressures of at least 200 Pascals and cell surface tensions of around 10 nN/µm. Using the CHA to compare mitotic cell rounding with induced cell rounding, it was observed that the intracellular pressure of mitotic cells is at least 3-fold higher than rounded interphase cells. To investigate the molecular basis of the mechanical changes inherent in mitotic cell rounding, inhibitors and toxins were used to pharmacologically dissect the role of candidate cellular processes. These results implicated the actomyosin cortex and osmolyte transporters, the most prominent of which is the Na+/H+ exchanger, in the maintenance of mechanical properties and intracellular hydrostatic pressure. Observations on blebbing cells under the cantilever supported the idea that the actomyosin cortex is required to sustain hydrostatic pressure and direct this pressure into cell shape changes. To gain further insight into the relationship between actomyosin activity and intracellular pressure, dynamic perturbation experiments were conducted. To this end, the CHA was used to evaluate the pressure and volume of mitotic cells before, during and after dynamic perturbations that included tonic shocks, influx of specific inhibitors, and exposure to pore-forming toxins. When osmotic pressure gradients were depleted, pressure and volume decreased. When the actomyosin cytoskeleton was abolished, cell volume increased while rounding pressure decreased. Conversely, stimulation of actomyosin cortex contraction triggered an increase in rounding pressure and a decrease in volume. Taken together, the dynamic perturbation results demonstrated that the actomyosin cortex contracts against an opposing intracellular pressure and that this relationship sets the surface tension, pressure and volume of the cell. The discussion section of this thesis provides a comprehensive overview of the physical basis of MCR by amalgamating the experimental results of this thesis with the literature. Additionally, the biochemal signaling pathways and proteins that drive MCR are collated and discussed. An exhaustive and unprecedented synthesis of the literature on cell rounding (approx. 750 papers as pubmed search hits on “cell rounding”, April 2012) reveals that the spread-to-round transition can be thought of in terms of a surface tension versus adhesion paradigm, and that cell rounding can be physically classified into four main modes, of which one is an MCR-like category characterized by increased actomyosin cortex tension and diminution of focal adhesions. The biochemical pathways and signaling patterns that correspond with these four rounding modes are catalogued and expounded upon in the context of the relevant physiology. This analysis reveals cell rounding as a pertinent topic that can be leveraged to yield insight into core principles of cell biophysics and tissue organization. It furthermore highlights MCR as a model problem to understand the adhesion versus cell surface tension paradigm in cells and its fundamentality to cell shape, mechanics and physiology.

No description available.

Following the discovery of the one-dimensional sequence of human DNA, much focus has been directed on microscopy and molecular techniques to learn about the spatial organization of chromatin in a 3D cell. The development of these powerful tools has enabled high-resolution, genome-wide analysis of chromosome structure under many different conditions. In this thesis, I focus on how the organization of interphase chromatin is established and maintained following mitosis. Mitotic chromosomes are folded into helical loop arrays creating short and condensed chromosomes, while interphase chromosomes are decondensed and folded into a number of structures at different length scales ranging from loops between CTCF sites, enhancers and promoters to topologically associating domains (TADs), and larger compartments. While the chromatin organization at these two very different states is well defined, the transition from a mitotic to interphase chromatin state is not well understood. The aim of this thesis is to determine how interphase chromatin is organized following mitotic chromosome decondensation and to interrogate factors potentially responsible for driving the transition. First, I determine the temporal order with which CTCF-loops, TADs, and compartments reform as cells exit mitosis, revealing a unique structure at the anaphase-telophase transition never observed before. Second, I test the role of transcription in reformation of 3D chromosome structure and show that active transcription is not required for the formation of most interphase chromatin features; instead, I propose that transcription relies on the proper formation of these structures. Finally, I show that RNA in the interphase nucleus can be degraded with only slight consequences on the overall chromatin organization, suggesting that once interphase chromatin structures are achieved, the structures are stable and RNA is only required to reduce the mixing of active and inactive compartments. Together, these studies further our understanding of how interphase structures form, how these structures relate to functional activities of the interphase cell, and the stability of chromatin structures over time.

Indiana University-Purdue University Indianapolis (IUPUI) / Mammalian target of rapamycin (mTOR) is a key regulator of protein synthesis. Dysregulation of mTOR signaling occurs in many human cancers and its inhibition causes arrest at the G1 cell cycle stage. However, mTOR’s impact on mitosis (M-phase) is less clear. Here, suppressing mTOR activity impacted the G2-M transition and reduced levels of M-phase kinase, Aurora A. mTOR inhibitors did not affect Aurora A mRNA levels. However, translational reporter constructs showed that mRNA containing a short, simple 5’-untranslated region (UTR), rather than a complex structure, is more responsive to mTOR inhibition. mTOR inhibitors decreased Aurora A protein amount whereas blocking proteasomal degradation rescues this phenomenon, revealing that mTOR affects Aurora A protein stability. Inhibition of protein phosphatase, PP2A, a known mTOR substrate and Aurora A partner, restored mTOR-mediated Aurora A abundance. Finally, a non-phosphorylatable Aurora A mutant was more sensitive to destruction in the presence of mTOR inhibitor. These data strongly support the notion that mTOR controls Aurora A destruction by inactivating PP2A and elevating the phosphorylation level of Ser51 in the “activation-box” of Aurora A, which dictates its sensitivity to proteasomal degradation. In summary, this study is the first to demonstrate that mTOR signaling regulates Aurora-A protein expression and stability and provides a better understanding of how mTOR regulates mitotic kinase expression and coordinates cell cycle progression. The involvement of mTOR signaling in the regulation of cell migration by its upstream activator, Rheb, was also examined. Knockdown of Rheb was found to promote F-actin reorganization and was associated with Rac1 activation and increased migration of glioma cells. Suppression of Rheb promoted platelet-derived growth factor receptor (PDGFR) expression. Pharmacological inhibition of PDGFR blocked these events. Therefore, Rheb appears to suppress tumor cell migration by inhibiting expression of growth factor receptors that in turn drive Rac1-mediate actin polymerization.

Objetivo: Verificar resposta após debridamento epitelial de córneas humanas. Métodos: Córneas normais foram submetidas a debridamento antes da cirurgia de enucleação. Realizou-se histologia, TUNEL, Ki67, SMA e microscopia eletrônica. Resultados: Seis córneas foram debridadas e preservadas entre ½ e 65 horas, apresentando apoptose nos ceratócitos do estroma anterior. Células estromais em proliferação foram observadas apenas no tempo de 65 horas. Miofibroblastos não foram encontrados. Uma córnea serviu de controle. Conclusões: Os eventos observados em córneas humanas após debridamento epitelial, apoptose e proliferação dos ceratócitos, foram semelhantes aos descritos em animais de experimentação / Purpose: To examine the early wound healing response to epithelial scrape in human corneas. Methods: Normal corneas had epithelial scrape prior to enucleation. Histology, TUNEL assay, Ki67, SMA and transmission electron microscopy were performed. Results: Epithelial scrape was performed in six corneas from ½ to 65 hours prior to preservation. Keratocyte apoptosis was detected in the anterior stroma in all scraped corneas. Keratocyte proliferation was detected exclusively 65 hours after scrape. No myofibroblast was detected. One cornea was not scraped (control). Conclusion: Results obtained in human corneas (keratocyte apoptosis and proliferation) were similar to animal models

It is known that fission yeast Schizosaccharomyces pombe maintains its nuclear envelope during mitosis and it undergoes an interesting shape change during cell division - from a spherical via an ellipsoidal and a peanut-like to a dumb-bell shape. However, the biomechanical system behind this amazing transformation is still not understood. What we know is, that the shape must change due to forces acting on the membrane surrounding the nucleus and the microtubule based mitotic spindle is thought to play a key role. To estimate the locations and directions of the forces, the shape of the nucleus was recorded by confocal light microscopy. But such data is often inhomogeneously labeled with gaps in the boundary, making classical segmentation impractical. In order to accurately determine the shape we developed a global parametric shape description method, based on a Fourier coordinate expansion. The method implicitly assumes a closed and smooth surface. We will calculate the geometrical properties of the 2-dimensional shape and extend it to 3-dimensional properties, assuming rotational symmetry. Using a mechanical model for the lipid bilayer and the so called Helfrich-Canham free energy we want to calculate the minimum energy shape while respecting system-specific constraints to the surface and the enclosed volume. Comparing it with the observed shape leads to the forces. This provides the needed research tools to study forces based on images.

Objetivo: Verificar resposta após debridamento epitelial de córneas humanas. Métodos: Córneas normais foram submetidas a debridamento antes da cirurgia de enucleação. Realizou-se histologia, TUNEL, Ki67, SMA e microscopia eletrônica. Resultados: Seis córneas foram debridadas e preservadas entre ½ e 65 horas, apresentando apoptose nos ceratócitos do estroma anterior. Células estromais em proliferação foram observadas apenas no tempo de 65 horas. Miofibroblastos não foram encontrados. Uma córnea serviu de controle. Conclusões: Os eventos observados em córneas humanas após debridamento epitelial, apoptose e proliferação dos ceratócitos, foram semelhantes aos descritos em animais de experimentação / Purpose: To examine the early wound healing response to epithelial scrape in human corneas. Methods: Normal corneas had epithelial scrape prior to enucleation. Histology, TUNEL assay, Ki67, SMA and transmission electron microscopy were performed. Results: Epithelial scrape was performed in six corneas from ½ to 65 hours prior to preservation. Keratocyte apoptosis was detected in the anterior stroma in all scraped corneas. Keratocyte proliferation was detected exclusively 65 hours after scrape. No myofibroblast was detected. One cornea was not scraped (control). Conclusion: Results obtained in human corneas (keratocyte apoptosis and proliferation) were similar to animal models