Researches on Hematopoietic Stem Cell (HSC) have been expanding that leads to an increase in our understanding of HSC normal behaviors and abnormal alterations. One of the most important issues in the research on HSCs is to understand the mechanism of the homing process of these cells to settle in their niche in the bone marrow and establish the production of various blood cell types after bone marrow transplantation. The cells first must come in contact with the endothelial cells. This contact is known as adhesion and occurs through a multi-step paradigm ending with transmigration to the bone marrow niche. The initial step of the homing, tethering and rolling of HSC, is mediated by P- and E-Selectins present on endothelial cell surface through their interactions with the ligands expressed on the surface of HSC. Thus, understanding the adhesion process and its contribution for efficient HSCs homing will have great impact on HSC therapy. The selectin – ligands interaction has been intensively studied using in vivo and in vitro approaches. However, the molecular mechanism involved by HSCs at single molecule level is poorly understood. Here in this study, a novel experimental method to unravel the molecular mechanisms of the Selectin-ligands interactions in vitro at the single molecule level is developed by combining microfluidics, epi-fluorescence microscopy and live cells. In this work, the new single-molecule imaging technique enabled us to directly visualize the nanoscale spatiotemporal dynamics of the membrane protein-ligand interactions under conditions of shear stress acting on the cells at the molecular level in real time. Using this method, we revealed that selectin ligands on membrane-tethers and slings show unique spatiotemporal dynamics that is distinct from those on the cell body. We demonstrated that the membrane tethers are formed from single microvilli on the cells, which provides a mechanism to spatially localize selectin ligands, PSGL-1 and CD44 on the tethers and slings. We also demonstrated that the selectin ligands show fast diffusional motion along the tethers and slings compared with that on the cell body due to the detachment of cell membranes from actin cytoskeleton during the formation of the tethers. Our results suggest that the spatial confinement of the selectin ligands together with the fast scanning of a large area by the selectin ligands increase the efficiency of selectin-ligands interaction during the rolling, resulting in slow and stable rolling of the cell on selectin. Our findings contribute significantly to molecular level understanding of the initial step of HSCs. This single-molecule imaging technique that we developed in this study will find wide applications in the molecular-level studies on cell-cell interactions including cancer cell metastasis.

Thesis advisor: John T. Fourkas / One can extract a tremendous amount of information about the organizational and dynamic states of molecules, in situ and in real-time, through highly sensitive and noninvasive single particle optical probing. The highly efficient, multi-photon excited luminescence from stabilized metal nanoclusters renders these species useful as optical probes that can be used in detecting single particle and molecular dynamics. We generate stable, and monodisperse samples of Ag nanoclusters as small as 1 nm in diameter, and find that through substitution of various stabilizer molecules we can precisely tune the size of the clusters over a 1-6 nm range of diameters, ensuring monodispersity and stability at every stage. These clusters also exhibit highly efficient, polarized luminescence upon two photon excitation at 800 nm and remain highly photostable, not exhibiting the deleterious blinking that occurs with many single-molecule fluorophores. In order to demonstrate the utility of these clusters as single-molecule probes, we track their emission polarization over long periods in deeply supercooled liquids such as 4'(octahydro-4,7-methano-5H-inden-5-yliden) bisphenol dimethyl ether (ODE). Our results suggest that these clusters can detect nanoscale dynamics with high sensitivity. / Thesis (BS) — Boston College, 2004. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Chemistry. / Discipline: College Honors Program.

AABBSSTTRRAACCTT Dimers and small aggregates as well as compact aggregates of Ag nanoparticles (NPs) were assembled and chemically anchored to supporting surfaces. The supporting surfaces were either glass slides or SiO2 - coated Cu or Au grids for TEM, both chemically functionalized by 3-aminopropyltrimethoxysilane (APTMS). Compact aggregates of Ag NPs incorporating protoporphyrin IX (PPIX) molecules were prepared by adsorption of chlorides in the presence of PPIX. Dimers and small aggregates of Ag NPs were assembled by selected molecular linkers: 4,4'-diaminoazobenzene (DAAB), 4,4'-diaminoterphenyl (DATP) and 5,10,15,20-tetrakis(4-aminophenyl)porphine (TAPP). The most efficient strategy of dimers and small aggregates preparation has been their assembling by a three - step procedure involving (i) attachment of isolated Ag NPs to the NH2 groups of APTMS functionalized TEM grid, (ii) attachment of molecular linker (with two functional NH2 groups in para position) to Ag NPs by a one terminal NH2 group, and (iii) attachment of Ag NPs to the second, free terminal NH2 group of the linker. In this procedure, the control over the perpendicular orientation of the bifunctional linker and its attachment by one terminal group to Ag NP surface has been accomplished by functionalization of Ag NPs by adsorbed citrate...

A espectroscopia de molécula única (SMS) é uma poderosa técnica para entender como a conformação da cadeia polimérica é modificada por superfícies e interfaces. No presente trabalho, a interação substrato-polímero foi modificada pela alteração da carga superficial do substrato pela modificação do caráter hidrofóbico-hidrófilico da superfície do substrato. A conformação planar / não planar assumida pelo poli (9,9 dioctilfluoreno) (PFO) foi utilizada para acessar os efeitos de substratos e inter-cadeias sobre a conformação do polímero. Usamos a combinação de diferentes tratamentos para alterar superfícies de quartzo inerte de super hidrofílico (ângulo de contato ∼ 0°) para hidrofóbico (ângulo de contato ∼ 80°) quase continuamente. As películas ultrafinas de polímero (<10 nm) e moléculas isoladas podem ser depositadas em diferentes superfícies do substrato por técnica de revestimento por centrifugação de forma controlada, que permitiu a investigação de processos foto físicos a um nível molecular único. Utilizou-se três técnicas espectroscópicas para caracterizar tais filmes ultrafinos, a saber: microscopia de fluorescência confocal equipada com imagens de fluorescência espectral, espectroscopia de absorção UV / Vis e espectroscopia de fotoluminescência dependente da temperatura. As propriedades de emissão e eficiência de filmes de PFO ultrafinos são fortemente afetadas pela presença da interface do substrato. No caso de uma única cadeia de PFO, a fase planar (energia mais baixa) é induzida pela forte interação superfície-polímero no caso da superfície hidrofílica carregada. Além disso, esta interação pode ser fortemente perturbada ou inteiramente destruída por interações entre cadeias. / Single molecule spectroscopy (SMS) is a powerful technique to understand how polymeric chain conformation is modified by surfaces and interfaces [1]. In the present work, substrate-polymer interaction was modified by changing substrate superficial charge by the modification of the hydrophobic-to-hydrophilic character of the substrate surface or by the deposition of charged self-assembling monolayers or polyelectrolytes as well. The planar/non-planar conformation assumed by poly (9,9 dioctylfluorene) (PFO) was used to access the effects of substrates and interchain interactions on the polymer conformation [2]. We used the combination of dif-ferent treatments to change inert quartz surfaces from superhydrophylic (contact angle ∼ 0°) to hydrophobic (contact angle ∼ 80°) almost continuously. Polymer ultrathin films (<10 nm) and isolated chains can be deposited on different substrate surface by spin-coating technique in a controlled way that allowed the investigation of photophysical processes at a single molecular level. We employed three spectroscopic techniques to characterize such ultrathin films, namely: confocal fluorescence microscopy equipped with spectral facilities, UV/Vis absorption spectroscopy and temperature dependent photoluminescence spectroscopy. The emission properties and efficiency of ultrathin PFO films are strongly affected by presence of the inert substrate interface. In the case of single PFO chain, the planar phase (lower energy) is induced by the strong surface-polymer interaction in the case of charged hydrophilic surface. Moreover, this interaction can be strongly perturbed or entirely disrupted by interchain interactions.

Nesta tese foi estudado o espalhamento Raman intensificado pela superfície (SERS) em regime de detecção de uma molécula em eletrodo de prata ativado por ciclos de oxidação e redução. Neste regime, de baixas concentrações, são observadas intensas flutuações de intensidade SERS as quais foram controladas neste substrato pela aplicação de potencial ao eletrodo, o que foi associado a alterações na concentração de moléculas adsorvidas na superfície do eletrodo. Além da dependência com o potencial aplicado, foram estudadas através de simulações Monte Carlo, a contribuição nestas flutuações da constante de adsorção das moléculas, do número de \"hot spots\" (regiões de altas intensificações SERS) e do tipo de \"hot spot\" (em termos de eficiência para detecção de espectros de uma molécula). Através destas simulações foram verificadas flutuações de intensidade muito semelhantes às observadas experimentalmente. Além das flutuações de intensidade foram também observadas flutuações de intensidades relativas, como por exemplo, das relações de intensidades anti-Stokes/Stokes, as quais foram interpretadas segundo um modelo de ressonância, através do qual foi possível estimar as energias de ressonância nos \"hot spots\". Alguns dos resultados indicaram a contribuição de ressonâncias finas, as quais foram interpretadas como resultado de interferências entre ressonâncias de plasmon de superfície. Interferências como estas foram demonstradas através de simulações pelo método DDA (\"Discrete Dipole appoximation\") em modelos simples de \"hot spots\" formados por nanobastões de Au. / In this thesis it was studied surface-enhanced Raman scattering (SERS) at single-molecule detection on Ag electrode activated by oxidation and reduction cycles. At this low concentration limit it was observed strong SERS intensity fluctuations that were controlled by the applied potential to the electrode and this control was associated to changes in surface concentration of adsorbed molecules. Furthermore, it was studied through Monte Carlo simulations the influence of adsorption constant, number of \"hot spots\" (regions of high SERS enhancements) and type of \"hot pot\" (in terms of efficiency for single-molecule detection). With such simulations, it was verified fluctuations of SERS intensities very similar to experimental observations. Besides absolute intensity fluctuations, we also observed fluctuations of relative intensities as, for instance, the. anti-Stokes to Stokes intensity ratios. These fluctuations were interpreted according to a resonance model, which made possible the estimative of resonance energies at the SERS \"hot spots\". Some of these results indicated the existence of sharp resonances that were interpreted as a result of interferences among surface plasmon resonances, which were demonstrated through DDA (Discrete Dipole Approximation) simulations in simple models of \"hot spots\" formed by Au nanorods

La synthèse des protéines est un mécanisme central de la vie cellulaire dont la compréhension est un enjeu du domaine biomédical. Les études en molécule unique permettent d’observer chaque système réactionnel individuellement et donnent accès à des évènements asynchrones difficilement observables en mesure d’ensemble, tels la traduction de protéines.Cette thèse présente les premiers résultats en molécule unique sur la traduction par un ribosome eucaryote (mammifère). Nous observons les systèmes traductionnels grâce à des marqueurs fluorescents liés à des oligonucléotides pouvant s’hybrider sur les séquences d’ARN traduites. L’observation de ces marqueurs est faite par microscopie de fluorescence en onde évanescente (TIRF), les ARN étant fixés sur une lamelle de microscope. En lisant l’ARN, le ribosome détache les marqueurs, et leurs instants de départs donnent des informations sur le passage du ribosome à différentes positions sur l’ARN. Cette méthode permet d’obtenir des données cinétiques sur un grand nombre de systèmes traductionnels en parallèle pouvant alors être interpolées par des lois de probabilité. Nous obtenons par cette méthode des mesures de la cinétique in vitro de l’élongation eucaryote et nous observons un délai dû à une initiation non-canonique. En effet, nous complexons le ribosome sur l’ARN grâce à une structure de type IRES. Dans nos conditions d’expérience, l’incorporation d’un acide aminé prend environ une seconde tandis que cette structure induit un retard à la traduction de plusieurs dizaines de secondes. Ces résultats ouvrent des perspectives d’étude cinétique dans des cas plus complexes tels le franchissement de structures secondaires de l’ARN. / Protein synthesis is a central mechanism of cellular life and understanding it is a challenge in biomedical research. Single molecule studies permit each reactive system to be observed individually and provide access to asynchronous events difficult to observe in ensemble experiments, such as protein translation.This thesis presents the first results on single molecule eukaryotic (mammalian) translation. We observe the translational systems using fluorophores linked to oligonucleotides annealed to the RNA translated sequences. The observation of these fluorophores is done by total internal reflection fluorescence microscopy, the RNA being attached to a microscope slide. When reading the RNA, the ribosome unzips the fluorescent oligonucleotides and their departure times provide information about the position of the ribosome at different locations on the RNA strand. This method provides kinetic data on a large number of parallel translational systems that can be fitted using probability laws.With this method, we measure the in vitro kinetics of eukaryotic elongation and we reveal a delay due to a non-canonical initiation of the ribosome. Indeed, in our experiments, the ribosome is initially complexed on an RNA structure called Internal Ribosome Entry Site. In our experimental conditions, each incorporation of an amino acid in the nascent protein takes about one second while the IRES structure induces a delay of several tens of seconds on the first incorporation. These results open new perspectives for kinetic studies in more complex configurations such as the passage of the ribosome through RNA secondary structures.

Silylamide ligands have been used throughout the Periodic Table since the 1960s. They have delivered landmark complexes by providing the first three co-ordinate f-element complexes, the first trigonal planar f-element complexes and the first near-linear f-element complexes. This area is reviewed in Chapter 2.Herein, this work presents the first uses of several novel bis-silylamide ligands developed at Manchester which take the form {N(SiR3)2} where R = Me, iPr or tBu to afford four novel ligands: N ʹ, {N(SiMe3)(SiiPr3)}; N**, {N(SitBuMe2)2}; N* {N(SitBuMe2)(SiiPr3)}; and N , {N(SiiPr3)2}. Group 1 and 2 complexes of all of these ligands are presented along with the previously reported N*ʹ [N*ʹ = {N(SitBuMe2)(SiMe3)}]; which show variable bonding motifs based on the steric bulk. The N** and N ligands have formed the bulk of the work presented and were used to stabilise the first trigonal planar actinide complex [U(N**)3], as well as the first near-linear Ln(II) (Ln = lanthanide) complexes [Ln(N )2] (Ln = Sm, Eu, Yb, Tm). Additionally the trigonal planar Ln(II) complexes [K(2.2.2-cryptand)][Ln(N**)3] (Ln = Sm, Eu, Yb, Tm) have also been synthesised to compare the physicochemical properties of trigonal planar and near-linear geometries on the same elements with similar ligands.

Doctor of Philosophy / Department of Chemistry / Daniel A. Higgins / This dissertation describes single-molecule tracking (SMT) studies for the quantitative characterization of one-dimensional (1D) solvent-filled surfactant-templated mesoporous silica (STMS) materials and other nanostructured materials, such as double-stranded DNA. SMT permits the simultaneous and quantitative assessment of the nanoscale and microscale morphologies and mass-transport properties of the materials with nanometer-scale spatial resolution. The efficiency and selectivity of catalytic reactions and chemical separations occurring in liquid-filled mesoporous materials are governed by the translational and orientational mobilities and surface interactions of the incorporated reagents and analytes. Polarization dependent SMT results demonstrate that the dye molecules used as probes of materials nanostructure are tightly confined within the one-dimensional (1D) pores of surfactant-templated mesoporous silica films. Spectroscopic single molecule tracking (sSMT) data reveal that the hydrophobic probe dyes are confined within nonpolar regions of the nanomaterials For this dissertation, surfactant templated mesoporous silica films were prepared by the spin coating of acid catalyzed tetramethoxysilane (TMOS)-based silica sols on glass substrates in the presence of Cetyltrimethylammonium bromide (CTAB). Cylindrical CTAB micelles formed during evaporation of the solvent acted as a structure directing template, forming nanometer-sized one-dimensional pores within the silica films. SMT experiments were performed using a wide-field fluorescence microscope that was sufficiently sensitive to allow detection of the fluorescence from individual dye molecules. A series of perylene diimide (PDI) dyes was employed for basic structural characterization of the silica materials. Single molecule fluorescence was recorded in the form of fluorescence videos. These videos revealed the presence of immobile dye molecules, along with those diffusing in one and two dimensions (1D and 2D). The 1D diffusing molecules provided basic evidence for the confinement mass transport of the dye molecules within the silica mesopores. Spectroscopic single molecule tracking (sSMT) studies served as an extension of basic SMT experiments and were employed to determine the location of the molecules. The polarity sensitive dye Nile Red (NR) was employed in these studies. It exhibits 1D diffusion, consistent with its confinement to the cylindrical pores, as was also the case for the PDI dyes. The sSMT data revealed that the majority of NR molecules were found in nonpolar environments having polarities similar to that of n-hexane. Single molecule emission polarization (SMEP) measurements were employed to explore the orientational confinement of the dyes. The results of these experiments demonstrated that the PDI and NR molecules diffuse with their long axes aligned parallel to the long axis of the pores. All of the dyes employed were found to be orientationally confined to ∼1 nm diameter pathways within the pores. The diffusion coefficient for the dyes was also shown to be ∼10^3 -fold smaller than in bulk solution. The results of the NR studies demonstrate that the dye molecules were confined to the hydrophobic cores of the micelles, and provide support for the conclusion that the PDI dyes are similarly confined. These studies afford an enhanced understanding of how nanostructuring of the pore-filling medium in solvent- and surfactant-filled mesoporous materials governs the mass transport and surface interactions of incorporated reagents and analytes. The dependence of molecular confinement on dye charge and structure was also explored in this dissertation. The confined translational and orientational motions of a series of four different PDI dyes diffusing along one dimension (1D) within individual cylindrical silica mesopores were investigated in these studies. Specifically, the motions of cationic and anionic PDI dyes were compared to those of two uncharged PDIs having different alkane tail lengths. All four dyes exhibited populations that were immobile, along with separate populations that diffused in either 1D or 2D. The anionic and cationic PDI dyes exhibited the largest and smallest populations, respectively, of immobile molecules, suggesting that electrostatic interactions between the charged dyes and the cationic surfactant head groups play a significant role in limiting molecular motion. The cationic and anionic PDI dyes also exhibit the largest populations of 2D diffusing molecules, suggesting they may more readily pass between the cylindrical micelles and through the silica pore walls. All four dyes also emit strongly polarized fluorescence as they move in 1D, indicating they are orientationally confined within the nanochannels. Nile Red dye was used to determine the dielectric constant, ε, of nonpolar microenvironments in double-stranded DNA (ds-DNA) single molecules both in aqueous buffer solution and when adsorbed on amine-modified chemical gradient surfaces. The value of ε within the DNA decreased with increasing buffer concentration. Values of ε ∼ 6.75 and ∼3.00 were obtained in 0.1 mM phosphate buffered saline (PBS) and in 10 mM PBS, respectively. Similar effects were observed upon adsorption to chemically graded amine-modified silica surfaces. Under 1 mM buffer, ε was measured to be ∼2.84 and ∼1.90 at the low amine (high silica), and high amine (low silica) ends of the gradient, respectively. An increase in the buffer concentration again led to a decrease in ε, but only at the low amine end. It is concluded that high buffer concentrations and binding to an amine surface cause condensation of the ds-DNA, forming less polar microenvironments within its structure. These results provide important knowledge of the factors governing the polarity of DNA microenvironments to which intercalators bind.

Prions are infectious agents that cause fatal neurodegenerative diseases in the brain. The wide-accepted protein-only hypothesis states that the misfolded form of prion protein (PrP) is the sole constituent of prions, and the self-propagating process of PrP is considered to play a central role in prion pathogenesis. Prions are believed to propagate when a PrP assembly enters a cell and replicates to produce two or more fibrils, leading to an exponential increase in PrP aggregate number with time. However, the molecular basis of this process has not yet been established in detail. This prion-like replication is also suggested to be the mechanism in the development of other notorious neurodegenerative disorders, such as Alzheimer's and Parkinson's disease. In this thesis, I use single-aggregate imaging to study fibril fragmentation and elongation of individual murine PrP aggregates from seeded aggregation in vitro. From fluorescence imaging of individual PrP aggregates on the coverslip surface, elongation and fragmentation of the PrP assemblies have been directly observed. PrP elongation occurs via a structural conversion from a proteinase K (PK)-sensitive to PK-resistant conformer. Fibril fragmentation was found to be length-dependent and resulted in the formation of PK-sensitive fragments. To gain more insights into the mechanism of the spread of PrP, the quantified kinetic profiles allows the determination of the rate constants for these processes through the use of kinetic modelling. This enables the estimation of a simple framework for aggregate propagation through the brain, assuming that doubling of the aggregate number is rate-limiting. In contrast, the same method was applied to measurement for α-Synuclein (αS) aggregation, which has been suggested to be prion-like and is associated with Parkinson's disease. While αS aggregated by the same mechanism, it showed significantly slower elongation and fragmentation rate constants than PrP, leading to much slower replication rate. Furthermore, the measurements in αS aggregation has been extended to the cellular environment, I use super-resolution imaging to study the amplification of endogenous αS aggregation in cells and the transcellular spread of αS. Endogenous αS showed a clear amplification in number of aggregates with time after seed transduction, and the newly-formed αS aggregates are likely to spread through cell-to-cell transmission. The proteasome was demonstrated to possess a novel disaggregase function for αS fibrils and thus produce more seeds for further replication. It partially explains that αS aggregation in cells was found to replicate at a substantially faster rate than that in vitro. Determining the nature of the oligomers formed during aggregation has been experimentally difficult due to the lack of suitable methods capable of detecting and characterising the low level of oligomers. To address this problem, I have studied the early formation of PrP oligomers formed during aggregation in vitro using various single-molecule methods. The early aggregation of PrP is observed to form a thioflavin T (ThT)-inactive and two ThT-active species of oligomers, which differ in size and temporal evolution. The ThT-active oligomers undergo a structural conversion from a PK-sensitive to PK-resistant conformer, while a fraction of which grow into mature fibrils. These results also enable the establishment of a kinetic framework for elucidating temporal evolution of PrP aggregation and the relationship between oligomers and fibrils. Overall, my research identifies fibril elongation with fragmentation are the key molecular processes leading to PrP and αS aggregate replication, an important concept in prion biology, and provides a simple framework to estimate the rate of prion and prion-like spreading in animals. The results also show that a diverse range of oligomers is formed and co-exist during PrP aggregation which differ both in their structure and properties and provides mechanistic insights into a prion aggregation. The work provides a new quantitative approach to describe the prion-like property in neurodegenerative diseases from a kinetic perspective that can be verified in extending studies in other proteins or in cells.

Le ribosome est un organite essentiel de la cellule qui assure la synthèse des protéines. C'est une structure très conservée, composée d'ARN et de protéines ribosomiques organisés en deux sous-unités. Les expériences de reconstitution in vitro du ribosome d'E. coli ont montré que l'assemblage est un processus coordonné impliquant de nombreuses interactions entre les différents constituants. En particulier, les premières étapes de l'assemblage de la grande sous-unité dépendent fortement de la fixation coopérative de cinq protéines ribosomiques à l'ARN 23S, mais les mécanismes moléculaires sous-jacents sont mal connus.Cette étude à l'échelle de la molécule unique vise à préciser ces mécanismes et porte sur un fragment constitué des hélices H18, H19 et H20 du domaine I de l'ARN ribosomique 23S contenant les sites de fixation des protéines uL24 et uL4. Ce fragment d'ARN a été préparé dans une configuration qui permet la mesure de force via un double piège optique. Les courbes de force obtenues ont permis de dresser une cartographie de la stabilité des structures du fragment d'ARN.Ces cartes ont été comparées en absence et en présence des protéines ribosomiques uL24 et/ou uL4, démontrant ainsi que le fragment d'ARN est stabilisé par la fixation des protéines uL24 et/ou uL4. Leur fixation est coopérative et la présence conjointe des deux protéines sur-stabilise les structures du fragment d'ARN.Ces résultats sont discutés dans la perspective de préciser le rôle du fragment d'ARN et des protéines ribosomiques uL24 et uL4 dans l'assemblage de la grande sous-unité du ribosome. / Ribosomes are essential organelles of the cell responsible for the synthesis of proteins. Their well conserved structure made of RNA and proteins is organized into two subunits. In vitro reconstitution of E. coli ribosomes showed that their assembly is a coordinated process which involves many interactions between the components. More specifically, the early stages of the large subunit assembly depend strongly on the cooperative binding of five ribosomal proteins to the 23S RNA. The underlying molecular mechanisms however remain poorly understood.The aim of this study is to shine new light on these mechanisms at the single molecule level. It focuses on a 23S ribosomal RNA fragment composed of the helices H18, H19 and H20 in domain I which encompasses the binding sites of the ribosomal proteins uL24 and uL4. This RNA fragment has been prepared in a dumbbell configuration and force versus displacement measurements have been performed using a dual optical trap. From these measurements, a map summarizing the mechanical stability of the RNA fragment has been determined.The maps obtained in absence and in presence of the ribosomal proteins uL24 and/or uL4 have been compared consequently demonstrating mechanical stabilization of the RNA fragment induced by the binding of uL24 and/or uL4. Moreover, their binding is cooperative and when both are present, the mechanical stabilization of the RNA fragment is enhanced.These results are discussed to specify the role of the RNA fragment and proteins uL24 and uL4 in the large ribosomal subunit assembly.