Investigation of improved immunostimulatory activity of D and K type CpG ODNs in liposomes

Ankara : The Department of Molecular Biology and Genetics and the Graduate School Engineering and Science of Bilkent University, 2013.Thesis (Master's) -- Bilkent University, 2013.Includes bibliographical references leaves 71-86.CpG ODNs are potent immunotherapeutic agents. In human, two major classes of CpG ODNs were shown to induce differential immune activation. D ODNs are strong IFNα inducers, thus promising antiviral agents, whereas K ODNs are effective against bacterial infections. However, their effects cannot be combined. When K and D type ODNs are used simultaneously, K ODN cancels D specific effect, a phenomenon known as K and D ODN dichotomy. The prime reason for this counter acting K ODN action was subcellular compartmentalization of K type CpG ODNs upon internalization. Besides, CpG ODNs have labile nature. When investigated in clinical trials, these nucleic acid based ligands are eliminated upon administration and displayed limited bio-availability due to nuclease digestion. Hence, efforts to protect in vivo performance, and increase stability and accumulation near target cells became a crucial task. Liposome technology offers a simple and mild approach to harbor these ODNs within membrane bilayers and protect them. We also reasoned that, if we use liposomes that alter subcellular fate of K and D ODNs, we can retain both K and D effect when liposomal ODNs are co-administered and the breadth of immunotherapeutic spectrum could be improved. This thesis was designed to understand and characterize different types of CpG ODNs loaded into different liposomes and aimed to determine their activities in different in vitro and in vivo settings. Our results revealed that when two different classes of clinically important CpG ODNs were encapsulated within proper liposome types, it is possible to recapitulate both K and D type ODN effect in PBMCs. Furthermore, in a vaccine model against H. felis, although initially did not induce significantly higher anti H.felis immunity, liposomal CpG ODNs improved persisting antibody levels for extended periods compared to free counterparts. Collectively, our results demonstrate that this platform allows more effective in vivo utilization of CpG ODNs and can be formulated to develop more efficient means to combat several health problems, ranging from cancer to allergy.Dereli, İhsanM.S

The enigmatic meiotic dense body and its newly discovered component, SCML1, are dispensable for fertility and gametogenesis in mice.

Meiosis is a critical phase in the life cycle of sexually reproducing organisms. Chromosome numbers are halved during meiosis, which requires meiosis-specific modification of chromosome behaviour. Furthermore, suppression of transposons is particularly important during meiosis to allow the transmission of undamaged genomic information between generations. Correspondingly, specialized genome defence mechanisms and nuclear structures characterize the germ line during meiosis. Survival of mammalian spermatocytes requires that the sex chromosomes form a distinct silenced chromatin domain, called the sex body. An enigmatic spherical DNA-negative structure, called the meiotic dense body, forms in association with the sex body. The dense body contains small non-coding RNAs including microRNAs and PIWI-associated RNAs. These observations gave rise to speculations that the dense body may be involved in sex body formation and or small non-coding RNA functions, e.g. the silencing of transposons. Nevertheless, the function of the dense body has remained mysterious because no protein essential for dense body formation has been reported yet. We discovered that the polycomb-related sex comb on midleg-like 1 (SCML1) is a meiosis-specific protein and is an essential component of the meiotic dense body. Despite abolished dense body formation, Scml1-deficient mice are fertile and proficient in sex body formation, transposon silencing and in timely progression through meiosis and gametogenesis. Thus, we conclude that dense body formation is not an essential component of the gametogenetic program in the mammalian germ line

Fabrika organizasyonunda Hillier algoritmasının güncelleştirilmesi için bir girişim

Bu çalışmada, fabrika yerleşimi için Frederick S. Hillier tarafından önerilen prosedürü daha efektif bir hale getirebilmek için çaba sarfedilmiştir. Prosedür, bölümler arasındaki materyal taşınımının fiatını en aza indirecek şekilde, bölümlerin yerlerinin bulunmasını sağlamaktadır. Bu amaçla, Hillier'in algoritmasından yola çıkılarak, bir bilgisayar programı yazılmıştır. Geliştirilen yazılımın daha ideal sonuçlar verebildiği görülmüştür. Programa girdi olarak, toplam departman sayısı, arzu edilen organizasyon tipi (satır ve sütun sayısı), bölümler arasındaki malzeme akışları, ve bir başlangıç çözüm verilmektedir. Ayrıca bölümler arasındaki uzaklıklar bilgisayar ekranından girilebildiği gibi istenildiği takdirde önceden hazırlanmış veri dosyalardan okunabilmektedir. İyileştirme, algoritma tarafından koyulan kuralların gerektirdiği biçimde, bölümlerin birbirleriyle ikili olarak yer değiştirmesiyle sağlanmaktadır. Program oldukça hızlıdır ve optimum çözüme yakın bir çözüm önerisi ile bitirilmektedir. Program Borland C++ programlama dili kullanılarak, IBM uyumlu bir bilgisayarda yazılmıştır. Programın kullanımı örneklerle açıklanmış, sonuçlar Hillier algoritmasının sonuçlarıyla karşılaştırılmıştır.In this study, an effort has been made to enhance the procedure suggested by Frederick S. Hillier for plant layout analysis. It considers the development of relative positions of a number of departments with the objective of minimising the total material flow between those departments. For this purpose, a computer program has been written by utilising the rules given in the well known Hillier's algorithm and it has been shown that the developed software yields more idealised layout approaching the optimum solution. Upon inputting total number of facilities, desired number of rows and columns in the final layout, weighting flows between the facilities, and an initial solution, the program either allows the user to give distances between the locations manually, or takes them from a file stored previously in the memory. The improvement is achieved by interchanging the facilities among each other as proposed by the rules constructed within the algorithm. The program is terminated when a final solution for the layout is achieved. It is ver}' fast and approaches to the optimal solution.The program is written in Borland C++ programming language on an IBM compatible PC. The use of the program is given through specific examples. The results are compared with those of Hillier Procedure

Alignment of Homologous Chromosomes and Effective Repair of Programmed DNA Double-Strand Breaks during Mouse Meiosis Require the Minichromosome Maintenance Domain Containing 2 (MCMDC2) Protein.

Orderly chromosome segregation during the first meiotic division requires meiotic recombination to form crossovers between homologous chromosomes (homologues). Members of the minichromosome maintenance (MCM) helicase family have been implicated in meiotic recombination. In addition, they have roles in initiation of DNA replication, DNA mismatch repair and mitotic DNA double-strand break repair. Here, we addressed the function of MCMDC2, an atypical yet conserved MCM protein, whose function in vertebrates has not been reported. While we did not find an important role for MCMDC2 in mitotically dividing cells, our work revealed that MCMDC2 is essential for fertility in both sexes due to a crucial function in meiotic recombination. Meiotic recombination begins with the introduction of DNA double-strand breaks into the genome. DNA ends at break sites are resected. The resultant 3-prime single-stranded DNA overhangs recruit RAD51 and DMC1 recombinases that promote the invasion of homologous duplex DNAs by the resected DNA ends. Multiple strand invasions on each chromosome promote the alignment of homologous chromosomes, which is a prerequisite for inter-homologue crossover formation during meiosis. We found that although DNA ends at break sites were evidently resected, and they recruited RAD51 and DMC1 recombinases, these recombinases were ineffective in promoting alignment of homologous chromosomes in the absence of MCMDC2. Consequently, RAD51 and DMC1 foci, which are thought to mark early recombination intermediates, were abnormally persistent in Mcmdc2-/- meiocytes. Importantly, the strand invasion stabilizing MSH4 protein, which marks more advanced recombination intermediates, did not efficiently form foci in Mcmdc2-/- meiocytes. Thus, our work suggests that MCMDC2 plays an important role in either the formation, or the stabilization, of DNA strand invasion events that promote homologue alignment and provide the basis for inter-homologue crossover formation during meiotic recombination

RAD51 and DMC1 foci persist in <i>Mcmdc2</i>^<i>-/-</i> oocytes.

<p>(<b>a, c, e)</b> Immunostaining of SYCP3 along with RAD51 (<b>a</b>), DMC1 (<b>c</b>) or γH2AX (<b>e</b>) on nuclear surface spreads of pachytene <i>Mcmdc2</i>^<i>+/+</i>, or zygotene-pachytene <i>Mcmdc2</i>^<i>-/-</i> oocytes. Oocytes were collected from the ovaries of littermate fetuses at 18dpc, which is a time point when most wild-type oocytes are in the late pachytene stage. RAD51 and DMC1 foci are largely absent from synapsed chromosomes in <i>Mcmdc2</i>^<i>+/+</i> oocytes. Both RAD51 and DMC1 foci are present in high numbers along the unpaired axes of <i>Mcmdc2</i>^<i>-/-</i> oocytes. (<b>e</b>) γH2AX is largely absent from the synapsed chromosomes of the <i>Mcmdc2</i>^<i>+/+</i> oocyte. γH2AX associates with chromatin throughout the nucleus in the <i>Mcmdc2</i>^<i>-/-</i> oocyte. Scale bars; 10μm. (<b>b, d</b>) Numbers of RAD51 (<b>b</b>) or DMC1 (<b>d</b>) foci are shown in <i>Mcmdc2</i>^<i>+/+</i> and <i>Mcmdc2</i>^<i>-/-</i> oocytes at 18dpc. Median numbers of foci are marked, and n corresponds to the number of analyzed oocytes in two pooled experiments. DMC1 and RAD51 foci numbers are significantly higher in <i>Mcmdc2</i>^<i>-/-</i> than in <i>Mcmdc2</i>^<i>+/+</i> oocytes (Mann Whitney test).</p

RAD51 and DMC1 foci persist in <i>Mcmdc2</i>^<i>-/-</i> <i>s</i>permatocytes.

<p>(<b>a, b, e</b>) Immunostaining showing SYCP3 together with RAD51 (<b>a</b>), DMC1 (<b>b</b>) or γH2AX (<b>e</b>) on nuclear surface spreads of pachytene <i>Mcmdc2</i>^<i>+/+</i>, late zygotene-pachytene <i>Mcmdc2</i>^<i>-/-</i>, <i>Spo11</i>^<i>-/-</i>, and <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/—</i>spermatocytes. RAD51 and DMC1 foci are present at comparatively high density along the axes of unsynapsed sex chromosomes (<b>a, b</b>, asterisk), and are largely absent from synapsed autosomes of <i>Mcmdc2</i>^<i>+/+</i> spermatocytes. Both RAD51 and DMC1 foci are present in high numbers along the unpaired axes of <i>Mcmdc2</i>^<i>-/-</i> spermatocytes. Absence of RAD51 and DMC1 foci is shown in <i>Spo11</i>^<i>-/-</i> and <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/—</i>spermatocytes. (<b>e</b>) γH2AX preferentially accumulates on the partially synapsed sex chromosomes of the <i>Mcmdc2</i>^<i>+/+</i> spermatocyte. γH2AX associates with chromatin throughout the nucleus in the <i>Mcmdc2</i>^<i>-/-</i> spermatocytes. γH2AX is largely restricted to the sex chromatin in wild-type pachytene spermatocytes, and to pseudo-sex bodies in <i>Spo11</i>^<i>-/-</i> and <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/—</i>spermatocytes. Scale bars; 10μm. (<b>c, d</b>) Numbers of RAD51 (<b>c</b>) or DMC1 (<b>d</b>) foci are shown in leptotene (lepto), early zygotene (e zygo) in <i>Mcmdc2</i>^<i>+/+</i> and <i>Mcmdc2</i>^<i>-/-</i>, late zygotene (l zygo) and early-mid pachytene (e-m pa) in <i>Mcmdc2</i>^<i>+/+</i> and zygotene-pachytene (zyg-pa) in <i>Mcmdc2</i>^<i>-/-</i> spermatocytes. Median numbers of foci are marked, and n corresponds to the number of analyzed spermatocytes in three pooled experiments. DMC1 and RAD51 foci numbers are significantly higher in zygotene-pachytene <i>Mcmdc2</i>^<i>-/-</i> spermatocytes than in late-zygotene or early-mid-pachytene <i>Mcmdc2</i>^<i>+/+</i> spermatocytes (Mann Whitney test).</p

MCMDC2 is not required for extensive non-homologous synaptonemal complex formation in the <i>Spo11</i>^<i>-/-</i> background.

<p>(<b>a</b>) SYCP3 (axis marker) and SYCP1 (synaptonemal complex marker) were detected by immunofluorescence on nuclear surface spreads of zygotene-pachytene <i>Mcmdc2</i>^<i>-/-</i>, <i>Spo11</i>^<i>-/-</i> or <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/—</i>spermatocytes. Whereas comparatively few synaptonemal complex stretches are detected in the <i>Mcmdc2</i>^<i>-/-</i>spermatocyte, extensive non-homologous synaptonemal complex formation is seen in the <i>Spo11</i>^<i>-/-</i> or <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/—</i>spermatocytes. Scale bars; 10μm (<b>b</b>) Quantification of SYCP1 stretch numbers in zygotene-pachytene spermatocytes with fully condensed chromosome axes of the indicated genotypes. The numbers of synaptonemal complex stretches is significantly higher in <i>Spo11</i>^<i>-/-</i> or <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/—</i>spermatocytes than in <i>Mcmdc2</i>^<i>-/-</i> (Mann Whitney test). The numbers of synaptonemal complex stretches are not significantly different in <i>Spo11</i>^<i>-/-</i> or <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/—</i>spermatocytes (p = 0.8639, Mann Whitney test). Median numbers of foci are marked, and n corresponds to the number of analyzed spermatocytes in two (<i>Spo11</i>^<i>-/-</i> or <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/-</i>) or three (<i>Mcmdc2</i>^<i>-/-</i>) pooled experiments.</p