Yes / In each generation, the germline is tasked with producing somatic lineages that form the body, and segregating a population of cells for gametogenesis. During animal development, when do cells of the germline irreversibly commit to producing gametes? Integrating findings from diverse species, we conclude that the final commitment of the germline to gametogenesis - the process of germ cell determination - occurs after primordial germ cells (PGCs) colonize the gonads. Combining this understanding with medical findings, we present a model whereby germ cell tumors arise from cells that failed to undertake germ cell determination, regardless of their having colonized the gonads. We propose that the diversity of cell types present in these tumors reflects the broad developmental potential of migratory PGCs. / This work was supported by the Howard Hughes Medical Institute where D.C.P. is an Investigator, and the Frontier Research Program from the Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology. P.K.N. is a recipient of the Hope Funds for Cancer Research Fellowship (HFCR-15-06-06) and an Early Career Fellowship from the National Health and Medical Research Council, Australia (GNT1053776).

In many organisms, the most fundamental event during embryogenesis is differentiating between germline cells and specialized somatic cells. In C. elegans, PIE-1 functions to protect the germline from somatic differentiation and appears to do so by blocking transcription and by preventing chromatin remodeling in the germline during early embryogenesis. Yet the molecular mechanisms by which PIE-1 specifies germline remain poorly understood. Our work shows that SUMOylation facilitates PIE-1-dependent germline maintenance and specification. In vivo SUMO purification in various CRISPR strains revealed that PIE-1 is SUMOylated at lysine 68 in the germline and that this SUMOylation is essential for forming NuRD complex and preserving HDA-1 activity. Moreover, HDA-1 SUMOylation is dependent on PIE-1 and enhanced by PIE-1 SUMOylation, which is required for protecting germline integrity. Our results suggest the importance of SUMOylation in the germline maintenance and exemplify simultaneous SUMOylation of proteins in the same functional pathway.

Cerca de 10% dos casos de câncer de mama e/ou ovário são caracterizados como hereditários, onde a presença de mutações germinativas no gene de suscetibilidade BRCA1 aumenta o risco de desenvolver esses cânceres durante a vida da mulher. O BRCA1 é um gene supressor tumoral envolvido na resposta de danos ao DNA, controle do ciclo celular, na remodelação da cromatina, ubiquitinação e regulação da transcrição. O presente estudo tem como objetivo central caracterizar as mutações do gene BRCA1 associadas a Síndrome Hereditária do Câncer de Mama e/ou Ovário (HBOC) em pacientes atendidos no Serviço de Aconselhamento Genético do Câncer do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (HCFMRP/USP). Os vinte e dois éxons codificantes do BRCA1 foram analisados utilizando o método de High Resolution Melting (HRM) para triagem de mutações pontuais, seguido pelo sequenciamento de DNA dos casos selecionados para validação. A técnica de MLPA (Multiplex Ligation-dependent Probe Amplification) também foi usada para detectar grandes deleções e duplicações. Uma vez confirmada a mutação, membros da família considerados de alto risco, serão investigados para a mutação específica, a fim de proporcionar-lhes um aconselhamento genético apropriado para a detecção precoce do câncer. No presente estudo, foram investigados 41 pacientes que preencheram os critérios para o teste genético de acordo com NCCN Clinical Practice Guidelines in Oncology v.1.2010. Um total de 21 mutações foram identificadas, duas das quais são patogênicas: a deleção dos éxons 17-18 e a deleção dos éxon 19. Ambas estão localizadas no domínio BRCT do gene BRCA1, essencial para a ligação de fosfoproteínas críticas para a ativação do complexo de reparo do DNA. Outra mutação, a S616del, foi tratada como patogênica, mas apresenta informações controversas em diferentes estudos. O trabalho também identificou uma nova mutação, Val1117Ile. Um estudo de haplótipos das mutações identificadas nos pacientes foi realizado e revelou que um dos haplótipos, denominado de 6, contendo quatro resíduos mutados (871Leu, 1038Gly, 1183Arg e 1613Gly) estava presente em 50% das pacientes. O estudo de associação com 82 indivíduos saudáveis, mostrou diferença significativa (p=0,026) nos pacientes, sugerindo assim um risco aumentado de HBOC. Adicionalmente, foi analisada a mutação germinativa R337H no gene p53 para os casos suspeitos de Síndrome de Li-Fraumeni. Em síntese, o presente estudo contribui com a identificação de uma nova mutação não-sinônina no gene BRCA1 e sugere que o haplótipo 871Leu-1038Gly-1183Arg-1613Gly possa conferir risco aumentado do câncer de mama e/ou ovário em pacientes diagnosticados com HBOC. / About 10% of cases of breast and/or ovary cancer are characterized as hereditary, where the presence of germline mutations in susceptibility BRCA1 gene increases the risk of developing these cancers during womans lifetime. BRCA1 is a tumor suppressor gene involved in DNA damage response, cell cycle control, chromatin remodeling, ubiquitination and transcriptional regulation. The present study aims to characterize BRCA1 gene mutations associated with Hereditary Breast/Ovary Cancer Syndrome (HBOC) in patients from the Cancer Genetic Counseling Service of the General Hospital of the Medical School of Ribeirão Preto, University of São Paulo (HCFMRP-USP). The twenty two coding exons of BRCA1 were analyzed using High Resolution Melting (HRM) method for the screening of point mutations, followed by DNA sequencing of the cases selected to validation. MLPA (Multiplex Ligation-dependent Probe Amplification) technique was also used to detect gross deletions and duplications. Once confirmed the mutation, family members most at risk will be analyzed for the specific mutation in order to provide them with an appropriate genetic counseling for early detection of cancer. In the present study, we investigated 41 patients that fulfilled the criteria for genetic testing according to NCCN Clinical Practice Guidelines in Oncology v.1.2010. A total of 21 mutations were identified, two of them are pathogenic: a deletion of exons 17-18 and a deletion of exon 19. Both of them are located in the BRCT domain of BRCA1 gene, impairing the binding of essential phosphoproteins critical to the activation of DNA repair complex. Another mutation, S616del, shows controversial information about its pathogenesis in different studies.The present study also describes a new mutation, Val1117Ile. A study of haplotypes of the mutations identified in patients was performed and revealed that one of the haplotypes, called 6, containing four mutated residues (871Leu, 1038Gly, and 1183Arg 1613Gly) was present in 50% of patients. The association study with 82 healthy subjects showed a significant difference (p = 0.026) in patients, thus suggesting an increased risk for HBOC. Additionally, the germline mutation R337H on p53 gene was also analyzed in the present study for suspected cases of Li-Fraumeni Syndrome. In summary, this study contributes to the identification of a new missense mutation in the BRCA1 gene and suggests that the haplotype-871Leu-1038Gly 1183Arg-1613Gly may confer increased risk of breast cancer and / or ovarian cancer in patients diagnosed with HBOC.

The longstanding belief that women are born with a finite ovarian reserve has been debated for over a decade, ever since the discovery, and subsequent isolation, of purported oogonial stem cells (OSCs) from adult mammalian ovaries. This rare cell population has now been reported in the mouse, rat, pig, rhesus macaque monkey and humans and, although a physiological role for the cells has not been proven, they do appear to generate oocytes when cultured in specific environments, resulting in live offspring in rodents. The primary aim of this thesis was to verify independently the existence of OSCs in human ovary and determine whether they could be isolated from a large animal model, the cow. The secondary aim was to investigate the cells’ in vitro potential, both to undergo neo-oogenesis and as a model for germ cell development. Putative bovine and human OSCs were isolated from disaggregated adult ovarian cortex using a previously validated fluorescence-activated cell sorting (FACS)-based technique, with cells sorted for externally expressed DDX4 (VASA). Freshly isolated and cultured cells were characterised by analysing their expression of pluripotency and germline markers, using RT-PCR, immunocytochemistry and Western blotting. The in vitro neo-oogenesis potential of the cells was explored by injecting fluorescently labelled cells into fragments of adult ovarian cortex and by forming aggregated artificial “ovaries” with putative OSCs and fetal ovarian somatic cells. Germ cell model experiments comprised treatment of cultured cells with BMP4 and/or retinoic acid (RA), with subsequent quantitative RT-PCR and immunocytochemistry analysis for downstream BMP4- and RA-response genes, and liposomal-mediated transfection of cells with a DAZL overexpression plasmid to assess their meiosis-related gene response. Scarce populations of putative OSCs were retrieved from 5 human samples (aged 13- 40 years) and 6 bovine samples. The cells were cultured long-term for up to 7 months and demonstrated consistent expression of several pluripotency-associated and germline markers at the mRNA and protein level, including LIN28, NANOG, POU5F1 (OCT4), IFITM3 (fragilis), STELLA, PRDM1 (BLIMP1), and C-KIT, indicating their early germline nature. Investigation of neo-oogenesis potential revealed that putative human OSCs were associated rarely with fetal somatic cells in primordial follicle-like structures, but could not be confirmed to have undergone oogenesis. However, like early germ cells, putative bovine and human OSCs were BMP4 and RA responsive, with both species demonstrating significant upregulation of expression of ID1 and bovine cells exhibiting a significant increase in MSX1, MSX2 and the meiotic marker SYCP3 in response to BMP4 and/or RA treatment. Cells could be successfully transfected to overexpress DAZL; however, no significant downstream gene expression changes were observed. This is the first report of putative bovine OSC isolation and corroborates a previous report showing putative human OSC isolation. Although the expression of both stem cell and germline markers indicates the cells have characteristics of OSCs, their capacity to enter meiosis and form functional oocytes has yet to be determined. Putative bovine OSCs, however, show promise as a novel model for investigating germ cell development. If their potential can be harnessed, then OSCs may have a role in clinical applications, for example in fertility preservation, in the future. Future experiments will examine the neo-oogenesis capabilities of the cells further and explore novel cell delivery systems for clinical use.

En fin de cytocinèse, le fin pont cytoplasmique qui relie les deux cellules filles est clivé au niveau d’une structure dense en microtubules, le midbody, et permet ainsi la séparation physique de leurs deux cytoplasmes. Les mécanismes cellulaires et moléculaires de ce processus, appelé abscission, sont très étudiés dans des modèles de cellules en culture. Cependant, ils restent encore mal connus dans le contexte d’un organisme en développement. L’ovogenèse de drosophile est un modèle de choix pour l’étude de la régulation développementale de l’abscission. En effet, des cellules à abscission complète (cellules souches germinales) et incomplète (cystes germinaux) sont situées côte à côte au sein de la même unité développementale, le germarium. Les cellules souches se divisent asymétriquement, pour donner une autre cellule souche et un cystoblaste individualisé. Celui-ci entre en alors en différenciation, un programme au cours duquel il réalise quatre cycles cellulaires synchrones au cours desquels la cytocinèse est incomplète. Un cyste germinal de seize cellules interconnectées est ainsi formé. La durée de l’abscission est régulée précisément et dépend du contexte développemental. Notre laboratoire a récemment montré que les kinases Aurora B et Cdk1/ Cyclin B sont des régulateurs de la durée d’abscission dans les cellules germinales de drosophile et en cellules en culture de vertébrés. Mon travail a consisté à explorer la fonction de la protéine Shrub, un membre du complexe ESCRT-III, au cours de l’abscission dans la lignée germinale femelle de drosophile. Nous avons montré que Shrb est localisé au midbody des cellules souches en fin de cytocinèse, et promeut l’abscission. En effet, nous avons montré qu’une réduction du niveau de Shrub dans la lignée germinale provoque un fort délai de l’abscission des cellules souches, supérieur à la durée de leur cycle cellulaire. La cellule souche et son cystoblaste restent donc connecté jusqu’à la mitose suivante, formant ainsi des structures de plusieurs cellules connectées, appelées stem-cyst . L’abscission tardive au sein du stem cyst libère un progéniteur binucléé qui entre en différenciation. En conséquence, des chambres ovariennes à 32 cellules, au lieu de 16, sont formées. De plus, la fonction de Shrub dans l’abscission semble être contrecarrée par Aurora B, puisqu’une réduction des niveaux d’Aurora B dans des hétérozygotes Shrub réduit le nombre de stem-cysts et de chambres à 32 cellules observés. Enfin, nous avons identifié un nouveau facteur impliqué lors de l’abscission, la protéine Lethal giant discs (lgd), dont la perte de fonction induit, comme celle de Shrub, la formation de stem-cysts. En accord avec son rôle dans l’abscission, nous avons montré que Lgd est localisé au midbody. Lgd est requis pour la fonction de Shrub dans la voie endosomale, mais son implication lors de la cytocinèse était inconnue. Nous avons montré qu’un niveau réduit de Lgd augmente le nombre de stem-cysts des hétérozygotes Shrub, indiquant que Lgd et Shrub fonctionnent ensemble pour l’abscission des cellules souches. De façon surprenante, un nombre réduit de chambres à 32 cellules est observé dans ces ovaires, suggérant une fonction antagoniste de Lgd sur Shrub dans les cystes germinaux. Dans ces cystes, une abscission tardive se produit, qui divise en deux cystes de 16 cellules les cystes de 32 cellules, et expliquant ainsi le paradoxe observé (plus de stem-cysts, mais moins de chambres à 32 cellules). / At the end of cytokinesis, a thin cytoplasmic intercellular bridge is cleaved to allow physical separation of the two daughter cells. This process is called abscission, and its cellular and molecular events have been extensively explored in yeast and isolated mammalian cells. However, how abscission is regulated in different cell types or in a developing organism remains poorly understood.Drosophila oogenesis is a great model to study how abscission is regulated developmentally, as within the same developmental unit, the germarium, we find cells undergoing abscission next to others where this process is blocked. Indeed, the germline stem cell (GSC) divides asymmetrically to give rise to another GSC and to an individualized cystoblast. This cell then enters a well-studied process of differentiation, where through four rounds of mitosis with incomplete cytokinesis, gives rives to a cyst of 16 interconnected cells. The duration of abscission, seems to be tightly regulated and dependent on the developmental context. Our lab has recently discovered that AurB and CycB/Cdk1 function as abscission timers in Drosophila GSC and isolated mammalian cells. Thus, my work consisted in exploring how this process is regulated in the Drosophila female germline.We showed that the ESCRT-III protein Shrb localizes to the midbody of the dividing GSC, functioning to promote abscission. Indeed, we found that reduced levels of Shrb resulted in the blockage, or strong delay, of abscission in the GSC and formation of a structure similar to a cyst. In these so called stem-cysts, the GSC keeps dividing while interconnected to its daughter cells. As a consequence, we saw the appearance of egg chambers formed of 32 cells, instead of 16. Furthermore, Shrb function in abscission seems to be counteracted by AurB, as reducing AurB levels in Shrb heterozygous resulted in decreased stem-cysts and 32-cell cysts. Finally, Lethal giant discs (lgd), required for Shrb function in the endosomal pathway, was also seen localizing at the midbody and regulating abscission in GSCs. Removing one copy of Lgd from Shrb heterozygous increased the number of stem-cysts, but surprisingly the number of 32-cell cysts was reduced. This paradoxical result was explained with the observation of late abscission events in mitotic cysts, which divided the 32-cell cysts in the middle, leading to the formation of two cysts of 16 cells.

The contents of nuclei are highly organized. Nuclear organization is facilitated by a dense protein network, called the nuclear lamina, which underlies the nuclear envelope. The nuclear lamina is composed of filamentous lamins and more than eighty lamin-associated proteins (LAPs). Among the first LAPs identified are LEM Domain (LEM-D) proteins, named after LAP2, emerin and MAN1. LEM-D proteins have many shared and unique functions that include providing structural support to the nucleus, regulating signal transduction pathways and gene expression, facilitating proper progression through the cell cycle and maintaining chromatin attachments at the nuclear periphery. Despite requirements for these processes in all cell types, loss of globally expressed LEM-D proteins causes tissue-restricted defects. Identification of the primary function in tissues susceptible to LEM-D protein loss is a persistent challenge in the nuclear lamina field. Research described here uses Drosophila as a model to understand LEM-D protein function. Loss of the Drosophila emerin homologue Otefin (Ote) causes a complex phenotype in the ovary wherein both somatic and germline cells are compromised. Using tissue-restricted expression experiments, it was determined that Ote function is only required in germline stem cells (GSCs) to maintain all cells in the ovary. Developmental, molecular and genetic analyses revealed that the primary defect in ote mutant ovaries is an early block in germline differentiation, followed by GSC death. Genetic rescue experiments revealed that both of these GSC defects are due to the activation of the DNA Damage Response (DDR) proteins ATR and Chk2. Interestingly, activation of ATR and Chk2 occurs independent of detectable canonical DDR triggers, including DNA damage. Immunohistochemical analyses suggest that Ote might be regulating chromatin condensation and/or heterochromatin organization in GSCs. Through studies of Ote, a rescue method was discovered that involves culturing animals at elevated temperatures. This novel rescue strategy, termed hyperthermia, acts independent of ATR or Chk2 inhibition. Interestingly, elevated temperatures leads to chromatin decondensation in Drosophila, suggesting that hyperthermia may rescue oogenesis by alleviating chromatin defects observed in ote mutant germ cells. Together, results from experiments discussed herein dissect a complex ovary phenotype to reveal the critical requirement for a nuclear lamina LEM-D protein. Investigations into Ote function have revealed several aspects of GSC biology. The ATR/Chk2 checkpoint activated in the absence of Ote uncovered a previously unidentified cause of female GSC death. Further, findings that ATR and Chk2 are activated in the absence of canonical triggers suggest that GSCs possess a system to monitor defects or changes in the nucleus that do not involve DNA damage. Therefore, studies of Ote function and ote mutant phenotypes have uncovered valuable insights into LEM-D protein function and revealed the existence of novel conditions required for GSC maintenance.

The studies in this thesis examined piglet gonocyte identification, isolation, purification, preservation and potential for initiation of spermatogenesis after transplantation into irradiated recipient testes. As a first step, we characterized a previously non-described auto-fluorescence in the piglet testis tissue. This auto-fluorescence mainly originated from granules among the testis interstitial cells, and we found that its interference with immuno-fluorescence can be overcome using Sudan black staining. We also showed that porcine gonocytes can be specifically labelled with the lectin Dolichos biflorus agglutinin (DBA). To optimize gonocyte isolation, we found that ~9-fold more live cells could be harvested by enzymatic digestion of testis tissues than with mechanical methods. However, the proportion of gonocytes (~7%) did not differ between the mechanical and enzymatic methods of testis cell isolation. We then developed a novel three-step strategy for isolation of gonocytes by combining enzymatic digestion and vortexing, resulting in a gonocyte proportion of ~40% (~5-fold more than that from conventional methods). For short-term preservation of testis cells, we found that the survival of testis cells under hypothermic conditions was dependent on the cell type, and affected by storage duration, temperature and medium used. More than 80% of live testis cells survived the 6-day hypothermic preservation period in 20% FBS-L15, without visible changes to the cell culture potential or gonocyte proportion. In another experiment where testis tissues were maintained under hypothermic conditions, we found that ~25% of testis cells could survive for 6 days if preserved in HypoThermosol-FRS solution (HTS-FRS), without morphological changes. To purify gonocytes, we showed that centrifugation of testis cells using 17% Nycodenz can lead to precipitation of gonocytes in pellets (with a purity of > 80%). We also found that pre-coating tissue culture plates with both fibronectin and poly-D-lysine can result in the negative selection of gonocytes (with a purity of up to 85%). We subsequently showed that further purification of gonocytes (to > 90%) could be achieved by combining the two latter approaches. To prepare recipients for germ cell transplantation, we used local irradiation of piglet testes which reduced testis growth, decreased seminiferous tubule diameters and completely eliminated spermatogenesis at 4 months post-irradiation. Compared with the absence of endogenous spermatogenesis in the control testes, spermatogenesis up to elongating spermatids was observed in the irradiated testes after gonocyte transplantation. In summary, we investigated several critical elements in the study and manipulation of gonocytes in a large animal model.

The studies in this thesis examined piglet gonocyte identification, isolation, purification, preservation and potential for initiation of spermatogenesis after transplantation into irradiated recipient testes. As a first step, we characterized a previously non-described auto-fluorescence in the piglet testis tissue. This auto-fluorescence mainly originated from granules among the testis interstitial cells, and we found that its interference with immuno-fluorescence can be overcome using Sudan black staining. We also showed that porcine gonocytes can be specifically labelled with the lectin Dolichos biflorus agglutinin (DBA). To optimize gonocyte isolation, we found that ~9-fold more live cells could be harvested by enzymatic digestion of testis tissues than with mechanical methods. However, the proportion of gonocytes (~7%) did not differ between the mechanical and enzymatic methods of testis cell isolation. We then developed a novel three-step strategy for isolation of gonocytes by combining enzymatic digestion and vortexing, resulting in a gonocyte proportion of ~40% (~5-fold more than that from conventional methods). For short-term preservation of testis cells, we found that the survival of testis cells under hypothermic conditions was dependent on the cell type, and affected by storage duration, temperature and medium used. More than 80% of live testis cells survived the 6-day hypothermic preservation period in 20% FBS-L15, without visible changes to the cell culture potential or gonocyte proportion. In another experiment where testis tissues were maintained under hypothermic conditions, we found that ~25% of testis cells could survive for 6 days if preserved in HypoThermosol-FRS solution (HTS-FRS), without morphological changes. To purify gonocytes, we showed that centrifugation of testis cells using 17% Nycodenz can lead to precipitation of gonocytes in pellets (with a purity of > 80%). We also found that pre-coating tissue culture plates with both fibronectin and poly-D-lysine can result in the negative selection of gonocytes (with a purity of up to 85%). We subsequently showed that further purification of gonocytes (to > 90%) could be achieved by combining the two latter approaches. To prepare recipients for germ cell transplantation, we used local irradiation of piglet testes which reduced testis growth, decreased seminiferous tubule diameters and completely eliminated spermatogenesis at 4 months post-irradiation. Compared with the absence of endogenous spermatogenesis in the control testes, spermatogenesis up to elongating spermatids was observed in the irradiated testes after gonocyte transplantation. In summary, we investigated several critical elements in the study and manipulation of gonocytes in a large animal model.

Potato production in tropical and subtropical countries suffers from damage caused by the potato tuber moth (PTM), Phthorimiaea operculella. Development of a germline transformation system and the identification of genes that are differentially expressed within the PTM midgut are the main goals of this research. We tested three components that are critical to genetic transformation systems for insects; promoter activity, marker gene expression, and transposable element function. We compared the transcriptional activities of five different promoters, hsp70, hsp82, actin5C, polyubiquitin and ie1, within PTM embryos. The ie1 promoter flanked with the enhancer element, hr5, showed a very high level of transcriptional activity compared with the other promoters. The expression of the enhanced green fluorescent protein (EGFP) was detected under UV-illumination within the embryonic soma demonstrating that it can be used as an effective marker gene for PTM. The transpositional activities of the Hermes, mariner and piggyBac transposable elements were tested in interplasmid transposition assays. The piggyBac element was shown mobile within the embryonic soma with a transposition frequency of 4.2 X 10-5 transposition/donor plasmid. The piggyBac mobility has been enhanced by incorporating a transactivator plasmid expressing the IE1 protein from the bacoluvirus Autographa californica nuclear polyhedrosis virus. Seven transformation experiments were performed. The experiments failed to produce a transgenic PTM. The insect midgut is a rich region of molecular targets involved in food processing that could be potentially used to design a new control strategy. The suppression subtractive hybridization (SSH) method was used to identify differentially expressed genes from the PTM midgut. From this subtracted library, 2984 clones were collected and screened. Of these clones, 637 clones are candidate differentially expressed genes within the PTM midgut. Sixty-nine cDNA clones were randomly selected for DNA sequencing. Tweleve clones were selected for further analysis using RT-PCR and Northern blot techniques. Eleven of the clones resulted in positive results for midgut expression. Five clones, showing homology with insect immune peptides, were used in the challenge experiment which revealed that these cDNAs are constitutively expressed in the midgut, as well as being up-regulated due to bacterial or viral challenge.

Antibodies are essential molecules that help to provide immunity against a vast population of environmental pathogens. This antibody conferred protection is dependent upon genetic diversification mechanisms that produce an impressive repertoire of lymphocytes expressing unique B-cell receptors. The advent of high throughput sequencing has enabled researchers to sequence populations of B-cell receptors at an unprecedented depth. Such investigations can be used to expand our understanding of mechanistic processes governing adaptive immunity, characterization of immunity related disorders, and the discovery of antibodies specific to antigens of interest. However, next generation sequencing of immunological repertoires is not without its challenges. For example, it is especially difficult to identify biologically relevant features within large datasets. Additionally, within the immunology community, there is a severe lack of standardized and easily accessible bioinformatics analysis pipelines. In this work, we present methods which address many of these concerns. First, we present robust statistical methods for the comparison of immunoglobulin repertoires. Specifically, we quantified the overlap between the antibody heavy chain variable domain (V H ) repertoire of antibody secreting plasma cells isolated from the bone marrow, lymph nodes, and spleen lymphoid tissues of immunized mice. Statistical analysis showed significantly more overlap between the bone marrow and spleen VH repertoires as compared to the lymph node repertoires. Moreover, we identified and synthesized antigen-specific antibodies from the repertoire of a mouse that showed a convergence of highly frequent VH sequences in all three tissues. Second, we introduce a novel algorithm for the rapid and accurate alignment of VH sequences to their respective germline genes. Our tests show that gene assignments reported from this algorithm were more than 99% identical to assignments determined using the well-validated IMGT software, and yet the algorithm is five times faster than an IgBlast based analysis. Finally, in an effort to introduce methods for the standardization, transparency, and replication of future repertoire studies, we have built a cloud-based pipeline of bioinformatics tools specific to immunoglobulin repertoire studies. These tools provide solutions for data curation and long-term storage of immunological sequencing data in a database, annotation of sequences with biologically relevant features, and analysis of repertoire experiments. / text