Transposable elements (TEs) are mobile genetic elements. They are a significant component of many eukaryotic genomes. They are involved in chromosomal rearrangement by serving as substrates for homologous recombination, in creating new genes through a process of TE "domestication", and in modifying and shuffling existing genes by transducing neighboring sequences (Lander et al., 2001). Therefore, both active and inactive TEs are potentially potent agents for genomic change (Kidwell and Lisch, 2001, 2002; Rizzon et al., 2002; Petrov et al., 2003). In the meantime, active TEs are being explored as useful tools for genetic transformation and possible gene drive mechanisms to deliver genes in natural populations (Ashburner et al.,1998; Alphey et al.,2002; Handler and O'Brochta, 2004). My thesis project focuses on AGH1, a novel DNA-mediated TE in Anopheles gambiae and related mosquitoes. I have studied its genomic structure, insertion polymorphism, evolution, and transposition activity. As part of the sequence and structural characterization of AGH1 in the A. gambiae genome, the boundaries of AGH1were determined. The TA target site duplications flanking AGH1 were verified by comparing a genomic sequence that had an AGH1 insertion with the sequence of a corresponding empty site. AGH1 has relatively long, 350bp, TIRs (Terminal inverted repeats). In addition to the transposase ORF (ORF1) that contains a DD34E catalytic motif, it contains an unusual ORF2 with unknown function. Phylogenic analyses clearly suggest that unlike most DD34E transposons that are similar to the Tc1 family, AGH1 belongs to a different clade that is related to the previously characterized fungal TE Ant and protozoan TEC1 and TEC2. Truncated AGH1 and AGH1-related MITE (Miniature inverted-repeat TE) families were also identified. AGH1 insertion polymorphism was studied using 4 natural populations that belong to two molecular forms of A. gambiae, M and S. AGH1 insertions showed considerable differences between M and S forms and the insertions of AGH1 are highly variable in two populations of M. These results are potentially significant in light of the hypothesis that M forms are newly derived incipient species that are only found in West Africa. PCR and sequencing results showed more than 99% sequence identity between AGH1 sequences in A. gambiae, A. arabiensis, and A. melas, which may indicate either purifying selection or recent horizontal transfer. To assess whether AGH1 is currently active, inverse PCR was performed which provided evidence for extrachromosomal circular AGH1 that may be a product of imprecise excision. RT-PCR detected transcripts for both intact and truncated transposase. Preliminary TE display experiments using genomic DNA isolated from different passages of an A. gambiae Sua1B cell line showed possible new insertions and deletions of AGH1 related elements, which may have been mobilized by AGH1. In summary, the structural and genomic characteristics of AGH1 and the phylogenetic relationship between AGH1 and other known transposons in the IS630-Tc1-mariner superfamily have been determined. Significant divergence was shown between M and S forms of A. gambiae according to AGH1 insertion patterns. Observations of high level of insertion polymorphism and low insertion frequency per site in M populations are preliminary indications that AGH1 may be active in some populations. AGH1 has at least been recently transposing and there are also indications for its current activity in A. gambiae cell lines. If AGH1 is indeed active, it has the potential to be used as genetic tools to study mosquito biology and to spread refractory genes into the field populations to help control mosquito-borne diseases. Although a few active DNA transposons have been discovered in different insects and are being used as tools to transform mosquitoes, no DNA active transposons have been reported in mosquitoes. It is our hope that active endogenous DNA transposons may present new features that will help us overcome some of the deficiencies of current transformation tools developed based on exogenous transposons. In addition, the discovery of an active DNA transposon will help us understand how TEs spread in natural populations of mosquitoes, which is critical if we are to use TEs to drive refractory genes into mosquito populations to control vector-borne infectious diseases. The differential insertion patterns of AGH1 in M and S populations are consistent with the hypothesis that the M and S forms of A. gambiae are in the process of incipient speciation. AgH1 showed much higher levels of insertion polymorphisms in two west African populations of the M molecular form compared to two east African S populations. Similarly, the maximum level of chromosomal differentiation is observed in west African dry savannah areas, while a much lower degree of chromosomal polymorphism is observed in east Africa. Therefore our insertion data support the hypothesis that the speciation process is likely to be originated in west Africa, probably as the result of the need of ecological flexibility created by the greater ecological variability of this region. From a biomedical perspective, this type of analysis is critical because the genetic differences between M and S forms may directly impact the effectiveness of mosquito control measure and perhaps disease transmission. / Master of Science

<p>Base excision repair (BER) is a DNA repair mechanism that involves the removal of single damaged bases from DNA and their excision as free bases. Another DNA repair mechanism known as nucleotide excision repair (NER) involves the removal of bulky lesions from DNA. Previous published reports have suggested a role for certain NER proteins in BER. Methylene blue (MB) is a type II photosensitizer, which upon excitation by visible light (VL) produces singlet oxygen that reacts with DNA to form 8hydroxyguanine (8-oxoG) lesions. The BER mechanism has been shown to preferentially remove 8-oxoG lesions from DNA. In the current work, the role of NER proteins in the BER of [MB+VL]-induced DNA damage was examined using a reporter gene assay. AdMCMVlacZ and AdHCMVlacZ are non-replicating adenoviruses that express the bacterial β-galactosidase ( β-gal) reporter gene under the control of the murine or human cytomegalovirus immediate early promoter, respectively. Host cell reactivation (HCR) of β-gal activity for a [MB+VL]-treated AdMCMVlacZ was examined in two repair-proficient normal Chinese hamster ovary (CHO) cell lines, NER-deficient mutants from rodent complementation groups (RCGs) 1 through 6, and the HER-deficient cell line EM9. Results show a decreased HCR capacity of [MB+VL]-induced DNA damage in the UV61 (RCG-6; CSB) cell line when compared to the repair-proficient normal AA8. This suggests an involvement of CSB in BER of [MB+VL]-induced damage. In contrast, the XRCCl-deficient EM9 showed an increased HCR capacity when compared to the parental AA8. This suggests a beneficial role for an XRCCl deficiency or for the specific gain-of-function gene mutation in the XRCCl gene for the repair of [MB+VL]induced damage. Similarly, the ERCCl-deficient UV20 (RCG-1) showed an increased HCR capacity when compared to the parental AA8. In contrast, another ERCCJdeficient cell line, 30PV, and the ERCCJ knock-out cell line, CH0-7-27, showed no significant increase in β-gal activity when compared to the parental CHO-Kl. The ability to induce BER of [MB+VL]-induced DNA damage was also examined. HCR of β-gal activity for [MB+VL]-treated AdHCMVlacZ or AdMCMVlacZ was examined in several CHO cell lines that were either untreated or treated with low levels of UVC or MB plus VL. Pre-treatment of NER-deficient UV61 and repair-proficient AA8 cells with UVC resulted in an enhanced HCR for [MB+VL]-treated AdHCMVlacZ, although the results were only significant for UV61. Similarly, an enhanced HCR of [MB+VL]-treated AdMCMVlacZ was observed in AA8 cells, suggesting that the repair of the [MB+VL]treated reporter gene is inducible by UVC. A significant enhancement in HCR of [MB+VL]-treated AdMCMV lacZ was not observed in AA8 cells following pre-treatment with MB, VL, or both, indicating that the detection of any induction in the repair of DNA damage from MB plus VL could not be made with the conditions of the HCR assay employed. As investigations into the effects of MB plus VL on whole cells have been limited, clonogenic survival of BER-and NER-deficient CHO cell lines was also observed following treatment with MB alone or MB plus VL. Results showed that the sensitivities of the NER-deficient CHO cell lines from RCGs 2, 4 and 5 and the BERdeficient EM9 cell line to MB alone were within the range obtained for the two repairproficient CHO normal cell lines, AA8 and K 1. In contrast, the UV20 cell line was more sensitive to MB alone compared to the normal cell lines. Additionally, both UV24 (RCG-3; XPB) and UV61 showed a decreased sensitivity toMB alone when compared to the normal AA8 cell line. The sensitivity of cells to MB plus VL was greater in the UV24, UV135 (RCG-5; XPG) and the XRCCl-deficient EM9 cell lines compared to that of the range obtained for the two repair-proficient normal cell lines, AA8 and Kl. Taken together with the results from the HCR assays, these results suggest that [MB+VL]induced DNA damage to cells and its repair do not play a major role in the survival of cells following treatment with MB plus VL. It appears likely that damage to cellular components other than DNA, such as protein, lipid and biological membranes, play a more important role in cell killing by MB plus VL.</p> / Thesis / Master of Science (MSc)

Group I introns are catalytic RNAs with the ability to splice out of RNA transcripts, often without the aid of proteins. These self-splicing introns have been reengineered to create ribozymes with the ability to catalyze reactions. One such ribozyme, derived from a Pneumocystis carinii group I intron, has been engineered to sequence specifically remove a targeted segment from within an RNA substrate, which is called the trans excision-splicing reaction.The two catalytic steps of the trans excision-splicing reaction occur at positions on the substrate known as the 5' and 3' splice sites. Strict sequence requirements at these sites could potentially limit the target choices for the trans excision-splicing ribozyme, so the sixteen possible base pair combinations at the 5' splice site and the four possible nucleotides at the 3' splice site were tested for reactivity. All base pair combinations at the 5' splice site allow the first reaction step (5' hydrolysis) to occur and several combinations allow the second step to occur, resulting in trans excision-splicing product formation. Moreover, we found that non-Watson-Crick base pairs are important for 5' splice site recognition and prevent product degradation via hydrolysis at other sequence positions. The sequence requirement at the 3' splice site is absolute, as guanosine alone produced complete product.To date, the experiments with the trans excision-splicing ribozyme have been conducted in vitro. The further development of this ribozyme as a biochemical tool and as a potential therapeutic agent requires in vivo reactivity. Thus, a prokaryotic system was designed and tested to assess the catalytic potential of the trans excision-splicing ribozyme. We show that the ribozyme successfully excised a single, targeted nucleotide from a mutated green fluorescent protein transcript in Escherichia coli. On average, 12% correction was observed as measured by fluorescence and approximately 1.2% correction was confirmed through sequence analysis of isolated transcripts.We have used these studies to further characterize trans excision-splicing ribozymes in vitro and to pave the way for future development of this ribozymereaction in vivo. These results increase our understanding of this ribozyme and advance this reaction as a biochemical tool with potential therapeutic applications.

Résumé : Les télomères sont des structures nucléoprotéiques spécialisées qui assurent la stabilité du génome en protégeant les extrémités chromosomiques. Afin d’empêcher des activités indésirables, la réparation des dommages à l’ADN doit être convenablement régulée au niveau des télomères. Pourtant, il existe peu d’études de la réparation des dommages induits par les ultraviolets (UVs) dans un contexte télomérique. Le mécanisme de réparation par excision de nucléotides (NER pour « Nucleotide Excision Repair ») permet d’éliminer les photoproduits. La NER est un mécanisme très bien conservé de la levure à l’humain. Elle est divisée en deux sous voies : une réparation globale du génome (GG-NER) et une réparation couplée à la transcription (TC-NER) plus rapide et plus efficace. Dans notre modèle d’étude, la levure Saccharomyces cerevisiae, une forme compactée de la chromatine nommée plus fréquemment « hétérochromatine » a été décrite. Cette structure particulière est présente entre autres, au niveau des régions sous-télomériques des extrémités chromosomiques. La formation de cette chromatine particulière implique quatre protéines nommées Sir (« Silent Information Regulator »). Elle présente différentes marques épigénétiques dont l’effet est de réprimer la transcription. L’accès aux dommages par la machinerie de réparation est-il limité par cette chromatine compacte ? Nous avons donc étudié la réparation des lésions induites par les UVs dans différentes régions associées aux télomères, en absence ou en présence de protéines Sir. Nos données ont démontré une modulation de la NER par la chromatine, dépendante des nucléosomes stabilisés par les Sir, dans les régions sous-télomériques. La NER était moins efficace dans les extrémités chromosomiques que dans les régions plus proches du centromère. Cet effet était dépendant du complexe YKu de la coiffe télomérique, mais pas dépendant des protéines Sir. La transcription télomériques pourrait aider la réparation des photoproduits, par l’intermédiaire de la sous-voie de TC-NER, prévenant ainsi la formation de mutations dans les extrémités chromosomiques. Des ARN non codants nommés TERRA sont produits mais leur rôle n’est pas encore clair. Par nos analyses, nous avons confirmé que la transcription des TERRA faciliterait la NER dans les différentes régions sous-télomériques. / Abstract : Telomeric DNA is made of short tandem repeats located at the ends of chromosomes and their maintenance is critical to prevent genome instability. DNA lesions constitute a serious risk to genome integrity. Thus, DNA repair mechanisms are required for continuous and unabridged cell divisions. The nucleotide excision repair (NER) pathway removes bulky DNA lesions such as UV-induced photoproducts, like the cyclobutane pyrimidine dimers (CPD). NER is divided in two sub-pathways: global genome repair (GGR) and the faster transcription-coupled repair (TCR), which only differ in how they recognize UV-induced lesions. In eukaryotes, NER must find and repair DNA lesions that are buried in nucleosomes. In the yeast S. cerevisiae, genes positioned close to telomeres are silenced by a heterochromatin-like structure that is formed by silent information regulator proteins (Sir). To determine if nucleosomes and chromatin in subtelomeric regions affect the efficiency of NER, we studied the repair of photoproducts in different telomere-associated regions in both, WT and SIR genes deleted cells (sirΔ). We found that NER efficiency was modulated by the presence of nucleosomes on the subtelomeric type X element. In addition, in absence of Sir proteins, NER efficiency increased and was not modulated by nucleosomes, indicating that nucleosome positioning was less defined in sirΔ cells. Remarkably, in telomeric restriction fragment, NER was less efficient at telomeres than in the subtelomere type Y’ element. We suggest that low NER efficiency at the very end of chromosomes results from attachment sites to the nuclear periphery. Our data indicate that NER in sub-telomeric chromatin is modulated by Sir proteins stabilized-nucleosomes, and that NER is inhibited in telomeric chromatin by the presence of YKu, independently from the presence of Sir proteins. It was recently shown that the chromosome ends are transcribed and a non-coding RNA, called TERRA, is produced. Currently the precise functions of TERRA are not understood. Our second goal is to help understand the function of TERRA. We think that transcription at the chromosome ends could facilitate the removal of DNA lesions from heterochromatin by TCR, which would prevent the formation of mutations and, ultimately, chromosome shortening. Our data showed that TC-NER is effective in Y’ element and the telomere. Without Sir proteins, TERRA transcription is found in a particular region at the end of the X element. The transcription of TERRA could improve the repair of UV-induced lesions.

Entre 100 et 140 millions de femmes, de petites filles et d’adolescentes sont excisées (Andro et Lesclingrand, 2007). Les risques sanitaires de l’excision sont élevés et concernent la santé reproductive, physique et psychologique des femmes. Les nouvelles migrations et l’augmentation des pays qui légifèrent l’excision ont contribué à l’internationalisation de l’excision et à la modification de son processus. On constate actuellement une tendance de l’excision à devenir une pratique clandestine et une perte de sa signification rituelle. En même temps, les mouvements de lutte internationaux, régionaux autant que nationaux prennent de l’ampleur et connaissent une période de mutation afin de contrer la nouvelle figure de l’excision. Le Burkina Faso ne fait pas exception. Le gouvernement burkinabé s’est clairement positionné en faveur du mouvement de lutte contre l’excision et met en place de nombreux dispositifs juridiques, politiques et économiques afin d’en soutenir les initiatives. En 2003, 77 % des femmes burkinabè âgées de 15 à 49 ans se déclaraient excisées. Parallèlement, on assiste à une diminution de la pratique chez leurs filles entre 1998 et 2003 et à une augmentation du nombre de Burkinabè se déclarant contre la pratique. Pourtant en 2003, environ 40 % des femmes ont excisé ou souhaitent exciser leurs filles et environ 24 % des hommes et 26 % des femmes sont encore favorables à la perpétuation de l’excision. Ce mémoire s’intéresse d’abord aux changements de pratique, de connaissance et d’attitudes par rapport à l’excision entre 1998 et 2003. Il s’intéresse ensuite aux déterminants socioculturels, démographiques et économiques favorisant la persistance de cette pratique au sein de la société burkinabé et aux obstacles rencontrés par les intervenants pour combattre l’excision sur le terrain. Pour ce faire la recherche associe méthodes quantitatives et qualitatives. Elle combine analyses statistiques des données des enquêtes démographiques de santé de 2003 et de 1998 et analyse des données d’entretiens collectées auprès d’acteurs sur le terrain entre le premier et le 10 octobre 2005. / Between 100 and 140 million women, young and teenage girls have undergone feminine genital cutting (Andro and Lesclingrand, 2007). The medical risks of excision are high and relate to the reproductive, physical and psychological health of women. New migrations and the increase of countries legislating female genital cutting contributed to the internationalization of the practice and to the changes in its process. The loss of the ritual significance of excision and the increase as a concealed practice have clearly been noticed. In the meantime, the international movements fighting it, regional as well as national, amplify and put changes together in order to counter the new figure of female genital cutting. Burkina Faso does not make exception. The Government has clearly positioned itself in favour of movements fighting excision; and has set up many legal, political and economic devices in order to support their initiatives. In 2003, 77% of Burkina Faso women aged between 15 to 49 years old were declared excised. In parallel, a reduction in the practice for their daughter between 1998 and 2003 is noted as well as an increase in the number of Burkina Faso people declaring themselves against the practice. However in 2003, approximately 40% of women have performed or would like their daughter to undergo the cut; and approximately 24% of men and 26% of women are still encouraging the perpetuation of excision. This thesis’ first concern is the changes in the practice, and the knowledge and attitudes regarding excision between 1998 and 2003. Secondly, the attention is drawn on the socio-cultural, demographic and economic determinants indulging the doggedness of this practice within Burkina Faso people and on the concrete obstacles workers encounter on the ground. Therefore this research associates quantitative and qualitative methods. It combines statistic analysis of the Demographic and Health Surveys of 2003 and 1998 data; and analyzes data collected from talks made between October 1 st and 10th, 2005 with fields’ workers.

Entre 100 et 140 millions de femmes, de petites filles et d’adolescentes sont excisées (Andro et Lesclingrand, 2007). Les risques sanitaires de l’excision sont élevés et concernent la santé reproductive, physique et psychologique des femmes. Les nouvelles migrations et l’augmentation des pays qui légifèrent l’excision ont contribué à l’internationalisation de l’excision et à la modification de son processus. On constate actuellement une tendance de l’excision à devenir une pratique clandestine et une perte de sa signification rituelle. En même temps, les mouvements de lutte internationaux, régionaux autant que nationaux prennent de l’ampleur et connaissent une période de mutation afin de contrer la nouvelle figure de l’excision. Le Burkina Faso ne fait pas exception. Le gouvernement burkinabé s’est clairement positionné en faveur du mouvement de lutte contre l’excision et met en place de nombreux dispositifs juridiques, politiques et économiques afin d’en soutenir les initiatives. En 2003, 77 % des femmes burkinabè âgées de 15 à 49 ans se déclaraient excisées. Parallèlement, on assiste à une diminution de la pratique chez leurs filles entre 1998 et 2003 et à une augmentation du nombre de Burkinabè se déclarant contre la pratique. Pourtant en 2003, environ 40 % des femmes ont excisé ou souhaitent exciser leurs filles et environ 24 % des hommes et 26 % des femmes sont encore favorables à la perpétuation de l’excision. Ce mémoire s’intéresse d’abord aux changements de pratique, de connaissance et d’attitudes par rapport à l’excision entre 1998 et 2003. Il s’intéresse ensuite aux déterminants socioculturels, démographiques et économiques favorisant la persistance de cette pratique au sein de la société burkinabé et aux obstacles rencontrés par les intervenants pour combattre l’excision sur le terrain. Pour ce faire la recherche associe méthodes quantitatives et qualitatives. Elle combine analyses statistiques des données des enquêtes démographiques de santé de 2003 et de 1998 et analyse des données d’entretiens collectées auprès d’acteurs sur le terrain entre le premier et le 10 octobre 2005. / Between 100 and 140 million women, young and teenage girls have undergone feminine genital cutting (Andro and Lesclingrand, 2007). The medical risks of excision are high and relate to the reproductive, physical and psychological health of women. New migrations and the increase of countries legislating female genital cutting contributed to the internationalization of the practice and to the changes in its process. The loss of the ritual significance of excision and the increase as a concealed practice have clearly been noticed. In the meantime, the international movements fighting it, regional as well as national, amplify and put changes together in order to counter the new figure of female genital cutting. Burkina Faso does not make exception. The Government has clearly positioned itself in favour of movements fighting excision; and has set up many legal, political and economic devices in order to support their initiatives. In 2003, 77% of Burkina Faso women aged between 15 to 49 years old were declared excised. In parallel, a reduction in the practice for their daughter between 1998 and 2003 is noted as well as an increase in the number of Burkina Faso people declaring themselves against the practice. However in 2003, approximately 40% of women have performed or would like their daughter to undergo the cut; and approximately 24% of men and 26% of women are still encouraging the perpetuation of excision. This thesis’ first concern is the changes in the practice, and the knowledge and attitudes regarding excision between 1998 and 2003. Secondly, the attention is drawn on the socio-cultural, demographic and economic determinants indulging the doggedness of this practice within Burkina Faso people and on the concrete obstacles workers encounter on the ground. Therefore this research associates quantitative and qualitative methods. It combines statistic analysis of the Demographic and Health Surveys of 2003 and 1998 data; and analyzes data collected from talks made between October 1 st and 10th, 2005 with fields’ workers.

The DNA glycosylases function in the first step of the base excision repair (BER) process, that is responsible for removing base lesions resulting from oxidation, alkylation or deamination. The DNA glycosylases that recognize oxidative base damage fall into two general families: the Fpg/Nei family and the Nth superfamily. Based on protein sequence alignments, we identified four putative Fpg/Nei family members as well as a putative Nth protein in Mycobacterium tuberculosis H37Rv, the causative agent of tuberculosis. While Fpg proteins are widely distributed among the bacteria and plants, Nei homologs are sparsely distributed across phyla, and are only found in γ-proteobacteria, actinobacteria and metazoans. Interestingly, M. tuberculosis H37Rv harbors two proteins (Rv2464c and Rv3297) from the Nei clade and two (Rv2924c and Rv0944) from the Fpg clade. All four Fpg/Nei proteins were successfully overexpressed by using a novel bicistronic vector, which theoretically prevented stable mRNA secondary structure(s) surrounding the translation initiation region (TIR) thereby improving translation efficiency. Additionally, MtuNth (Rv3674c) was also overexpressed in soluble form. The substrate specificities of the purified enzymes were characterized in vitro with oligonucleotide substrates containing single lesions. Some were further characterized by gas chromatography/mass spectrometry (GC/MS) analysis of products released from γ-irradiated DNA. MtuFpg1 (Rv2924c) has a substrate specificity similar to that of EcoFpg and recognizes oxidized purines. Both EcoFpg and MtuFpg1 are more efficient at removing spiroiminodihydantoin (Sp) than 7,8-dihydro-8-oxoguanine (8-oxoG); however, MtuFpg1 has a substantially increased opposite base discrimination compared to EcoFpg. The Rv0944 gene encodes MtuFpg2, which contains only the C-terminal domain of an Fpg protein and has no detectable DNA binding activity or DNA glycosylase/lyase activity and thus appears to be a pseudogene. MtuNei1 (Rv2464c) recognizes oxidized pyrimidines not only on doublestranded DNA but also on single-stranded DNA. It also exhibits uracil DNA glycosylase activity as well as weak activity on FapyA and FapyG. MtuNth recognizes a variety of oxidized bases, such as urea, 5,6-dihydrouracil (DHU), 5-hydroxyuracil (5- OHU), 5-hydroxycytosine (5-OHC) and methylhydantoin (MeHyd) as well as FapyA, FapyG and 8-oxoadenine (8-oxoA). Both MtuNei1 and MtuNth excise thymine glycol (Tg); however, MtuNei1 strongly prefers the (5R) isomers of Tg, whereas MtuNth recognizes only the (5S) isomers. The other Nei paralog, MtuNei2 (Rv3297), did not demonstrate activity in vitro as a recombinant protein, but when expressed in Escherichia coli, the protein decreased the spontaneous mutation frequency of both the fpg mutY nei triple and nei nth double mutants, suggesting that MtuNei2 is functionally active in vivo recognizing both guanine and cytosine oxidation products. The kinetic parameters of the MtuFpg1, MtuNei1 and MtuNth proteins on selected substrates were also determined and compared to those of their E. coli homologs. Since pathogenic bacteria are often exposed to an oxidative environment, such as in macrophages, our data, together with previous observations, support the idea that the BER pathway is of importance in protecting M. tuberculosis against oxidative stress, as has been observed with other pathogens .

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Breast fibroadenomas account for approximately 25% of all lesions in asymptomatic women, resulting in large health care costs every year. There are 3 different variations of the disease: simple, juvenile giant and multiple. Patients may have different management and treatment options available to them depending on which variation they have. Of particular interest are female adolescents, who are at most risk for developing these lesions. With this age group not only is it important to pursue options that are minimally invasive and effective, but there are psychosocial implications to consider regarding the cosmetic changes that may occur with the disease, as well as generalized anxiety over having a breast lump. These issues are important to consider for physicians when recommending a treatment or management option. After a systematic review of all options available, it appears the best management method is the conservative treatment as it minimizes invasive intervention and operates on the principle that 10-40% of lesions regress on their own; however, there may be times that adolescents are uncomfortable with this treatment due to anxiety and other uneasiness about having a lesion remain in their breasts, despite the low chance of malignancy associated with breast fibroadenomas. Minimally invasive procedures are being developed in order to minimize possible iatrogenic injury to the developing breasts as well as maintain efficiency and good cosmesis post-procedure. Cryoablation is a minimally invasive technique utilizing extreme cold temperatures for lesion excision that is not currently widely used, however it has great potential to replace traditional open surgical excision.

Replication of HIV-1 is inhibited by azidothymidine (AZT), which leads to chain termination and inhibition of DNA synthesis. Resistance to AZT is frequently the result of mutations that increase the ability of RT to remove the chain-terminating nucleotides after they have been incorporated. It has been proposed that RNase H cleavage of the RNA template can occur when RT is stalled near the site of chain termination and contributes to the inhibition by causing the dissociation of the primer-template before the chain terminator can be excised. Mutations in the connection and RNase H domains of RT have been shown to increase excision. It has long been known that resistance to thymidine analogs is conferred by the mutations M41L, D67N, K70R, L210W, T215F/Y and T219Q/E in RT and that this resistance is suppressed by the additional presence of the M184V mutation. Changes in excision activity on DNA templates have been observed with these mutant RTs, but effects on RNase H cleavage resulting in indirect effects on excision activity is also possible with RNA templates. We used a 5'-labeled -3'-chain-terminated DNA primer annealed to either a DNA or RNA template to evaluate primer rescue activities, a 5'-labeled RNA template to evaluate RNA cleavage activity and a biotin-tagged chain-terminated oligodeoxynucleotide to monitor primer-template dissociation. We first investigated differences between RNA and DNA templates when the primers were chain terminated and observed a correlation between RNase H activities and template/primer (T/P) dissociation. An inverse correlation was observed between excision rescue rates and RNase H cleavages leading to T/P dissociation. We observed that the chain terminator (i.e. AZTMP or ddAMP) affected RNase H cleavages and excision rates with RNA template and dNTPs. When we investigated mutations in the N-terminal domain of RT associated with nucleoside reverse transcriptase inhibitor (NRTI) resistance we found that primer rescue was decreased when M184V was present in combination with thymidine analog mutations (TAMs) and the template was RNA with either ATP or PPi as excision substrate. RNase H cleavage at secondary cleavage sites (-7, -8) was substantially reduced with M41L/T215Y RT in comparison with wild type RT, and primer-template dissociation was decreased. In contrast, when M184V was present, RNase H cleavage at the secondary cleavage sites and dissociation of the primer-template occurred at higher levels and excision rescue was decreased. The ability of RT to rescue an AZT terminated primer in the presence of the 184V mutation was restored when the RNase H activity was inactivated by the RNase H negative mutation E478Q. Electromobility shift assay (EMSA) analysis of AZT-resistant mutant RT with M184V showed an increased Kd for formation of the ternary complex. These results suggest that RNase H-mediated RNA-DNA template-primer dissociation is influenced by mutations associated with thymidine analog resistance, and that suppression of resistance to nucleoside RT inhibitors by M184V may be partly explained by effects on RNase H cleavage that decrease the time available for excision to occur. This is the first time that mutations in the polymerase domain are shown to affect excision rescue through an RNase H-dependent mechanism.