<p>Spermatogenesen är i många organismer en av de mest dramatiska förvandlingar som en cell kan genomgå – en vanlig, rund, diploid cell omvandlas till en nålformad, haploid cell med ett tätt packat cellmaskineri. I bananfluga, <i>Drosophila melanogaster</i>, innebär denna process flera karaktäristiska stadier. Ett av dessa är det primära spermatocyt-stadiet, som infaller innan cellen påbörjar meios-delning. Stadiet karaktäriseras av en uppluckrad kromatinstruktur i cellens kärna och ovanligt höga transkriptions- och translationshastigheter, för att producera allt det mRNA och de proteiner som behövs senare under spermatidomvandlingen. Två av de proteiner som uttrycks i höga nivåer i primära spermatocyter är ThioredoxinT (TrxT) och Painting of fourth (POF).</p><p>Thioredoxiner är små proteiner som har som funktion att reducera disulfidbryggor i andra proteiner, en mekanism som används i många olika fysiologiska sammanhang. I denna avhandling visar jag att <i>TrxT</i>-genen kodar för ett testikelspecifikt thioredoxin som binder specifikt till Y-kromosom-loopar i primära spermatocyter. <i>TrxT</i>-genen ligger precis bredvid <i>deadhead</i> (<i>dhd</i>), en gen som kodar för ett hon-specifikt thioredoxin som är lokaliserat till cellkärnorna i flugans äggstockar. Ett tredje thioredoxin i <i>Drosophila</i> är det allmänt uttryckta Thioredoxin-2 (Trx-2). Jag har upptäckt att flugor som saknar Trx-2 är livsdugliga, men att de lever kortare än vildtypsflugor, medan flugor med extra mycket Trx-2 har en ökad tålighet mot oxidativ stress. Slående nog är en total avsaknad av alla tre thioredoxiner inte förenat med letalitet, tvärtemot vad man skulle kunnat vänta sig. Alla tre thioredoxiner finns i de olika <i>Drosophilider</i> som undersökts och den ovanliga genorganisation som delas av <i>TrxT</i> och <i>dhd</i> är generellt konserverad.</p><p>Gen-namnet <i>Painting of fourth</i> härstammar från upptäckten att POF binder till (”målar”) <i>Drosophilas</i> kromosom 4. Jag visar i min avhandling att POFs bindning till den fjärde kromosomen är bevarad i olika <i>Drosophila</i>-arter och att POF kolokaliserar med både ett protein och en histon-modifiering, som är förknippade med doskompensation, i arter där POF också binder till hanens X-kromosom. POF uttrycks överallt i både honor och hanar, men i väldigt höga nivåer i hanens testiklar. Jag visar här att POF finns i cellkärnan hos primära spermatocyter, men också i kärnan på mognande spermatider, och att avsaknad av POF in hanens könsceller orsakar en global nedreglering av gener som ligger på kromosom 4. Kombinationen av mina POF- resultat tyder på att POF har en viktig funktion i det första kända fallet av genreglering av en hel autosomal kromosom.</p> / <p>The process of spermatogenesis is in many organisms one of the most dramatic cellular transformations - a normal round diploid cell is ultimately transformed into a needle shaped haploid cell with tightly packaged cell machinery. In <i>Drosophila melanogaster</i> this process involves several characteristic stages, one of these being the primary spermato-cyte stage, which is the stage prior to meiosis. This stage is characterized by a loose chromatin structure in the nucleus and exceptionally high rates of transcription and translation to produce essentially all the mRNAs and proteins that are needed later during spermatid formation. Two proteins that are expressed in high levels in primary spermatocytes are ThioredoxinT (TrxT) and Painting of fourth (POF).</p><p>Thioredoxins are small thiol proteins that reduce disulfides in other proteins, a mechanism that is utilized in many different contexts. In this thesis I show that the <i>TrxT</i> gene encodes a testis-specific thioredoxin that specifically associates to Y-chromosome loops in primary spermatocytes. <i>TrxT</i> is located right next to <i>deadhead</i> (<i>dhd</i>), a gene that encodes a female-specific thioredoxin that specifically locates to nuclei in the ovaries. A third thioredoxin in <i>Drosophila</i> is the ubiquitously expressed Thioredoxin-2 (Trx-2). I have found that flies lacking Trx-2 are viable but have shorter life spans than wild type flies, while over-expression of Trx-2 mediates an increased resistance to oxidative stress. Interestingly, a lack of all three thioredoxins does not result in lethality, contrary to what could be expected. All three thioredoxins are conserved among <i>Drosophilids</i> and the striking genomic organization of <i>TrxT</i> and <i>dhd</i> is generally conserved.</p><p>The gene name <i>Painting of fourth</i> originates from the finding that POF stains the 4th chromosome of <i>Drosophila</i> in a banded pattern on salivary gland chromosomes. I show in this thesis that POF binding to the equivalent of the 4th chromosome is conserved in genus <i>Drosophila</i> and that POF co-localizes with both a protein and a histone modification associated with dosage compensation in species where POF also binds the male X. POF is expressed ubiquitously in both males and females, but at very high levels in male testes. I show that POF is present in nuclei of primary spermatocytes, but also in nuclei of maturing spermatids and that a lack of POF in the male germline causes a global down-regulation of chromosome 4 genes. These results combined suggest a function of POF in the first known case of chromosome-wide gene regulation of an autosome.</p>

Spermatogenesen är i många organismer en av de mest dramatiska förvandlingar som en cell kan genomgå – en vanlig, rund, diploid cell omvandlas till en nålformad, haploid cell med ett tätt packat cellmaskineri. I bananfluga, Drosophila melanogaster, innebär denna process flera karaktäristiska stadier. Ett av dessa är det primära spermatocyt-stadiet, som infaller innan cellen påbörjar meios-delning. Stadiet karaktäriseras av en uppluckrad kromatinstruktur i cellens kärna och ovanligt höga transkriptions- och translationshastigheter, för att producera allt det mRNA och de proteiner som behövs senare under spermatidomvandlingen. Två av de proteiner som uttrycks i höga nivåer i primära spermatocyter är ThioredoxinT (TrxT) och Painting of fourth (POF). Thioredoxiner är små proteiner som har som funktion att reducera disulfidbryggor i andra proteiner, en mekanism som används i många olika fysiologiska sammanhang. I denna avhandling visar jag att TrxT-genen kodar för ett testikelspecifikt thioredoxin som binder specifikt till Y-kromosom-loopar i primära spermatocyter. TrxT-genen ligger precis bredvid deadhead (dhd), en gen som kodar för ett hon-specifikt thioredoxin som är lokaliserat till cellkärnorna i flugans äggstockar. Ett tredje thioredoxin i Drosophila är det allmänt uttryckta Thioredoxin-2 (Trx-2). Jag har upptäckt att flugor som saknar Trx-2 är livsdugliga, men att de lever kortare än vildtypsflugor, medan flugor med extra mycket Trx-2 har en ökad tålighet mot oxidativ stress. Slående nog är en total avsaknad av alla tre thioredoxiner inte förenat med letalitet, tvärtemot vad man skulle kunnat vänta sig. Alla tre thioredoxiner finns i de olika Drosophilider som undersökts och den ovanliga genorganisation som delas av TrxT och dhd är generellt konserverad. Gen-namnet Painting of fourth härstammar från upptäckten att POF binder till (”målar”) Drosophilas kromosom 4. Jag visar i min avhandling att POFs bindning till den fjärde kromosomen är bevarad i olika Drosophila-arter och att POF kolokaliserar med både ett protein och en histon-modifiering, som är förknippade med doskompensation, i arter där POF också binder till hanens X-kromosom. POF uttrycks överallt i både honor och hanar, men i väldigt höga nivåer i hanens testiklar. Jag visar här att POF finns i cellkärnan hos primära spermatocyter, men också i kärnan på mognande spermatider, och att avsaknad av POF in hanens könsceller orsakar en global nedreglering av gener som ligger på kromosom 4. Kombinationen av mina POF- resultat tyder på att POF har en viktig funktion i det första kända fallet av genreglering av en hel autosomal kromosom. / The process of spermatogenesis is in many organisms one of the most dramatic cellular transformations - a normal round diploid cell is ultimately transformed into a needle shaped haploid cell with tightly packaged cell machinery. In Drosophila melanogaster this process involves several characteristic stages, one of these being the primary spermato-cyte stage, which is the stage prior to meiosis. This stage is characterized by a loose chromatin structure in the nucleus and exceptionally high rates of transcription and translation to produce essentially all the mRNAs and proteins that are needed later during spermatid formation. Two proteins that are expressed in high levels in primary spermatocytes are ThioredoxinT (TrxT) and Painting of fourth (POF). Thioredoxins are small thiol proteins that reduce disulfides in other proteins, a mechanism that is utilized in many different contexts. In this thesis I show that the TrxT gene encodes a testis-specific thioredoxin that specifically associates to Y-chromosome loops in primary spermatocytes. TrxT is located right next to deadhead (dhd), a gene that encodes a female-specific thioredoxin that specifically locates to nuclei in the ovaries. A third thioredoxin in Drosophila is the ubiquitously expressed Thioredoxin-2 (Trx-2). I have found that flies lacking Trx-2 are viable but have shorter life spans than wild type flies, while over-expression of Trx-2 mediates an increased resistance to oxidative stress. Interestingly, a lack of all three thioredoxins does not result in lethality, contrary to what could be expected. All three thioredoxins are conserved among Drosophilids and the striking genomic organization of TrxT and dhd is generally conserved. The gene name Painting of fourth originates from the finding that POF stains the 4th chromosome of Drosophila in a banded pattern on salivary gland chromosomes. I show in this thesis that POF binding to the equivalent of the 4th chromosome is conserved in genus Drosophila and that POF co-localizes with both a protein and a histone modification associated with dosage compensation in species where POF also binds the male X. POF is expressed ubiquitously in both males and females, but at very high levels in male testes. I show that POF is present in nuclei of primary spermatocytes, but also in nuclei of maturing spermatids and that a lack of POF in the male germline causes a global down-regulation of chromosome 4 genes. These results combined suggest a function of POF in the first known case of chromosome-wide gene regulation of an autosome.

Rearranjos cromossômicos estruturais estão potencialmente associados ao desenvolvimento de doenças genéticas devido à disrupção, inativação ou alteração da dosagem gênica. O objetivo deste projeto foi realizar a caracterização genômica de duas pacientes e seus familiares portadores de rearranjo cromossômico estrutural envolvendo o braço curto do cromossomo 4 e o braço longo do cromossomo 12, associando técnicas de citogenética clássica (bandamento GTG), citogenética molecular (FISH) e citogenômica (array-CGH), para definição diagnóstica e maior conhecimento sobre os fatores envolvidos na correlação cariótipo-genótipo-fenótipo. Foram avaliados seis indivíduos, duas pacientes, primas em primeiro grau que apresentavam alterações fenotípicas, assim como seus familiares, portadores de translocação aparentemente equilibrada e fenótipo normal. Apesar das duas pacientes apresentarem alteração cromossômica comum, derivativo do cromossomo 4 [der(4)], foram observados achados fenotípicos distintos. A investigação permitiu a definição do diagnóstico de deleção 4p16 e trissomia 12qter para as duas pacientes com fenótipo alterado e cariótipo 46,XX,der(4)t(4;12)(p16;q24.3), a definição precisa dos pontos de quebra em 4p16.3 e 12q24.31->q24.33, assim como a determinação da origem parental do rearranjo e a definição do diagnóstico citogenético final de quatro portadores de translocação aparentemente equilibrada e cariótipo t(4;12)(4pter->4p16.3::2q24.31->12qter;12qter->12q24.31::4p16.3->4pter),direcionando o aconselhamento genético para a família. Nas duas pacientes, a técnica de array-CGH (Plataforma 2x400K, Agilent®) detectou uma diferença sutil de tamanho entre as perdas e ganhos referentes aos cromossomos envolvidos no rearranjo, sendo diagnosticado em P1 uma perda de 2.707.221 pb na citobanda 4p16.3, além de um ganho de 12.405.205 pb em 12q24.31->q24.33. A paciente 2 apresentou uma perda de 2.710.969 pb em 4p16.3 e um ganho de 12.393.885 pb em 12q24.31->q24.33. Ambas as regiões de desequilíbrio genômico incluem genes que podem ser relevantes para manifestação fenotípica observada nas pacientes, entre eles: WHSC1, NELFA, LETM1, FGFRL1 e SPON2. Os resultados da investigação citogenômica indicaram, ainda, a presença de translocação equilibrada nos quatro indivíduos portadores, não sendo detectadas perdas e/ou ganhos genômicos nas regiões dos pontos de quebra cromossômica. Os resultados obtidos na investigação do padrão de metilação dos genes FGFRL1 e SPON2 não permitiram afirmar que uma provável repressão da expressão gênica devido ao imprinting materno e paterno esteja associada às características fenotípicas distintas observadas nas duas pacientes. Embora tenha sido possível a indicação de genes correlacionados ao fenótipo das pacientes, a correlação entre a alteração genética e o fenótipo das mesmas pode depender da ação sinérgica dos mais de 190 genes envolvidos neste rearranjo cromossômico estrutural familiar. / Structural chromosomal rearrangements are potentially associated with the development of genetic disorders due to disruption, inactivation or gene dosage alterations. The objective of this project was to perform the genomic characterization of a familial structural chromosomal rearrangement involving the short arm of chromosome 4 and the long arm of chromosome 12 in two patients and carriers. The experimental approach involved using a combination of classical cytogenetic techniques (GTG banding), molecular cytogenetics (FISH) and cytogenomics (array-CGH), to provide a diagnostic definition and a better understanding of how changes in the karyotype and genotype may be associated with the phenotype. Six individuals were evaluated, two patients with phenotypic abnormalities, as well as the carriers of an apparently balanced 4p;12q translocation with normal phenotypes. Although the two patients showed a common chromosomal abnormality, the derivative chromosome 4 [der (4)], they presented distinct phenotypic findings. The investigation provided a definition of the diagnosis of 4p16 deletion and trisomy 12qter for the two patients with abnormal phenotypes and a karyotype 46,XX,der(4)t(4;12)(p16;q24.3). In addition a precise definition of the breakpoints at 4p16.3 and 12q24.31->q24.33, and the parental origin of the rearrangement was determined. A precise definition of the cytogenetic diagnosis of four carriers with an apparently balanced translocation and karyotype t(4;12)(4pter->4p16.3::2q24.31->12qter; 12qter 12q24.31->4pter::4p16.3), facilitated the genetic counseling for the family. In both patients, the array-CGH technique (2x400K Platform, Agilent®) detected a subtle difference in size between losses and gains in the chromosomal regions involved in the rearrangement. Patient 1 presented a loss of 2,707,221 bp in the cytoband 4p16.3, and a gain of 12,405,205 bp in 12q24.31->q24.33. Patient 2 had a loss of 2,710,969 bp in 4p16.3 and a gain of 12,393,885 bp in 12q24.31 -> q24.33. Both regions of genomic imbalance included genes that may be relevant to phenotypic findings observed in our patients, including: WHSC1, NELFA, LETM1, FGFRL1 and SPON2. Genomic findings also confirmed the presence of a balanced translocation in four carriers, with no genomic losses and/or gains in the regions of chromosome breakpoints. The results of the investigation of the methylation pattern of FGFRL1 and SPON2 genes could not demonstrate that repression of gene expression due to maternal and paternal imprinting was associated with the distinct phenotypes observed in the two patients. Although it has been possible to indicate genes related to the phenotype of the patients, the correlation between the genetic alteration and phenotype may depend on the synergistic action of multiple genes from more than the 190 involved in this familial chromosomal rearrangement.

Rearranjos cromossômicos estruturais estão potencialmente associados ao desenvolvimento de doenças genéticas devido à disrupção, inativação ou alteração da dosagem gênica. O objetivo deste projeto foi realizar a caracterização genômica de duas pacientes e seus familiares portadores de rearranjo cromossômico estrutural envolvendo o braço curto do cromossomo 4 e o braço longo do cromossomo 12, associando técnicas de citogenética clássica (bandamento GTG), citogenética molecular (FISH) e citogenômica (array-CGH), para definição diagnóstica e maior conhecimento sobre os fatores envolvidos na correlação cariótipo-genótipo-fenótipo. Foram avaliados seis indivíduos, duas pacientes, primas em primeiro grau que apresentavam alterações fenotípicas, assim como seus familiares, portadores de translocação aparentemente equilibrada e fenótipo normal. Apesar das duas pacientes apresentarem alteração cromossômica comum, derivativo do cromossomo 4 [der(4)], foram observados achados fenotípicos distintos. A investigação permitiu a definição do diagnóstico de deleção 4p16 e trissomia 12qter para as duas pacientes com fenótipo alterado e cariótipo 46,XX,der(4)t(4;12)(p16;q24.3), a definição precisa dos pontos de quebra em 4p16.3 e 12q24.31->q24.33, assim como a determinação da origem parental do rearranjo e a definição do diagnóstico citogenético final de quatro portadores de translocação aparentemente equilibrada e cariótipo t(4;12)(4pter->4p16.3::2q24.31->12qter;12qter->12q24.31::4p16.3->4pter),direcionando o aconselhamento genético para a família. Nas duas pacientes, a técnica de array-CGH (Plataforma 2x400K, Agilent®) detectou uma diferença sutil de tamanho entre as perdas e ganhos referentes aos cromossomos envolvidos no rearranjo, sendo diagnosticado em P1 uma perda de 2.707.221 pb na citobanda 4p16.3, além de um ganho de 12.405.205 pb em 12q24.31->q24.33. A paciente 2 apresentou uma perda de 2.710.969 pb em 4p16.3 e um ganho de 12.393.885 pb em 12q24.31->q24.33. Ambas as regiões de desequilíbrio genômico incluem genes que podem ser relevantes para manifestação fenotípica observada nas pacientes, entre eles: WHSC1, NELFA, LETM1, FGFRL1 e SPON2. Os resultados da investigação citogenômica indicaram, ainda, a presença de translocação equilibrada nos quatro indivíduos portadores, não sendo detectadas perdas e/ou ganhos genômicos nas regiões dos pontos de quebra cromossômica. Os resultados obtidos na investigação do padrão de metilação dos genes FGFRL1 e SPON2 não permitiram afirmar que uma provável repressão da expressão gênica devido ao imprinting materno e paterno esteja associada às características fenotípicas distintas observadas nas duas pacientes. Embora tenha sido possível a indicação de genes correlacionados ao fenótipo das pacientes, a correlação entre a alteração genética e o fenótipo das mesmas pode depender da ação sinérgica dos mais de 190 genes envolvidos neste rearranjo cromossômico estrutural familiar. / Structural chromosomal rearrangements are potentially associated with the development of genetic disorders due to disruption, inactivation or gene dosage alterations. The objective of this project was to perform the genomic characterization of a familial structural chromosomal rearrangement involving the short arm of chromosome 4 and the long arm of chromosome 12 in two patients and carriers. The experimental approach involved using a combination of classical cytogenetic techniques (GTG banding), molecular cytogenetics (FISH) and cytogenomics (array-CGH), to provide a diagnostic definition and a better understanding of how changes in the karyotype and genotype may be associated with the phenotype. Six individuals were evaluated, two patients with phenotypic abnormalities, as well as the carriers of an apparently balanced 4p;12q translocation with normal phenotypes. Although the two patients showed a common chromosomal abnormality, the derivative chromosome 4 [der (4)], they presented distinct phenotypic findings. The investigation provided a definition of the diagnosis of 4p16 deletion and trisomy 12qter for the two patients with abnormal phenotypes and a karyotype 46,XX,der(4)t(4;12)(p16;q24.3). In addition a precise definition of the breakpoints at 4p16.3 and 12q24.31->q24.33, and the parental origin of the rearrangement was determined. A precise definition of the cytogenetic diagnosis of four carriers with an apparently balanced translocation and karyotype t(4;12)(4pter->4p16.3::2q24.31->12qter; 12qter 12q24.31->4pter::4p16.3), facilitated the genetic counseling for the family. In both patients, the array-CGH technique (2x400K Platform, Agilent®) detected a subtle difference in size between losses and gains in the chromosomal regions involved in the rearrangement. Patient 1 presented a loss of 2,707,221 bp in the cytoband 4p16.3, and a gain of 12,405,205 bp in 12q24.31->q24.33. Patient 2 had a loss of 2,710,969 bp in 4p16.3 and a gain of 12,393,885 bp in 12q24.31 -> q24.33. Both regions of genomic imbalance included genes that may be relevant to phenotypic findings observed in our patients, including: WHSC1, NELFA, LETM1, FGFRL1 and SPON2. Genomic findings also confirmed the presence of a balanced translocation in four carriers, with no genomic losses and/or gains in the regions of chromosome breakpoints. The results of the investigation of the methylation pattern of FGFRL1 and SPON2 genes could not demonstrate that repression of gene expression due to maternal and paternal imprinting was associated with the distinct phenotypes observed in the two patients. Although it has been possible to indicate genes related to the phenotype of the patients, the correlation between the genetic alteration and phenotype may depend on the synergistic action of multiple genes from more than the 190 involved in this familial chromosomal rearrangement.